Arch Dermatol Res (1983) 275:310-314 Archives of (cid:14)9 Springer-Verlag 1983 Adenosine Deaminase in Human Epidermis from Healthy and Psoriatic Subjects H. Koizumi 1 H. Iizuka 2, T. Aoyagi 1, and Y. Miura 1 1 Department of Dermatology, Hokkaido University, School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo, Japan 2 Department of Dermatology, Asahikawa Medical College, Asahikawa, Japan Summary. Adenosine deaminase, which catalyzes the irreversible hydrolytic deamination of adenosine and deoxyadenosine to inosine and deoxyinosine respec- tively, plays an important role in the degradation of adenine nucleotide and purine nucleotide salvage path- way metabolism. We investigated human epidermal adenosine deaminase activity using a radiochemical method, which enabled us to measure the adenosine deaminase activity of protein samples as small as several micrograms. We measured adenosine de- aminase activity of microdissected pure epidermis of the healthy skin and the psoriatic affected and un- affected skin. It was shown that psoriatic affected epidermis had increased adenosine deaminase activity compared with the healthy epidermis (P < 0.05) and the unaffected epidermis (P < 0.01). There was no differ- ence in enzyme activity between healthy and psoriatic unaffected epidermis. The increased adenosine de- aminase activity in the psoriatic affected epidermis may reflect the accelerated salvage pathway of the nucleic acid metabolism probably associated with the hyper- proliferative condition of the psoriatic epidermis. Key words: Adenosine deaminase - Human epidermis - Psoriasis Introduction Adenosine deaminase catalyzes the irreversible hy- drolytic deamination of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. This enzyme plays an important role in the degradation of adenine nucleotides. In human tissue adenosine cannot be directly degradated to adenine in contrast to guanosine and inosine, which are degradated by direct deribosy- Offprint requests to : H. Koizumi (address see above) lation resulting in the formation of guanine and hypoxanthine respectively. Adenosine is first catalyzed to inosine by adenosine deaminase. Because of the irreversibility of this reaction and the relatively low enzyme activity, this enzyme reaction seems to be one of the rate limiting steps of adenosine degradation [1,2]. The product, inosine, is then converted to hypoxan- thine, which is either converted to IMP by hypo- xanthine-guanine phosphoribosyl transferase [2], a final rate limiting enzyme of the purine nucleotide salvage pathway, or catalyzed to xanthine and uric acid by xanthine oxidase [3- 5]. From the investigation of severe immunodeficiency disease which is defective in adenosine deaminase [6], it has been suggested that detoxification of adenosine and deoxyadenosine might be another significant function of adenosine deaminase. The cytotoxic and cytostatic effects of adenosine and deoxyadenosine are generally potentiated by inhibitors of adenosine deaminase. Several mechanisms of toxicity have been proposed. First, adenosine or deoxyadenosine causes dATP accu- mulation, which is a strong inhibitor of ribonucleotide reductase and causes inhibition of DNA synthesis [7]. Second, access of deoxyadenosine inactivates S-adeno- sylhomocysteine hydrolase. Absence of this enzyme causes interference with the critical methylation- dependent processes such as the synthesis, maturation or function of DNA [8]. It has been reported that in adenosine deaminase- defective lymphocytes cyclic AMP level is increased [9]. Since adenosine is known to stimulate the adenylate cyclase system of epidermis [10], adenosine deaminase might be significant for the regulation of cell pro- liferation through the cyclic AMP system. Adenosine deaminase also plays an important role in cell matu- ration such as human monocyte [11]. These findings suggest that adenosine deaminase might play a significant role in the nucleic acid metab- olism of the epidermis where the significance of salvage
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`H. Koizumi et al. : Adenosine Deaminase in Human Epidermis 311 pathway has been emphasized recently 12, 13. These findings also suggest that adenosine and deoxyaden- osine metabolism in the epidermis is important for cell proliferation and maturation. However, because of the low enzyme activity in the epidermis 14, 15, the nature and activity of the enzyme in skin disorders remain unknown at present. Although Kalcker's method of adenosine deaminase determination, which employs the decrease in OD at 265 nm, is simple and popular 16, the sensitivity is relatively low and we cannot detect the adenosine deaminase activity of small biopsy samples. In this report we attempt to characterize the biochemical nature of the epidermal adenosine de- aminase using the microanalytical method. Then we determine the adenosine deaminase activity of normal and psoriatic pure epidermis obtained by microdissec- tion technique. Materials and Methods Chemicals (2-3H) adenosine was purchased from New England Nuclear (Boston, MA, USA). TLC plates cellulose F were obtained from Merck (Darmstadt, FRG). Coformycin was a generous gift from Dr. Umezawa of the Institute of Microbial Chemistry, (Tokyo, Japan) 18-21. Adenosine, inosine, AMP, hypoxanthine, adenine and reagents for scintillation were obtained from Wako Pure Chemical Industries (Osaka, Japan). All other reagents used were of the highest purity commercially available. All chemicals were freshly prepared before each experiment. Preparation of the Enzyme Healthy human epidermis was obtained from intermediate split thickness skin obtained by free hand dermatome under systemic anesthesia or 0.2ram thickness by Castroviejo keratome. Two slices of 5 mm square epidermis were homogenized in glass homogenizer in I ml 50mM phosphate buffer, pH 7.1. The homog- enate was centrifuged at 1,500 g for 10 rain and the supernatant was used for the enzyme source. All procedures were performed at 4~ Adenosine Deaminase Assay Adenosine deaminase activity was quantitated by measuring the formation of 3H-inosine. Adenosine deaminase was assayed by mixing 10gl enzyme source and reaction mixture which contained 340gM 3H-adenosine and 50mM phosphate buffer, pH 7.1. Incubation was carried out for 30 rain at 37 ~ C, and the reaction was stopped by 8 N formic acid. The sample was then developed by thin layer chromatography and the radioactivity of the inosine fraction was measured by liquid scintillation counter. Protein was determined by the method of Lowry et al. 22 using bovine serum albumin as a standard. Thin Layer Chromatography All samples were analyzed by ascending chromatography on TLC plates cellulose F. The plates were developed at room tem- perature with the two systems of solvents for the separation of the purine analogs. The first system of the chromatogram was developed with 1 M ammonium acetate 23, which separated inosine, AMP, adenosine plus hypoxanthine and adenine. The second system was developed with a mixture composed of 60 parts butanol, 20 parts methanol, 20 parts water and 1 part ammonium hydroxide, which separated adenosine, adenine, inosine plus hypoxanthine and AMP 24. An appropriate mixture of standard materials and 1 gl of each sample were chromatographed and the plates were examined under ultraviolet light. The developed chromatograms of the experimental samples were cut at right angles to the direction of migration. Each fraction cut out was put into the scintillation vial arid mixed with 100 gl 1 M LiC1, and radioactivity was measured by liquid scintil- lation counter. The first system of the chromatogram required about 1.5 h and it sharply separated each purine analog, but hypoxanthine was developed on the same fraction as adenosine. Adenosine was catalyzed to inosine by adenosine deaminase and then inosine to hypoxanthine by purine nucleoside phosphorylase. If the con- centration of purine nucleoside phosphorylase is high, hypoxanthine is produced in incubation mixture, and adenosine deaminase activity cannot be measured correctly. The second system required 4 h, and inosine and hypoxanthine were developed on the same fraction. In these two systems, the counts of the adenine fraction were not increased as compared with the blank control. It seems that there is no conversion of adenosine to adenine in epidermis. The conversion of adenosine to AMP was only about 5 % in these system s. In keratome sliced healthy epidermis the difference in the counts of inosine between these two systems was only about 15 % under the assay condition described. The difference might represent the iconversion of inosine to hypoxanthine, which was larger in the psoriatic epidermis. We used the second system for studying the enzyme niature and for comparing the psoriatic epidermal and healthy epiderr~al adenosine deaminase activity. Preparation of Microdissected Pure Epidermis The skin samples were obtained by 7 mm punch from affected and unaffected areas of psoriasis under local anesthesia with 0.5 % lidocaine. The healthy skin samples were provided by plastic skin surgeons. The pure epidermis was obtained as described previously 25. Each sample washed with saline was placed in and frozen on the cryostat adaptor in less than 10 s. The samples were cut 20 ~tm thick. After lyophilization the epidermis and dermis were dissected under a stereomicroscope and the keratin layers were removed. The pure epidermal tissues weighing approximately 30 gg were used for each assay. The statistical significance was determined by Student's t-test. Results Using epidermal homogenate as an enzyme source we investigated the biochemical nature of adenosine de- aminase and established the assay conditions. The time course showed that the activity linearly increased for 60min, and then gradually decreased (Fig. 1). The boiled enzyme had no adenosine deaminase activity. The tissue dependency curve showed that the enzyme activity was proportional to the amount of tissue (Fig. 2). Consequently, the incubation in th e following experiments was 30min and about 7gg protein was used as the enzyme source. The K~ value for adenosine was 3.3 x 10-SM, which is almost identical to the values reported by several investigators 26, 27 (Fig. 3). Optimum pH was around 7 (Fig. 4.). Coformycin 18- 21, a tight binding inhibitor of adenosine deaminase, inhibited adenosine
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`312 H. Koizumi et al. : Adenosine Deaminase in Human Epidermis 150-- 300- 04 A w / /o /o I 2O v I I I 4O 6O 80 Time (rain) 5- Fig. 1. Time course of adenosine deaminase activity. Keratome sliced epidermis was homogenized and centrifuged and used as the enzyme source. Samples of 7.81xg protein were assayed at 37~ The adenosine deaminase activity was determined as nmol/mg protein ((cid:14)9 The boiled extract was assayed for adenosine deaminase activity at 37~ for 30min (O) 30- e- E -6 E O- O 20- 10- 0 r~) O I I I I I I I I v 0 1 2 3 4 5 6 7 8 Protein ()Jg) Fig. 2. Relation between the amount of sample tissue and adenosine deaminase activity. The homogenate was used as the enzyme source. The protein of the enzyme source was determined, and the enzyme source was diluted with 50 mM phosphate buffer, pH 7.1. The enzyme activity was measured 4- r a. E ~2- E -6 E 1- O /' 3- ~ km for /~ 2- /- adenosine / ~' / , , , - " Ix10 5 2xi05 3xi0 5 11S 04 i i I 0 50 100 150 Adenosine (~M) Fig.3. The effect of different concentrations of adenosine. The homogenate was used as the enzyme source. Adenosine deaminase activity was measured as described in 'Materials and Methods' c- o en E -6 E r O 4- 3- 2- 1- 0 I ( 1 I I I i 4 5 6 7 8 9 10 pH Fig. 4. pH dependence of adenosine deaminase. The homogenate was used as the enzyme source. 50 mM acetate buffer (pH 4- 5), 50 mM phosphate buffer (pH 6-- 8), 50 mM borate-NaOH buffer (pH 9-10) were used r Q. E -6 E
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`H. Koizumi et al. : Adenosine Deaminase in Human Epidermis 313 e- o E .c_ E 1- O E r- 0-~ I i I i i i I i 0 0.02 0.2 2 20 200 2000 20000 Coformycin nM) Fig. 5. Inhibition of adenosine deaminase by coformycin. Coformycin was added in the reagent mixture simultaneously with adenosine and skin extract (O). Coformycin and enzyme were preincubated for 30 min at 37~ and the enzyme activity was assayed ((cid:12)9 Final concentration of adenosine for the enzyme assay was 170 gM deaminase activity markedly (Fig. 5), and 50% in- hibition of the enzyme activity was obtained at the concentration of 2 x 10-9M of coformycin when sub- strate, coformycin and the enzyme were mixed simul- taneously. However, after 30 min preincubation of the enzyme and coformycin, almost complete inhibition was obtained at the concentration of 2 (cid:141) 10-11 M. The data are consistent with the tight binding nature of coformycin to the enzyme 20. The inhibition constant (~) is reported approximately 1 - 10 x 10- ~1 M 21. In the case of the tight binding inhibitor the association and dissociation reaction may be so slow that proper inhibitory constants can only be obtained after pre- incubation of the enzyme and the inhibitor. Thus the microanalytical method of adenosine deaminase assay enables us to determine the adenosine deaminase activity of protein samples as small as a few micrograms. Since a significant amount of dermal component was contaminated in the skin slice samples, we used microdissected pure epidermis and determined the adenosine deaminase activities of the healthy epidermis and the psoriatic unaffected and the affected epidermis. Ten psoriatic patients and four healthy controls were investigated (Fig. 6). The mean adenosine deaminase activity + 1 SD in the psoriatic affected epidermis was 0.78 _+ 0.28nmol/min/mg dry weight and that of unaffected epidermis was 0.45 _+ 0.14 nmol/ E: 1.0- ,_03 lU 13 E -- 0.r- E O E e- $ IP r I i I w Psoriasis Psoriasis Healthy affected unaffected epidermis epidermis epidermis Fig.6. Adenosine deaminase activity in the microdissected pure epidermis of ten psoriatic patients and four healthy controls. Adenosine deaminase assay was started by mixing approximately 30gg dry weight pure epidermis with 10gl of the reagent mixture containing 340 gM (2-3H) adenosine in 50 mM phosphate buffer, pH 7.1, and incubated for 30min at 37~ Vertical bars indicate mean adenosine deaminase activity min/mg dry weight. The difference was statistically highly significant (P< 0.01). The mean adenosine deaminase activity in the normal controls was 0.43 _+ 0.18 nmol/min/mg dry weight. The difference was also statistically significant (P< 0.05) against psoriatic involved epidermis. There was no difference in the enzyme activity between healthy and psoriatic unaffected epidermis. In summary, it was shown that psoriatic affected epidermis had about 180 % increased adenosine de- aminase activity compared with the healthy epidermis and the unaffected epidermis. Discussion A microanalytical method is now available to de- termine the adenosine deaminase activity of small protein skin samples. Two systems of thin layer chromatography can be used. However, the am- monium acetate system was not adequate for the samples which have high adenosine deaminase activity such as psoriatic affected epidermis because of the increased conversion of in(cid:12)9 to hypoxanthine due to purine nucleoside phosphorylase. Although it required 4-5h for development, the butanol-methanol-am- monium hydroxide-water system was better in such cases.
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`3 t4 H. Koizumi et aI. : Adenosine Deaminase in Human Epidermis In psoriatic affected epidermis the germinative cell cycle time is shortened 28, and RNA content is increased 29. Psoriatic scales are also known to contain large amounts of RNA and DNA with RNase and DNase. Several enzyme activities of purine nuc- leotide salvage pathway are reported to be increased in psoriatic affected epidermis 4, 30. The increased adenosine deaminase activity reported in this study seems to be another finding of increased enzyme activity of nucleotide metabolism in the psoriatic affected epidermis. The physiological significance of increased adenosine deaminase activity remains un- known at present. Our data indicate that adenosine deaminase activities of healthy and psoriatic unaffected epidermis were about the same (Fig. 6). Previously we reported that adenosine deaminase activity in periph- eral lymphocytes of psoriatic patients was not increased 17. Therefore, the increased adenosine deaminase activity in the psoriatic affected epidermis seems to be a secondary phenomenon, which may reflect the accele- rated salvage pathway of nucleic acid metabolism associated with the hyperproliferative state of psoriasis affected epidermis. Acknowledgement. This study was supported in part by grant 56770502 from the Ministry of Education, Japan. References 1. Iizuka It, Koizumi H, Kamigaki K, Aoyagi T, Miura Y (1981) Two forms of adenosine deaminase in pig epidermis. J Dermatol 8:91-95 2. Koizumi H, Iizuka H, Aoyagi T, Miura Y (1982) Two forms of adenosine deaminase in pig epidermis (iI). J Dermatol 9: 29 - 35 3. Ogura R, Koga H, Kumano S (1980) Purification and characteri- zation of hypoxanthine/guanine phosphoribosyl-transferase in bovine snout epidermis. J Invest Dermatol 75 : 240-- 243 4. Kizaki H, Matsuo I, Sakurada T (1977) Xanthine oxidase and guaninase activities in normal and psoriatic epidermis. Clin Chim Acta 75 : 5- 8 5. Tomonari K, Fujiyoshi M, Matsuzaki T, Kumano S, Ogura R (1980) High pressure liquid chromatographic analysis for uric acid in the epidermis. Proc Japn Soc Invest Dermatol 5:91 -92 6. Giblett ER, Anderson JE, Cohen F, Pollara B, Meuwissen HF (1972) Adenosine deaminase deficiency in two patients with severe impaired cellular immunity. Lancet 2:1067--1069 7. Donofrio J, Coleman MS, Hutton JJ, Daoud A, Lampkin B, Dyminski J (1978) Overproduction of adenine deoxynucleosides and deoxynucleotides in adenosine deaminase deficiency with severe combined immunodeficiency disease. J Clin Invest- 62:884-887 8. Herstffield MS, Kredich NM (1980) Resistance of an adenosine kinase-deficient human lymphoblastoid cell line to effects of deoxyadenosine on growth, S-adenosylhomocysteine hydrolase inactivation, and dATP accumulation. Proc Nat Acad Sci USA 771 : 4292 -- 4296 9. Schmalsteig FC, Nelson JA, Mills GC, Monahan T, Goldman AS, Goldblum RM (1977) Increased purine nucleotides in adenosine deaminase-deficient lymphocytes. J Pediatr 91:48- 51 10. Iizuka H, Adachi K, Halprin KM, Levine V (1976) Adenosine and adenine nucleotide stimulation of skin (epidermal) adenylate iBiochim Biophys Acta 444: 685- 693 cyclase. 11. Fischer D, Van Der Weyden MB, Snyderman R, Kelley WN (1976) A role for adenosine deaminase in human monocyte maturation. J Clin Invest 58 : 399- 407 12. Delapp NW, Karasek MA (1976) Importance of pyrimidine nucleotide salvage pathways for DNA synthesis in skin. J Invest Dermatol 66: 306-- 312 13. Davison PM, Karasek MA (1978) Nucleic acid metabolism in the skin. Int J Dermatol 17:605-615 14. Ogura R, Koga H, Kinoshita M, Takashima Y (1978) Distribution of nucleic acid metabolism in cow snout epidermis. Br J Dermatol 98 : 405- 410 15. Matsuo I, Ohkido M, Kizaki H, Sakurada T (1977) Purine metabolism in epidermis. In- Seiji M, Bernstein tA (eds) Biochemistry of cutaneous epidermal differentiation. Univ Park Press, Baltimore, pp 148-156 16. Kalcker HM (1947) Differential spectrophotometry of purine compounds by means of specific enzymes. III. Studies of the enzyme of purine metabolism. J Biol Chem 167:461-475 17. Iizuka H, Koizumi H, Kimura T, Miura Y (1980) Adenosine deaminase activity in peripheral lymphocytes of psoriasis and Sezary's syndrome. J Dermatol 7:165-169 18. Nakamura H, Koyama G, Iitaka Y, Ohno M, Yagisawa N, Kondo S, Maeda K, Umezawa H (1974) Structure of cofor- mycin, an unusual nucleoside of microbial origin. J Am Chem Soc 96:4327-4328 19. Cha S (1975) Tight-binding inhibitors. I. Kinetic behavior. Biochem Pharmacol 24: 2177- 2185 20. Cha S, Agarwal RP, Parks RE (1975) Tight-binding inhibitors. II. Non-steady state nature on inhibition of milk xanthine oxidase by allopurinol and alloxanthine and human erythrocytic aden- osine deaminase by coformycin. Biochem Pharmaco124 : 2187- 2197 21. Agarwal RP, Parks RE (1977) Potent inhibition of muscle 5'-AMP deaminase by the nucleoside antibiotics coformycin and deoxycoformycin. Biochem Pharmacol 26:663- 666 22. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193 : 265- 275 23. McBurney MW, Whitmore JGF (1974) Mutant of chinese hamster cells resistant to adenosine. J Cell Physiol 85 : 87-100 24. Carson DA, Seegmiller JE (1976) Effect of adenosine deaminase inhibition upon human lymphocyte blastogenesis. J Clin Invest 57:274- 282 25. Iizuka H, Umeda K, Koizumi H, Aoyagi T, Miura Y (1981) Epinephrine-induced cyclic AMP accumulation in the psoriatic epidermis. Acta Derm Venereol (Stockh) 61:391 - 395 26. Akedo H, Nishihara H, Shinkai K, Komatsu K, Ishikawa S (1972) Multiple forms of human adenosine deaminase. Biochim Biophys Acta 276: 257- 271 27. Daddona PE, Kelley WN (1977) Human adenosine deaminase. Purification and subunit structure. J Biol Chem 252:110-115 28. Pullmann H, Lennartz K J, Steigleder GK (1977) Disturbance of DNA-synthesis in early psoriasis. Arch Dermatol Res 258 : 211 - 218 29. Mier PD, McCabe MGP (1963) The distribution of phosphorus in the lesions of eczema, psoriasis and seborrheic dermatitis. Br J Dermatol 75:354- 357 30. Matsuo I, Ohkido M, Kizaki H, Sakurada T (1977) Pnrine metabolism in psoriatic epidermis. Japn J Dermatol 87:617- 620 Received January 1, 1983
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