Conference on the Structure-Function Relationships of Pituitary Growth Hormone: A Report Jack

1. Kostyo

and

Alfred

E. Wilhelmi

A

CONFERENCE on the structure-function relationships of pituitary growth hormone was held at the headquarters of The Kroc Foundation in Santa Ynez Valley, California, on January 21-24, 1975. The purpose of the conference was to consider current experimental and clinical studies on the relationship between the structure of growth hormone and its growthpromoting, diabetogenic, and insulin-like properties. Twenty-two scientists attended. Formal sessions dealt with (1) the biologic activity of growth hormone in blood, (2) chemical and biologic properties of naturally-occurring fragments of growth hormone, (3) the biologic properties in animals and man of fragments of growth hormone produced by chemical and enzymatic means, and (4) the properties of synthetic fragments of growth hormone. BACKGROUND

OF

THE

PROBLEM

Alfred E. Wilhelmi opened the conference with a review of past work on the structure-function relationships of growth hormone and an outline of current approaches being used to explore the problem. Prior to 30 yr ago the leading question for students of the pituitary was: 1s there a growth hormone? Our colleague Choh Hao Li, with Evans and Simpson, answered that question affirmatively in 1944 by the isolation from bovine pituitary glands of a simple protein with the properties of a globulin that promoted growth in the hypophysectomized rat and was free of all the other known hormonal activities of the pituitary. The beginning of our quest dates from that discovery. Meanwhile, great improvements in the methods of isolation have made extensive investigation of growth hormone practical, culminating in the isolation of human growth hormone on a scale sufficient for the study and treatment of a great many people. The problem facing us now arises from the phenomenon of specificity: only primate and human growth hormones are effective in man. We must rely upon the limited supply of human pituitaries. The important question is: Can we find a simpler method of reaching the goal of an adequate supply of growth hormone for use in man? At present, we know something about the amino acid composition of the growth hormones of eight species of animals. From comparative immunochemical and biochemical studies, such as those of Hayashida and his colleagues, we know something more about the relatedness of the growth hormones of many more kinds of animals. Evidence of this kind, and the evidence from more careful physico-chemical studies of several growth hormones, now From the Department of Physiology. Division of Basic Health Sciences, Atlanta. Ga. Received for publication June 13.1975. Reprint requests should be addressed IO Dr. Jack L. Kostyo. Department sion of Basic Health Sciences, Emory University. Atlanta. Ga. 30322. 0 1976 by Grune & Stratton, Inc. Metabolism,

Vol. 25, No.

1 (January),

1976

Emory

University,

of Physiology,

Divi-

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makes it plain that these proteins in different vertebrates comprise a family of simple proteins of similar size (with monomer weights of about 20,00022,000) and similar amino acid composition. There are differences in detail (e.g., in isoelectric point), but it is generally agreed that the vertebrate pituitary growth hormones, like the cytochromes, the hemoglobins, and other like proteins in different species, are more like one another than they are unlike. This makes the question of the specificity of the growth hormones all the more interesting. Bovine and porcine growth hormone are ineffective in man. Pickford and Wilhelmi showed that the growth hormone of a teleost fish, the hake, was inactive in the rat but quite active in the hypophysectomized minnow, Fundulus heteroclitus. Knobil and Greep showed that bovine growth hormone was not active in hypophysectomized monkeys, but monkey growth hormone was. The isolation of human growth hormone by Li and Papkoff and by Raben was soon followed by the observation that human and primate growth hormones are active in man. One interesting feature of this specificity is its one-way aspect. Human, monkey, bovine, ovine, and porcine growth hormone are all active in the hypophysectomized minnow, but the growth hormones of at least two teleost fishes are not active in the most widely used test animal, the rat. Human and monkey growth hormone, and all of the other known mammalian growth hormones, are active in the rat, but only primate growth hormones are active in primates, including man. In the light of the similarity of the growth hormones and of the ability of the rat to respond to most of them, it is possible to subscribe to the hypothesis of the common core which states that there is an active portion of the molecule common to all the growth hormones, and that it is the rest of the molecule that may interfere with its ability to interact in a given species. This is the optimistic hypothesis which we perforce adopt: that we can prepare, either by partial degradation of the hormone or by selective synthesis of part of the molecule, a small unit which would be despeciated, still retaining the activity of the native hormone. A less optimistic hypothesis has to do with the target requirements. Let us say that a given species requires just characters A and B at the target. Then in other species, an evolutionary advance leads to requirements A, B, and C. Finally, in a further advance another group of animals have evolved A, B, C, and D as target requirements. This would account for both the specificity of the hormones and its one-way character. There is yet another aspect of specificity. Suppose that there is indeed an active sequence common to all the hormones. This would be comparable to the catalytic site of an enzyme. There then may be different points for binding to effecters and binding to the target (substrate). The experience of enzymologists is that all of these sites are seldom arranged in a short linear sequence but most often are brought to an appropriate focus by folding of the molecule as it organizes itself into the active protein. It is therefore possible that the common core, when it is discovered, may indeed be the operational bit. The problem then is to devise modifications of it which will yield the appropriate fit for the target: whatever human cell growth hormone acts upon. Yet another possibility is that, given that we can isolate a simple common

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core, it may be found that the rest of the molecule is required to protect it from rapid destruction. It would be necessary to learn how to protect the simple active moiety so that its half-life in the organism is sufficient to enable the active bit to carry out its job. Knowledge of the amino acid sequence of human, ovine, and bovine growth hormone and of a good part of the porcine hormone reveals a high order of homology among them. The parts of the hormone essential to its activity have been studied in two ways, by examining the effect of chemical reagents on the native hormone, and by using reagents such as cyanogen bromide that cleave the molecule at specific peptide bonds. Eric Reid, later confirmed by Li and his colleagues, observed that acetylation of the amino terminal group of bovine growth hormone has no effect on its activity, but that extensive acetylation of the c-amino groups of the lysines destroys the activity. Geschwind and Li showed that the activity of growth hormone is not affected by converting the lysine t-amino groups to guanido groups. More recently Canfield has shown that polyalanyl residues can be added to c-amino groups of lysine in human growth hormone without significant loss of activity. Li and his colleagues have shown that selective substitution of the tryptophan residue in human growth hormone with 2-nitrophenyl sulfenyl chloride or with 2-hydroxy-5nitrobenzyl bromide causes no loss of activity in the tibia test but almost complete loss of crop sac-stimulating activity. Mills has observed that treatment of bovine and porcine growth hormone with an excess of sulfite, followed by treatment with iodoacetate or iodoacetamide, leads to opening of one of the two disulfide bonds with no loss of activity. If, however, both disulfide bonds are opened and carboxymethylated, then bovine, porcine, and human growth hormone all lose activity completely. Bewley and Li, however, have shown that complete reduction of human growth hormone with dithiothreitol, followed by carbamidomethylation, does not lead to loss of activity. In the case of porcine growth hormone, however, Mills has found that this cannot be done. Treatment of growth hormones by cyanogen bromide results in cleavage at the three methionyl bonds (in the case of human growth hormone), breaking the molecule into four pieces. The largest of these pieces exhibited some activity in stimulating protein synthesis in the liver and the diaphragm of the rat. In the weight-gain test, however, this large fragment was inactive. Another long-established line of growth hormone study has been to observe the effects of partial enzymatic degradation of the molecule. Li and his colleagues first showed that bovine growth hormone retained its activity after partial digestion with chymotrypsin. Indeed, it was claimed that this “chymotryptic core” was active in man, but the observation could not be confirmed. This line of study was carried further by Elsair and his colleagues, who achieved finer control over the hydrolysis by utilizing the pancreatic trypsin inhibitor of Kunitz. Their digests were still active in the rat, and they claimed that the whole digest was active as well in human subjects. Very large doses, not well tolerated by the patients, were used. The most thorough-going work of this kind has been carried out by Sonenberg and his co-workers. Carefully controlled lo-min digestion of bovine growth hormone (bGH) with trypsin cleaves the molecule into two pieces, the smaller of which, representing residues 96-133 of bGH, has

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some activity in the tibia test. There is evidence that this fragment is also active in man. Recently, Li and his colleagues have repeated some of this work and have also concluded that peptide 96-133 shows some activity in the tibia test. It is useful to remember that the order of the activity is about 2%-6x of the activity of the native hormone, so far as it can be estimated at all; the dosee response curves are not parallel, the curve for the native hormone reference preparation being much steeper. Another line of study of enzymatic effects upon growth hormone emerged from several laboratories almost at once, but U. J. Lewis deserves credit for the first detailed report of work which showed that the electrophoretic heterogeneity of purified preparations of growth hormone could be attributed to contamination with proteolytic enzymes. Mildly alkaline solutions of the hormone change progressively with time, the native hormone being transformed into more acidic components. These changes can be arrested by treating the preparations with inhibitors of proteolytic enzymes such as diisopropyl phosphofluoridate, Later, Stanley Ellis and his colleagues showed that the principal protease contaminating growth hormone preparations was very likely plasmin. The interesting point about these enzymatic changes in growth hormone is that, at least in the early stages, they are without effect on the biologic activity of the preparation. Sonenberg and his co-workers showed that the different electrophoretic components of bGH were all equally active. Chrambach and Lewis have observed that some of the more acidic products of proteolysis of human growth hormone (hGH) by plasmin are several times as active as the native precursor in the tibia and pigeon crop sac tests. The report that the action of plasmin upon growth hormone did not result in loss of activity led us to try deliberately to digest hGH with plasmin. The whole digest is fully active. It can be separated into several parts by exclusion chromatography on Sephadex. A major piece, peak 2, lacking only the hexapeptide 135140, is fully active. It can be resolved into two pieces, 1-134 and 141-191, by reduction and carbamidomethylation; the larger, amino-terminal piece is still active. Independently, Li and his colleagues have carried out similar experiments and have confirmed what we have found. Another approach to the problem, made possible by knowledge of the amino acid sequence of growth hormone, is to synthesize small peptides comprising likely short sections of the molecule. Chillemi and Pecile have synthesized peptides 88-124 and 125-156 of growth hormone, both of which have a little activity in the tibia test. Niall has prepared peptide 96-134 of hGH, corresponding to the bovine peptide AI1 of Sonenberg. It was inactive in the weight gain test and in some of Kostyo’s in vitro tests. Blake and Li have prepared the N-acetylated peptide 96-136. It has a low order of activity in the tibia test. In fact, no one seems to be able to produce a peptide that does not have a little activity in the tibia test. The approach by synthesis of small peptides, guided by the results of systematic degradation of the molecule, is probably the most promising line of attack on the problem of the scarcity of hGH. Finally, a line of study of great importance is the examination of the question, What is circulating growth hormone? Is it what we isolate from the pituitary? Is what we see (immunochemically) in the plasma identical to what

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we isolate from the pituitary? Is there some change in growth hormone taking place during or after secretion that is essential for its activity? If the last is the case, then we should make every effort to identify the real hormone, which may reveal the secret of high activity and simplified structure. BIOLOGIC

ACTIVITY

OF GROWTH

HORMONE

IN THE BLOOD

Although much is known about the chemistry and biologic properties of the growth hormone contained in the pituitary, there is little understanding of the nature of the growth hormone that circulates in the blood. Recently, circulating growth hormone has been investigated by several groups using immunochemical methods, and their work has suggested the presence of several forms of growth hormone in the blood. Besides what appears to be the native growth hormone molecule, other substances of larger molecular size than the native hormone have been found which cross-react with antibodies to native growth hormone. Nothing is known about the biologic activity of any of these forms of circulating growth hormone. Early attempts to measure the biologic activity of growth hormone in the plasma of animals and man resulted in a paradoxical finding, namely that much more biologic activity was detected than can now be accounted for by estimates made by radioimmunoassay. Another paradox is the finding in the rat that stress and insulin-induced hypoglycemia, which deplete the pituitary of growth hormone, do not produce a rise in immunoassayable growth hormone in the blood. Taken together, these findings suggest that growth hormone may be modified, during the process of secretion or in the bloodstream, to a form that has enhanced biologic activity but does not crossreact with antibodies to native growth hormone. The first session of the conference was devoted to a consideration of recent work on the nature of circulating growth hormone. Stanley Ellis (Ames Research Center, Moffatt Field, Calif.) described studies done in collaboration with Richard E. Grindeland on growth hormone in the blood of man and the rat. Human blood plasma was concentrated and then assayed by the tibia test. Approximately 200 times more activity was found than predicted on the basis of the amount of hGH detected in the plasma by radioimmunoassay. The plasma of an acromegalic patient did not show such a wide discrepancy between biologic and immunologic activity, the ratio between the two approaching one. Concentrated plasma of normal rats had approximately 50 times mare activity in the tibia test than predicted from the amount of rat growth hormone (rGH) found by radioimmunoassay. In contrast, the plasma of rats bearing a transplantable pituitary tumor (MtTW5) which secretes large amounts of growth hormone showed a much better correlation between biologically active and immunoreactive growth hormone (BA:RIA = 1.4: 1). Further, when concentrated plasma was administered to hypophysectomized rats along with an antiserum against native rGH, the plasma still stimulated growth of the tibia1 cartilage. In control experiments, the ability of purified rGH or rat pituitary extracts to stimulate tibia1 growth was suppressed by the antiserum. It is concluded that there is a growth hormone present in small amounts in the circulation that reacts with antibodies to native (pituitary) growth hormone and another growth hormone present in the blood at high

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concentrations that is not immunoreactive but detectable by bioassay. The latter substance is presumably produced from the former by some transformation occurring in the pituitary or in the peripheral circulation. Ellis next presented experiments dealing with the question of where the presumed transformation of growth hormone takes place. Adult anesthetized rats were bled either from the jugular vein or the heart, and the ratio of bioassayable to immunoassayable growth hormone in the plasmas was determined. The discrepancy between biologic activity and immunologic activity was greater in blood samples taken from the jugular vein that in those taken from the heart, suggesting that native growth hormone is being modified somewhere in the head, perhaps in the pituitary itself. Ellis then described efforts to concentrate and isolate the biologically active form of growth hormone in the plasma, using either whole human plasma or Cohn fraction IV of human plasma. Inert protein was precipitated by metaphosphate, using a step-wise lowering of pH. Cohn fraction IV yielded a product having a specific activity of 180 ng equivalents of growth hormone per mg with a recovery of 40%. The discrepancy between activity in the tibia test and in the radioimmunoassay was still preserved. Another preparation of lower specific activity made from whole plasma, administered to hypophysectomized rats, caused a significant gain in body weight. It was possible to produce preparations having activity in the tibia test but having no detectable crossreactivity with antibodies to growth hormone. A concentrated preparation of whole plasma made by the metaphosphate precipitation technique was subjected to chromatography on Sephadex G-200 in ammonium bicarbonate buffer. Radioimmunoassayable growth hormone was detected only in fractions having apparent molecular weights above 180,000, while biologic activity was found in all fractions having apparent molecular weights of 34,000 or greater. Thus, the lower molecular weight fractions appeared to be biologically active but did not react with antibodies to native growth hormone. A similar separation of activities was achieved when concentrated plasma samples were submitted to free-flow electrophoresis. A fraction derived from whole human plasma by precipitation between 2.0 and 3.2 M ammonium sulfate from the supernatant solution remaining after treatment with metaphosphate was subjected to electrofocusing. Bioassayable activity, and traces of radioimmunoassayable activity as well, was found at a p1 of 4.9-5.4. This agrees with the observation of Friesen et al. that radioimmunoassayable hGH also concentrates in this p1 range. Lastly, Ellis remarked that the plasma concentrates contain little, if any, somatomedin as measured by the embryonic chick cartilage assay for this substance. Jack L. Kostyo (Emory University, Atlanta, Ga.) then summarized work done with Charles R. Reagan and Jennifer Stewart that is also directed toward the question of the correlation between the biologic activity and the immunologic activity of the growth hormone in blood. The blood used for these studies was obtained from rats bearing the transplantable somatotrophic-mammotrophic tumor MtTW15. It contains substantial amounts of growth hormone (2-175 rg/ml plasma), as measured by radioimmunoassay. Using a sensitive in vitro bioassay for growth hormone, the biologic activity of growth hormone

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in the plasma of these animals could be measured without having to concentrate it. The bioassay used is based upon the ability of growth hormone in vitro to stimulate the uptake of 3-O- methyl glucose into the isolated diaphragm of the hypophysectomized rat, an expression of the hormone’s insulin-like activity. It was shown that purified rGH was active at concentrations ranging between 0.04 and 1.0 pg/ml of medium. Normal rat plasma at dilutions of lo-50-fold or hypophysectomized rat plasma at dilutions as low as four-fold were not active. In contrast, the plasma of tumor-bearing rats at dilutions of 30-200-fold stimulated 3-O-methyl glucose uptake by the diaphragm. The magnitude of the responses obtained were those expected from the amount of rGH present in the plasma added to the medium, as measured by radioimmunoassay. These responses were abolished with an antiserum against purified rGH, indicating that the biologically active material in the plasma is related to growth hormone and not to other insulin-like components of the plasma. It was shown that the biologic activity of the plasma could be abolished by theophylline, which blocks the stimulatory effect of native growth hormone on 3-O-methyl glucose uptake into the diaphragm but not that of agents such as insulin or somatomedin. The 3-O-methyl glucose assay gives a linear log-concentration/response with purified rGH in the range of 0.04-l .O pg/ml of incubation medium. This permitted a quantitative comparison of the amounts of biologically active and immunologically active growth hormone present in the blood of tumor-bearing rats. The ratio of biologically active to immunoreactive growth hormone present in a number of plasmas was found to be essentially 1: 1. A good correlation between biologic activity and immunologic activity of the plasma growth hormone in the tumor-bearing rat was also found when the plasma was assayed for ability to stimulate cartilage metabolism in hypophysectomized rats. The end-point measured was the stimulation of in vitro thymidine incorporation into isolated costal cartilage. When plasma was administered to the test animals in amounts that by radioimmunoassay contained sufficient rGH to bracket the doses of native rGH needed to give a threshold response, a threshold response was indeed obtained with the calculated dose of the plasma. This response was abolished by the concomitant administration of antibodies to rGH. These experiments support the conclusion that there is a good correlation between the biologic activity and immunologic activity of the rGH in the blood of the tumor-bearing animal. The possibility that circulating growth hormone may undergo some chemical transformation is not ruled out by these experiments, since a modification in structure may occur which does not interfere with its ability to cross-react with the antibodies used in these studies. Kostyo speculated upon explanations for the differences between his findings with blood of tumor-bearing animals and those of Ellis. One possibility was that the difference might be due to methodology, in particular, that the discrepancy between biologic and immunologic activity is seen when plasma is concentrated (for the tibia test) rather than when it is diluted, as in the in vitro bioassay system. On the other hand, if transformation of native growth hormone to a biologically active but nonimmunoreactive entity occurs only in the

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pituitary gland (or during the process of secretion), as Ellis suggests, then it is possible that the tumor lacks the capacity to transform growth hormone. The result would be that the growth hormone circulating in the tumor-bearing rat would behave biologically and immunologically like the molecule isolated from the pituitary gland. CHEMICAL AND BIOLOGIC NATURALLY-OCCURING FRAGMENTS

PROPERTIES OF OF GROWTH HORMONE

This session of the conference was concerned with a discussion of the identification and properties of the modified forms of growth hormone occurring in pituitary extracts and more purified hormone preparations. Urban J. Lewis (Scripps Clinic, La Jolla, Calif.) described his extensive studies on modified forms of growth hormone found in extracts of human pituitary glands. Presumably, these are hormone derivatives present in the glands prior to extraction or formed during storage of the glands or during the initial stages of extraction. Lewis illustrated the various forms of hGH detected in extracts of pituitary tissue by disc gel electrophoresis at pH 4 and pH 10. These include deamidated growth hormone, components LYI, (Ye, 03, and ,f3, and a very fast electrophoretic component. Components (Y, and a2 were equipotent with native hGH in the tibia assay. Component cy2consists of amino acid residues 1- 134 coupled to residues 141- 191 by the disulfide bridge between residues 53 and 165. Component CX~,which exhibited enhanced activity (threefour-fold) in the tibia assay, was found to consist of residues l-134 joined to residues 147-191 by the intrachain disulfide bridge. Component ,!3, which also had considerable activity in the tibia assay, is a mixture of as yet unidentified peptides. The very fast electrophoretic component, consisting of acidic material of low molecular weight, was inactive in the tibia assay. These fragments of growth hormone were also active in the pigeon crop sac assay for prolactin, component (Yeagain showing more activity than native hGH. Lewis suggested that the diabetogenic property of hGH preparations is not a property of the native hormone molecule or of any of these large fragments, but is due to a slightly smaller molecular weight component which is usually isolated with hGH. This diabetogenic component is very potent in producing glucose intolerance of long duration in the dog. Whether it is a portion of hGH remains to be determined. Component a3 is particularly interesting, since it appears to be considerably more active than native growth hormone and thus could have potential value in alleviating the shortage of hGH. Unfortunately, the yields of component (Yefrom different pituitary extracts are variable and generally not better than 5% of the total growth hormone that can be isolated from the extracts. Component (Ye is also interesting because of the nature of the enzymatic cleavages that result in its formation, presumably from component CY~.The removal of hexapeptide 135-140 from hGH to form component ti2 and the removal of pentapeptide 141-145 may result from the action of plasmin contained in the pituitary gland. The removal of the phenylalanine residue at position 146, however, must be accomplished by some other enzyme. Dr. Lewis has attempted to produce component a3 by digestion of hGH with plasmin, but the product is usually component cx2 (l-134 + 141-191).

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The intrachain disulfide bridges of component a3 have been reduced, and the products consisting of two peptides, Fl (residues 1-134) and F2 (residues 141191), have been isolated. Peptide Fl has 5510% the activity of native hGH in the tibia and pigeon crop sac assays, while peptide F2 is inactive. Attempts to recombine peptides Fl and F2 have been unsuccessful largely because of aggregate formation. Peptides Fl and F2 have also been isolated in the reduced and alkylated form and found to behave like Fl and F2 in the tibia and crop sac assays. Lewis also reported on the heterogeneity of various purified hGH preparations revealed by SDS gel electrophoresis. The principal components appear to have molecular weights of 45,000, 24,000, 22,000, and 20,000. In addition, a number of minor components of smaller size were also observed. The 45,000 molecular-weight component is presumably a dimer of the native hormone. The component of apparent molecular-weight 24,000 is the native molecule with a single cleavage between residues 139 and 140, which makes the molecule behave on the SDS gel as if it were larger than it is. The component of apparent molecular-weight 20,000 appears to be the native hormone with residues 39-41 missing. Lewis also described attempts to isolate these components from saline extracts of human pituitaries by Sephadex chromatography. Assay of the purified components in the pigeon crop sac assay suggested that the native hormone may have quite low prolactin activity. The intrinsic prolactin activity of purified hGH preparations may be due primarily to enzymatically modified forms, since their activities in the crop sac assay are considerably greater than that of undegraded hGH. Finally, Lewis illustrated the marked heterogeneity seen in various purified preparations of hGH when they are submitted to isoelectric focusing. Even a preparation that appeared to be homogeneous by SDS gel electrophoresis was found to contain three components in addition to deamidated forms. Lewis stressed the great difficulty of isolating chemically homogenous human growth hormone. Andreas Chrambach (NICHHD, NIH, Bethesda, Md.) continued the discussion of the heterogeneity of growth hormone by demonstrating the many common components revealed by disc gel electrophoresis in a variety of widelyused hGH preparations. He suggested that this heterogeneity may be typical for the native state of growth hormone and not caused by the purification process, since these same components occur in the material secreted by cultured pituitary adenomas. Chrambach stressed the importance of having reliable and rigidly standardized methods for the separation, identification, and definition of the components in hormone preparations. He described his extensive efforts to establish appropriate methods using analytical gel electrophoresis and isoelectric focusing. With respect to the heterogeneity revealed in purified hGH preparations with isoelectric focusing, Chrambach does not find additional components with this technique, in contrast to the experience reported by Lewis. Chrambach suggested that differences in the methods used by the two laboratories might account for this. Chrambach also reviewed his group’s work on the effects of digestion of hGH with plasmin. By careful monitoring of the digestion mixture by gel electrophoresis, the transformation of the major components of the starting ma-

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terial into component E was demonstrated. Chrambach stressed the importance of monitoring the digestion process by gel electrophoresis, since, in his experience, the rate of digestion varied with different lots of the enzyme. Component E, like component (Yedescribed by Lewis, showed enhanced activity in the tibia test and the pigeon crop sac assay. Component E, however, is not identical to (Ye(which is presumably equivalent to Chrambach’s component D) but may be a modified form of the latter. Chrambach indicated that his group plans to isolate substantial amounts of component E using isoelectric focusing or preparative electrophoresis in stacking gels. The latter technique appears to be quite promising for this application, judging from Chrambach’s description of model experiments using this technique. Chrambach also commented upon the extent to which deamidation contributes to the heterogeneity of growth hormone preparations and enzymatitally degraded hormone preparations. It was his opinion that growth hormone is quite resistant to deamidation, and he cited evidence for the failure of hGH to deamidate during digestion with plasmin or upon incubation at 50°C under alkaline conditions. CHEMICAL

AND

ENZYMATIC FRAGMENTATION STUDIES IN ANIMALS

OF GROWTH

HORMONE:

Three sessions of the conference were concerned with the chemical and biologic properties of fragments of growth hormone produced by cleavage of the native molecule with specific reagents or enzymes. The first two sessions focused on the evaluation of these fragments by animal assays for biologic activity. Martin Sonenberg (Memorial Sloan-Kettering Cancer Center, New York City) summarized work carried out in collaboration with Norbert I. Swislocki and other colleagues on the isolation and characterization of fragments of bGH produced by limited (10 min) digestion with trypsin. He first reviewed the characterization of one of the cleavage products, TBGH-d, which is fully active in the tibia test and in in vitro assays on isolated rat adipose tissue and which shows some anabolic and diabetogenic activity in human subjects. When TBGH-d was treated with 50% acetic acid, it dissociated into two peptides, one comprising residues l-95 + 134-191 (AI) and another consisting of residues 96-133 (AH). Sonenberg then described his extensive characterization of the immunologic, biologic, and physico-chemical properties of these two peptides. Both fragments cross-react slightly with antibodies to native bGH, fragment AI cross-reacting to a greater extent (0.16%) than fragment AI1 (0.001%). Both fragments also cross-reacted with antibodies to rGH, and AI1 showed some cross-reaction with antibodies to hGH, which is of some interest in view of the fact that native bGH does not cross-react with hGH antibodies. Both fragments also had some activity in the tibia test and weight-gain test and were active in vitro on isolated rat adipose tissue. Fragment AH is more active than AI. Estimates of its activity relative to native bGH are of the order of 57&10%. A precise estimate of potency cannot be made, since the fragment does not give a dose-response curve that is parallel to the standard in the tibia test. With re-

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spect to molecular conformation, AI appears to have an a-helix content of approximately 25% (as compared to 50% a-helix for both native bGH and TBGH-d), while AI1 appears to be a random coil. Sonenberg commented on reasons for the reduced activity of fragment AI1 relative to native growth hormone. Considering that the fragment is dissociated from TBGH-d using 35%-50x acetic acid, chemical modification of the peptide might account for the reduced activity. On the other hand, AI1 may lack a portion of the native molecule needed for proper binding to the receptor on target cells. Sonenberg described experiments carried out with David Donner involving the binding of native hormone and fragment AI1 to isolated hepatocytes. The binding constant for hGH in this system is in the range of 10e9M, while that for fragment AI1 is lo-“M. A point of some interest is that there does not appear to be a good correlation between the loss of biologic activity of AI1 in hypophysectomized rats and the loss of binding affinity for the isolated hepatocyte. Biologic activity is approximately one-twentieth that of the native material, while binding affinity is three orders of magnitude less. Sonenberg described experiments involving the recombination of AI and AII. The two fragments, recombined in 1 M acetic acid, behaved as one component on gel filtration, and the product appeared to have a greater content of a-helix than AI does alone. The product also had greater activity than expected from simple addition in the tibia assay and in vitro on isolated rat adipose tissue. Whether this enhanced activity is related to an improvement in binding to receptors remains to be established. Sonenberg also commented upon work done with Bruce Merrifield on the synthesis of a nonapeptide of bGH representing residues 125-133. This peptide was also produced from the natural fragment AI1 by cyanogen bromide cleavage at the methionyl residue at position 124. Both the synthetic and natural nonapeptide had slight activity in the tibia test but no activity in the weight gain test in hypophysectomized rats. John B. Mills (Emory University, Atlanta, Ga.) next described work carried out with Charles R. Reagan, Jack L. Kostyo, and Alfred E. Wilhelmi on fragments of hGH produced by plasmin digestion. Digestion of hGH with human plasmin yielded a product having undiminished activity in the weight-gain test. Enhanced activity was not observed as described earlier by Chrambach. The major component of the digests, apparently responsible for much of their biologic activity, was isolated by gel filtration. It consists of residues 1-134 connected to residues 141-191 by the intrachain disulfide bridge. This component, designated peak 2, is equivalent to component (Y* described by Lewis. It was fully active in the weight-gain test and in a variety of other in vivo and in vitro tests for growth hormone. Aside from a component emerging ahead of peak 2 on gel filtration that seems to be an aggregate with reduced biologic activity, other components of the digests were found to be variable in character and inactive. The disulfide bonds of peak 2 were reduced and carbamidomethylated, and the two peptides formed were separated by gel filtration. The fragment comprising residues I-134 was designated peptide 2a and that consisting of residues 141-191 was called 2/3 (these peptides are equivalent to the reduced forms El and F2 described by Lewis). Both fragments were essentially inactive

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in the weight-gain test and when tested for the ability to stimulate thymidine uptake by rat costal cartilage upon intraperitoneal administration. In contrast, peptide 2a was highly active in vitro in stimulating leucine incorporation into the isolated rat diaphragm and in stimulating glucose oxidation in isolated rat adipose tissue. Peptide 20 was inactive in these assay systems. When peptide 2~y was administered intravenously in the rat cartilage metabolism test, one preparation was found to be as active as native hGH. Several other preparations had lo%-30% of the activity of native hGH in this assay system. From these experiments, Mills concluded that the peptide may be considerably more labile in vivo than native hGH. Mills described an alternative method of preparing the peptide by reducing and carbamidomethylating hGH prior to digestion with plasmin. Reduced and carbamidomethylated hGH (RCAM-hGH) retains the full biologic properties of the native hormone. Plasmin digests of RCAM-hGH were also fully active in the weight-gain test and in stimulating thymidine uptake into rat costal cartilage when administered intraperitoneally. Thus, these digests do not show the in vivo lability characteristic of peptide 2a. Mills then described efforts to isolate the active component(s) from the digests by gel filtration. A peak designated R2 was found to contain material that is fully active in the weight gain, cartilage metabolism, and in vitro adipose tissue assays. Peak Rl, which elutes ahead of R.2, appears to be an aggregate with reduced biologic activity. Other peaks (R3-R5) contain many peptides, including the usual hexapeptide consisting of residues 135-140. Thus, plasmin attacks RCAM-hGH and hGH in a similar manner, but the digestion of RCAM-hGH appears to be more extensive. Preliminary information suggests that the material in peak R2 is structurally similar or identical to peptide 2a. Choh Hao Li (University of California, San Francisco, Calif.) next described recent work on fragments of bGH, oGH, and hGH carried out with L. Grif, T. A. Bewley, and A. J. Rao, with technical assistance of D. Gordon, J. Knorr, and J. D. Nelson. They have isolated and characterized two fragments produced by limited tryptic cleavage of bGH and oGH. These consist of residues 96-133 (equivalent to the AI1 peptide of bGH of Sonenberg) and residues 151-191. Li reported that peptide 96-l 33 was resistant to hydrolysis with leucine aminopeptidase. Further, both the bovine and ovine versions of this peptide were only weakly active in the tibia assay and gave dose-response curves that were not parallel to the bGH reference standard. In this respect, these peptides behaved like the peptide AI1 isolated by Sonenberg. Li described the isolation of two peptides produced by plasmin digestion of hGH followed by reduction and carbamidomethylation of the intrachain disulfide bridges. These two fragments, consisting of residues l- 134 and residues 141-191, are identical to the peptides (2c~ and 2@) described by Mills. Li indicated that peptide 1-134 had about 14% the activity of native hGH in the tibia assay, an observation which is consistent with the experience reported by Mills, namely that this peptide had little activity in vivo when administered subcutaneously or intraperitoneally. Peptide 14 I- 19 I also showed some activity (about 6%) in the tibia assay. Peptide 1-134 also had some ability to stimulate ornithine decarboxylase activity in the liver, but peptide 141- 191 was inactive in this regard. When both peptides were tested for the in vitro ability to stimu-

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late lipolysis in isolated rabbit adipose cells, they had very low activity in contrast to native hGH, which was quite effective. In the local pigeon crop sac assay, peptide 1-134 had about 10% the activity of the native hormone, while peptide 141-191 was virtually inactive. Li showed that these two peptides retained some immunochemical identity to hGH but had lost a considerable fraction of their immunoreactivity. Peptide 1-134 of hGH was also subjected to fragmentation with cyanogen bromide, resulting in cleavage of the molecule at the methionyl residues in positions 14 and 125. A large fragment consisting of residues 15-125 was isolated from the reaction mixture. (This peptide is similar to the “fragment A” produced by cyanogen bromide cleavage of reduced and S-aminoethylated hGH, described recently by Nutting, Kostyo, Mills, and Wilhelmi.) Conformational analysis revealed that while precursor peptide 1-134 had an a-helix content of 40x, peptide 15-125 had no detectable helical structure. When 300 pg of peptide 15-125 were administered to hypophysectomized rats, some increase in tibia1 epiphyseal width was detected, but there was some question whether this small response was indeed reflecting growth-hormone-like activity. The peptide did appear to have approximately 5% the activity of native hGH in the pigeon crop sac assay for prolactin activity. Finally, Li described the reactivity of his various hGH fragments in the pregnant rabbit liver radio-receptor assay, carried out by Michael Aubert. Compared to the native hormone, the whole plasmin digest of hGH, peptide 1-134 and peptide 141-191, showed a descending order of reactivity in this system. In contrast, peptide 15-125 was virtually unreactive. This comparison was of considerable interest in view of the biologic properties and activities attributed to these fragments by the various groups that have studied them. This session of the conference concluded with a presentation by Ardis Lostroh (Stanford University, Stanford, Calif.) of studies done with Maurice E. Krahl on diabetogenic fragments of growth hormone. The work is based upon the hypothesis that native growth hormone is not itself diabetogenic, but rather is a precursor of a diabetogenic factor generated in the animal, presumably by proteolytic cleavage. The time-lag in the expression of the diabetogenie effect of growth hormone in in vivo experiments and for the development of the inhibitory phase of the action of GH on glucose metabolism in isolated tissue systems might reflect, in part, the time required for the production of the diabetogenic factor. To test this hypothesis, Lostroh and Krahl digested growth hormone with proteases (pepsin, trypsin, chymotrypsin) and then tested the products from each digestion for the ability to produce acute glucose intolerance. An acute assay was developed since, if the hypothesis is correct, once formed, the diabetogenic fragment of growth hormone should have a prompt inhibitory action on glucose uptake and utilization by the peripheral tissues. Lostroh described in detail the procedure used to prepare and test diabetogenie materials from ovine growth hormone (NIH-GH S-10). The hormone was digested with pepsin for 16 hr at 18”-19°C at pH 3.7, using a hormone:enzyme ratio of 14O:l. Under these conditions, an average of one peptide bond was cleaved. The digests were chromatographed on Bio-Gel P-6 columns in 0.5 M acetic acid; only the rising side of the final peptide peak to emerge from the column contained the diabetogenic material. The assay for diabetogenic activity

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uses the hereditary obese female (ob/ob) mouse. In the assay, the animals were given an injection of dexamethasone and fasted for 6 hr. Then a dose of test material was administered subcutaneously, followed 15 min later by a second subcutaneous dose of test substance and glucose (1 mg/g body weight) intraperitoneally. Twenty-five minutes later, blood samples were taken for the determination of blood glucose concentration. Ovine growth hormone subjected to the chromatographic procedure does not impair glucose tolerance in this acute assay, but striking effects are produced by the chromatographic fraction of the peptic digests of oGH described above. The diabetogenic material in the peptic digests of oGH appears to be a slightly basic peptide of approximately 2000 molecular weight, judging from its behavior on Bio-Gel columns. Lostroh indicated that the material does not cross-react with antibodies against hGH. Lostroh also reported that a diabetogenic substance had been produced by digestion of hGH (NIH-HS-1523D) with pepsin. The active component in the peptide digests of hGH behaved like the diabetogenic material prepared from oGH when subjected to chromatography on Bio-Gel P-6. Of particular interest was the observation that the diabetogenic material produced from hGH, in contrast to that made from oGH, caused prolonged glucose intolerance (up to 6 wk in one mouse) in several test animals. Lastly, Lostroh indicated that a synthetic peptide representing the C-terminal 25 amino acid residues of hGH was not hyperglycemic in her test animals, nor were substantial doses either of synthetic linear ovine somatostatin or of a synthetic peptide representing residues 95 134 of the hGH sequence. During the discussion period of this session, Jon H. Levine (Medical University of South Carolina, Charleston, S.C.) presented evidence raising some question about the usefulness of the effect of growth hormone on hepatic ornithine decarboxylase activity as a bioassay for growth hormone. In the course of work on oGH, Levine, with R. Bartou, L. A. Holladay, W. E. Nicholson, and D. Puett, found that when crude oGH (NIH GH S-10) was purified by Sephadex chromatography, the purified material consisting mainly of oGH dimer had only 25%-30’% the activity of the crude material in stimulating ornithione decarboxylase activity in livers of hypophysectomized rats (4-hr treatment period). Approximately 50% of the crude oGH preparation consisted of high-molecular-weight material that differed from oGH in its amino acid composition. However, this high-molecular-weight material appeared to be as good a stimulator of hepatic ornithine decarboxylase activity as dimeric oGH. Interestingly, it had little activity in the weight-gain test in the hypophysectomized rat. Levine also reported that oGH would stimulate ornithine decarboxylase activity in the adrenal and kidney of the hypophysectomized rat, but that the high-molecular-weight fraction of crude oGH was not as effective in stimulating the renal enzyme as the dimer of oGH. Lastly, when the high-molecular-weight material was subjected to chromatography on Sephadex G-200, four fractions were obtained that had differential effects on ornithine decarboxylase activity in liver and kidney and had no effect on the enzyme in the adrenal. Thus, it would appear that the ability of growth hormone preparations to stimulate ornithine decarboxylase activity may not be a biologic activity intrinsic only to the hormone present in the

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preparations, and that changes in the kidney enzyme rather than in the liver enzyme more accurately reflect the growth hormone activity of any preparation. CHEMICAL

AND

ENZYMATIC

FRAGMENTATION STUDIES IN MAN

OF GROWTH

HORMONE:

This session of the conference was devoted to a discussion of the problems associated with the evaluation of the activity of growth hormone preparations and derivatives in man and to reports of recent findings in this area. Daniel Rudman (Emory University, Atlanta, Ga.) described efforts to devise quantitative methods for the evaluation of the various biologic properties of growth hormone in human test subjects. From a quantitative point of view, the most reliable assay, in Rudman’s opinion, is the ll-day elemental balance study, in which the effects of growth hormone (during a 7-day period) on the retention of nitrogen, phosphorous, sodium, potassium, calcium, and chloride are measured. Rudman indicated that reasonable dose-response relationships can be obtained in such balance studies on a given patient, provided that several factors are controlled. First, the nutritional history of the test subject must be considered. A period of dietary adaptation should be used to bring the subject into nitrogen balance, so that essentially zero nitrogen balance and a stable body weight prevail during the control period. Also, Rudman recommended that both the dose of the hormone and metabolic responses should be normalized for the metabolic mass of the test subject, the best index of the latter being body weight i. Further, he indicated that in his experience, various lots of hGH gave different responses in a given patient, despite the fact that equivalent amounts of hormone (International Units) were administered. Perhaps this reflects the error involved in the assignment of relative potency to growth hormone preparations using the growth assay in hypophysectomized rats. Rudman therefore recommended that if growth hormone fragments were to be tested in a human subject, that individual’s responsiveness to native hGH should be calibrated first with the same preparation of hormone from which the fragments were derived. He also pointed out the advantage of measuring the effects of growth hormone on the balance of a variety of elements in addition to nitrogen, namely that insight can be gained regarding the effects of the hormone on various compartments of the body. For example, the balances of sodium, potassium, and chloride yield information on the expansion of the extracellular fluid compartment, while measurement of calcium and phosphorous reflect effects on bone. Assuming that the elemental composition of the body compartments remains stable during the assay period, one can use the method of Reifenstein and Albright to estimate the proportion of weight gain produced by the hormone in each of the body compartments. Lastly, with regard to assay procedures, Rudman indicated that attempts to use the effects of growth hormone on free fatty acid mobilization, plasma amino acid levels, and glucose tolerance for quantitative bioassays of the hormone in man were not successful. At best, these effects, which require substantial amounts of native hGH, can only be used as qualitative tests. Rudman illustrated the use of these bioassay procedures for the calibration of the responsiveness of a variety of test subjects and for the assessment of the activity of growth hormone derivatives. The test subjects used included normal

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children and adults, individuals with growth-hormone deficiencies, individuals with muscular dystrophies, and children with short stature having normal levels of circulating growth hormone. Compared to children with growth-hormone deficiencies, normal children were less responsive to given doses of hGH, and normal adults were slightly less responsive than normal children. In contrast, adults with myotonic and limb-girdle dystrophies were hyperresponsive to hGH, showing responses typical of growth-hormone-deficient children. Interestingly, children with Duchenne dystrophy showed negative elemental balances in response to hGH and exacerbation of their disease so that the tests had to be stopped. Children with short stature, normal circulating growth hormone levels, and retarded bone age responded to hGH in a fashion intermediate between normal and growth-hormone-deficient children, while short children with normal bone age were found to be quite insensitive to hGH. Using the method of Reifenstein and Albright to calculate the distribution of weight gain following the administration of hGH, Rudman found that, in growth-hormone-deficient children and adults with muscular dystrophy, about one-half of the daily weight gain was due to the formation of protoplasm, about one-half to the formation of extracellular fluid, and less than 1% to the deposition of bone. In short children with retarded bone age, most of the weight gain in response to hGH was accounted for by the accumulation of protoplasm. Children with chronic renal disease and short stature were found to gain weight with hGH treatment, but there is little or no nitrogen retention. An analysis of their response indicated primarily the formation of extracellular fluid. Lastly, Rudman described efforts to evaluate the activity of plasmin digests of hGH and RCAM-hGH (prepared by Mills) in individuals whose responsiveness to native hGH had been calibrated. Both the plasmin digests and RCAM-hGH had activity in the elemental balance assay comparable to that of the native hGH preparations from which they were derived. Further, the plasmin digests of hGH retained the diabetogenic property of the native hormone, as well as the ability to mobilize free fatty acids and to depress the level of amino acids in the plasma. Rudman also indicated that large doses of pGH and a plasmin digest of pGH were without effect in elemental balance assays in growth-hormone-deficient children. Martin Sonenberg continued the discussion of the clinical evaluation of growth hormone and growth hormone derivatives by describing his studies on the assay of tryptic fragments of bGH. Sonenberg indicated that his approach to the assay of these materials in human subjects differed from that of Rudman in the sense that a qualitative response to the test substance was sought rather than a quantitative estimate of potency relative to a standard. He stressed the value of the qualitative approach in providing a means to screen hormone derivatives rapidly. Sonenberg first illustrated results obtained with fraction TBGH-d of a limited tryptic digest of bGH, a material which is fully active in the rat. In human hypophysectomized subjects, this material produced positive nitrogen balance, decreases in urinary nitrogen and creatine, increases in urinary calcium and hydroxyproline, and inhibited the hypoglycemic effect of insulin. In hypophysectomized diabetic subjects, TBGH-d raised the fasting blood sugar and

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blunted the glucose tolerance test, although other aspects of their disease were not affected adversely by the test material. Patients receiving an unfractionated tryptic digest of bGH (lo-60 min) had increased blood urea nitrogen, urinary glucose, and ketonuria. Sonenberg also summarized results obtained with peptide AI1 of bGH (residues 96-133) in four teenage children with growth deficiency and a 5-yr-old child with growth retardation but a normal level of circulating growth hormone. Doses of peptide AI1 administered ranged from 5 to 100 mg/day over a 4-5-day period, the usual dose being 5 mg/day for 5 days. Within 24-48 hr of the administration of the peptide, a decrease in blood urea nitrogen was usually noted. Increases in plasma fibrinogen and urinary calcium excretion were also observed to occur during the treatment period, but the increase in calcuria was sometimes delayed, in contrast to the prompt change seen when hGH is administered to similar test subjects. Peptide AI1 also increased the excretion of uric acid, although it had no effect on the level of uric acid in the serum. Of particular interest was that peptide AI1 had no consistent effect on nitrogen balance. In discussing this result, Sonenberg pointed out that even hGH did not produce positive nitrogen balance in three of the five test subjects used. Lastly, Sonenberg demonstrated marked effects of peptide AH on decreasing insulin sensitivity in his subjects. This demonstration of human responsiveness to a 38residue fragment of the bovine hormone provoked considerable discussion of the desirability, if sufficient material were available, of determining whether peptide AH can produce growth in human subjects. Maria I. New (The New York Hospital-Cornell Medical Center, New York, N.Y .) then raised an issue that received much discussion, namely whether any of the metabolic responses used in clinical assays of growth hormone activity are reliable predictors of the extent to which a child will grow on longterm hGH treatment. According to New, one of the principal difficulties is that a given patient may not respond to hGH treatment with detectable changes in all of those aspects of metabolism characteristically affected by the hormone. For example, blood urea nitrogen might decrease in a child receiving hGH, yet no change might occur in other metabolic features, such as nitrogen and phosphorous retention, calcuria, blood fibrinogen level, blood glucose level, or insulin sensitivity. She has also found that some children receiving hGH fail to respond with an increase in plasma somatomedin level, suggesting that the latter is also not a reliable indicator of the metabolic effectiveness of growth hormone. Further, she has observed that some patients grow in response to chronic hGH treatment and yet fail to have consistent acute metabolic responses to the hormone. Finally, she noted that more consistent metabolic and growth responses are seen in young children, which would make them the preferred assay subject’s for growth hormone fragments were it not for the difficulties involved in carrying out elaborate investigative procedures on the young child. SYNTHETIC

FRAGMENTS

OF GROWTH

HORMONE

The final session of the conference dealt with current efforts to synthesize various portions of the amino acid sequence of the human growth hormone molecule. The session opened with a brief review by the chairman, Robert E.

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Fellows (Duke University, Durham, N.C.), of past efforts to synthesize peptide hormones and the potential advantages offered by this approach to the study of structure-function relationships, a principal one being that specific regions of the primary structure can be produced for biologic evaluation by this procedure, in contrast to the uncertain outcome of the enzymatic fragmentation approach. Also, in the case of hGH, the large-scale synthesis of a biologically active fragment of the molecule would appear to be the only ultimate solution to the problem of providing an adequate supply of the hormone. Geoffrey W. Tregear (Howard Florey Institute, Melbourne, Australia) then presented work done in collaboration with Hugh Niall and other colleagues on the solid-phase synthesis of a number of small fragments of hGH, Since fragments produced from the large disulfide loop of native growth hormone by enzymatic cleavage appear to retain some biologic activity, portions of this region of the molecule were selected for synthesis. Also, since the growth hormone molecule appears to contain repeating homologous amino acid sequences, these regions were chosen for synthesis. Tregear then decribed the synthesis of the following peptides: residues l-35; residues 78-103; residues 88-124 (also synthesized by Chillemi and co-workers); residues 95-134; and residues 135-172. Peptide 88-124 was prepared in both the free and blocked forms, the latter being blocked with a pyroglutamyi group at the N-terminus and by forming an amide at the C-terminus. Peptide 95- 134 was also blocked at the N-terminus with an acetyl group and with an amide at the C-terminus. Both basic and acid conditions were used in the purification of various batches of peptide 88- 124 and peptide 95- 134 in the hope of determining optimum conditions for the preservation of biologic activity. After careful purification and chemical characterization, these synthetic peptides were tested for the ability to stimulate growth in hypophysectomized rats, thymidine incorporation into rat costal cartilage, leucine incorporation into isolated rat diaphragm, glucose utilization in isolated rat diaphragm, glucose utilization in isolated rat adipose tissue, and lipolysis in isolated rat adipocytes. Unfortunately, none of the peptides was found to have activity in these assay systems. Also, the peptides failed to compete with native hGH in the radio-receptor assay using pregnant rabbit liver cell membranes. From these findings, Tregear concluded that none of the peptides contain the structural features required for biologic activity. In order to produce a biologically active peptide, he believes that it will be necessary to synthesize a much larger fragment from the 1- 134 region of the hGH sequence, perhaps a peptide comprising residues 80-129, for example. Tregear concluded his presentation with a discussion of the difficulties and uncertainties involved in the preparation of a pure peptide of this size in good yield. He did express the optimistic opinion, however, that this could probably be achieved with present methodology. Choh Hao Li gave a progress report on the efforts of his group to synthesize peptide fragments of the hGH sequence. He first described work done with James Blake involving the synthesis, purification, and characterization of a peptide comprising residues 95- I36 of the hGH molecule. In the tibia assay, this peptide had some activity at very high doses, but it did not give a doseresponse curve parallel to that of native hGH. Hence, it was not possible to

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arrive at an estimate of potency. Li also reported that this peptide had some 50% the activity of the native hormone in the complement fixation assay. Li then presented current work with J. Blake on the synthesis of the peptide consisting of residues 140-191 of the hGH sequence. The half-cystine residue of the peptide was S-carbamidomethylated. Li indicated that peptide 140- 19 1 had a low order of activity in the tibia assay, behaving like the natural peptide 141191 produced by cleavage of the native hormone with plasmin. Also, like natural 141-191, the synthetic peptide was weakly reactive in the complement fixation assay. The session concluded with remarks by John M. Stewart (University of Colorado, Denver, Colo.) on the general problems faced in attempting the solid-phase synthesis of peptides of substantial size, these problems being of obvious concern to those interested in synthesizing fragments of hGH that will be large enough to have significant biologic activity. Like the previous speakers, Stewart stressed the difficulties involved in obtaining quantitative coupling of amino acids to the growing peptide and quantitative removal of side-chain blocking groups. Even with current improvements in materials and techniques, Stewart believes it is only possible, when synthesizing large peptides, to produce mixtures rather than a fairly homogeneous product. Thus, sophisticated purification procedures must be developed and strict criteria for chemical homogeneity must be applied if one wishes to produce a uniform product. Another major difficultly with no obvious solution is that one cannot predict that a given amino acid sequence can be synthesized readily with available technology. Finally, Stewart predicted that further technological developments will be necessary, and particularly, much careful and critical work will be required to produce large biologically active hGH peptides in substantial amounts. SUMMARY

AND

CONCLUSIONS

The following is a summary of the major issues raised and some of the conclusions reached during the conference. (1) The circulating form of growth hormone which interacts with target cells to produce biologic effects may be a modified or altered form of the molecule that is isolated from pituitary glands. Transformation of the hormone to the active, circulating form may take place during the process of secretion or in the circulation itself. At the moment, there is some question whether the biologically active circulating form of growth hormone can cross-react with antibodies to native pituitary growth hormone. (2) It is now apparent that highly purified growth hormone preparations are quite heterogeneous, containing a number of different cleavage products and other charged isomeric forms of the native molecule. This degradation of the native molecule is presumed to result either from processes occurring in the pituitary gland itself or from the procedures used to isolate and purify the hormone. Improved methods of isolation, purification, and identification will be required to resolve this issue. Some of the degraded forms of the hormone appear to have greater activity in the tibia and pigeon crop sac assays than the native molecule. This suggests that appropriate enzymatic cleavage of the mole-

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cule may result in the enhancement of biologic activity. This is a point worthy of further exploration. (3) Growth hormone has been digested with a variety of enzymes resulting in the production of certain fragments that have retained substantial biologic activity in a number of assays in animals and man. A small peptide of bGH consisting of residues 96- 133 has been shown to have some metabolic activity in human subjects, suggesting that a common active core may exist in the various species of growth hormone. The smallest fragment of hGH that has retained substantial activity, particularly in vitro or when administered intravenously, consists of residues 1-134. The diabetogenic property of the hormone has been retained in what appears to be a small, as yet unidentified, fragment produced by digestion of the native hormone with pepsin. It is not known whether this fragment is derived from the portion of the amino acid sequence imparting the growth-promoting property to the hormone. (4) Attempts to produce synthetic fragments of growth hormone having significant biologic activity have so far yielded rather disappointing results. The peptides produced have been either inactive or marginally effective when given in massive doses. It is difficult to interpret the positive bioassay results because the responses obtained are not typical of those given by the native hormone. As better methodology evolves, it is hoped that larger fragments of the primary structure will be synthesized than those made heretofore and that these will possess substantial biologic activity. (5) At the moment, there is considerable uncertainty about the validity and meaning of the various animal biologic assays used to assess the activity of growth hormone preparations and derivatives. This seems particularly true for the tibia assay. Does it reflect the growth promoting property of the hormone? What is the meaning of marginal effects produced in this assay with massive doses of test material? Also, a major difficulty in assessing the potency of fragments of growth hormone in the various assays is that they may, as has already been noted, give dose-response curves that are not typical of those obtained with the native hormone. (6) There also appears to be some uncertainty about the assays used to evaluate the activity of growth hormone and growth hormone derivatives in human subjects. While elemental balance studies appear to be the best procedure for the quantitative estimation of biologic activity in the human, they are difficult, painstaking, and not readily accomplished on very young subjects. Further, there does not appear to be a good correlation between an individual’s acute metabolic responses to hGH and his ability to grow on chronic treatment with the hormone. (7) A bewildering nomenclature has evolved for the many fragments and derivatives of growth hormone produced and isolated during the past few years, as any reader of this report will discover. Clearly a single systematic nomenclature for these substances should be established.