lnosine D i- and Triphosphate Synthesis in Erythrocytes and Cell Ex rac S BERNARD0 S. VANDERHEIDEN Eastern Pennsylvania Psychiatric Institute, Philadelphia, Pennsylvania I9129

ABSTRACT The ability to synthesize inosinetriphosphate was demonstrated in blood cells as well as in a variety of tissue extracts in spite of the presence of ITP pyrophosphohydrolase. A t the expense of having sub-optimal conditions, an assay system was selected that completely repressed the hydrolyzing enzyme, thus permitting the accumulation of ITP. In an attempt to define the biosynthetic pathway of ITP, and since guanylate kinase has been implicated in the formation of ITP, the rate of synthesis of ITP and GTP in cell extracts was compared. The comparison of the specific activities of the [’Cl-labeled hypoxanthine and guanine moieties of the inosine and guanosine phosphates formed during incubation with [8- 14C1-inosineand [8-14C1-guanosinerespectively, revealed striking differences in the relative rates of isotope incorporation. Tentative mechanisms are proposed to explain these differences. The data obtained thus far does not discard the possibility that ITP may be formed by stepwise phosphorylation and (or) by direct pyrophosphorylation of IMP. The inability to demonstrate the synthesis of ITP in cellular preparations has been attributed to the presence in cytoplasm of inosinetriphosphate pyrophosphohydrolase (ITPH) an enzyme that does not permit accumulation of the nucleotide (Vanderheiden, ’75). The first indication of the capacity of cells to synthetize ITP came to light with the discovery of a genetic trait in which high concentrations of ITP appeared in human erythrocytes of some individuals (Vanderheiden, ’65). Studies with ITPH deficient cells permitted the first demonstration of synthesis of ITP in vitro (Vanderheiden, ’67).It was subsequently observed that the ability of erythrocytes to “synthetize” ITP varied from individual to individual, and that it was inversely proportional to ITPH activity (Vanderheiden, ’69a). I t has recently been shown however, that ITP apparently accumulates in all erythrocytes under certain experimental conditions (Henderson et al., ’77). Although the mechanism of synthesis of IPT has not been elucidated, Agarwal et al. (‘71) postulated its synthesis by formation of IDP from IMP by guanylate kinase (EC 2.7.4.8) followed by transphosphorylation J. CELL. PHYSIOL. (1979)99: 287-302.

by nucleosidediphosphate (NDP) kinase (EC 2.7.4.6). On the other hand, based on specific activity measurements of the I4C-labeledhypoxanthine moiety of the inosine phosphates formed during incubation with [8- I4C1-inosine by ITPH deficient cells, Vanderheiden (‘69b) postulated an ATP: IMP pyrophosphorylation. The present studies describe factors that contribute to the accumulation of ITP in human erythrocyte preparations. In addition, evidence is presented for the ability of the cytosol fraction of cells of various tissues to synthetize ITP, regardless of the original ITPH activity. In an attempt to define the biosynthetic pathway of ITP, the rate of synthesis of ITP and GTP in cell extracts is compared. Furthermore, the relative rates of [8- 14C1-labeledhypoxanthine and guanine incorporation into the respective inosine and guanosine phosphates was compared. The method used for determination of synthetizing activity, in addition to its high sensitivity, permits simultaneous determination of anabolic as well as catabolic enzyme prodReceived Feb. 17, ’78. Accepted Jan. 23, ’79. I Because of its high specificity (Vanderheiden, ‘79). the author prefem the name of inosine triphosphate pyrophosphohydrolase.

287

288

BERNARD0 S. VANDERHEIDEN

paper for high-voltage electrophoretic separation. Inosine and hypoxanthine remain close to the point of application and are not effectively separated. However, other undesirable MATERIALS and interfering components are elimated by ITP was purchased from Fluka AG. Other electrophoresis. The twin band containing hybiochemicals were obtained from Boehringer poxanthine and inosine was then eluted; the Mannheim Corp. [8-I4C1-ITPand [8-14C1-IMP eluate was lyophilized and taken up in 50 p1 of (29 mCi/mM) were purchased from Schwarz- water. An aliquot (40 p1) was applied on acidMann. [8-14C1-GMP(58 mCi/mM) and [8-14C1- washed Whatman No. 1 paper, and the cominosine (28 mCi/mM) were obtained from pounds were separated by ascending chromaAmersham-Searle Corp. [8-14C1-guanosine (58 tography with a mixture of n-butanol: formic mCi/mM) was purchased from ICN Chemical acid (99%):water (77:10:13, v/v/v) (Markham and Radioisotope Division. Ficoll-Paque was and Smith, '49). Hypoxanthine and inosine purchased from Pharmacia Fine Chemicals. concentrations of the eluates from the paper Out-dated blood was supplied by the American chromatograms were determined enzymatically (Jorgensen, '74; Coddington, '74). Red Cross Blood Bank in Philadelphia. Labeled compounds were counted in a liquid METHODS scintillation spectrometer at 35% efficiency A. Whole blood studies using a modified Bray's scintillation solvent 1. Labeled inosine (Vanderheiden, '71). The concentration of Blood from individuals with ITPH deficient compounds is expressed in pmoles/g Hb, and erythrocytes was incubated with [8-I4C1-ino- the specific activity in counts/min/pmole of sine (6.8 pCi/lO ml blood) a t 37" for varying compound. intervals of time. Suitable aliquots (5-10 ml) were removed a t the times indicated, cooled, 2. Labeled guanosine and centrifuged 30 minutes a t 2,000 rpm. An The method is essentially t h a t described for aliquot of plasma was removed and saved for labeled inosine (section 1) except that blood subsequent analysis. A control sample (not in- with normal ITPH erythrocytes was used. cubated) was processed along with the rest of Ammonia from the commercial sample of the samples. Following removal of residual [I*CI-labeledguanosine was removed by bubplasma and white cells, the packed red cells bling with nitrogen gas to pH 7.0. An equal were washed three times with 10 volumes of volume of 1.7%NaCl solution was added prior cold saline solution. All operations were car- to addition to the blood (6.7 pCi/lO ml blood). ried out a t 4". The acid-soluble phosphate Labeled compounds from plasma were not esters from erythrocytes were obtained by studied. Radioactivity measurements were trichloroacetic acid extraction as described carried out as previously described Wanderpreviously (Vanderheiden and Boszormenyi- heiden, '72). Nagy, '65). Separation was accomplished by combination of high-voltage paper electroB. Studies with cell extracts phoresis and paper chromatography. Detection and quantitation was carried out as de- 1. Determination of synthesis of nucleoside triphosphates scribed by Vanderheiden ('64, '68). Labeled compounds from plasma (inosine The method used consists of a n adaptation and hypoxanthine) were obtained by ex- of the microassay for determination of ITPH traction with a n equal volume of 10%trichlo- activity (Vanderheiden, '72). [I4C1-labelednuroacetic acid, centrifugation for ten minutes cleoside monophosphates are used as subat 2,000 rpm, and filtration of the supernatant strates and the products of the reaction are solution using Whatman No. 4 filter paper. separated by high-voltage paper electrophoreThe protein precipitate was washed three sis. The labeled nucleotides are then counted times with a small volume (3-5 ml) of 5% tri- directly by liquid scintillation spectrometry. chloroacetic acid. The combined supernatant The final concentration of compounds in the solutions were extracted four times with 2 to 3 enzyme reaction mixture is as follows: 100 volumes of ether. Excess ether was removed mM potassium phosphate buffer pH 8.0, 2.2 with N Z Finally, the extracts were lyoph- mM MgCl,, 5 mM CaCl,, 5 mM ATP, and 1mM ylized to dryness, taken up in 0.20 ml of water IMP or GMP, and the corresponding [I4C1-laand 0.16 ml was applied on Whatman 3MM beled nucleoside monophosphate (0.02 pCi per

ucts formed when crude extracts are used in conjunction with the [14C1-labelednucleoside monophosphates.

IDP AND ITP SYNTHESIS IN CELLS

50-p1 sample). The enzyme source (10-30p1) is incubated a t 30" in a stoppered micro test tube with the corresponding volume of assay mixture so t h a t the total volume is 50 pl. The reaction is stopped by addition of 10 p1 of 2% trichloroacetic acid. The sample is applied and analyzed as derscribed previously (Vanderheiden, '72). Calculations. The total cpm of each sample is correlated to the total number of nmoles of labeled nucleotide present in the assay mixture. A control sample without enzyme is treated in a similar manner in order to correct for possible breakdown products of the labeled substrate. The total cpm of the labeled compounds detected after electrophoretic separation of the control sample is related to the nmoles of the labeled nucleotide present in the original mixture. The nmoles of each labeled product is proportional to its cpm. The calculations can be summarized as follows: Let X A , XB, . . . X N represent the net cpm of compounds A, B, . . . N in the control sample, and YA,YB . . . YN the net cpm of compounds A, B, . . . N a t the end of the enzymatic reaction. The number of nmoles of A, B, . . . N in the control and experimental samples are, respectively:

where C is the number of nmoles of nucleotide in the reaction mixture, in this case 20. The net change of substrate or formation of product is given by:

[ -C- . Y A ]

-

[ ~C .

X A ] =AA

ZY

For a simple case of conversion of A to B, AA should correspond to AB. This type of calculation can easily be incorporated into a programmable calculator for rapid resolution. Protein concentration was determined by the method of Lowry et al. ('51). Hemoglobin was determined using Drabkin's reagent. Enzyme specific activity is expressed in terms of pmoles of nucleotide produced/hr/g Hb or protein. 2. Preparation of hemolyzates Human blood, fresh heparinized or ACD stored blood was centrifuged for ten minutes at 2,000 rpm. All operations were carried out at 4'. Plasma and white cells were discarded

289

and the packed cells were washed three times with saline solution. The cells were hemolyzed with one or two volumes of distilled water, and dialyzed overnight versus a buffer solution containing 2 mM MgC12, 1 mM 2-mercaptoethanol, 0.2 mM Na EDTA, and 1 mM Tris-HC1, the mixture adjusted to pH 7.1 (Buffer A). The dialyzate was centrifuged for one hour a t 23,000 x g. The clear supernatant solution was removed by suction for subsequent use.

3. Preparation of tissue extracts The cytosol was obtained as previously described (Vanderheiden, '75). Perfusion with saline was omitted except in the case of the rat. 4. Preparation of platelet extracts

Whole blood collected in 3.1%Na citrate in siliconized tubes was centrifuged at 150 x g for 15 minutes at 4". The platelet rich plasma was centrifuged once more (polyethylene tubes) a t 150 x g for 15 minutes. The supernat a n t solution was then centrifuged a t 2,000 x g for 20 minutes. The resulting supernatant solution was discarded. The precipitate was washed three times with saline solution. One volume of cold distilled water was added to the washed platelets. The mixture was homogenized for one minute in a teflon homogenizer and dialyzed overnight versus buffer A. Finally, the dialyzate was centrifuged a t 23,000 x g for one hour to remove cell debris. 5. Preparation of leukocyte extracts Leukocytes were isolated as described by Bertino e t al. ('63). The packed cells were suspended in 9 volumes of cold distilled water, homogenized in teflon homogenizers for one minute, and dialyzed overnight versus buffer A. The dialyzate was centrifuged at 23,000 x g for one hour to remove cell debris. 6. Preparation of lymphocytes

Lymphocytes were isolated using FicollPaque as outlined by the manufacturer following the method of Beyum ('68). The extracts were prepared in the same manner as that of leukocytes. RESULTS AND DISCUSSION

In order to determine the sequence of events Unless otherwise indicated, cell extracts were dialyzed against buffer A (see Preparation of hernolyzates). Therefore, the enzyme reaction mixture, in addition to the components mentioned above, contained 0.6 mM 2-mercaptoethanol,0.12 mM Na EDTA and 0.6 mM Tris-HC1.

290

BERNARD0 S. VANDERHEIDEN

30

60

90

120

TIME (min.)

Fig. 1 Incubation of EDTA blood with 18-1El-inosine.ITP pyrophosphohydrolase deficient blood collected in Na EDTA anticoagulant (approx. 4-5 mM) was allowed to stand overnight at 4°C. The blood was then incubated at 37" with [8-1C1-inosine (10 pCi/lO ml). Aliquots were removed at the times indicated and the inosine phosphate concentrations and specific activities were determined as described in the text. x - X IMP, A-A IDP, 0-0 ITP, 0-0 IMP IDP + ITP.

+

leading to the synthesis of ITP, the pathway of [8-l4C1-inosine incorporation was followed during in vitro incubation of whole blood from subjects with ITPH deficient erythrocytes. The concentration and specific activity time curves of the ["CI-labeled nucleotides are shown in figure 1. Under these conditions (blood collected in Na EDTA anticoagulant and allowed to stand overnight a t 4" prior to incubation), a rapid increase in the concentration of IMP is observed after 15 minutes incubation followed by a decrease and a leveling off after 45 minutes. IDP shows a slow and progressively increasing level after a rapid initial surge in concentration. ITP, on the other hand, shows a gradual increase peaking at 45 t o 60 minutes with a subsequent decrease and leveling off a t 90 minutes. The overall total hypoxanthine nucleotide concentration is threefold its original value a t 15 minutes, decreases in the next 15 minutes, and finally levels off to twice its original concentration. The specific activity values, on the other hand, show an almost linear increase for IMP during the first hour followed by a sudden drop and leveling after two hours. Both IDP and ITP show a gradual increase leveling after 60 to 90 minutes incubation.

The main feature of the relative specific activities is that ITP is consistently and significantly higher than IDP. In another experiment, the incubation with [8-14C1-inosinewas extended to six hours. The results are shown in figure 2. Under these conditions (fresh heparinized blood), the specific activity of ITP is a t all times, significantly higher than that of IDP. Even though the intracellular total inosine phosphate concentration fluctuates in prolonged incubation, the cell apparently can regulate and maintain a range of values that does not oscillate significantly during the duration of the experiment. It is interesting to note, that the rate of incorporation of the [ 14C1-labeledprecursors into IMP, as well as IDP and ITP increases as time progresses, perhaps as a result of the gradually increasing concentration of hypoxanthine in the red cell and plasma, as shown in figure 3,or (and) as a result of changes in the levels 3The determination of concentration and specific activity of intracellular hypoxanthine and inosine is technically difficult if not impossible, since contaminating plasma compounds cannot be removed without subjecting erythrocytes to repeated saline washes. In the case of hypoxanthine, transport across the membrane is very rapid (Lassen and Overgaard-Hansen, ' 6 2 ) , therefore, considerable loss of the compound would result. In the ca8e of inosine, no information IS available on its rate of transport since it is actively metabolized (Lieu et al., '71).

291

IDP AND ITP SYNTHESIS IN CELLS

i

I

2

4

vals of time) incorporated into the hypoxanthine and guanine nucleotides respectively, were 15:l in the case of IMP and GMP, 1:4 in the case of IDP and GDP, and 1:12 in the case of ITP and GTP. The GMP concentration remains unchanged during the length of the experiment. GDP, on the other hand, shows a transient increase after one hour, peaking a t two hours and leveling down after three hours. GTP shows a n apparent cycling after two hours with peaks a t three and five hours. If one assumes a homogeneous pool of t h e individual nucleotides (i.e., no compartmentalization, or binding of the nucleotides with a protein or enzyme), and a sequence of reactions without branching or cycling, the relative order of reactions leading to the formation of ITP and GTP is established by the order of the relative specific activities of t h e various purine derivatives. For the inosine series:

6

TIME (hrs.)

Fig. 2 Prolonged incubation of fresh heparinized ITP pyrophosphohydrolase deficient blood with [8-'FI-inosine. Sixty-five milliliters of fresh heparmized blood was incubated with 45 pCi of l8-'Tl-inosine (28 mCi/mM). Aliquots were removed at the times indicated and the concentration and specific activity of the inosine phosphates were determined as described in the text. X-X IMP, A-A IDP, 0-0 ITP, 0-0 IMP + IDP + ITP.

of other metabolites, as well as changes in pH during prolonged incubation. For example, in the absence of glucose, one can expect a decrease in ATP concentration with a concomit a n t increase in intracellular inorganic phosphate, which would have a stimulating effect on the production of PP-ribose-P (Hershko et al., '69) resulting in an overall higher rate of exchange or incorporation of hypoxanthine into IMP, IDP, and ITP. In the case of incubation of fresh heparinized blood with [8-14C1-guanosine,a different and unexpected pattern of incorporation was obtained. Figure 4 shows the rate of incorporation of the labeled guanine moiety into t h e guanosine phosphates and their concentrations a t the times indicated. The total dpm incorporated after six hours incubation was of similar magnitude for both the inosine and guanosine phosphates, 224 X lo3and 152 x lo3respectively. However, the distribution of dpm in the mono-, di-, and triphosphates varied considerably. The overall average ratios of dpm (from the various inter-

- - - - Is

[Hxl

IMP

ITP

IDP.

And for the guanosine series: Gs

[Gul

GDP (or GTP)

GTP (or GDP)

-

GMP.

In the case of ITP, the sequence would suggest the existence of a pyrophosphorylating enzyme. In the case of the guanosine series, the pathways are highly unlikely, therefore, some type of nucleotide-protein complex must exist. In order to account for the data obtained, the following series of reactions are proposed as a tentative mechanism for the synthesis of inosine and guanosine phosphates. represents hypoxanthine or guanine and 8 - R , the corresponding nucleoside; E, protein or enzyme. X-R

+

pi nucleoside phosphorylase

(2)

X + P-R-PP + E

(3)

E-XMP

(4)

E-XMP

+ ATP

(5)

E-YDP

(6)

E-XDP + ATP

(7)

XTP

HGPRT

X *

E-XMP + PPi

*

E+XMP

iEEi:Mp.

Ezse

+ R-I-P

E-XDP E

+ ADP

+ YDP

XTP

i ADP i E

YDP

+ Pi

Reaction 3, representing the dissociation of an enzyme (or protein) - guanosine (or inosine) monophosphate complex, is by necessity, the slowest of the guanosine series of reactions, thus accounting for the relatively low

292

BERNARD0 S. VANDERHEIDEN

-- I:

I

TIME (hrs)

Fig. 3 Plasma hypoxanthine and inosine. Specific activity and concentrations of plasma hypoxanthine and inosine from ITP pyrophosphohydrolase deficient erythrocytes were determined on blood incubated with inosine, 0-0 hypoI8-WI-inosine. See figure 2 and text for experimental details. Specific activity: A-A xanthine. Concentration in moles/lO ml blood: A-A inosine, 0-0 hypoxanthine.

specific activity of GMP. The dissociation is also considerably lower for the GMP complex than for the IMP analog. Reaction 5, on the other hand, representing the dissociation of the enzyme (or protein) GDP (or IDP) complex, is the slowest of the inosine series of reactions, accounting for t h e low specific activity of IDP. The dissociation is also relatively lower for the IDP complex than for the GDP analog. Reaction 7 is by necessity, the rate limiting reaction particularly in the case of the inosine series. It appears that the relative rates of reactions 5 and 6 in the guanosine series are subject to regulatory mechanisms, possibly by the effect of changes in the concentration of GDP (mass action?). This is evident in figure 4 a t intervals of one and two hours, where the relative specific activities of GTP and GDP are reversed. Ideally, t h e comparison of biosynthetic pathways of ITP and GTP by determination of the incorporation of labeled purines in whole

cells should be carried out with the same blood, i.e., ITPH normal erythrocytes. However, there are some inherent difficulties. In the first place, ITP and IDP concentrations in normal erythrocytes are extremely low, thus requiring the processing of relatively large amounts of blood in order to obtain significant values; and secondly, the presence of ITPH activity in the red cells would contribute to a recycling effect, as ITP synthesized is promptly hydrolyzed to IMP.4 In order to overcome this difficulty, blood with ITPH deficient erythrocytes was used for the labeled inosine experiment. If, on the other hand, the comparison of the incorporation of both purines were to be carried out using ITPH deficient blood, one could not disregard the possible effect of its relatively high intracellular concentration of both IDP and ITP on the incorporation of guanine into the guanosine phosphates, particularly if ' Since human erythrocyte ITPH is highly specific for ITP, showing less than 1%hydrolytic activity towards GTP, (Vanderheiden, "79) no recycling would be expect& in the case of GTP.

293

IDP AND ITP SYNTHESIS IN CELLS

2

4

6

TIME (hrs)

Fig. 4 Prolonged incubation of fresh heparinized ITP pyrophosphohydrolase normal blood with [E-'TIguanosine. Sixty-five milliliters of fresh heparinized blood was incubated with 45 pCi of [8-'T]-guanosine (58 mCi/mM). Aliquot8 were removed at the times indicated and the concentration and specific activity of the guanosine phosphates were determined as described in the text.

the enzymes (and the pathways illustrated in reaction 1 through 7) are responsible for the synthesis of GTP. It is hard to know whether the proposed series of reactions is compatible with existing knowledge of the enzymes involved, namely hypoxanthine-guanine phosphoribosyl transferase (HGPRT) (EC 2.4.2.81,GMP kinase and NDP kinase. Perhaps, the only pertinent information available is the evidence for the binding of GMP to HGPRT (Krenitsky and Papaioannou, '69). It is premature nevertheless, to discard the possibility that ITP (and GTP) may also be formed by direct pyrophosphorylation, a s there is no evidence as yet for an IDP or GDP protein or enzyme complex, particularly in the case of the respective monophosphate kinases. Although it had been suggested that ITP accumulation in erythrocytes reported by Blair and Dommasch ('69) and Zachara and Lewandowski ('74) was due to a probable ITPH deficiency (Vanderheiden, '75), more recent reports show that human erythrocytes can accumulate ITP under certain experimental

conditions (Henderson et al., '77). Differences in ITPH activity accounted for individual variations in ITP accumulation. The data of Zachara ('74) suggest that the intact normal erythrocyte has a potential rate of synthesis of ITP of approximately 1.5 kmole/hr/g Hb, when whole cell suspensions of stored blood are incubated a t 37" in 20 mM inosine, 2 mM pyruvate and 100 mM inorganic phosphate. Somewhat analogous results were reported recently by Henderson et al. ('77). It appears, therefore, that given the proper experimental conditions (inhibitory to ITPH), it might be possible to demonstrate the synthesis of ITP in cell free extracts. Since human erythrocyte ITPH has an absolute requirement for Mg++ (Vanderheiden, '70), and shows optimal activity a t 50 mM concentration, one would expect considerably less activity of the enzyme a t concentrations of Mg++ endogenous to the red cell, approximately 2 mM. In addition, Ca++ inhibitory to ITPH (Vanderheiden, '79) may contribute to the accumulation of ITP. It was necessary then to select optimal con-

294

BERNARD0 S. VANDERHEIDEN TABLE I

Effect of metals on ITPand GTPsynthesis mM MgCI,

2 4 5 10

0.026 0.181 0.100 0.046 0.018

0

~

ITP nmolea

'

~~~~

1.2 mM MgC12

5 mM CaC1,

5 mM COCI?

5 mM MnC1,

ITP nmoles

GTP nmolea

GDP nmoles

+ +

+

-

-

-

0.117 0.242 0.021 2 0.030 0.096 ' 0.211 ' 0.169 ' 0.010 0.094 '

0.80 0.56 1.04 0.77 2.42 13.7 6.13 14.3 14.0

0.17 0.06 0.11 3 0.10 0.31 4.40 1.47 4.29 4.29

+ ++ -

-

+

-

-

+

-

+

-

-

-

-

-

+

+

For additional experimental details, see METHODS. ' Non-dialyzed fresh human hemolyzate, 0.8 mg Hb,4.5-hour incubation. Dialyzed (buffer A) fresh human hemolyzate, 2 mg Hb, 1-hour incubation. Dialyzed (buffer A) cat bram cortex cytosol, 3.7 fig, 15-minute incubation. Dialyzed (modified buffer A, no MgCI,) hemolyzate of atored h l d . 3.4 mg Hb,5-hour incubation. Dialyzed (modified buffer A, no MgCl,) hemolyzate of stored blood, 3.4 rng Hb,10-minute incubation.

ditions for ITP accumulation. In preliminary experiments with hemolyzates, a reaction mixture containing 1.2 mM MgCl,, a 1:5 ratio of IMP to ATP (1 and 5 mM) and 100 mM potassium phosphate buffer pH 8.0, gave best accumulation of ITP. This basic assay mixture was used on subsequent experiments therefore, in attempts to optimize further the enzyme assay system. It was observed that using fresh heparinized hemolyzate of blood with normal ITPH activity as the source of the enzyme, and in a system containing 1mM IMP, 5 mM ATP, and MgCl, concentrations ranging from 1.2-50 mM, or in the presence of 5 mM CaC1, with either 1 mM IMP or 5 mM ATP, there was no detectable [14CI-IMPproduced when ITPH activity was measured using L14C1-ITPsubstrate (Vanderheiden, '72). The fact that ATP and IMP are inhibitory to ITPH has already been reported (Chern et al., '69). It is fortuitous then, t h a t under the conditions of assay, two of the obligatory substrates for the synthesis of ITP are inhibitory to its breakdown by ITPH. Table 1shows t h e effect of MgC1, concentration on the formation of ITP. The effect of other cations in the presence and absence of MgC1, on the formation of ITP and GTP is also ilGstrated. As can be observed, Mg++does not

appear to be an absolute requirement for the synthesis of the nucleotides, but there is considerable activation of the system at 2 mM MgC1,. At higher concentration of Mg++however, there is a decrease in the accumulation of ITP, probably due to increased activity of ITPH. In the presence of 1.2 mM MgCl,, Cat+ promotes accumulation of ITP, while Cot+ and Mn++diminish it. In contrast, Cat+ decreases formation of GTP, Co++activates it and MnC+ has no significant effect. Since ITPH is highly specific for ITP (less than 1%activity with GTP as substrate [Vanderheiden, '791 ), it is safe to assume that the effect of cations on GTP synthesis is not due to increased or decreased GTP pyrophosphohydrolytic activity of the enzyme. Moreover, (not shown in table 1) whether ITP or GTP was being measured, no significant accumulation of nucleoside diphosphate occurred except as indicated. Although 5 mM CaCl, in the presence of 1.2 mM MgC12 is inhibitory to GTP synthesis, ITP accumulation is favored under these conditions. Table 2 shows t h e results obtained when Ca++or Mg++ concentrations are kept constant and the concentration of the second cation is varied. As can be observed. best ITP accumulation is obtained in the combination

295

IDP AND ITP SYNTHESIS IN CELLS TABLE 2

Effect of Mg++andCa++concentrationon ITPand GTPsynthesis 1.2 mM MgC1,

5 mM CaC1,

mM MgCI,

ITP

’

GTP

mM CaC1,

ITP ‘

0 1 2 3 5 10

0.20 0.36 0.40 0.46 0.42 0.26

nmoles

nmoles

0.39 0.46 0.26 0.23 0.04 0.04

1.2 2.2 4.2 6.2 8.2 10.2

GTP

0.73 0.74 0.84 0.80 0.77 0.73

0.73 0.81

-

0.82 0.59 0.42

Cat cerebellum cytosol, dialyzed versus buffer A. For additional experimental details see METHODS. ‘ Six-hour incubation, 12 p g protein. 2Ten-minute incubation, 2.4 g g protein.

GTP /*

2-0.2

In

-0 aJ

E

0

~o---o-~-o 10 60

20 120

30 ( 0 ) I80 “1

T/M€ fmin/ Fig. 5 Time course of inosine and guanosine di- and triphosphate formation. The final enzyme reaction mixture contained 1 mM inosine- or guanosine monophosphate, 0.02 pCi of the respective [‘TI-labeled nucleotide, 5 mM ATP, 2.2 mM MgCl,, 5 mM CaCl,, 100 mM K phosphate buffer, pH 8.0,in a total volume of 50 pl. The reaction wa8 started by addition of low1 of dialyzed cell extract (see footnote 2 on p. 289).and stopped by addition of 10 pl of 2% trichloroacetic acid. Values represent average of two determinations. ITP and IDP, ox brain cytosol, 23 pg protein; GTP and GDP, dialyzed fresh human hemolyzate, 126 pg Hb.For additional experimental details see METHODS.

of 5 mM CaC1, and 2.2 mM MgC12, or 3 mM CaC1, and 1.2 mM MgCl,, the latter system being slightly better for GTP formation. It was necessary to establish that under the chosen experimental conditions, the relationships between ITP and GTP formation were linear with respect to time as well as with respect to enzyme concentration. Figures 5 and 6 show that under the conditions described,

near linearity exists in both cases. There is a difference, however, between the inosine and guanosine nucleotides formation. The curves for the formation of GTP and GDP are normal under the conditions described (i.e., when the rate of NDP kinase is lower than the rate of GMP kinase). The curves for ITP and IDP show a n abnormal characteristic, namely an intercept with a positive value. This was a

296

BERNARD0 S. VANDERHEIDEN

0.I

.,’

/

P100

200

300 ( G )

PROTEIN (.gl Fig. 6 Protein concentration and inosine- and guanosine triphosphate formation. Reaction mixture as described in figure 5. The reaction was started by addition of the proper aliquots of dialyzed cell extracts (see footnote 2 on p. 2891,and was stopped with 10 fi1 of 25%trichloracetic acid. Values represent average of two determinations. ITP and IDP, ox brain cytosol, 1-hour incubation. GTP and GDP, dialyzed fresh human hemolyzate, 15-minute incubation. For additional experimental details see METHODS.

consistent observation in time and protein concentration curves obtained in repeated studies using either hemolyzate preparations or brain extracts. It suggests the presence of a secondary reaction for ITP formation (other than NDP kinase), perhaps by dismutation of IDP a reaction first described by Joklik (’55) and reported not to be carried out by animal tissue preparations. 2 IDP

-

IMP

+ ITP.

However, in the absence of ITPH activity or under conditions where ITPH activity is repressed, the formation of ITP by irreversible dismutation of IDP has been demonstrated in our laboratories (results not shown). In the case of ITP formation, the rate of NDP kinase is greater than the “IMP kinase,” accounting for the leveling off of IDP in the two sets of curves. It must be emphasized that the assay system finally selected for the measurement of ITP formation is optimal only in that i t pre-

vents its breakdown by ITPH present in the cell extracts. It is not necessarily optimal for ITP synthesis. This was demonstrated with ITPH-free preparations. Table I11 shows the effect of MgC12and combinations of Mg++and Ca++ions on ITP and GTP synthesis carried out by preparations with no ITPH activity. As can be observed, in the presence Mg++,a lowering of Ca++concentration results in an increase in synthesis of ITP (or combined ITP IDP), comparatively more than in the case of GDP). GTP (or combined GTP In the absence of Ca++and a t 21 mM MgC12, in the case of the protein fraction, there is a 400% increase of ITP synthesis when compared to the standard assay system (2.2 mM MgC12 and 5 mM CaC12).On the other hand, GTP was only 20% higher, while combined GTP and GDP formation was lower by 30%.In the case of the ITPH deficient hemolyzate, the difference was not as marked in the absence of Ca++. Based on the properties of erythrocyte

+

+

297

IDP AND ITP SYNTHESIS IN CELLS TABLE 3

Synthesis of inosine and guanosine nucleotides in ITPH-freepreparations Mg"

Ca"

ITP

mM

IDP nmoles

GTP Sp. Act. (I) (ITP + IDP)

GDP nmoles

5.0 2.2 2.0

2.2 2.2 3.0 21.0

0.32 0.66 0.75 1.32

ITPH-free protein fraction 0.03 11.5 1.01 0.09 24.4 1.00 0.10 27.3 1.17 0.12 46.8 1.27

5.0

2.2 21.0

0.22 0.29

ITPH deficient hemolyzate 0.065 0.19 0.50 0.065 0.23

Sp. Act. ( G ) (GTP + GDP)

X (l)/(G)

7,470 7,750 8,140 5,060

0.15 0.32 0.34 0.92

' 2.82 2.96 2.99 1.30 0.034

35.3

0.54

' Fraction of non-hemoglobin protein of erythrocytes from normal ITPH blood, obtained from Whatman DEAE 32 column eluting between 10 and 20 mM NaC1, just prior to elution of ITPH, and corresponding to fraction containing inorganic pyrophosphatase activity as determined by qualitative test (Vanderheiden,'79)as well as ITP synthetizingactivity. Assay for ITP, 3 hours 10.2 /"g protein; GTP, 15 minutes 2.0 pg protein. * ITPH activity of erythrocytes = 0 (see table 5); assay for ITP 1.5 mg Hb, 60 minutes; GTP 0.06 mg Hb, 15 minutes.

guanylate kinase, Agarwal et al. ('71) postulate the formation of ITP via this enzyme (and NDP kinase) even though the Kmvalue (1.3 x M) is considerably higher than the normal concentration of IMP in human erythrocytes (1-2 x loe5 M) (Vanderheiden and ZarateMoyano, '76), and even though the Vm, with IMP is about 0.2% that with GMP. A comparison of the relative rates of synthesis of ITP and GTP under different experimental conditions may give indications as to whether the same enzymes are responsible for the synthesis of the two nucleotides. Differences in the ratio of the rates of synthesis under various but comparable experimental conditions, may suggest two different enzyme systems or perhaps, two distinct biosynthetic pathways (tables 3,5). If indeed, the synthesis of ITP and GTP is carried out by the same enzymes, one would expect activation and (or) inhibition to be relative in magnitude and direction for the two cases. This was not the case, however, in the experiments with ITPHfree preparations shown in table 3. Let us assume t h a t ITP and GTP are formed only via guanylate kinase, coupled to NDP kinase. The comparisons of the rate of synthesis of ITP and GTP would be meaningful only in those cases where guanylate kinase is the rate limiting enzyme. In cases where NDP kinase activity is rate limiting and accumulation of the corresponding nucleoside diphosphate ensued, a n estimate of the rate of formation of the triphosphates can be obtained by adding the values of the corresponding diand triphosphates. In this particular case, the

comparison of the rate of synthesis of ITP and GTP would be subject to the assumption that under identical experimental conditions, effects caused by allosterism, feedback inhibition, or abortive-complex formation (Mourad and Parks, '66) are non-existent for the two enzymatic reactions under consideration. There is no evidence, however, to exclude these possibilities. Furthermore, the possibility of interference by the presence of related enzymes such as nucleoside diphosphate phosphohydrolase (EC 3.6.1.6),5'-nucleotidase (EC 3.1.3.5), nucleosidase (EC 3.2.2.1), or nonspecific phosphohydrolase cannot be overlooked. The rate of synthesis of ITP by human erythrocyte preparations was compared under different experimental conditions. The results are shown in table 4. Dialyzed hemolyzate of stored blood showed a 5-fold increase in the ability to synthetize ITP when compared to t h a t of fresh blood from the same subjects. The highest rate of formation of ITP was obtained with hemolyzate of blood stored in citrate-dextrose-phosphate solution, a rate close to that reported by Zachara ('74) with erythrocyte suspensions of blood stored in acid-citrate-dextrose solutions. However, as pointed out previously, the selection of assay conditions were determined by ITPH activity, so it is conceivable that a n even higher rate of accumulation of ITP can be achieved at optimal conditions, providing ITPH does not interfere. Under similar experimental conditions, no significant difference was found in the rate of synthesis of ITP when a dialyzed hemolyzate

298

BERNARD0 S. VANDERHEIDEN TABLE 4

ITPand IDPsynthesis by human erythrocyte preparations ITP

IDP

Preparation g m o l e s k d g Hb

Assay @ndltions

1. Dialyzed hemolyzate Fresh blood

0.15 (0.13-0.20)

0.16 (0.09-0.37)

Stored blood

0.80 (0.40-1.8)

1.1 (0.22-2.9)

I

lmMIMP, 5mM ATP, 100mMKPO,pH8.0, 5 mM Cat+,2.2 mM Mg+', 1 hour, 30" Same as above

2. Stored blood 15%erythrocyte suspension

1.5

n.r.

20 mM inosine, 20 mM Na pyruvate, 100 mM KPO,pH7.5, 150mMNaC1,2hour~,37~. ACD storage 15-45days. 4".

3. 10%erythrocyte suspension

0.03-0.15'

n.r.

Fischer's medium, 25 mM PO, pH 7.4,lOO pM hypoxanthine, 2 hours, 37"

4. 10%erythrocyte suspension Before storage

0.035 '

n.r.

0.152

n.r.

Krebs Ringer, 25 mM PO, pH 7.4,lOOgM hypoxanthine, 2 hours, 37".Sample stored 24 hours, 22". Same as above

After storage

For additional experimental details see METHODS. I Blood of the same three subjects stored in citrate, dextrose, phosphate 20 days, 4' Mean value of three samples and ranges. Duplicate determinations. Calculated from data of Zachara ('74). 'Calculated from data of Henderson et al. ('77).

of blood with normal ITPH activity (table 4) was compared to that of an ITPH deficient blood sample (table 3). This suggests that high ITP concentration found in ITPH deficient erythrocytes is due to the absence of the pyrophosphorolytic activity and not to a variation in the rate of synthesis of ITP as proposed by Agarwal et al. ('71). Frazer et al. ('75) were unable to detect ITP accumulation in leukocytes when incubated with [8-14C1-hypoxanthinein modified Fischer's medium. It is evident now that in order to demonstrate ITP synthesis, the conditons of assay require complete repression of ITPH activity. Under these conditions, it was possible to show that in addition to erythrocytes, leukocytes, lymphocytes as well as platelets have the ability to synthetize ITP, even though their ITPH activity is significantly higher than that of erythrocytes. Table 5 shows the rates of formation of ITP (and IDP) as well as that of GTP (and GDP) of erythrocytes, leukocytes, lymphocytes, and platelets, compared to their respective ITPH activities. As can be observed, the rate of formation of ITP is considerably higher in leukocytes (lymphocytes) than in erythrocytes. It must be pointed out too, that in contrast to erythrocytes, the normal leukocyte showed considerable accumula-

tion of IDP as well as GDP. In the case of the ITPH deficient leukocytes, there is significant accumulation of IDP but not of GDP. The data is insufficient to determine whether the phenomenon is due to a difference in the properties of the guanylate kinase or those of the NDP kinase or both. It was thought of interest to determine whether tissues other than blood cells also showed the ability to synthetize ITP. For this purpose, tissues from various organs of the rat were utilized. The results are shown in table 6. As can be seen, ITP synthesis was demonstrated in the cytosol of most tissues with the exception of liver, lung, and spleen. GTP synthesis however, was demonstrated in all tissues. It is possible t h a t the conditions of assay were not suitable for demonstration of ITP synthesis in the three tissues cited, possibly due to differences in the properties of ITPH which may not be completely repressed under the conditions described, and (or) to the interference caused by the presence of relatively high activity of nucleoside diphosphate phosphohydrolase (IDPase) (EC 3.6.1.61, 5'-nucleotidase (EC 3.1.3.5), and nucleosidase (EC 3.2.2.1) which has already been demonstrated (Vanderheiden, '75), or by non-specific phosphohydrolase activities.

299

IDP AND ITP SYNTHESIS IN CELLS TABLE 5

Synthesis of inosine and guanosine phosphates in human blood cells ITP

'

IDP

'

GTP '

GDP

ITPH % ( I ) l ( G )'

pmoleshrlg Hb or protein

Erythrocytes Normal (5) ITPHdeficient (1)

0.13f0.018 0.14

0.033f0.015 0.077

Leukocytes Normal (4) ITPH deficient (1)

0.68f 0.09 0.58

3.68 23.12 1.8

181 2 1 7 6 192

Lymphocytes Normal (2)

0.32f0.14

1.82 20.35

149 2 82

257

f

Platelets Normal (3)

0.172 0.08

0.43 20.22

194 2 83

101

f

5 5 . 3 2 4.4 33

4.562 1.9

1.02

108f 0

45

0.28 0.62

8 3 3 2 981 0

1.73 1.23

50

2,820%1,710

0.53

61

1,970f1.240

0.20

71 2 1 0 6 1.8

Numbers in parentheses represent the number of samples. All determinations were carried out in duplicate. Experimental conditions: 1 1 hour, 30" (erythrocytes 0.3-1.2 mg Hb;leukocytes 0.7-13 pg; platelets 170 pg one hour, 16-60 pg four hours; lymphocytes 4-6 pg four hours). Fifteen minutes, 30" (erythrocytes 0.06-0.28mg Hb; leukocytes 0.34-2.1 Wg; platelets 8-48 pg; lymphocytes 2-3 pg). Fifteen minutes, 30" (erythrocytes 0.5-0.6mg Hh; leukocytes 2-17 pg; platelets 170 pg; lymphocytes 2-3 pg). For additional experimental details see Vanderheiden ('72). (I), specific activity of ITP IDP; (G), specific activlty of GTP GDP. See also table 3.

+

+

TABLE 6

Synthesis of inosine andguanosine nucleotides in rat tissues ITP

IDP

GTP

0.08520.063 0.22 20.10 0.002 f0.001 0.11 f0.05 0.02620.016 n n 0.02920.005 0.28

n n 0.14f0.04 1.2 f0.54 n 0.4420.13 n 0.39f0.47 n

n

n

0.11 fO.056 0.015f0.004

n n

GDP

pmolehr/glprotein ~

Adrenal Brain Erythrocytes Heart Kidney Liver Lung Muscle Ovary Spleen Testes Thymus

'

608f113 1,040f240 8 8 2 190 7 3 0 2 33 500f 47 5 6 0 2 48 530% 72 5 3 2 6.3 550 3 5 0 2 80 1,090f 54 420f 49

61 2 2 4 130 2 1 0 160 f 6 5 55 f 1.9 44 f 7.3 52 f 3.7 66 f 1 9 9 . l f 8.2 123 450 f 1 4 120 f 4.9 52 f 1 4

n, not detected. Average values and standard deviations of three rats, except ovary (I), and testes (2) For expenmental details see METHODS. ' Specific activity in terms of Hb.

The demonstration of the ability of tissues to synthetize ITP is evidence for the natural occurrence of ITP in tissues such as heart, liver, and muscle reported as early as 1960 (Dianzani-Mor, '60; Davey, '62; Cain et al., '63) and for a long time considered questionable. In conclusion, evidence is presented for the first time for the presence in the cytosol of a variety of tissues, of an enzyme system responsible for the synthesis of di- and triphosphates of inosine at the expense of ATP and inosine monophosphate.

The method used for the determination of synthetizing activity, in addition to its high sensitivity (0.05nmoles), permits the simultaneous determination of anabolic as well as catabolic enzyme products formed when crude extracts are used in conjunction with the I4C-labeled nucleoside monophosphates. The comparison of the rate of synthesis of ITP as well as IDP, with that of GTP and GDP, using cell extracts in a variety of comparable experimental conditions, did not provide unequivocal evidence to conclude that

300

BERNARD0 S. VANDERHEIDEN

ITP is formed strictly via guanylate kinase and NDP kinase. The comparison of the specific activities of IMP, IDP, and ITP with those of GMP, GDP, and GTP in experiments of blood incubated with either [8-14C1-inosineor [8-14C1-guanosine,although showing different patterns of relative incorporation of the labeled compounds, did not clarify whether different biosynthetic pathways exist for the two nucleotides. Interpretation of the data obtained by measurements of the specific activity of the [8-14-C1-hypoxanthinemoiety of inosine mono-, di-, and triphosphates formed by incubation of ITPH deficient blood with [8- I4C1-inosine, assuming no binding of the nucleotides to proteins, suggests that ITP may be synthetized by pyrophosphorylation. It must be pointed out t h a t the presence of IDP does not necessarily imply its formation by guanylate kinase. If ITP is formed by pyrophosphorylation, the presence of NDP kinase and ADP would lead to the formation of IDP. It is not unlikely t h a t both biosynthetic pathways may exist in the cell and that the system responsible for pyrophosphorylation may be affected when cell integrity is destroyed. ACKNOWLEDGMENTS

The author is indebted to Mrs. Connie Andrews, Frances Bryce, Kay Rush, and Portia Russell for their technical assistance. This work was supported in part, by US. Public Health Grants AM-11116 and HE-13631. LITERATURE CITED Agarwal, R. P., E. M. Scholar, K. C. Agarwal and R. E. Parks, Jr . 1971 Identification and isolation on a large scale of guanylate kinase from human erythrocytes. Biochem. Pharmacol., 20: 1341-1354. Bertino, J. R., R. Silver, M. Freeman, A Alenty, M. Albrecht, B. W. Gabrio and F. M. Huennekens 1963 Studies on normal and leukemic leucocytes. IV. Tetrahydrofolate dependent enzyme systems and dihydrofolic reductase. J. Clin. Invest., 42: 1899-1907. Blair, D. G. R., and M. Dommasch 1969 Formation of inosine triphosphate and IIC1-labeled 2.6-diaminopurine ribonucleoside di- and triphosphates in stored human erythrocytes. Transfusion, 9: 198-202. BByum, A. 1968 Separation of leucocytes from blood and bone marrow. Scand. J. Clin. Lab Invest., Vol. 21 (Suppl. 97): 1.108. Cain, D. F., M. J. Kushmerick and R. E. Davies 1963 Hypoxanthine nucleotides and muscular contraction. Biochim. Biophys. Acta, 74: 735-746. Chern, C. J., A. B. MacDonald and A. J. Morris 1969 Purification and properties of nucleoside triphosphate pyrophosphohydrolase from red cells of t he rabbit. J. Biol. Chem., 244: 5489-5495. Coddington, A. 1974 Inosine. In: Methods of Enzymatic

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