Polyamines in mammalian biology and medicine.

Polyamines in Mammalian Biology and Medicine H. Guy Williams-Ashman, Zoe N. Canellakis Perspectives in Biology and Medicine, Volume 22, Number 3, Spring 1979, pp. 421-453 (Article) Published by Johns Hopkins University Press DOI: https://doi.org/10.1353/pbm.1979.0013

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POLYAMINES IN MAMMALIAN BIOLOGY AND MEDICINE* H. GUY WILUAMS-ASHMAm AND ZOE N. CANEUAKISt

Spermine, spermidine, and putrescine are three aliphatic amines of widespread biological occurrence whose molecular structures (fig. 1) were established over 50 years ago. Yet until past the middle of this century these polyamines were seldom allotted more than a line or two in the textbooks and were generally considered only as curios of physiological chemistry. In the interim, research on polyamines has blossomed into an astonishingly vigorous enterprise.

H3N(CHj)4NH3 Putrescine

H3N(CH2)4NH2(CH2)3NH3 Spermidine

H3N(CH2)3NH2(CH2)4NH2(CH2)3NH3

Spermine Fig. 1.—The molecular structure of the three major polyamines in mammalian cells. All

of the primary and secondary amine groups are shown in the protonated form, as occurs under physiological conditions.

Some major results of recent investigations may be highlighted (Note 1). AU prokaryotes and eukaryotes manufacture putrescine and sper-

midine, whereas spermine production is largely confined to nucleated eukaryotic cells. A number of pathways for the biosynthesis and ?Studies from the authors' laboratories were supported by NIH research grants HD04592 and CA-16359. We are grateful to Ms. D. WaIz for excellent help in the preparation of the manuscript. This essay is dedicated to the memory of Chandrakant V. Dave. tBen May Laboratory for Cancer Research, Departments of Biochemistry and of Phar-

macological and Physiological Sciences, University of Chicago, Chicago, Illinois 60637.

fVeterans Administration Hospital, West Haven, Connecticut 06516, and Departments of Pharmacology and Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.

© 1979 by The University of Chicago. 0031-5982/79/2203-0072$01.00

Perspectives in Biology and Medicine · Spring 1979 \ 421

metabolism of polyamines have been uncovered in animals, plants, and microorganisms. Various polyamine biosynthetic enzymes are exqui-

sitely regulated by innumerable agents that stimulate cell growth and

differentiation, with resulting fluctuations in cellular polyamine concentrations. In cell-free systems, polyamines form tight, noncovalent com-

plexes with many biomolecules, and especially with various nucleic acids, thereby influencing many reactions involved in the formation, turnover, and postsynthetic modification of DNA, RNA, and protein molecules. Spermine, spermidine, and putrescine also directly modulate, or participate in, other diverse metabolic processes and exert stabilizing effects on cell and organelle membranes. And clinical studies suggest that

alterations in polyamines in some human body fluids may aid in the diagnosis or prognostic evaluation of certain diseases. Despite all this, many challenging questions in polyamine physiology remain unanswered. Here we shall dwell on several of these problems in the context of mammalian biology and medicine, with emphasis on neglected lines of research.

I. Conformationally Flexible Polycations As its epithet implies, spermine was originally discovered in semen. Notwithstanding the large amounts of polyamines in the seminal plasma

of certain species, these substances are preeminently of intracellular significance in mammals. Polyamine levels in blood plasma and many other body fluids except semen are extremely low. Spermidine and spermine

concentrations in most tissues lie within the range of 0.1-2 /¿mol per gram fresh weight; notable exceptions are the pancreas and the prostate gland in which higher values are encountered in some creatures. The

spermidine/spermine ratio is frequently, but not invariably, highest in cells undergoing rapid growth or differentiation, and tends to drop in many tissues with advancing age or as a result of involution of many hormone-dependent organs precipitated by hormonal deprivation. Intracellular putrescine concentrations are strikingly lower than those of spermidine and spermine: values of less than 0. 1 /nmol of putrescine per gram are usually observed in resting mammalian cells, and hardly ever exceed thrice this value in stimulated or malignant tissues. The levels and turnover rates of the three amines in higher animal cells can fluc-

tuate enormously—often independently of each other and sometimes in a polyphasic fashion—in response to hormones, drugs, viral infections, and other factors.

Assessment of polyamine functions in mammalian cells would obviously be facilitated by knowledge of their intracellular distributions. Un-

fortunately, technical difficulties have stood in the way of obtaining trustworthy data. Many attempts to study the associations of polyamines 422 I H. Guy Williams-Ashman and Zoe N. Canellakis ¦ Polyamines in Biology and Medicine

with subcellular fractions of homogenized tissues have been vitiated by uncontrollable redistribution artifacts. The only chemically reactive groups in polyamine molecules are primary and secondary amine functions, and this has thwarted development of specific histochemical tech-

niques. Likewise, autoradiographic experiments involving exposure of

cells to exogenous radioisotopically labeled polyamines are hard to interpret because interconversions of putrescine, spermidine, and sperm-

ine as well as their transformations to other substances occur with

celerity, and also because endogenous polyamines already bound to intracellular structures may not always be freely exchangeable. It is difficult to draw the line between fact and fantasy with regard to many

reports that polyamines in vivo may be preferentially bound to struc-

tures such as chromatin, nucleoli, and cytoplasmic ribosomes. Needless to add, the intracellular districts where any substance may be predominantly sequestered may not necessarily represent the chief sites of its physiological actions. Two physicochemical attributes of polyamines are germane to their interactions with cellular constituents. The first is that, under physio-

logical circumstances, all of the primary and secondary amino groups of putrescine, spermidine, and spermine are very largely protonated, so that they exhibit net charges close to 2+, 3 + , and 4+, respectively. Second, rotations around the carbon-carbon and carbon-nitrogen bonds impart considerable conformational flexibility on these polycations.

Certain fairly extended conformations of spermidine and spermine are such that the protonated nitrogens of the trimethylene portion(s) can interact with adjacent phosphate groups on one chain, while the tetra-

methylene portion bridges to a phosphate on the other strand of double helical regions of a nucleic acid. When it associates firmly with some regions of certain crystalline tRNA molecules, spermine may assume a more hooked conformation. The secondary and tertiary structures

of all sorts of polynucleotides can be profoundly affected by polyamines.

It is these effects of polyamines on nucleic acid conformations, rather than on the requisite enzymes, that seem to account for many of the direct in vitro actions of polyamines on various DNA and RNA polymerases, methylases, nucleotidyl transferases, and hydrolases, and on reactions involving tRNA, ribosomal RNA, and mRNA molecules that

contribute to polyribosomal protein synthesis. The specificity as well as the kinetics of some of these reactions can be modulated by spermidine and spermine in vitro. For instance, spermidine both enhances the cleavage of polyuridylate and provokes an otherwise negligible hydrolysis

of polycytidylate by RNases from several sources. And polyamines can selectively influence the copying of one strand of DNA templates by RNA polymerases, and also the translation of specific mRNAs in isolated polyribosomal systems. The multitudinous effects that polyamines exert Perspectives in Biology and Medicine ¦ Spring 1979 | 423

on cell-free systems that synthesize, degrade, and modify nucleic acids, or utilize them for protein biosynthesis, may be either positive or negative in direction, depending not only on the concentrations of spermidine

and spermine but also on many other factors, including ionic strength, pH, temperature, the conformational potentialities of the polynucleo-

tides, and, very importantly, the ambient levels of Mg ions. Although requirements for Mg++ usually cannot be totally replaced by polyamines, the direct effects of spermidine and spermine on such macromolecular biosynthetic processes often exhibit a strong dependency on Mg++.

A key distinction between metallic and organic polyamine cations should be emphasized. Even if they undergo intracellular re-

compartmentalizations, Mg++ and other metal cations within cells are ultimately derived from the circulation, and their levels are contingent

upon their diffusion or transport across biological membranes. By contrast, polyamines are fabricated within the cells that contain them by

enzymatic reactions regulated by many factors; this permits adjustment of intracellular polyamine concentrations to meet physiological needs. There are attractions to the belief that many of the functions of

polyamines may relate to their serving as preferred cations for complexing with nucleic acids. Thus, in vitro effects of polyamines on RNA polymerase reactions in isolated nuclei, and on RNA stability, have been construed as germane to the roughly parallel accumulation of RNA and spermidine observed in many tissues stimulated to grow and differ-

entiate. However, as considered below, other lines of evidence do not

support a cause-and-effect relationship between RNA or protein synthesis and new polyamine production in some types of stimulated cells, although polyamine regulation of progression through the DNA synthetic (S) phase of the cell cycle may possibly occur. Nonetheless it is

striking that the only types of mammalian cells containing little or no polyamines are either those which have no (functional) nuclei (mature

erythrocytes and platelets) or those in which nuclear DNA replication and RNA transcription as well as extramitochondrial protein synthesis are irreversibly switched off, as in ejaculated spermatozoa.

Nevertheless, whereas disavowal of the importance of polyamine con-

trol of key steps in polynucleotide and protein synthesis is absurd, there is insufficient evidence to prove that these macromolecular biosynthetic

domains represent the really paramount sites of polyamine action in all mammalian cells. Thus, some of the in vitro effects of spermidine and spermine on diverse nucleic acid polymerase reactions, especially when

studied with naked nucleic acid templates, may be little more than testtube artifacts from a physiological standpoint, despite their inherent enzymological interest. Considerations of polyamines acting as counter ions for nucleic acids in vivo must be tempered by the fact that nuclear

DNA and ribosomal RNA in cells are largely complexed by unique pro-

424 I H. Guy Williams-Ashman and Zoe N. Canellakis ¦ Polyamines in Biology and Medicine

teins of the chromatin and ribosomes, respectively. Again, the possible influence of polyamines on generation of ribo- and deoxyribonucleoside triphosphate precursors of RNA and DNA is virtually unexplored, as are potential polyamine requirements for the actions of mammalian proteins that specifically unwind DNA strands or otherwise affect DNA conformations. Many other reactions related to nucleic acid

synthesis and functions can be added to this list. Furthermore, the properties of many enzymes and substrates of carbohydrate, fat, and amino acid metabolism, and also of certain biomembranes, can be influenced

profoundly by physiological concentrations of spermine and spermidine. Recent studies also hint that posttranslational modification of

proteins via covalent attachment of polyamines promoted by transamidating enzymes may be physiologically significant. Expansion of the big inventory of biochemical processes directly affected by polyamines

will surely not diminish in the near future. And as we shall discuss later,

the heightened polyamine accumulation accompanying cell development might occasionally represent an epiphenomenon masking a more functional increase in production of 5'-methylthioadenosine, the byproduct of spermidine and spermine synthesis. To inquire "What is the biological function of polyamines?" is almost like asking the same question about potassium. Maximal effects of spermine on biochemical processes in cell-free systems are frequently manifest at lower molar concentrations than are

required for spermidine, doubtless for reasons of charge and conformation. An intriguing but neglected conundrum is whether spermine can exert qualitatively specific regulatory actions which are not elicited by

spermidine. To put it in another way: Is spermine a luxury biomolecule whose biological properties can be entirely replaced by spermidine? A potentially fruitful way to attack this problem would be to develop mutant cells with specific defects in the different steps of polyamine biosynthesis, particularly with regard to the spermine synthase reaction. Unfortunately, although well-characterized mutants of yeasts and bacteria that are deficient in various polyamine biosynthetic enzymes are available, comparable mutants of mammalian cells are not. And we are not

aware of any mammalian cell lines for which spermine but not spermidine or putrescine can act as a specific growth factor. Pathways for the biosynthesis and turnover of polyamines must now be overviewed before we can consider attempts to alter cellular polyamine levels by drugs that depress polyamine formation.

II. Biosynthetic Pathways Polyamines can be regarded as metabolic derivatives of the amino

acids L-ornithine and L-methionine. Putrescine, the precursor of spermidine and spermine, is formed in mammalian cells by direct dePerspectives in Biology and Medicine ¦ Spring 1979 | 425

carboxylation of ornithine. (Additional pathways for putrescine production occur in certain bacteria and higher plants.) Spermidine and spermine, respectively, contain one and two aminopropyl groups derived

from methionine and attached to amine moieties of putrescine. The only well-established pathway for mammalian polyamine biosynthesis involves the four linked enzymatic processes summarized in figure 2. The decarboxylases catalyzing the first two reactions are usually rate limiting for spermidine and spermine synthesis and are very tightly regulated at the levels of protein turnover and by other means. In nearly all mammalian cells, the four enzymes are localized almost exclusively in the soluble part of the cytoplasm. Before we can talk about the enzymology and regulation of each of the reactions in figure 2, the origin of the two principal precursors, L-ornithine and S-adenosyl-L-methionine, must be briefly considered.

The enzyme arginase catalyzes hydrolysis of L-arginine to L-ornithine

and urea. There is considerable evidence that mammalian arginases play an important role in the provision of ornithine for polyamine biosynthesis. However, in those many tissues which do not detoxify ammonia

via operation of a complete urea cycle yet exhibit appreciable arginase

activity, it is unclear how far ornithine abstracted as such from blood plasma is utilized as a precursor of polyamines.

In order to participate in spermidine and spermine biosynthesis, L-methionine must first be converted into S-adenosyl-L-methionine METHIONINE + ATP

NH1 NH2(CH2)JCH-COOh ORNITHINE

NH2(CHt)4NH2

HOOC

PUTRESCINE OH

OHOH

adenosylmethionine

OH

DECARBOXYLATED ADENOSYLMETHIONINE

(D

NH2(CH2I4NH(CH2I3NH SPERMIDINE decarboxylated

adenosylmethionine OH

OH

NH2(CH2), NH(CH1I4NH(CH1I5NH2

METHYLTHI0ADEN0SINESPERMINE

Fig. 2.—Biosynthesis of putrescine, spermidine, and spermine in mammalian tissues


(AMe)1 as catalyzed by methionine adenosy!transferase, which employs ATP as the second substrate. At least in liver, the supply of methionine is

probably rate limiting for AMe formation, although methionine adenosyltransferase activities are influenced by a variety of factors, including age and hormones. There is evidence that intracellular AMe concentrations are not of central importance in the regulation of hepatic polyamine metabolism. Besides serving as a precursor of spermidine and spermine, AMe is the methyl group donor for about 100 known transmethylating enzymes, many of which are powerfully inhibited by their reaction product S-adenosylhomocysteine. However, the latter substance has no significant influence on any of the mammalian polyamine

biosynthetic enzymes except spermine synthase, which it inhibits at fairly high concentrations (Note 2). Apart from being utilized in polyamine synthase, methyltransferase, and other reactions via its conversion to AMe, methionine plays a dual role in protein biosynthesis, since it is not

only inserted into internal positions in innumerable proteins, but also is

involved in initiations of the synthesis of all protein chains directed by mRNA templates. DECARBOXYLATION OF ORNITHINE

The ornithine decarboxylase (ODC) reaction is catalyzed by pyridoxal phosphate-requiring enzymes from which this coenzyme easily dissociates. The ODC activity of most resting adult mammalian tissues, with the notable exception of the prostate, is extremely low. Increases in ODC

activity of up to several hundredfold occur during the early phases of cellular responses to all sorts of hormones and a huge array of other

agents that stimulate cell growth or differentiation. A remarkable attribute of ODCs in mammalian cells is their very fast rate of turnover, as

indicated by rapid decline in activity following treatment with inhibitors of protein biosynthesis. In many tissues, the apparent half-life of ODC is

less than 20 min, which is shorter than that of any other enzyme as yet examined. The half-life values for ODC depend on many factors, including the particular type of cell, the stage of induction of the enzyme, and pharmacologic influences.

Several groups have reported the separation of multiple forms of ODC from tissues such as regenerating or drug-treated liver and normal or malignant cells grown in culture. There are reports that different

'The abbreviations used are: AMe, S-adenosyl-L-methionine; ODC, ornithine decarboxylase; ODCIF, ornithine decarboxylase inactivating factor; AMeDC,

S-adenosylmethionine decarboxylase; MTA, 5'-methylthioadenosine; DFMeOrn, a-difluoromethylornithine; MGBG, methyl glyoxal bis(guanylhydrazone), or l,l'-[(methylethanediylidene)dinitrilo]-diguanidine; MBAG, 1, l'-[(methylethanediylidene)dinitrilo]bis(3-aminoguanidine); GABA, •y-aminobutyrate; a-MO, a-methylornithine, NADPH, reduced nicotinamide adenine dinucleotide phosphate.

Perspectives in Biology and Medicine ¦ Spring 1979 | 427

forms of ODC can vary in their affinities for pyridoxal phosphate (but hardly in their molecular sizes) and that their relative proportions in

certain cell types alter in response to growth stimulants. Even though definitive proof is lacking, a likely explanation for such apparent multi-

plicities of ODCs is that they result from posttranslational modifications. There is no evidence for the existence of true isoenzymic variants of mammalian ODCs that are encoded by entirely different genes, as oc-

curs in some bacteria. Despite experimental pointers to participation of cAMP-dependent protein phosphokinases in the induction of ODC under certain biological circumstances, this may relate to ODC biosynthesis rather than catalytic activity, and we are cognizant of only unsuccessful attempts to demonstrate ODC regulation at the level of phos-

phorylation of the enzyme protein. It is pointless to speculate about many other imaginable posttranslational modifications of ODC in this context, if only because pertinent studies have been hampered by difficulties in obtaining sufficiently large amounts of the enzyme in a homogeneous form. It should be pointed out that in some recent studies on the separation of apparent multiple forms of mammalian ODCs rela-

tively high concentrations of pyridoxal phosphate were added to all of the fractionation media for stabilization purposes, a maneuver which

could well produce artifacts, because formation of Schiff bases might

occur with many lysine residues that are not involved in coenzyme bind-

ing or catalytic action. Specific ODC antibodies developed in several institutions have been

used to correlate immunoreactive protein with decarboxylase activity

measurements in tissue extracts. Z. N. Canellakis and Theoharides

found that enhancement of ODC activity in cultured rat HTC hepatoma

cells evoked by dibutyryl cAMP or dexamethasone, as well as in rat liver

after partial hepatectomy, was accompanied by rises in labeled leucine incorporation into protein precipitable by high-titer antibodies mono-

specific to rat liver ODC. It was also observed (i) the same antibodies

cross-reacted with rat thyroid ODC and (ii) thyroid tissue stimulation by thyrotrophin plus isobutylmethyl xanthine in vitro concordantly elevated ODC activity and the corresponding immunoprecipitable protein. Employing an immunoadsorption technique, Hölttä likewise demonstrated parallelism in ODC activity enhancement and immunoreactive enzyme protein in liver following partial hepatectomy or somatotropin

treatment, and also during rapid decay of ornithine decarboxylation in regenerating livers after cycloheximide injection. Again, Obenrader and Prouty reported that their antibodies to a purified preparation of rat liver ODC cross-reacted with the enzyme from other tissues including prostate and kidney; immunotitration experiments indicated that dur-

ing the cycloheximide-induced decline of ODC in thioacetamide-treated 428 I H. Guy Williams-Ashman and Zoe N. Canellakis ¦ Polyamines in Biology and Medicine

rat river, the apparent half-life of decarboxylase activity was slightly

shorter than that of antigen (Note 3). The latter discrepancy accords with the notion that activities of ODC

or other enzymes in tissues reflect a balance between the rates of their synthesis and degradation, assuming that loss of catalytic power without

disappearance of immunoreactivity represents an initial step in the

intracellular breakdown. There is substantial evidence that protein

structure is an important determinant of protein half-lives in animal cells; that many proteins in their native conformations are less susceptible to proteolysis than their denatured counterparts; and that enzymes can often be protected against proteolytic attack by their substrates, coenzymes, or inhibitors. However, the detailed mechanisms of degradation of individual enzymes or other proteins in mammalian cells remain

poorly understood, especially from the standpoints of what processes are rate limiting and the extent to which lysosomal or other proteinases are involved. Experiments indicating a requirement for ATP for protein

breakdown in vivo are among the many phenomena that still defy cogent explanation. It is frequently remarked that enzymes like ODC which turn over with extreme rapidity would be expected to exhibit the greatest activity increases following nonspecific enhancement of tissue protein synthesis. But this cannot be the only factor that determines the

specificity and extent of ODC induction because, as considered below,

AMe decarboxylase also exhibits a remarkably high turnover rate yet does not rise in many tissues responding to some agents that greatly elevate ODC.

Crude and purified ODC preparations from many mammalian tissues are strongly activated by low molecular weight thiol compounds. With the enzyme from regenerating liver and prostate, saturating concentra-

tions of dithiols such as dithiothreitol appear to elicit greater decarboxylase activities in comparison with monothiols like glutathione and 2-mercaptoethanol. Gel nitration and sucrose gradient analyses indicate that in the absence of thiols rat prostate ODC polymerizes to larger but catalytically inactive forms that regain decarboxylase activity

on addition of dithiothreitol. Nothing is known about the nature of intermolecular or intramolecular disulfide linkages that may be present in native or denatured ODC. Glutathione can activate mammalian ODC

in vitro. But whether glutathione or other natural low molecular weight thiols are involved in maintaining ODC sulfhydryl groups in vivo by thiol-disulfide interchanges, and whether such putative reactions may be enzyme catalyzed, needs to be settled. Of related interest is recent evi-

dence suggesting that what has often been called the NADPHdependent "protein disulfide reductase" may be identifiable with the thioredoxin-thioredoxin reductase system. The latter is involved in Perspectives in Biology and Medicine ¦ Spring 1979 | 429

other biological reductions, particularly the ribonucleotide reductase

systems that convert ribonucleoside diphosphates to deoxyribonucleoside diphosphates, a key reaction in the generation of de-

oxyribonucleoside triphosphate substrates for DNA synthesis. It would

be interesting to determine whether thioredoxin and its reductase can regulate mammalian ODC activity in a manner similar to low molecular

weight thiols and to examine alterations in the sulfhydryl-disulfide status of the enzyme as possible initial steps in its intracellular degradation (Note 4).

Mammalian ODCs do not exhibit allosteric behavior, being remarkably insusceptible to direct regulation by small biomolecules other than thiols. These enzymes are not affected by many substrates participating in neighboring biochemical pathways, or by various nucleotides that profoundly affect bacterial ODC. Putrescine and, to a lesser extent, spermidine are feeble direct competitive inhibitors of animal ODCs, but the inhibition constants for these amines are sufficiently high to suggest that the inhibitions are physiologically insignificant. By contrast, in both

intact animals and cultured cells, the natural polyamines, as well as some synthetic congeners, potently prevent the rise in ODC evoked by many,

although not all, stimuli. Certain synthetic diamines that are particularly

active in this respect, such as 1,3-diaminopropane, when injected intra-

peritoneally cause rapid decline in the high ODC activity in regenerating liver and other tissues. The mechanisms of such "repression" of ODC by amines in vivo are obscure. The active amines promote the accumulation

in some cells of as yet not fully characterized protein(s) of roughly 25,000 daltons, discovered by E. S. Canellakis and his co-workers, and

designated as "ODC antizyme." This material inhibits ODC noncompetitively with respect to either L-ornithine or pyridoxal phosphate, the inhibitions being reversed at high ionic strengths. The existence of such

amine-induced ODC antizyme material has been substantiated in a number of laboratories. However, formation of ODC antizyme in response to appropriate amines has not always been detected under con-

ditions where ODC activity is greatly lowered; the kinetics and extents of ODC antizyme accumulation do not always accord with the notion that this alone accounts for ODC repression by diamines in living cells (Note 5). Other explanations have been proposed, including interactions of

ODC-repressing amines with putative cell surface receptors which somehow alter ODC turnover, or inhibitory effects of the amines on the translation of ODC mRNA by cytoplasmic polyribosomes. Another even-

tuality would be covalent incorporation of the amines into ODC by the transglutaminase-catalyzed reactions considered later. Regardless of its mechanistic basis, amine-induced ODC repression may contribute to the multiphasic oscillations of ODC activities in cultured cells during phases of the cell cycle, in liver after partial hepatec-


tomy, and in other biological situations as a consequence of transient

fluctuations in intracellular putrescine concentrations. A rapid synthesis of the enzyme undoubtedly occurs in many instances of ODC induction. But it is unclear if this is always contingent upon prior production of new ODC mRNA molecules, even though the rise in decarboxylase activity can frequently be prevented by administration of

inhibitors of DNA-directed RNA synthesis together with the inducing agent. There are in fact many indications that ODC biosynthesis can be separately regulated at both the transcriptional and translational levels. Icekson and Kaye have reported that rat ventral prostate cytosol contains an "ODC inactivating factor" (ODCIF) that specifically inactivates ODC apoenzyme and whose actions are nullified by pyridoxal phos-

phate. The ODCIF is heat labile, acts in the presence of thiols, and is clearly distinct from ODC antizyme material discussed earlier. The nature of this enzyme-like ODCIF and its role in ODC turnover in vivo are mysterious (Note 6). S-ADENOSYLMETHIONINE DECARBOXYLASE (AMeDC)

The second step in polyamine biosynthesis (fig. 2) is the loss of CO2 from AMe to yield a product containing an "active aminopropyl" moiety. Mammalian AMeDCs do not require metal ions but are directly stimulated by putrescine and a few related aliphatic amines. Putrescine markedly enhances AMeDC at physiological concentrations of this amine. In all mammalian tissues examined so far except human prostate, sper-

midine also acts as an activator of AMeDC, although it is less active than putrescine in this respect. Spermine, which does not stimulate the enzyme, inhibits the enhancement by putrescine. The mechanism by which putrescine and other amines activate AMeDC in vitro is unsettled;

although affinity for AMe is increased by stimulatory amines, the kinetics are not classically allosteric. There is considerable evidence that rises in tissue putrescine occasioned by ODC inductions contribute to ex-

pected increases in decarboxylation of AMe in vivo. Pyruvate covalently linked to the enzyme is the prosthetic group of mammalian AMeDC (Note 7). Convincing evidence for the existence of multiple forms of the

enzyme is lacking.

Besides being directly regulated by putrescine and spermidine, AMeDC activities are controlled at the level of enzyme turnover. The apparent half-life of this decarboxylase is almost as short as that of ODC

(usually less than 40 min). Although ODC and AMeDC activities can occasionally rise coordinately in tissues responding to certain hormones (e.g., during androgen-induced rat prostate growth), large increases in ODC in many stimulated tissues are often not accompanied by any pro-

nounced elevation in AMeDC.

Perspectives in Biology and Medicine · Spring 1979

43 1

SPERMIDINE AND SPERMINE SYNTHASES

These two distinct and completely separable enzymes transfer an aminopropyl group from decarboxylated AMe to putrescine and spermidine, resulting in the respective synthesis of spermidine and spermine. When estimated under optimal conditions in soluble extracts of all tissues that have been examined, the activities of the two synthases are considerably higher than those of the polyamine biosynthetic decarboxylases. Their apparent half-lives are relatively much longer. Apparently, no dissociable or bound cofactors are needed for spermidine or spermine synthases. Putrescine, a substrate for spermidine synthase, is a competitive inhibitor of spermine synthase. ALTERNATE PATHWAYS OF POLYAMINE BIOSYNTHESIS

Certain bacteria and higher plants contain L-arginine decarboxylases together with other enzymes that convert the reaction product, agmatine, into putrescine. This "agmatine pathway" for putrescine pro-

duction does not operate in mammalian cells, from which arginine decarboxylases are absent.

An interesting new pathway for spermidine synthesis in Micrococcus and Rhodopseudomonas species was recently described by Tait (Note 8). This involves condensation of /3-aspartyl-semialdehyde with putrescine to form a Schiff base, which is then reduced enzymatically to "carboxy-

spermidine" with NADPH as hydrogen donor. Another enzyme requiring pyridoxal phosphate decarboxylates the "carboxy-spermidine" to

yield spermidine. This mechanism of spermidine biosynthesis, utilizing carbons and nitrogens of aspartic acid for generation of the aminopropyl portion of spermidine, stands in contrast to the pathways for spermidine and spermine synthesis in many gram-negative bacteria and

animal cells in which methionine is the ultimate source of aminopropyl groups. It is well established that /3-aspartyl-phosphate (formed from aspartate and ATP) is converted biologically into /3-aspartylsemialdehyde that can in turn be utilized for homoserine and lysine biosynthesis. However, a precursor function for /3-aspartylsemialdehyde for spermidine formation was not suspected until very recently. It would be worthwhile to hunt for this pathway in mammalian

tissues and to see whether an analogous mechanism for spermine syn-

thesis might also be present. Isotopically labeled spermine and spermidine injected into rats undergo considerable conversion to spermidine and putrescine, re-

spectively. These "back conversions" of polyamines to their biosynthetic precursors are increased in liver after partial hepatectomy or treatment with growth hormone, thioacetamide and carbon tetrachloride, but the reaction mechanisms are obscure. It is conceivable that the spermidine


and spermine synthase reactions can operate in the reverse directions. However, their equilibrium constants have not been determined. The eventuality of a dismutation between two molecules of spermidine to give one molecule each of spermine and putrescine has also been

hypothesized, but not as yet demonstrated. Conversions of spermine to spermidine, and of spermidine to putrescine, are catalyzed by an oxidase

associated with liver peroxisomes that is considered below, and which may well promote these reactions in vivo.

III. Methylthioadenosine As illustrated in figure 2, stoichiometric formation of 5 '-meth-

ylthioadenosine (MTA) takes place in the spermidine and spermine synthase reactions. The inevitable tendency has been to regard MTA as

"metabolic garbage" produced by these two key enzymes of polyamine biosynthesis. Nevertheless it is imaginable that biological situations exist in whch formation of MTA represents the paramount functions of these

synthases. The MTA does not accumulate significantly in a large number of mammalian tissues, including those such as rat ventral prostate that harbor and secrete large quantities of spermidine and spermine. In 1969 Pegg and Williams-Ashman found an enzyme in prostate and liver that

phosphorolytically cleaves MTA into free adenine and 5-methylthioribose-1 -phosphate (fig. 3). This enzyme, whose existence in several additional tissues was recently corroborated by other investigators, is not identical with purine nucleoside Phosphorylase, or any other known animal enzymes that split the N-ribose linkages of nucleosides or nucleotides either phosphorolytically or hydroIytically. Among other functions MTA Phosphorylase almost certainly facilitates metabolic recycling

of adenine derived from the AMe molecules used for spermidine and spermine synthesis.

Relatively little is known about the physiological significance of MTA and its metabolites. It is an inhibitor of a few transmethylases but does not influence the enzymes of polyamine formation in mammalian tissues except spermine synthase, which it inhibits at high concentrations. 5' -methylthioadenosine5-methy I thioribose-1 -phosphate + inorganic phosphate+ adenine Methylthioadenosine Phosphorylase

5-methyl thioribose-1 -phosphateV

"CH_S" +· [ribose-1 -phosphate]

"Methylrhiolase" Fig. 3.—Enzymatic fate of 5'-methylthioadenosine


Toohey has published some fascinating observations suggesting that certain malignant cell lines require methylthio groups for proliferation in vitro while others do not. He also showed cells that grew without methyl-

thio group supplementation exhibited marked MTA Phosphorylase activity whereas those lines requiring exogenous methylthio groups were deficient in the enzyme. A second enzyme system designated "methylthiolase" which apparently liberates methylthio groups from 5-methylthioribose- 1 -phosphate (fig. 3) was found to be about equally

active in all the malignant cell lines examined, regardless of any requirement for an exogenous source of methylthio groups for cell division. It was concluded that this requirement in some cell lines was due to MTA Phosphorylase deficiency, and that the latter enzyme is vital for the production of methylthio or related groups involved in cell division. If these hypotheses turn out to be valid, then correlative studies of increased polyamine formation in relation to mammalian cell proliferation must take into account the possible relevance of MTA and hence methylthio group formation, since the spermidine and spermine synthase reactions represent major avenues of methylthioadenosine production in mammalian tissues. Evaluation of these ideas might be aided by applica-

tion of the 7-deaza analog of methylthioadenosine, which was recently synthesized by Coward and shown to be a competitive inhibitor of MTA Phosphorylase (Note 9).

IV. Selective Drug Inhibition of Polyamine Formation ENZYMOLOGICAL ASPECTS

Drugs capable of penetrating cells that selectively inhibit polyamine biosynthetic enzymes might be exploited to alter tissue polyamines in a predictable manner and thus perhaps cast new light on the biological functions of putrescine, spermidine, and spermine. Most attention has been given to ODC inhibitors. Among the first compounds found to be active in this respect was a-hydrazino-ornithine (fig. 4) which competes

with the ornithine substrate as a fairly strong competitive inhibitor of mammalian ODC; unfortunately this drug also readily reacts nonenzymatically with pyridoxal phosphate. Inhibition by another drug,

a-methyl-ornithine (a-MO), is not overcome by excess pyridoxal phosphate, but this compound is only a rather weak competitive inhibitor of

ODC. Administration of the two aforementioned substances increases

the apparent half-life of ODC in various tissues, perhaps because their combination with the ornithine-binding site on ODC retards the rate of intracellular degradation of the enzyme, although other mechanisms are not excluded. Both drugs can diminish accumulation of putrescine in


tissues stimulated by ODC-inducing agents. More active competitive inhibitors of ODC are írans-3-dehydro-ornithine and iram-l,4-diamino-

2-butene, which are unsatured analogs of ornithine and putrescine, re-

spectively. Bey, Mamont, and their co-workers have recently reported that a-difluoromethyl-ornithine (DFMeOm) is a very potent and irreversible

ODC inhibitor. Its probable mode of action is as follows: the drug forms a Schiff base with enzyme-bound pyridoxal phosphate, which after catalytic removal of CO2 leads to loss of a fluorine atom; the resulting very reactive intermediate then covalently combines with the ODC protein. Thus, DFMeOrn can be looked upon as a catalytic irreversible or "suicide" inhibitor by virtue of its containing a latent reactive group that

H2N-CH2-CH2-CH2-C-NH2

H2N-CH-CH=CH-CH2-NH

COOH

trans- 1 ,4-diamino-2 -butène H

ornithine

H2N-CH-CH2-CH2-C-NH2 C

CH3

III

CH

H2N-CH2-CH2-CH2-C-NH2

5-hexyne- 1 ,4-diamine

COOH

H

a-methyl -ornithine (a-MO)

H2N-CH2-CH=CH-C-NH2 C III CH

CHF2

H2N-CH2-CH2-CH2-C-NH2 COOH

trans-hex-2-en -5-yne-l,4-diamine

a-difluoromethy l-ornithine (DFMeOm) H

H2N-CH2-CH2-CH2-C-NHNH2

H0N-OL-CH=CH -C-NH0 ¿ ¿ ? 2

COOH

COOH

a-hydrazino-ornithine

tra ns-3-dehydro-orni thine

H-N

CH3NH2

,NHNHo H2NHN.CH3^??p??p2

C-NH-N= C - CH=N-NH-Cn\;-NH-N= C-CH =N-NH-C,, NH HN* HN^ NH MGBGMBAG

Fig. 4.—Some inhibitors of polyamine biosynthetic decarboxylases

Perspectives in Biology and Medicine ¦ Spring 1979

435

is unmasked at the enzyme's active site as a result of catalytic turnover. Two acetylenic congeners of putrescine (5-hexyne-l-4-diamine and iran5-hex-2-en-5-yne-l,4-diamine; see fig. 4) are similarly potent irreversible inhibitors of ODC. These substances do not inhibit pyridoxal

phosphate-requiring glutamate or aromatic amino acid decarboxylases (Note 10).

The cytotoxic drug methyl glyoxal bis(guanylhydrazone) or MGBG (fig. 4) is a very potent inhibitor of putrescine-activated AMeDC but is

without direct influence on ODC, or on spermidine and spermine synthases. The MGBG inhibition of mammalian AMe is much more pro-

nounced when saturating levels of putrescine or other activating aliphat-

ic amines are present. In other words, putrescine not only activates the enzyme but also makes it more susceptible to inhibition by MGBG.

Nonetheless, MGBG inhibits competitively with respect to the AMe sub-

strate rather than the putrescine activator. The inhibition constant for MGBG with putrescine present is less than 1 µ.?. By contrast, the drug MBAG (fig. 4), a close analog of MGBG, inhibits putrescine-activated AMeDC irreversibly (Note 11).

Treatment of mammalian organisms or cultured cells with MGBG can

result in more than 10-fold enhancement of AMeDC activities as de-

termined after dialysis of tissue extracts to remove endogenous MGBG. This MGBG-induced increase of AMeDC is accompanied by extensive

prolongation of the apparent half-life of the enzyme. Thus it appears that under some circumstances binding of MGBG to mammalian AMeDC renders the enzyme less susceptible to intracellular degrada-

tion. However, other mechanisms, including interruption by the drug of intracellular ATP synthesis, may come into play, because MGBG causes

extensive damage to mitochondria in living cells. Since ATP seems to be required for the breakdown of some proteins in living cells, the puzzling increase in ODC as well as AMeDC activities evoked in vivo by MGBG

may possibly be related to its effect on energy production (MGBG does

not inhibit or bind to mammalian ODC). It must also be remembered

that MGBG in somewhat high concentrations directly inhibits certain DNA polymerases and also diamine oxidase. As already discussed, MGBG appears to compete with the sites on

AMeDC that bind the AMe substrate rather than the putrescine ac-

tivator. No specific inhibitors of the amine activator binding site on

AMeDC have as yet been uncovered: substances such as trans-1,4diamino-2-butene and 1 ,4-diamino-2-butyne have been found to activate

the enzyme in a putrescine-like fashion. A search for drugs that would be inhibitory in this regard might be rewarding, as would the development of specific inhibitors of spermidine and spermine synthases, even though the latter enzymes do not appear to be rate limiting for polyamine biosynthesis in many mammalian tissues.

436 I H. Guy Williams-Ashman and Zoe N. Canellakis · Polyamines in Biology and Medicine

BIOSYNTHETIC INHIBITORS AS PROBES OF POLYAMINE FUNCTIONS

In attempts to analyze polyamine functions in living cells, inhibitors of ODC and AMeDC have been widely used to block new polyamine production. It is vital in evaluating the results to remember that, although the turnover of putrescine in many mammalian cells is fairly rapid, spermidine and spermine once formed turn over remarkably slowly. For

this reason, it is impossible to effect a quick and extensive reduction of polyamines in resting nondividing tissues by inhibitors of ODC and AMeDC, applied singly or in combination, even though these drugs

depress de novo formation of polyamines induced by many stimulatory agents.

Extensive investigations by Morris and co-workers employing a-MO

and MGBG as inhibitors suggest that enhanced synthesis of RNA and protein evoked by lectins in cultured lymphocytes is not dependent on de novo formation of polyamines. However, the polyamine biosynthetic inhibitors, especially when added together, markedly depressed lectininduced incorporation of thymidine into DNA with lengthening of the S-phase of the cell cycle, and the actions of the drugs were overcome by

addition of putrescine, spermidine, and spermine. Other workers using ODC competitive inhibitors and MGBG as probes have concluded that a certain minimum level of polyamines is necessary for continued traverse

of cell cycles, although the drug effects were dependent on the types of

cultured cells examined. Very recently, Mamont and his colleagues reported that the irreversible ODC inhibitor DFMeOm almost completely prevented accumulation of putrescine and spermidine during prolifera-

tion of cultured rat hepatoma and mouse leukemia cells elicited by addition of fresh medium. The effects of DFMeOrn were accompanied by marked decrease of cell growth that was prevented by addition of pu-

trescine and spermidine. Nevertheless, growth inhibition by DFMeOrn was pronounced only after a 24-hour lag period, perhaps because the stationary cells before dilution with fresh medium contained sufficient

amounts of putrescine and spermidine to permit progression through one cell cycle. Moreover, prolonged treatment (8 days) with DFMeOrn

did not totally arrest cell growth, which suggests either that intracellular spermine (which was not decreased by the drug) can partially fulfill the functions of putrescine and spermidine for growth, or that these particu-

lar tumor cells have a residual growth that is independent of polyamines (Note 12).

V. Metabolic Transformations of Polyamines Conversion of putrescine, spermidine, and spermine into a consider-

able array of metabolites occurs in mammalian organisms. Oxidative and conjugation reactions feature prominently in this regard. Some of the Perspectives in Biology and Medicine ¦ Spring 1979 ) 437

known polyamine metabolites found in tissues or urine are depicted in figure 5. The available data are too sparse to arrive at any definitive

picture of the biological importance of polyamine metabolites or their

relationship to polyamine turnover in vivo. It is possible that some of the compounds already isolated are simply biotransformation intermediates

that do not fulfill any functional roles, or that certain of the enzymes that transform polyamines may primarily act on quite different and more

physiologically important types of substrates. The very low tissue levels of polyamine metabolites suggests that their functional value, if any, may

be qualitatively different from that of spermidine and spermine. The only acetylated polyamine reported to be present in mammalian cells is mono-N-acetylputrescine. The acetyl-CoA dependent N-acetyl

transferase catalyzing its formation is extensively associated with nuclear

chromatin in a variety of tissues. The two isomers of mono-N-acetyl spermidine are found in urine. Spermidine and spermine are

monoacetylated by chromatin-associated enzymes and can be rapidly deacetylated by cytoplasmic enzymes. Small amounts of free ¦y-glutamyl putrescine are found in brain. Mechanisms of formation of this amide Metabolites of Putrescine

H2N(CH)3COOHCH CONH(CH ) NH2 y-aminobutyrate (GABA)N-acerylpurrescine

OHOHNH0

I

II2

H NCH CHCH2CH NHH NCH CHCH NH(CH ) CHCOOH 2(3)hydroxy putrescinehypusine Metabolites of Spermidine H N(CHJ NH(CHJ NHCOCHCH CONH(CH ) NH(CH ) NH 2 242 3 3 3 2423 2 1

8

N -acetyl spermidineN -acetyl spermidine

H2N(CH )4NH(CH2)2COOHHOOC(CH2)3NH(CH2)3NH2 Putreanineisoputreanine Metabolites of Spermine

HOOC(CH2)2NH(CHJ4NH(CH) COOH Spermie acid Fig. 5.—Some metabolites of polyamines in mammalian organisms


conjugate of glutamate are considered later in the context of transglutaminase catalyzed reactions. Noteworthy is the fact that glutathionyl spermidine (in which the aminopropyl group of spermidine is present in

amide linkage with the carboxyl group of the glycine moiety of

glutathione) is produced by E. coli but has never been found in mammalian tissues or urine.

Diamine oxidases that are especially active in kidney, placenta, and small intestine of some species catalyze the formation of -y-aminobutyraldehyde from putrescine. The latter substance can undergo cyclization or be further oxidized to y-amino butyrate (GABA). Another

pathway for the production of GABA from putrescine, discovered by Seiler, involves an initial formation of mono-N-acetyl putrescine fol-

lowed by its oxidation by mitrochondrial monoamine oxidase to N-acetyl-GABA, and subsequent deacetylation. It is well known that

GABA is present in fairly high concentrations in brain and spinal cord, where it probably functions as an inhibitory neurotransmitter and also appears to stimulate protein synthesis. Glutamate decarboxylase appears

to be the principal enzyme responsible for GABA synthesis in the central nervous system, and the neurophysiological importance of pathways for

GABA formation from putrescine needs to be clarified. Nakajima's laboratory has established that small amounts of putreanine and spermic acid are present in brain. They appear to be de-

rived from spermidine and spermine, respectively. Likewise hypusine, isoputreanine, and 2(3)-hydroxyputrescine, all of which may be derived in vivo from polyamines (fig. 5), have been isolated from tissues and/or urine. The enzymatic basis of the formation of these derivatives, as well as their physiological importance, remains to be worked out (Note 13).

Two enzymes of mammalian origin that attack spermidine and spermine have been characterized extensively (fig. 6). The first is a

polyamine oxidase from the blood plasma of ruminants that catalyzes the oxidative deamination of the aminopropyl groups of spermidine and spermine to the corresponding mono- and di-aldehyde products (the aminoaldehydes readily react with nucleic acids and proteins and are remarkably cytotoxic). The presence of this enzyme in ruminant serum, and even as a contaminant of bovine serum albumin preparations, can

be a nasty experimental nuisance in studies on cultured mammalian cells exposed to media containing such proteins, as both the polyamine aldehyde oxidation and the hydrogen peroxide products of the amine oxidase action may evoke metabolic changes that can be mistaken for

effects of exogenous spermidine and spermine. The second mammalian polyamine oxidase shown in figure 6 was recently discovered by Hölttä to be associated with liver peroxisomes. This enzyme catalyzes the re-

lease of 3-aminopropionaldehyde from spermidine and spermine. The Perspectives in Biology and Medicine ¦ Spring 1979 | 439

H2N(CH2)3NH(CH2)4NH2 + O2 + H2O Spermidine Y

H

JC(CH2)2NH(CH2)4NH2 + NH3 + H3O3

H2N(CH2)3NH(CH2)4NH(CH2)3 NH2 + 2 O2 + 2H2O Spermine Y

O

y0

VC(CHJNH(CHJ NH(CHJ-C^ + 2NH, + 2 H0O0 H/ 2¿ 2422\H3 2 2

H2N(CH2)3NH(CH2)4NH2 + O3 + H3O Spermidine Y

H2N(CH2)4NH2 + H2N(CH2)2CHO + H3O3 3-aminopropionaldehyde

H2N(CH2)3NH(CH2)4NH(CH2)3NH2 + O3 + H3O Spermine Y

H2N(CH2)3NH(CH3)4NH3 + H3N(CH2J3CHO + H3O3 3-aminopropionaldehyde Fig. 6.—Enzymatic oxidation of polyamines. Top, oxidation of polyamines by ruminant plasma amine oxidase. Bottom, oxidation of polyamines by oxidase associated with rat

liver peroxisomes. Note that at high pH values the dialdehyde oxidation product of sper-

mine oxidation by the ruminant plasma enzyme can undergo nonenzymatic degradation to yield acrolein and other products.

oxidase may be involved in conversions of spermine to spermidine, and of spermidine to putrescine, that have been demonstrated in living rats (Note 14).

There are reports that blood plasma and urine contain as yet chemically unidentified conjugates of spermidine and putrescine from which

these amines can be recovered after hydrolysis with strong HCl at high temperatures. Some of these polyamine conjugates may conceivably rep-

resent peptides or proteins to which the amines are attached covalently. VI. Enzymatic Covalent Incorporation of Polyamines into Proteins

For 20 years it has been recognized that polyamines can become attached covalently to certain proteins. These reactions are catalyzed by

transglutaminases (transamidases), a family of enzymes present both extracellularly and intracellularly in mammals. This mode of postsynthetic covalent modification of proteins by polyamines has been demon-

strated unequivocally only in cell-free enzyme systems, and its potential

physiological significance has been almost completely ignored (Note 15).

Transglutaminases catalyze Ca++-dependent formation of isopeptide (amide) linkages between various primary amino groups attached to "amine-donor" molecules (which can be of either high or low molecular weight) and the ?-carbonyl function of y-carboxamide groups of specific polypeptide-bound glutamine residues in selected proteins. These enzymes do not utilize as "amine-acceptor" substrates either free glutamine or -y-glutamyl compounds such as glutathione in which the a-amino and a-carboxyl groups are not present in peptide linkage or its equivalent: this distinguishes transglutaminases from other enzymes that utilize the y-carboxamide moiety of free or bound glutamine as substrates, such as glutaminases, and also the y-glutamyltransferase that acts on glutathione. Transglutaminases appear to act via a modified double dis-

placement mechanism. The first step involves the formation of a thioester bond between a specific cysteine thiol group at the active site of the enzyme and a polypeptide-bound •y-glutamyl carbonyl group, with liberation of ammonia from the •y-carboxamide of the reacting glutaminyl residue. The second step is aminolysis of the thio-ester bond by the

primary amine substrate, yielding a polypeptide-bound y-glutamylamide with concomitant regeneration of the sulfhydryl group at the enzyme's active site, as illustrated in figure 7. The only transglutaminase-catalyzed reactions whose physiological importance have been established unequivocally involve the establishment of e-(y-glutamyl)lysine cross-links in proteins. This entails utiliza-

tion of the e-amino groups of certain lysine residues in appropriate proteins as the amine donors. Cross-bridges of this type were first shown in the cross-linking of mammalian fibrin molecules by fibrinoligase or Perspectives in Biology and Medicine ¦ Spring 1979 | 441

/

HN

+

/ CHCH0CH0CONH0 2 2 2

OC

HS-Enz

transglutaminase

^ glutamine residue on amine acceptor

polypeptide

¦* NH3 «4/

/

HN

\

/ CHCH2CH2CO-S-EnZ OC

\

RNH,

amine donor

/

V

HN. OC

CHCH2Ch2CONHR

HS-E nz

\

/

HN.

CHCH2CH2CONH(CH2)4NH2

OC \

Peptide-bound y-glutamyl putrescine

/

\

HNCO

S CHCH2CH2CONH(CH2)4-CH^'NH

OC" \

/

e—(y—glutamyl) lysine cross bridge between polypeptide chains Fig. 7.—Transglutaminase-catalyzed reactions (top) and some reaction products (bot-

tom).

Factor XIIL the active form of a plasma zymogen Factor XIII, which is converted into fibrinoligase by the actions of thrombin and Ca++. Other forms of extracellular transglutaminases secreted by the coagulating (an-

terior prostate) gland catalyze the polymerization of seminal vesicle secretion proteins that is the basis of the postejaculatory clotting of semen.

Additional forms of transglutaminase are present intracellularly in most tissues. The enzymes in hair follicles and in epidermis (where they are involved in formation of e-(y-glutamyl)lysine linkages present alongside disulfide bridges in certain hair and epidermal proteins) exhibit different properties from those of liver and erythrocyte transglutaminases. The molecular basis of the heterogeneity of mammalian intracellular enzymes of this class is not understood. Relatively few glutamine-containing mammalian proteins appear to be effectively utilized as amine-acceptor substrates by transglutaminases. Acceptor activity seems to depend more on the conformation of the entire polypeptide than on the amino acid sequence adjacent to a reactive glutamine residue. Besides many synthetic primary amine com-

pounds, many (though by no means all) low molecular weight amines

present in animal cells can serve as amine-donor substrates. Since reactivity of various amines depends on many factors, including species and organ sources of the enzyme, the protein acceptor substrate, and temperature, it is impossible to rank the naturally occurring amines in

any precise order. Nevertheless, at the concentrations at which they occur in mammalian tissues, putrescine, spermidine, and spermine are

among the best substrates for transglutaminases from liver, coagulating gland, and other sources. It is easy to imagine that attachment of polyamines to specific intracellular structural, catalytic, or regulatory proteins by transglutaminase

action could profoundly alter their biological properties. Among the

proteins reported to act as polyamine acceptors in cell-free systems are rhodopsin, aldolase, certain erythrocyte membrane proteins, fibrin, and

homogeneous guinea pig liver transglutaminase itself. Does postsyn-

thetic modification by polyamines of these or any other proteins take place within living cells? Hardly any data bearing on this important question are available. For a variety of technical reasons, such reactions

could easily have been overlooked. Two points need emphasizing in this regard. First, the small amounts of free ?-glutamyl-putrescine that Nakajima found in brain tissue probably do not originate from hydrolysis of proteins to which putrescine was attached covalently via transglutaminase catalysis; rather the substance seems to be formed as a

result of putrescine exchanging, in place of more active amino acid substrates, with the y-glutamyl group of glutathione in reactions promoted by the quite different enzyme -y-glutamyltransferase. Second, free or polypeptide-bound ?-glutamyl-polyamine linkages are not split by a

Perspectives in Biology and Medicine ¦ Spring 1979 \ 443

wide spectrum of proteinases, or by any other known hydrolytic enzymes.

Beil and Williams-Ashman (unpublished data) have recently shown that the putrescine covalently incorporated into dimethylated rat seminal vesicle secretion proteins by coagulating gland transglutaminases is indeed present mainly in the form of peptide-bound-y-glutamylputrescine, as indicated by quantitation of free y-glutamyl-putrescine in proteolytic digests of the reaction product. In addition, a second adduct

is formed which, after separation following proteolytic hydrolysis, has properties indicative of the bis-(y-glutamyl)amide of putrescine. This suggests that once putrescine has been incorporated into the protein acceptor substrates in the form of peptide-bound -y-glutamyl-putrescine, the other free primary amino group can further react as a trans-

glutaminase substrate with other glutamine residues so as to form cross-bridges between peptide chains. Very recent model experiments

by Schrode and Folk have shown that both primary amino groups of a number of diamines and polyamines (including putrescine, spermidine, and spermine) can similarly form cross-links between polypeptides in

reactions catalyzed by both liver transglutaminase and blood plasma fibrinoligase (Note 16). Whether comparable processes operate in healthy or diseased (e.g., cystic fibrosis) organisms is a tantalizing problem.

VII. Clinical Investigations Following the announcement by Russell in 1971 that polyamines in acid-hydrolyzed urine are abnormally high in cancer patients, a great deal of attention has been given to polyamines in human body fluids in

relation to the etiology, diagnosis, and therapeutic handling of a gamut

of human diseases. There have been two major impediments to medical studies on polyamines. The first is that there is a dearth of information

on the polyamine content of many human tissues in healthy individuals, particularly with regard to the influence of age, sex, nonspecific stresses,

renal clearance values, and the effects of tissue anoxia and autolysis

(findings obtained with autopsy material as commonly processed must be

viewed with caution). Second, there arise methodological problems. For example, the values for spermine in human urine in some early studies

employing methods based on one-dimensional electrophoretic polyamine fractionations were later shown to be grossly overestimated

because of comigration of impurities. Only recently have there become available adequately sensitive and specific chromatographic procedures for polyamine determinations on small biopsies of human tissues, and

especially on blood plasma, in which spermidine, spermine, and putrescine are present in the free form in less than micromolar concentrations.


The extremely sensitive radioimmunoassay methods for free polyamines in body fluids just developed by Bartos and Bartos is a signal advance (Note 17). It is important to remember that (a) polyamines in urine are present largely in the form of acid-labile conjugates, and that most re-

ported values for human urinary polyamines have been determined after hydrolysis with hot strong acid; and (b) more than 90 percent of the total polyamines in human blood are within the cells, the concentra-

tions of free polyamines in blood plasma being very low indeed. For this reason, little meaning attaches to clinical correlative studies on polyamines in unfractionated blood. CANCER

A large body of data indicates that, although growth rates and anaplasia of a wide spectrum of experimental malignant tumors are not directly proportional to their polyamine contents or their polyamine biosynthetic decarboxylase activities, all rapidly proliferating and highly dedifferentiated cancer cells as yet examined manufacture and accumu-

late spermidine and spermine at least as vigorously as their normal cells of origin, and often exhibit elevated putrescine levels. Tumor-bearing

animals and patients frequently have elevated levels of polyamines in

various extracellular fluids. This seems in many instances to result from

tumor-cell destruction rather than from massive overproduction and/or excretion of polyamines by undamaged tumors cells. Although heightened urinary excretion of polyamines is evident in many patients with advanced malignant tumors, this appears to be

neither a specific attribute of cancer nor a consistent index of the clinical status of a number of malignancies, including those of the lung, stomach, pancreas, and female mammary gland. However, increases in urinary spermine in Burkitt's lymphoma and colorectal carcinoma are

frequently striking, and serum polyamines are often increased in patients with some advanced malignant hematological tumors. The value

of estimations of urinary or serum polyamines in assessing the status of prostate neoplasms merits further study. Also worthy of follow-up are

the preliminary observations of Abdel-Monem that the ratio of N1- to N8-acety!spermidines may be increased in the urine of some cancer patients.

Successful surgical ablation or chemotherapy of tumors often results in diminution of urinary polyamines several weeks later. Contrariwise, swifter increases in serum or urinary polyamines have been reported to

occur within 1-2 days after beneficial responses to chemotherapy of some hematological tumors and may possibly provide a telling index of massive tumor-cell death.

Marion's laboratory has established that putrescine and spermidine Perspectives in Biology and Medicine ¦ Spring 1979 | 445

levels in cerebrospinal fluid are frequently increased in patients with advanced medulloblastoma or glioblastoma, but less often in as-

trocytoma or pituitary tumors, in comparison with CSF polyamines in a variety of nonmalignant diseases of the central nervous system.

Polyamine determinations in CSF indeed appear to be valuable for both the diagnosis of certain brain tumors and evaluation of their responses to chemotherapy. This seems to represent the most promising clinical application of polyamine analysis as yet apparent (Note 18). OTHER DISEASES

A rapidly proliferating literature attests to the growing interest in polyamines in relation to a large number of human pathological con-

ditions besides cancer, and especially cystic fibrosis, psoriasis, certain muscle dystrophies, and various nonmalignant blood diseases. In many instances, abnormalities in urinary and sometimes serum polyamine profiles have been reported, but it is too early to tell whether this is of

profound etiologic or diagnostic significance. Biopsies from patients with psoriasis often exhibit manyfold increases in ODC and AMeDC

activities in comparison with uninvolved epidermal tissue. Conceivably psoriasis might be amenable to successful treatment by topical application of specific inhibitors of polyamine biosynthetic enzymes.

It is noteworthy that no pathological states attributable to deficiencies in polyamine production, or to abnormal formation of polyamine metabolites, have ever been described.

VIII. Sexual Physiology SEMINAL FLUID

Large amounts of spermine, and in some instances of spermidine as well, are found in the semens of certain mammalian species. In healthy

young men, for example, the average concentration of seminal spermine is almost 3 mM, whereas putrescine and spermidine are present at less than 0.3 mM. Most of the spermine in human semen originates from prostatic secretion, which together with secretions from die epididymis,

seminal vesicles, and bulbourethral glands comprise the seminal plasma

added to spermatozoa at ejaculation. The relative contributions of these

liquids from various male accessory sex organs to whole seminal plasma

can vary extensively from one normal ejaculate to another. This is a major reason why human seminal spermine concentrations are so variable. In the rat, the secretion of the ventral lobe of the prostate normally contains more than 5 mM spermine and spermidine; lesser amounts are also secreted by the dorsolateral prostate whereas the anterior prostate

446 I H. Guy Wilhams-Ashman and Zoe N. Canellakis ¦ Polyamines in Biology and Medicine

and vesicular fluids are virtually devoid of polyamines. Assertions that the prostatic fluids and seminal plasmas of mammals in general are very

rich in polyamines are erroneous. In mice, rabbits, and guinea pigs, for example, hardly any spermine and spermidine are secreted by the prostate or seminal vesicles. Likewise in the dog (which is devoid of seminal vesicles and bulbourethral glands), the semen and prostatic fluid contain

very little polyamines. Accurate assessment of the polyamine levels inside ejaculated spermatozoa is difficult in those species in which the seminal plasma are rich in these substances because of their adsorption to sperm cell surfaces; but it seems that the values are tiny. That spermatozoa in the undiluted semens of some species are bathed in a sea of spermidine and/or spermine has naturally incited speculation

on the reproductive functions of seminal polyamines. There are many reports of both positive and negative effects of spermine on motility and metabolism of washed spermatozoa from several species. Evaluation of these effects, which depend on species and experimental conditions, is not easy, especially because of the well-known deleterious effects of dilu-

tion on sperm motility and lack of data regarding the specificity of these actions of spermine. Similar questions arise concerning the physiological

significance of reports that polyamines can influence fructolysis by washed spermatozoa, adenylate cyclase reactions in spermatozoal extracts, and sperm head acrosomes.

It must be remembered that in most mammals ejaculation is extraordinarily brisk and is completed within a few seconds, so that under coital

circumstances there is only a short time of contact between spermatozoa

and polyamines in seminal plasma before the semen becomes diluted with female genital secretions. Moreover, to postulate that all substances in seminal plasma, regardless of their concentrations, must be of functional significance for any aspect of sperm physiology is as unreasonable

as assuming that all constituents of blood plasma are meaningfully related to erythrocyte physiology. Indeed, it may well be that output of large quantities of polyamines by the prostate in some species may be

epiphenomenal in the sense that the prime importance of spermidine

and spermine relates to metabolic events within prostatic epithelial cells, and that polyamines may be nonspecifically secreted, together with many

other low molecular weight substances in the cytoplasm. Polyamines in seminal plasma are obviously not essential (rather than facilitatory) for sperm transport and fertilization, since washed spermatozoa from many

species after suspension in polyamine-free media are employed successfully in artificial insemination on a widespread scale. It is possible, of course, that in some species seminal spermine represents a biochemical vestige that was of reproductive importance in evolutionary ancestors (Note 19).

Williams-Ashman and co-workers have shown that the postejaculatory


clotting of rat and guinea-pig semens, which in these species is responsible for the formation of a vaginal plug after coitus, involves the

cross-linking of seminal vesicle secretion proteins via the formation of e-(y-glutamyl)lysine cross-bridges catalyzed by transglutaminases secreted by the coagulating gland, a lobe of the prostate (see Sec. VI and above). Putrescine, spermidine, and spermine can be covalently in-

corporated into vesicular secretion proteins during the clotting process by competing with protein lysyl residues as amine-donor substrates. This accords with observations that millimolar concentrations of polyamines inhibit the semen coagulation process. Perhaps spermine in seminal plasma is of value in preventing excessive clotting of protein in the urethra during or after ejaculation which if unimpeded might cause urinary retention.

Formation of characteristic crystalline salts of spermine with picric or

flavianic acids was at one time used in tests for semen stains in forensic

medicine, but this has been superseded by more sensitive procedures based on the activity of the exceedingly active prostatic acid phosphatase

present in human seminal plasma. Seminal spermine estimations in men with otherwise normal prostate function could possibly be exploited in screening for congenital defects in spermine production or anthropological variants.

Human (but not rat) seminal plasma readily oxidizes polyamines. This

was first demonstrated by Zeller in 1941 and has been investigated more

recently by Hölttä, Jänne and their co-workers. The relative rates of oxidation of spermine, spermidine, putrescine, and also histamine remained fairly constant throughout extensive purification of an active seminal plasma protein fraction, but whether more than one enzyme is

involved is not entirely clear. The products of spermine oxidation by human seminal plasma appear to be aldehyde derivatives but have not

been rigorously characterized. There is evidence that they can exert toxic actions on spermatozoa. Moreover, aminoaldehyde products of polyamine oxidation formed by enzymes from other sources can nonenzymatically form covalent adducts with nucleic acids. This has car-

cinogenic and mutational implications. But there is no evidence for the

reaction of these polyamine oxidation products in seminal plasma with the highly condensed DNA in the head pieces of spermatozoa, although reactions with the sperm cell plasma membranes might occur. The

human seminal plasma polyamine oxidase(s) utilize molecular oxygen and give rise to hydrogen peroxide as one of the reaction products. In addition to the fact that hydrogen peroxide is itself toxic to spermatozoa,

this may be germane to reports from the laboratories of Sheth and Jänne that substantial putrescine-dependent AMeDC activity is present in human seminal plasma, but not in blood serum. The seminal AMeDC may be an artifact. For Suresh and Adiga have recently shown that 448 I H. Guy Williams-Ashman and Zoe N. Canellakis ¦ Polyamines in Biology and Medicine

extracts of Lathyrus sativus seedlings readily decarboxylate AMe in the absence of Mg++ when putrescine is added, as a result of hydrogen peroxide formation by a diamine oxidase in the same preparations (the AMeDC of this plant, like many of its bacterial counterparts, requires

Mg+ + but not putrescine as a cofactor). It has been shown that hydrogen

peroxide causes extensive nonenzymatic release of CO2 from the carboxyl group of AMe via reactions that are greatly enhanced by peroxidases and pyridoxal phosphate (Note 20). POLYAMINES AND THE FUNCTIONAL DIFFERENTIATION OF REPRODUCTIVE ORGANS

Marked changes in tissue polyamine concentrations, and in the activity of ODC and often of AMeDC as well, have been reported to accompany the growth and functional differentiation evoked by gonadotrophins in ovary and testis, by estrogens in uterus, and by androgenic hormones in

male genital glands. The very large upsurges in ODC in ovary caused by

lutropin (LH) provide the basis for a sensitive bioassay for this hormone

developed by Nureddin. The pioneer studies of Maudsley and Kobayashi disclosing an extremely active polyamine metabolism in the maternal and especially the fetal part of the placenta during the later phase of rat pregnancy have been widely confirmed. The functional

importance of polyamines in all of these reproductive tissues remains obscure (Note 21).

Dramatic elevation of ODC and AMeDC, more or less in concert with

rises in spermidine and RNA content, are seen in rat mammary gland over the last 6 days of pregnancy. Oka observed comparable increases in spermidine and polyamine biosynthetic decarboxylases in mouse mam-

mary gland expiants during induction of milk secretion by a mixture of insulin, prolactin, and glucocorticoid hormone. Exogenous spermidine mimicked the effects of the glucocorticoid component of the hormonal triad; the steroid itself induced AMeDC. Addition of MGBG to the cul-

ture medium, with resultant inhibition of AMeDC, prevented the rise in spermidine levels and synthesis of specific milk constituents due to hormonal stimulation. The effects of MGBG were overcome by spermidine

addition. These findings hint of involvement of spermidine in maintenance of lactation and of AMeDC in some actions of glucocorticoid hormones on mammary epithelial cells. Comparable evidence for polyamine involvement in the progesterone regulation of glycogen synthesis by endometrial expiants was recently published by Feil and co-

workers. They noticed that progesterone-stimulated glycogen deposition was accompanied by an increase in AMeDC and in the conversion of spermidine to putrescine. Inhibition of AMeDC by addition of MGBG also blocked progesterone-induced glycogen synthesis, and these effects


were reversed by exogenous spermidine (in this instance it was shown

that penetration of MGBG into the cells was not impeded by spermidine) (Note 22).

IX. Epilogue The gross neglect of mammalian polyamine physiology that prevailed for so long has been superseded by many spectacular advances over the last sesquidecade. Prominent among these are mapping of pathways for polyamine synthesis and metabolism, development of an array of specific inhibitors of polyamine biosynthetic enzymes, and demonstration of impressive changes in polyamines and especially ornithine decarboxylase in cells responding to all sorts of hormones and other stimulants of growth

and differentiation. Nevertheless, chiaroscuro pervades the field. A wide variety of in vitro actions of polyamines on macromolecular biosynthetic processes and other metabolic reactions have been uncovered. But the physiological significance of many of these effects remains problematic, although regulation of key reactions of nucleic acid and protein synthe-

sis seems to be centrally related to polyamine functions in living cells. The aphorism " 'for example' is not proof" likewise applies to many hypotheses concerning the extracellular functions of polyamines in semen. Increases in the polyamine content of various extracellular fluids can accompany extensive tumor-cell death in some cancer patients, espe-

cially during chemotherapy; however, the clinical utility of polyamine determinations on body fluids in many neoplastic and other diseases remains equivocal. At all events, it is safe to predict that the increasing pace of polyamine research will shortly shed greater light on these and

other challenging problems related to the normal functions of polyamines and their disturbances in disease. NOTES AND REFERENCES

Note 1. References to most of the studies discussed in this essay are provided

in the following reviews and books. 1.H. Tabor and C. W. Tabor. Adv. Enzymol., 36:203, 1972.

2.C. W. Tabor and H. Tabor. Annu. Rev. Biochem., 45:285, 1976.

3.S. S. Cohen. Introduction to the polyamines. Englewood Cliffs, N.J.: Prentice-Hall, 1971. Also Nature 274:209, 1978.

4.U. Bachrach. Function of naturally occurring polyamines. New York: Academic Press, 1973.

5.H. G. Williams-Ashman, A. E. Pegg, and D. H. Lockwood. Adv. Enzyme Regul., 7:291, 1969.

6.H. G. Williams-Ashman, J. Jänne, G. L. Coppoc, M. E. Geroch, and A. Schenone. Adv. Enzyme Regul., 10:225, 1972. 7.H. G. Williams-Ashman. In: E. Run and S. Grisolia (eds.). Biochemical

regulatory mechanisms in eukaryotic cells, p. 245. New York: Wiley Interscience, 1972.


8.H. G. Williams-Ashman, A. Corti, and B. Tadolini. Ital. J. Biochem., 25:5, 1976.

9.A. Raina and J. Jänne. Med. Biol., 53:121, 1975.

10.D. H. Russell and B. G. M. Durie. Polyamines as biochemical markers of normal and malignant growth. New York: Raven, 1978. 11.R. A. Campbell, D. R. Morris, D. Bartos, G. D. Daves, and F. Bartos

(eds.). Advances in polyamine research, vols. 1 and 2. New York: Raven, 1978.

12.J. Jänne, H. Pösö, and A. Raina. Biochim. Biophys. Acta, 473:241, 1978.

References cited in other notes are either to very recent publications, or to

articles in other fields.

Note 2. Inhibitory effects of S-adenosyl homocysteine and 5'-methylthioadenosine on polyamine synthases are described in: 13.H. H. Hibasami and A. E. Pegg. Biochem. Biophys. Res. Commun., 81:1398, 1978.

Note 3. For studies on ODC antibodies, see:

14.Z. N. Canellakis and T. C. Theoharides. J. Biol. Chem., 251:1781 and 4436, 1976.

15.S.J. Scheinman, G. N. Burrow, T. C. Theoharides, and Z. N. Canellakis. Life Sci., 21:1143, 1977.

16.E. Hölttä. Biochim. Biophys. Acta., 399:420, 1975. 17.M. K. Obenrader and W. F. Proutv. J. Biol. Chem., 252:2866, 1977. Note 4. Thiol requirements for mammalian ODC are considered by:

18.J. Jänne and H. G. Williams-Ashman. J. Biol. Chem., 246:1725, 1971.

19.S. J. Friedman, K. V. Halpern, and E. S. Canellakis. Biochim. Biophys. Acta, 261:181, 1971.

For the relationship of the thioredoxin reductase-thioredoxin system to protein disulfide reductases, see:

20.F. Tietze. Arch. Biochem. Biophys., 138;177, 1970. 21.C. A. Apffel and J. E. Walker. J. Natl. Cancer Inst., 51:575, 1973.

22.P. Maness and A. Orengo. Biochim. Biophys. Acta, 429:182, 1976. 23.A. Holmgren. J. Biol. Chem., 252:4600, 1977.

Note 5. Studies on "ODC antizyme" are described in: 24.J. S. Heller, W. F. Fong, and E. S. Canellakis. Proc. Natl. Acad. Sci. USA, 73:1858, 1976.

25.P. McCann, C Tardif, and P. S. Mamont. Biochem. Biophys. Res. Commun., 75:948, 1977.

26.A. E. Pegg, C. Conover, and A. Wrona. Biochem. J., 170:651, 1978.

27.A. Kallio, M. Lofman, H. Pösö, and J. Jänne. FEBS Lett., 79:195, 1977.

28.E. S. Canellakis, J. S. Heller, D. Kyriades, and K. Y. Chen: see Note 1, reference 11, vol. 1, p. 17. Note 6.

29.I. Icekson and A. M. Kaye. FEBS Lett., 61:54, 1976. Note 7.

30.A. E. Pegg. FEBS Lett., 84:33, 1977, and A. E. Demetriou, M. S. Cohn,

C W. Tabor, and H. Tabor. J. Biol. Chem. 253:1684, 1978.


Note 8.

31.G. H. Tait. Biochem. Soc. Trans., 4:610, 1976.

Note 9. Studies on 5'-methylthioadenosine Phosphorylase and its metabolic

significance are described in: 32.A. E. Pegg and H. G. Williams-Ashman. Biochem. J., 115:241, 1969. 33.D. L. Garbers. Biochim. Biophys. Acta, 523:82, 1978.

34.J. I. Toohey. Biochem. Biophys. Res. Commun., 78:1273, 1977 and 83:27, 1978.

35.J. K. Coward, N. C. Motóla, and J. D. Mover. J. Med. Chem., 20:500, 1977.

Note 10. Earlier work on ODC inhibitors is overviewed in reference 8. For

preparation of a-difluoromethylornithine and unsaturated putrescine derivatives and their inhibition of ODC, see:

36.B. W. Metcalf, P. Bey, C. Danzin, M.J.Jung, P. Casara, and J. P. Vevert.

J. Amer. Chem. Soc, 100:2551, 1978.

37.P. S. Mamont, M. -C. Duchesne, J. Grove, and P. Bey. Biochem. Biophys. Res. Commun., 81:58, 1978.

Note 11.

38.A. E. Pegg. J. Biol. Chem., 253:539, 1978. Note 12. Use of polyamine biosynthetic enzyme inhibitors in the analysis of polyamine functions in animal cells is considered in reference 37 and in: 39.D. R. Morris: See Note 1, reference 11, vol. 1, pp. 105 and 181. 40.A. B. Pardee, R. Dubrow, J. L. Hamlin, and R. L. Kletzien. Annu. Rev. Biochem., 47:715, 1978.

Note 13. Polyamine metabolites in mammalian organisms are discussed in: 41.T. Nakajima. J. Neurochem., 20:735, 1973. 42.T. Noto, T. Tanaka, and T. Nakajima. J. Biochem. (Jap.), 83:543, 1978.

43.H. Konishi, T. Nakajima, and I. Sano. J. Biochem. (Jap.), 81:355, 1977.

44.T. Nakajima, Y. Kakimoto, M. Tsuji, and H. Konishi. J. Neurochem., 26:115, 1976.

45.N. Seiler and M.J. Al-Therib. Biochem. J., 144:29, 1974.

46.M. Tsuji and T. Nakajima. J. Biochem. (Jap.), 83:1407, 1978. Note 14. Polyamine oxidizing enzymes in ruminant blood serum and rat liver

are, respectively, considered in:

47.U. Bachrach. Ann. N.Y. Acad. Sci., 171:939, 1970.

48.E. Hölttä. Biochemistry, 16:91, 1977. Note 15. Transglutaminase action on polyamines and other amine substrates is

discussed in:

49.J. E. Folk and J. & Finlayson. Adv. Protein Chem., 31:1, 1977.

50.L. Lorand. Ann. N.Y. Acad. Sci., 202:6, 1972.

51.H. G. Williams-Ashman, J. Wilson, R. E. Beil, and L. Lorand. Biochem. Biophys. Res. Commun., 79:1192, 1977. Note 16.

52.J. Schrode and J. E. Folk. J. Biol. Chem., 253:4837, 1978.


Note 17.

53.F. Bartos, D. Bartos, D. P. Grettie, R. A. Campbell, L. J. Marton, R. G.

Smith, and G. D. Daves. Biochem. Biophys. Res. Commun., 75:915, 1977.

54.F. Bartos, D. Bartos, A. M. Dolney, D. P. Grettie, and R. A. Campbell. Res. Commun. Chem. Pathol. Pharmacol., 19:295, 1978.

Note 18. Studies on polyamines in neoplastic diseases are considered in Note

1, references 10, 11, and 12, and in:

55.M. M. Abdel-Monem and K. Ohno. J. Pharm. Sci., 66:1195, 1977. 56.L.J. Marton, O. Heby, V. A. Levin, W. P. Lubich, D. C. Crafts, and C. B.

Wilson. Cancer Res., 36:973, 1976. 57.S. S. Cohen. Cancer Res., 37:939, 1977.

Note 19. For reviews on polyamines in semen, see:

58.H. G. Williams-Ashman and D. H. Lockwood. Ann. N.Y. Acad. Sci., 171:882, 1970. 59.H. G. Williams-Ashman. Invest. Urol., 2:605, 1965.

60.T. Mann. The biochemistry of semen and of the male reproductive tract. New York: Wiley, 1964. Note 20.

61.A. N. Thakur, Sheth, and S. S. Rao. Clin. Chim. Acta, 55:377, 1974.

62.J. Jänne, E. Hölttä, P. Haaranen, and K. Elfving. Clin. Chim. Acta, 48:393, 1973.

63.E. Hölttä, P. Pulkkinen, K. Elfving, and J. Jänne. Biochem. J., 145:373, 1975.

64.M. R. Suresh and P. R. Adiga. Eur. J. Biochem., 79:511, 1977.

65.G. L. Coppoc, P. Kallio, and H. G. Williams-Ashman. Int. J. Biochem., 2:673, 1971. Note 21.

66.A. Nureddin. Biochem. Med., 17:67, 1977. 67.D. V. Maudsley and Y. Kobayashi. Biochem. Pharmacol., 26:121, 1977. Note 22.

68.T. Oka and J. W. Perry. J. Biol. Chem., 249:7647, 1974; and Note 1, reference 11, vol. 1, p. 59. 69.P. D. Feil, A. E. Pegg, L. M. Demers, and C. W. Bardin. Biochem. Biophys. Res. Commun., 75; 1, 1977.

Perspectives in Biology and Medicine ¦ Spring 1979

453

Polyamines: from molecular biology to clinical applications.

Phospholipases in biology and medicine.

Ultrasonics in medicine and biology.

Molecular biology in medicine.

Mouse medicine and human biology.

Acoustic droplet vaporization in biology and medicine.

Accelerating precision biology and medicine with computational biology and bioinformatics.

Organometallic compounds in medicine and biology.

Ultrasound in Medicine and Biology Clinical Prize.

Alkaline phosphatases in biology and medicine.

Chlorins as photosensitizers in biology and medicine.

Developmental biology: impact on medicine.

Biology of Heme in Mammalian Erythroid Cells and Related Disorders.

Bioarcheology: Medicine, Biology, and Forensic Sciences.

The Future of Vascular Biology and Medicine.

Mammalian synthetic biology: emerging medical applications.

The MicroRNA Biology of the Mammalian Nucleus.

The molecular biology of mammalian glucose transporters.

Polyamines and mammalian hormones. Part II: Paracrine signals and intracellular regulators.

Data Acquisition and Processing in Biology and Medicine.

Carbon nanotubes in medicine and biology - safety and toxicology.

Coherent and quasi-coherent optical methods in biology and medicine.

Polyamines and mammalian hormones. Part I: Biosynthesis, interconversion and hormone effects.

Gasotransmitters in Biology and Medicine: Molecular Mechanisms and Drug Targets.