144

Biochimica et Biophysica Acta, 381 (1975) 144--156

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27555 I N C O R P O R A T I O N OF H Y P O X A N T H I N E INTO ADENINE AND GUANINE N U C LEOTI DES BY HUMAN P L A T E L E T S

GEORGES E. RIVARD, JOHN D. MCLAREN and ROBERT F. BRUNST Division o f Hematology-Oncology and Department o f Biochemistry, Childrens Hospital o f Los Angeles, Los Angeles, Calif. 90027 (U.S.A.)

(Received June 24th, 1974)

Summary 1. Incubation (1--4 h) of normal hum a n washed platelets (5--11 • 10 ~ per ml) with [8 - 1 4 C ] h y p o x a n t h i n e at a concentration of 10 -s M resulted in a linear incorporation of radioactivity into adenine and guanine nucleotides. 2. Washed platelets from patients with Lesch--Nyhan syndrom e, deficient in h y p o x a n t h i n e : guanine phosphoribosyltransferase, failed to demonstrate any significant incorporation of [ 8 - 1 4 C ] h y p o x a n t h i n e but did incorporate [8-~ 4 C] adenine like normal platelets under the same incubation condition. 3. These findings are taken to indicate that normal platelets have the enzymes necessary for salvage of h y p o x a n t h i n e and that h y p o x a n t h i n e : guanine phosphoribosyltransferase is the obligatory first step in this pathway.

Introduction The role of adenine nucleotides in platelet function has been extensively studied during the last decade [ 1] . "De n o v o " purine nucleotide synthesis in human platelets has n o t been d e m o n s t r a t e d in spite of good trials [ 2 ] . However, other mechanisms exist by which platelets could maintain their "metabolic p o o l " of nucleotides. T w o general pathways have been described [3] for the synthesis of ribonucleotides f r om purine bases or nucleosides that are derived f r o m endogenous catabolism, dietary a n d / o r therapeutic sources. These pathways are: (a) phosphoribosyltransferase reactions, in which free bases condense with P-Rib-P-P to f or m ribonucleotides in a single step or (b) nucleoside phosphorylase reactions in which free bases react with ribose 1-phosphate to form ribonucleosides which can then be p h o s p h o r y l a t e d via kinase reactions to yield ribonucleotides. Abbreviation: P-Rib-P-P, 5-phosphoribosyl-l-pyrophosphate.

145 Holmsen and Rozenberg demonstrated that exogenous adenine is incorporated into AMP via adenine phosphoribosyltransferase [2], and that adenosine is incorporated into AMP via adenosine kinase [4]. Ireland and Mills [5] established evidence that a small proportion of the radioactivity from [8 -14C]inosine could be found in adenine nucleotides after incubation with washed human platelets. Until recently [6,7], the salvage pathway in human platelets for h y p o x a n t h i n e had not been demonstrated. It is the purpose of this paper to further establish the existence of hypoxanthine phosphoribosyltransferase in human platelets, and its obligatory role in the incorporation of h y p o x a n t h i n e into adenine and guanine nucleotides. Materials and Methods Purine bases, nucleosides and nucleotides were purchased from Sigma Chemical Company, St. Louis, Mo. Phosphoenolpyruvate and pyruvate kinase (E.C. 2.7.1.40) were also obtained from Sigma Chemical Company. [8-14C] Adenine, Code NEC-005H, spec. act. 61.4 Ci/mole was supplied by New England Nuclear, Boston, and [8-14C]hypoxanthine, Code 1623-41, spec. act. 53.7 Ci/mole was supplied by Schwarz Bio-Research, New York. High-voltage electrophoresis in citrate buffer, at pH 3.75 showed a trace of radioactive contamination (less than 0.1%) of this hypoxanthine; before being used it was purified with the anion-exchange resin chromatography system to be described below.

Platelet preparations 9 vol. of blood were anticoagulated with 1 vol. of 3.8% sodium citrate and centrifuged at 200 × g for 10 min at room temperature, to obtain platelet-rich plasma. Healthy adult volunteers and three patients with Lesch--Nyhan syndrome were used as sources of blood samples. Platelets were counted with an MK-4 Haema-Count Platelet Counting System (General Science Corp., Bridgeport, Conn.). Erythrocyte and leukocyte contamination of platelet-rich plasma was evaluated by visual counting and f o u n d to be one of each type of cell per 5000 platelets. Washed platelets were prepared according to the technique of Walsh and resuspended in a calcium-free Tyrode solution at pH 7.3 [8]. "Platelet lysates" were prepared by centrifuging platelet-rich plasma at 1000 × g for 15 min, removing the supernatant platelet-poor plasma and adding 0.2 ml of deionized water to the platelet pellet. This mixture was frozen and thawed 10 times, vortexing between each cycle.

Incubation of platelets A D u b n o f f shaking incubator kept at 37°C and 20 cycles/min was used after the addition of [8 - 1 4 C ] h y p o x a n t h i n e at different concentrations to a platelet suspension containing between 4 • 108 and 11 • 108 platelets per ml or to a platelet-rich plasma preparation containing between 2 • 108 and 4 • 108 platelets per ml. The plastic incubation vial was closed after introducing a 02 --CO2 (95 : 5, v/v} gas mixture. At various intervals of time, 1-ml aliquots of the mixture were transferred to plastic tubes containing 4 ml of ice-cold calcium-free Tyrode solution, and centrifuged for 15 min at 1000 × g; the super-

146 natant was quantitatively removed for counting. 3 ml of ice-cold deionized water was added to the ptatelet pellet and vortexed for 1 min', after which it was frozen in a glycerol--solid CO2 mixture. Immediately after thawing, the platelet lysate was added dropwise to 3 ml of ice-cold 15% trichloroacetic acid while being vortexed. This mixture was then subjected to five cycles of freeze--thawing in glycerol--solid CO2. After storing on melting ice for 1 h, the preparation was centrifuged at 12 000 × g for 10 min, the supernatant was removed (saved) and the pellet was washed with 2 ml of 7.5% trichloroacetic acid. This suspension was recentrifuged, and the supernatant combined with the previous supernatant. The solid pellet was kept for counting. Trichloroacetic acid was removed from the supernatant by five extractions using 3 vol. of diethyl ether per extraction. The platelet extracts (aqueous layer) was lyophilized, redissolved in 0.2 ml of deionized water and chromatographed w~thout further treatment. The combined radioactivity lost in the trichloroacetic acid pellet and the extracting ether represented less than 0.1% of the total radioactivity of the incubation mixture. For experiments in which column chromatography was not to be used, the extraction was done either by (1) adding 1 vol. of an ice-cold, freshly prepared mixture, consisting of 1 part 100 mM EDTA and 9 parts 96% ethanol, to a platelet-rich plasma or a platelet suspension, or (2) by adding a mixture of 50% ethanol-5 mM EDTA to a platelet pellet.

Thin-layer chromatography Two-dimensional chromatography [9] was performed on Brinkman M-N Polygram Cel 400, 0.1 m m thickness microcrystalline cellulose pre-coated plastic sheets. The positions of the carrier substances were visualized with ultraviolet (254 nm) light, outlined with a pencil, and the thin-layer plates were xeroxed to provide a permanent record. Autoradiography of selected experiments was performed by placing the thin-layer plates over clinical X-ray films for a period of 1--3 weeks. The spots of k n o w n markers as well as the origin were cut out, placed in 5 ml of scintillation liquid, and counted, thereby giving the relative radioactivity of the different separated metabolites.

Ion-exchange column chromatography The a u t o m a t e d liquid column chromatography system used has been described in detail elsewhere*. A 2.8 mm by 75 mm column loaded with Aminex A-25 anion-exchange resin, chloride form (Bio--Rad, Richmond, Calif.) was eluted with 75 ml of buffer delivered by a Cheminert CMP-2 pump (Chromatronix, Berkeley, Calif.) at a rate of 24 ml/hr. Compounds were eluted by a linear gradient of NH4 C1 (0.05--0.3 M) adjusted to pH 10.0 with NH4 OH. The same gradient at pH 8.2 was used to separate XMP from IMP and adenylosuecinic acid from GDP. However, at this pH, the other mono-, di- and triphosphates were not sufficiently separated, so the gradient at pH 8.2 was used only to look for the presence of XMP and adenylosuccinic acid. Radioactivity of the eluate was monitored by a Nuclear Chicago Liquid Scintillation System Model

* Robert

F. B r u n s t , d i s s e r t a t i o n ,

University of Southern

California, 1974.

147 Unilux III (Des Plaines, Ill.) equipped with an anthracene flow-cell. Counts were simultaneously printed, and changes in counts were plotted via a ratemeter Model 432 A (Baird Atomic, Cambridge, Mass.) on a multichannel Honeywell Electronic 112 recorder {Fort Washington, Penn.). After passing through the liquid scintillation counter, the eluting stream was delivered to an automatic asher Model DA-25 (Hauptman, Culver City, Cafif). The ashed material was reacted with a m m o n i u m molybdate, sulfuric acid and ascorbid acid according to an adaptation of Brewer's technique [10]. In the presence of phosphate, reduced phosphomolybdate complexes were formed and their absorbance read at 820 nm by a Spectronic 70 spectrophotometer {Bausch and Lomb, Rochester, New York) equipped with a flow-cell. The absorbance was recorded on the same graph as the radioactive tracing. Commercial standards were used to determine the R F for each c o m p o u n d of interest. A constant time delay between the detection of radioactivity by the scintillation counter and the detection of phosphate by the spectrophotometer permitted the identification of the radioactive peaks by the commercial standards.

E n z y m a t i c assays A modification of the technique described by Cartier and Hamet [11] was used to assay the phosphoribosyltransferases. The concentrations of the different substances in the incubation mixtures were as follows: 20 pl of platelet lysate (2--8 • 10 7 platelets), Tris--HC1 buffer 69 mM (pH 7.4), MgC12 6.25 mM, P-Rib-P-P 1.4 mM, and [8 -1 a C ] h y p o x a n t h i n e 3.54 • 10 -s M (57.7 Ci/mole). This mixture (160 pl) was incubated at 37°C for 20 min and the reaction stopped by adding 50 pl of 4 M formic acid. The respective nucleotide monophosphates were separated from the bases and nucleosides by thin-layer chromatography {microcrystalline cellulose) using carriers for ultraviolet detection. The solvent systems used where: n-propanol--25% ammonia--water (6 : 3 : 1, v/v) for adenine phosphoribosyltransferase and isopropanol : saturated ammonium sulphate : water (2 : 79 : 19 v/v) for h y p o x a n t h i n e phosphoribosyltransferase. The carriers were located on the chromatogram under ultraviolet fight (254 nm), the spots cut out, placed in scintillation liquid and counted. The results were expressed in nmoles of the respective nucleotide monophosphates produced per min per 101 ~ platelets. Results

Incubation of normal washed platelets with [8- ~* C] hypoxanthine Fig. 1 depicts a typical anion-exchange chromatogram of an extract from normal washed platelets that had been incubated with [8-14 C] h y p o x a n t h i n e at a concentration of 10 -s M for 4 h. 27% of the radioactivity was f o u n d in the trichloroacetic acid extract of the platelet pellet, and of this intracellular radioactivity, 84% was in the adenine nucleotides, 8.6% in the guanine nucleotides, 4.5% in IMP and 2.9% in hypoxanthine. No radioactivity was detected in IDP, ITP, XMP or adenylosuccinic acid. Inosine and h y p o x a n t h i n e had a similar R F with both types of gradients and were, therefore, counted together. Thin-layer chromatography demonstrated that inosine represented less than 1% of the counts in the trichloroacetic acid extract.

148

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The incorporation of radioactive hypoxanthine into total adenine nucleotides (AMP, ADP and ATP) and guanine nucleotides (GMP, GDP and GTP) was linear during the 4-h-incubation period (Figs 2 and 3). The maximal level of incorporation was reached at an initial extracellular hypoxanthine concentration between 3.74 and 7.74 • 10 -s M (Table I). Ten times greater incorporation of radioactivity into adenine nucleotides as compared to guanine nucleotides was noted. The authenticity of the adenine and guanine nucleotides was further confirmed by a peak-shift experiment. A platelet extract (from about 108 g-J

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TIME OF INCUBATION F i g . 2. T o t a l r a d i o a c t i v e a d e n i n e n u c l e o t i d e s ( A M P + A D P + A T P ) i n n m o l e s p e r 1 0 1 l t r i c h l o r o a c e t i c a c i d - e x t r a c t e d p l a t e l e t s a f t e r 1, 2 a n d 4 h or i n c u b a t i o n o f w a s h e d p l a t e l e t s (x = 1 0 - 1 0 8 a n d • = 5.5 • 1 0 8 p l a t e l e t s / m l ) f r o m t w o n o r m a l v o l u n t e e r s w i t h 1 0 -5 M [ 1 4 C ] h y p o x a n t h i n e . Data obtained by the column chromatography system.

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TABLE I THE EFFECT OF INCREASING CONCENTRATION OF HYPOXANTHINE ON ITS UPTAKE AND CONVERSION TO ADENINE AND GUANINE NUCLEOTIDES [8-14C] Hypoxanthine in increasing concentration was incubated with washed platelets (5.8--10 • i 0 8 platelets/ml) at 37°C for 4 h and the incorporation of radioactivity into adenine and guanine nueleotides was determined. The amount of nucleotides produced from the hypoxanthine is given in nmoles per 10 1 l platelets, and represents the averages obtained from three experiments. The percentages of incorporation of the total hypoxanthine (%) were calculated using the mean platelet count (6.64 • 108 platelet/ml) of the incubation mixtures. Initial hypoxanthine concentration (X 1 0 - 5 M )

Hypoxanthine converted to nucleotides (in 10 -9 nmoles per 1011 platelets)

0.187 1.13 1.87 3.74 7.74 15.7

44.9 232.5 263.1 298.4 317.9 317.6

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(%)

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4.4 27.6 29.4 31.1 37.6 41.6

(%)

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