THE JOURNAL OP INVESTIGATIVE DERMATOLOGY Vol. 4s, No. Z Copyright 1957 by The Williams & Wilkins Co. Fri nte,1 in U.S.A.
PURINE AND PYRIMIDINE METABOLISM IN HUMAN EPIDERMIS* JEAN DE BERSAQUES, MD. The continuous cellular renewal occurring inthine, which contained 5% impurity, and for uric the epidermis requires a very active synthesisacid, which consisted of 3 main components. The reaction was stopped after 1—2 hours in- and breakdown of nuclear and cytoplasmiecubation at 37° and the products were spotted on nucleic acids. Data on the enzyme systemsWhatman 1 filter paper sheets. According to the participating in these metabolic processes arereaction products expected, a choice was made of rather fragmentary (1—9) and some are, inat least 2 among the following solvents, all used terms of biochemical time, in need of up- in ascending direction: 1. isoamyl alcohol—5% Na2HPO4 (1:1), dating. In some other publications (10—18), 2. water-saturated n-butanol, the presence and concentration of various in- 3. distilled water, termediate products is given. 4. 80% formic acid—n-hutanol——n-propanol— In this paper, we tried to collect and supple- acetone—30% trichloro-aeetic acid (5:8:4: ment these data by investigating the presence 5:3), 5. n-butanol——4% boric acid (43:7), or absence in epidermis of enzyme systems 6. isobutyrie acid—water—ammonia 0.880—ver- that have been described in other tissues. sene 0.1M(500:279:21:8), This first investigation was a qualitative one, 7. upper phase of ethyl acetate—water—formic and some limitations were set by practical acid (12:7:1), 8. t-butanol——88% formic acid—water (14:3:3). considerations. Also we cannot be certain that The radioactive spots were identified by auto- the results are valid for every type of epider-radiography on X-ray film and quantitated with a mis, most experiments having been performedPanax radioehromatogram scanner. Their locali- with normal plantar epidermis and with pso-zation was compared with that of reference sub- riatic and tumoral tissue. It will be seen thatstances run on the same paper. For a few reactions, the radioactive CO2 formed the results were qualitatively comparable.was trapped with 0.3 M KOH and counted in a Quantitatively, higher activities were some-liquid scintillation counter. times found in psoriatie epidermis or scales. Because crude tissue was used, the simulta- RESULTS neous occurrence of several reactions and the The results of the various experiments are influence of inhibitors cannot be ruled out.summarized in Table I. Hence, a negative result does not establish an absolute value. COMMENTS The main pathway towards the formation MATERIALS AND METHODS of purine nueleotides starts from small mole- Small fragments of frozen lyophilized epidermiscules (glutamine, bicarbonate, formate, gly- were incubated with about 20 l reaction mixture containing a buffer (mostly 0.1Mtris pH 72, ineine, aspartate, and ribose-5-phosphate) and rare cases 0.05 M phosphate pH 7.1), the radio-leads to the formation of inosine-5'-monophos- active substrate, other substrates, and activatorsphate. The purine nueleosides and bases are or inhibitors. The radioactive products were:not intermediates on that synthetic route. How- adenine-8-'4C, hypoxanthine-84C, xanthine-8-°4C, ever, numerous enzymatic reactions have been guanine-8-14C hydrochloride, uric aeid-2-'4C, ura- described involving the synthesis, interconver- cil-2-°4C, thymine-2-14C, thymidine-2-14C, eytosine- 2-14C and orotie acid-6-'4C. All were 98% or more sion, and breakdown of bases, nucleosides, and ehromatographically homogenous, except for xan-nueleotides. Some of these reactions are re- garded as salvage pathways permitting re- This work was supported by a grant from theutilization of purines. The breakdown reactions Fund for Scientific Medical Research, Belgium. Presented at the Twenty-seventh Annual Meet-lead to uric acid, the common metabolic prod- ing of The Society for Investigative Dermatology,uct of purines. These various reactions are Inc., Chicago, Ill., June 26, 1966. * From the Department of Dermatology, Uni-given schematically in Fig. 1. versity of Gent, Belgium. The synthesis of pyrimidine nueleotides 169 170 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
TABLE I'
Radioactive Other sobstratea, Radioactive producta substrate activators Buffer identified Solventa H Tris H 3,5 X Tris X 1,3 UA Tris UA(?) 1, 4 T NADPH Tris 002 * U NADPH Tris 002 *
2. A Tris,P04 A,H 1,6 0 Tris G,X 1,4,6 3.a A RiP Tris A, H, HIlt 1, 2, 3, 4, 5, 6 H RiP Tris H,HR 1,2,4,5,6 0 RiP Tris G,GR 1,2,4,5,6 X RiP Tris X,XR 1,2,4,5,6 UA RiP Tris UA(?) 1, 2, 4, 5, 6 A dRiP Tris A, H, HdR 1, 3, 4, 7 H dRiP Tris H, HdR 1, 4, 7 0 dRiP Tris 0, GdR 1, 4 X dRiP Tris X, XdR(?) 1, 4 UA dRiP Tris UA(?) 1, 4 T RiP Tris T,TH 1,2,5,7,8 U RiP Tris U,UR 1,2,5,7,8 0 RiP Tris 0 1,2,7 0 RiP Tris 0 1,7 T dRiP Tris T, TdR 4, 7 TdR P04 T, TdR 4, 7 U dRiP Tris U, UdR 1, 4, 7 0 dRiP Tris 0 1,4 0 dRiP Tris 0 1, 4 b A PRPP,Mg P04 A, AMP, H, 1,3,4,6,7 HR, IMPt 0 PRPP, Mg P04 0, OR, GMP 1, 4, 6, 7 II PRPP,Mg P04 H,HR,IMP 1,4,6,7 X PRPP,Mg P04 X 1,4,6,7 UA PRPP,Mg P04 UA 1,6 U PRPP,Mg P04 U 1,4,6,7 T PRPP,Mg P04 T 1,4,6,7 0 PRPP,Mg P04 0 1,4,6 0 PRPP, Mg P04 0, UR, UMP 1, 3, 4, 6, 7, 8
4. TdR ATP, Mg Tris T, TdR 1, 6, 7 A RiP, ATP, Mg Tris A, H, HR 1, 6 H RiP, ATP, Mg Tris H, HR 1, 6, 8 0 RiP, ATP, Mg Tris G, OR 1, 6 U RiP, ATP, Mg Tris U, UR 1, 6, 7, 8 *The'00 liberated was trapped in a KOH seal and counted. t The identity of the various products was confirmed in reaction mixtures containing hypoxanthine or inosine as competitive inhibitors. UA(?) As the uric acid used was quite impure, the reaction products could not be identified with certitude; however, the pattern obtained did not appear to be different from unreaeted uric acid. XdR(?) The identification of deoxyxanthosine in some reactions is only tentative, as tbis unstable product was not available as a marker and no ehromatographic data were at hand. PURINE AND PYRIMIDINE METABOLISM 171
dAMP AIp:::::::::: iivt it. AdR /AR-.—h+HR\ HdR A UAR 1. I C 'X__UAAll OdR/\\\GR__'\ XR/ XdR I \1I 'IJ dGMP4 GMP XMP
Fm. 1. Metabolism of purines1
dCMP 1JMP ...>dUMP>TMP OMP I Cdtt /CR UR\ UdR TdR TRIOR C >15 T 0 1. 1 dihydroU dihydroT I I Fio. 2. Metabolism of pyrimidincs' —+:reactionsfound in the present experiments. —s: reactions described in other tissues, not found in the present experiments. —-+:reactionsdescribed in other tissues, not studied in the present experiments. starts from aspartate and carbamyiphosphate,urate. Its presence was described in rat and leading to the formation of orotate. The otherguinea pig skin (1, 4, 9); it was not found in pyrimidine-5'-phosphates are derived fromhuman epidermis (1, 7), a result confirmed in orotidine-5'-monophosphate.Both oxidativethe present experiments. Urate oxidase cata- and reductive degradation pathways have beenlyzes the oxidation of uric acid to allantoin. It described for uracil and thymine, but onlywas not found in human epidermis. the latter is thought to be of importance in The presence of IMP-dehydrogenase was not animals, The various reactions involving py-investigated. The reductive degradation of rimidines are given schematically in Fig, 2. uracil and thyminc leads first to the formation The enzymes responsible for the reactionsof a dihydrodcrivative and further of f9-alanine shown in Fig. 1 and 2 can be divided intoor /3-amino-isobutyric acid, ammonia and several groups: CO,. The latter originates from the number 2 carbon atom of the pyrimidine ring. The re- 1. Oxidoreductases action proceeds much faster in the presence of Xanthine oxidase catalyzes the oxidation ofNADPH. In these conditions, radioactive CO2 hypoxanthine to xanthine and of xanthinc towas recovered from uracil- and thymine-2-14C incubated with epidermis; the reaction, how- 1 Abbreviationsused in Table I and Figures 1 and 2. ever, was not very active. A: adenine; H: hypoxanthine; G: guanine; X: xanthine; UA: uric acid; All: allantoin; C: 2. Hydrolases acting on C—N bonds, other cytosine; U: uracil; 0: orotic acid. than peptide bonds AR: adenosine; HR: inosine; etc. AdR: deoxyadenosine; HdR: dcoxyinosine; etc. AMP: adenosine-5'-monophosphate; IMP: mo- Adenine and guanine deanñnases act on sine-5'-monophosphate; etc. adenine and guanine, yielding respectively hy- dAMP: dcoxyadenosine -5'-monophosphate; poxanthine and xanthine. dGMP: deoxyguanosinc-5'-monophosphatc; etc. RiP: ribose-1-phosphatc, dRiP: deoxyribosc-1- Adenine deaminase is absent from rat skin phosphate; NADPH: reduced nicotinamide-(3), bnt present in human epidermis (7, 8). adenine dinucleotide phosphate; Tris: tris(hy-In our experiments deamination of adenine droxymethyl)aminomethane-HC1 buffer; P04: phosphate buffer. occurred very rapidly. Guanine deaminase was 172 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY described in rat skin (3); it was also presentwere not formed from hypoxanthine-'4C. In our in human epidermis. Adenosine deaminase wasexperiments the correspondingnucleotides not studied in the present experiments, but itswere synthetized from adenine, guanine, hy- presence in psoriatie scales and rat skin waspoxanthine, and orotic acid; with the latter demonstrated earlier by means of paper chro-substrate the radioactive product formed was matography and differential spectrophotometryidentified as uridine-5'-monophosphoric acid, of unlabeled compounds (2, 5). It was notwhose mobility it had in 6 different solvents. certain whether a guanosine deaminase wasNo reaction was found with xanthine and nra- also present in rat skin. The observed forma-cil. Enzymes for cytosine, thymine, and uric tion of xanthine from guanosine could be ex-acid are not known in other tissues and were plained by a pathway involving first the forma-not found. tion of guanine (2). The presence of AMP-deaminase was not 4. Transf erases transferring phosphate groups investigated. Nueleoside phosphokinases catalyze the for- Cytosine deaminase was not present. mation of a nucleotide from its nucleoside and require the participation of ATP. They have 3. Trcnsf erases transferring pentose groups been described for adenosine, guanosine, mo- a) Nucleosidoses—Furine nucleoside phos-sine, uridine, and thymidine. In the present phorylase catalyzes the equilibrium between aexperiments, and except for the use of thymi- nucleoside + inorganic phosphate and the cor-dine-'4C, they could only be studied indirectly, responding purine base + ribose- or deoxyri-coupled with the action of the nueleoside phos- bose-i-phosphate. The equilibrium favors thephorylase, in a system containing the radioac- synthesis of the nueleoside. This reaction, al-tive base, ribose-i-phosphate, and ATP. With ready mentioned or studied in previous papersneither substrate was nucleotide formation dem- (5, 6), occurred with hypoxanthine, guanine,onstrated. and xanthine, giving both ribonucleosides and These results are at variance from those of deoxyribonucleosides. Inosine and deoxyinosineauthors who found incorporation of labeled were formed from adenine, after a preliminarythymidine into nuclear DNA in vivo; the dis- deamination to hypoxanthine; adenine nucleo-crepancy is probably due to the different ex- sides were not found. As far as could be ascer-perimental conditions. tained, no reaction occurred with uric acid. Thymine and uracil reacted with deoxyribose- 5. Hydrola.ses acting on phosphoric 1-phosphate to yield thymidine (5) and deoxy- monoesters uridine, with ribose-i-phosphate to yield thy- The presence of acid phosphatase in epider- mine riboside and uridine. The responsiblemis is well known; this enzyme also catalyzes enzyme is thought to be different from purinetransphosphorylations. Neutral 5'-nucleotidase nucleoside phosphorylase. No synthetic reac-is apparently absent from epidermis. The ac- tion was found when starting with cytosine ortivity of these enzymes could not be revealed orotie acid. directly in the present experiments, but they Nucleoside hydrolases were not detected. Ifwere probably responsible for the appearance an apparent transribosidase reaction took placeof the nucleoside in a reaction mixture con- when a base was incubated with a nucleoside,taining a base (hypoxanthine, guanine, orotic the probability was strongly in favor of theacid) and phosphoribosylpyrophosphate. action of the phosphorylase. The numerous reactions involving mono-, di- b) Pyrophosphorylases.—Enzymes yieldingand trinucleotides were outside the scope of directly ribonucleotides from a base and phos-this paper. phoribosylpyrophosphate play an important part in the salvage of nucleic acid bases; they Our experiments demonstrate the presence in are thought to be specific for the respectivehuman epidermis of a fairly complete array of bases. The presence of this class of enzymes wasenzyme systems for the metabolism of purine described in epidermis by Schwarz (7, 8), whoand some pyrimidine bases and nucleosides. detected adenylie and guanylie nucleotidesHowever, some enzymes are apparently lacking. after incubation with adenine-'4C. NucleotidesIt is too early to speculate on the significance PURINE AND PYRIMIDINE METABOLISM 173 of these findings, particularly in view of the freiwerdendcn Purine. Arch. Klin. Exp. Derm., 216:427,1963. fact that our in vitro systems may not cor- 8. Schwarz, B.: Zur Genese von Imidazol-Deriva- respond to the situation in vivo, and that the ten der wasserldslichen Substanzen epider- enzyme activities used for the cellular metabo- maler Hornschicht. In-vitro-Untersuchungcn mit 24C Adenin. Zt. Physiol. Chem., 339: lism are not known. Further quantitation of 110, 1964. these activities and of the various intermedi- 9. Westerfeld, W. W. and Richert, D. A.: The ate products is needed to elucidate these prob- xanthine oxidase activity of rat tissue. Proc. Soc. Exp. Biol. Med., 71:181,1949. lems more fully. 10. Bollinger, A. and Cross, R.: Quantitative stud- ies on some water-soluble organic constitu- SUMMARY ents associated with vertebrate keratin. Aust. J. Exp. Biol. Med. Sci., 30:181,1952. The enzymes of purine and pyrimidine base11. Bollinger, A. and Cross, R.: Uric acid content of keratinous tissues as determined by the and nuclcoside metabolism present in human unease method. Aust. J. Exp. Biol. Med. epidermis were studied in in vitro systems. Sci.,30: 395, 1952. 12. Bollinger, A. and Cross, R.: Non-keratins of REFERENCES avian and mammalian skin flakes. Aust. J. Exp. Biol. Med. Sci., 32: 747, 1954. 1. Barry, C., Bunbury, B. and Kennaway, B. L.:13. Bollinger, A. and Gross, R.: Water-soluble The effect of arsenic upon some oxidation- compounds (non kcratins) associated with reduction systems. Biochem. J., 22: 1102, the skin flakes of the human scalp. Aust. J. 1928. Exp. Biol. Med. Sci. 34: 219, 1956. 2. Block, W. aod Johnson, D.: Studies of the en-14. Braun-Falco, 0. and Salfeld, K.: Ueber den zymes of purine metabolism in skin. II. Cehalt wkssriger Extrakte von Callus und Nocleoside deaminases of rat skin. J. Invest. Psoriasis-Schuppen an freien Purincn und Derm., 23: 471, 1954. freiem Phosphat. Arch. KIm. Exp. Derm., 3. Block, W. and Johnson, D.: Studies of the en- 208: 395, 1959. zymes of purinc metabolism in skin. I.15. Hodgson, C.: Nucleic acids and their de- Cuanase activity of rat skin. J. Biol. Chem., composition products in normal and patho- 217: 43, 1955. logical horny layers. J. Invest. Derm., 39: 4. Block, W. and Johnson, D.: Factors influenc- 69, 1962. ing xanthine oxidase activity in rat skin.16. Sempinska, E.: Badania chromatograficzne Arch. Biochem., 66: 137, 1955. hydrolizatow lusek i nukleoproteidow z 5. Dc Bersaques, J.: Nucleosidase in human epi- lusek choryck na luszczyce. Acta Biochim. dermis and in normal and abnormal scales. Pol., 2: 21, 1955. J. Invest. Dcrm., 38:133,1962. 17. Wheatley, V. R. and Farber, E. M.: Studies 6. Hershey, F. B. and Mendle, B. J.: Quantitative on the chemical composition of psoriatic histochemistry of burned and normal skin. scales. J. Invest. Derm., 36: 199, 1961. Surg. Forum, 6: 745, 1954. 18. Wheatley, V. R. and Farber, B. M.: Chemis- 7. Schwarz, B.: Untersuchungcn zum Schicksal try of psoriatic scales. II. 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