CYCLE IN HUMAN EPIDERMIS*

Transcription

1 EVALUATION OF THE ENZYMES OF THE KREBS-HENSELEIT CYCLE IN HUMAN EPIDERMIS* ROBERT G. CROUNSE, M.D.'.' AND SIMON ROTHBERG, Pu.D. Urea, one of the chief nitrogenous by-products of mammalian protein and amino acid metabolism, is synthesized in mammalian liver via the Krebs-Henseleit cycle (1). The details of the enzymatic pathways of this cycle have been established in several laboratories, particularly those of Cohen (2 6) and Ratner (7 1), and are summarized in Figure 1. Arginase, which catalyzes the cleavage of arginine to ornithine and urea, is widely distributed in nature (11). The has been found in many mammalian tissues other than the liver, such as kidney (12), breast (13), testes (14) and skin (15). Arginase is found only in the epidermal portion of human skin (16, 17). In the abnormal stratum corneum (scales) from the cutaneous disorder psoriasis arginase activity is markedly increased (18, 19). The reason for extra-hepatic localization of arginase is not clear. In none of these tissues has the complete Krebs-Henseleit cycle been demonstrated, though in some of them, sueh as kidney, arginine synthetase is present as well as arginase. In this tissue the two s may function as a partial cycle, in which amino-nitrogen of aspartic acid may be transferred to citrulline, and thence to urea. Therefore, the amount of urea so formed is dependent upon the quantity of pre-formed citruffine available to the tissue. In addition, only one half of the nitrogen in the urea so formed will be derived directly from aminonitrogen. The present experiments were undertaken to determine the presence or absence of the other s of the Krebs-Henseleit cycle in human epidermis, in an attempt to explain the role of arginase in this tissue. * From the Dermatology Section, General Medicine Branch, National Cancer Institute (National Institutes of Health, Public Health Service, Department of Health, Education and Welfare), Bethesda, Md. 1 Present address, Department of Dermatology, Miami University School of Medicine, Jackson Memorial Hospital, Miami, Florida. 2 This work was performed while a Public Health Research Fellow of the National Cancer Institute. Received for publication June 3, MATERIALS AND METHODS All reagents used in these experiments were obtained commercially. Uniformly labelled arginine-c' with a specific activity of 6.88 hc//mole was supplied by Nuclear-Chicago Corp., Chicago, Ill. Radioactive ornithine and citrulline were derived from the arginine by incubation with whole liver homogenate, known to contain the s of the Krebs-Henseleit cycle. The three radioactive amino acids were separated by high voltage electrophoresis on Whatman No. 3 paper (2 volts, ph 6.5, 5 minutes) (Fig. 2). Epidermis of fresh and frozen surgical specimens of abdominal skin was separated from the dermis by the stretch method (2). Weighed amounts of the wet tissue were homogenized in.1% cetyltrimethylammonium bromide (9 parts to 1 part tissue) in an all glass (Potter-Elvehjem) homogenizer immersed in ice. Supernatant and residue fractions were obtained by centrifugation at 2 x G for 15 minutes at 5 C. Homogenates of rabbit liver, of wet weights equivalent to the epidermis (8 4 mg./reaction), were prepared in the same manner. Ornithine transcarbamylase and arginine synthetase were each assayed by two different methods. Method I utilized an excess of substrate at optimum ph according to modifications of the technics of Brown and Cohen (21). Method II utilized as substrates tracer amounts of arginine- C', ornithinec14, and citrulline-c'4. Ornithine Transcarbamylase (Method I) Duplicate aliquots of both supernatant and residue fractions of epidermal homogenates were incubated at 38 C. for periods ranging from 15 minutes to 4 hours with 45 micromoles of glyclyglycine buffer, (ph 8.) 1 micromoles of carbamyl phosphate (dilithium salt), and 1 micromoles of L-ornithine (total volume 1.1 ml.). The enzymatic reactions were terminated by the addition of 1.25 millimoles of perchloric acid to a final volume of 1.6 ml. Controls included boiled tissue, omission of tissue, omission of substrate, omission of carbamyl phosphate, and addition of perchioric acid prior to incubation. Rabbit liver homogenates were incubated simultaneously to test the adequacy of the assay system. Combined liver and epidermal extracts were used to exclude

2 288 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY I Carbamyl Phosphate Synthetase CO2+NH4 +2ATP II Ornithine Transcarbamylase acetylgiutamate Mg H2N C--P3H2 + 2ADP + Pi (carbamyl phosphate) H2N C P3H2 + H2N CH2 CH2 CH2 t!h H2N C,.?OH HN CH2 CH2 CH2CH + Pi (carbamyl phosphate) III Synthetase (1-ornithine) (1-citrull ne) H2N C=?H a) HN CH2 CH2 CH2 CH + H2N CH + ATP CH2 H (1-citrulline) (1-aspartic acid) condensing Mg HN_?_.NH_tH_CH2_COH + AMP + PPi HN CH2 C142 CH2 CH CH (1-argi ninosuccinate)?h NH.1 NH CH CH2CH b) HN CH2 CH2 CH2 CH IV Arginase lh2 (1-argininosuccinate) cleavage l lh2 HN CH2 CH2 CH2 CH + CH (1-arginine) (fumarate) b H CH2 CH2 CH2 CH CH + H2o H2N CH2 CH2 CH2 H CH + H2N C (1-arginine) (1-ornithine) (urea) FIG. 1. Steps in the Krebs-Henseleit Urea Cycle the possibility of inhibition of the by epidermal tissue. Citrulline produced enzymatically was quantitated by reaction with diacetyl monoxime according to the method of Archibald, (22) modified by Ratner (23), and the optical density of the product determined at 49 millimicrons. The optical density varied from time to time, necessitating preparation of a separate standard reference curve for each experiment. Ornithine Transcarbamylase (Method II) Uniformly labelled ornithinie-c'4 was added to aliquots of the tissue preparations with 2 micromoles of phosphate buffer (ph 7.) and 1. micromole of carbamyl phosphate (dilithium salt), (Total volume.325 ml.) and incubated at 38 C; the aforementioned controls were utilized. The reactions were terminated at four hours by the addition of 1.2 millimoles of 112S4 to give a final volume of.425 ml. Aliquots (.25 ml.) of the reaction mixtures were spotted on Whatman No. 3 paper and electrophoresed at 2 volts, ph 6.5, for 45 minutes. The areas representing arginine, ornithine, and citrulline were identified with appropriate markers (Fig. 2) and eluted. The total eluates were placed at infinite thinness with several washes on copper discs and their radioactivity measured in a windowless gas-flow system. In addition, to exclude the possibility that epidermis might require ornithine that had been activated by the arginase reaction, arginine.ci4 was employed as substrate. The tissue preparations were pre-incubated for 2 hours at 38 C. with 775 micromoles of MnCl2 for activation of arginase, and then incubated with 2 micromoles of phosphate buffer, 1. micromoles of carbamyl

3 'a Arp KREBS-HENSELEIT CYCLE IN EPIDERMIS 289 S C;t A;g, 'C S 8 'A FIG. 2. Composite electrophoresis paper demonstrating separation of substrates and products of the Krebs-Henseleit cycle. The anode is to the left, the cathode to the right. This paper was run at 2 volts for 45 minutes in a ph 6.5 buffer. The amino acids (approximately.25 imoles of each) were loaded at the origin as follows; A. aspartic acid; B. argininosuccinic acid; C. citrulline; D. arginine; E. ornithine; F. a mixture of all of these amino acids. phosphate, and.1 micromoles of arginine-c'4 for periods up to four hours. Rabbit liver homogenates were again run in parallel, and in addition combined liver and epidermal extracts were assayed to exclude inhibition by the epidermal tissue. Substrate and products were separated by high voltage electrophoresis, eluted, plated, and counted as described above. Synthetase (Method I) The overall reaction of arginine synthetase was measured by incubation of the homogenized tissue extracts for one hour at 38 C. with 4 micromoles of phosphate buffer, 2.5 micromoles of MgSO4, 2.5 micromoles of aspartic acid, 2.5 micromoles of ATP, and 2.5 micromoles of citrulline, each prepared at ph 7. (Total volume.475 ml..) The reactions were terminated by the addition of 1.25 millimoles of perchloric acid, final volume of.975 ml., and aliquots of the mixtures were electrophoresed as previously described. was identified by staining with.4% ninhydrin in acetone, and ornithine was identified by a modification of the Chinard reaction (24). Synthetase (Method II) Incubations were performed in a manner similar to that described above, except that smaller amounts of reactants were utilized, and citrulline- 14 was added as substrate. The incubation times ranged from two to six hours. Electrophoretic separation, elution, and counting were performed by the usual methods. In addition, preliminary experiments to assess the activity of both the condensing and the cleavage s of the overall arginine synthetase reaction were performed. Argininosuccinic acid markers were electrophoresed with the reaction mixtures, and the areas representing this compound were eluted and counted. The usual controls were included, and combined extracts were employed to detect possible inhibition of the liver by epidermal tissue. RESULTS Ornithine Transcarbamylase No citruffine in excess of controls was formed by epidermis in the assays for ornithine transcarbamylase by Method I (Table I). These results are contrasted to those obtained with liver preparations run in parallel, which indicated the production of approximately.72 micromoles of citrulline per mg. wet weight tissue per 15 minutes, after correction for non-enzymatic production of citrulline. No inhibition of rabbit

4 29 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY liver ornithine transcarbamylase by epidermal tissue could be demonstrated in combined tissue experiments, even when the weight of epidermis was twenty times that of rabbit liver (Table II). Incubation of epidermal tissue with radioactive ornithine for four hours did not result in labelling of citrulline or arginine. Similarly, when radioactive arginine was used as substrate, no labelling of citruffine occurred, in contrast to the results obtained with liver tissue (Table III). Again, no inhibition of the liver by epidermal tissue could be demonstrated. Synthetase Electrophoresis of aliquots of the incubation mixtures following completion of the reactions by Method I demonstrated that large amounts of arginine (and ornithine resulting from endogenous arginase activity) were formed in the liver preparations, but that no arginine (or TABLE I Omit hi me transcarbamylase assay (method I) System D (49 mu.) Citrul Reaction Citrul- 1ine pmoles/ mg. Tissue/iS mm. Complete* with liver ho mogenate supernatant 1:2 dilution minus tissue boiled tissue acid treated time zero Complete* with epidermal homog. supernatant undiluted minus tissue boiled tissue acid treated time zero Complete* with epidermal homog. residue undiluted boiled tissue acid treated time zero * Tissue preparation representing 8.4 mg liver or epidermis, respectively, plus 45 micromoles glycylglycine buffer (ph 8.), 1 micromoles of carbamyl phosphate, and 1 micromoles of L-ornithine. Omission of carbamyl phosphate resulted in no enzymatic citrulline synthesis. ** Corrected for non-enzymatic citrulline production. TABLE II Ornithine transcarbamylase assay (method I) inhibition experiment System Complete* minus tissue acid treated time zero Complete** minus tissue acid treated time zero Complete*** minus tissue acid treated time zero (49 mu.) Citrul- Citiuiline line zmo1es/ ijmoles/ Reactiosue/15 mg. Tis- mm * Liver homogenate supernatant 1:2 dilution. ** Liver homogenate supernatant 1:2 plus Epidermal homog. supernatant 1:4. *** Liver homogenate supernatant 1:2 plus Epidermal homog. supernatant undiluted. ornithine) was formed in the epidermal tissue incubations. Similarly, no labelling of arginine or ornithine occurred in the experiments with epidermal tissue in which radioactive citrulline was employed, though incubation with liver tissue in equivalent amounts resulted in considerable labelling of these compounds (Table IV), as well as labelling of argininosuccirtic acid. Preliminary experiments indicated that argininosuccinic acid was labelled in experiments with epidermal tissue, the percentage of recoverable counts proportionate to time (two, four, and six hours). The apparent presence of the condensing of the overall arginine synthetase system is to be further substantiated. DISCUSSION The absence in human epidermis of both ornithine transcarbamylase and the overall arginine synthetase System suggests that arginase may have some function in this tissue other than its role in the Krebs-Henseleit cycle. Some correlation has been found between increased arginase activity and increased rates of cellular growth in both normal (25) and neoplastic (26) tissues. Similarly, the corniiied cells desquamated from the hyperplastic epidermis in the cutaneous

5 KREBS-HENSELEIT CYCLE IN EPIDERMIS 291 TABLE III Ornithine Transcarbamyas e Assay (Method!l)* Citrulline 5.1% Ornithine 24.7% Citrulline Ornithine 3.8% Rabbit Liver Homogenate Supernatant Rabbit Liver Homogenate Residue Citrufline Ornithine 7.1% Citrulline Ornithine 4.1% Epidermis Homogenate Supernatant Epidermis Homogenate Residue Citrulline 16.3% Ornithine 25.9% Combined Supernatants TABLE IV synthetase assay (method II)* Tissue Epidermis Liver Liver + Epidermis % of Counts Recovered in and Ornithine Above Controls** * The incubations included citrulline-c'4, 2. micromoles aspartic acid, 2. micromoles ATP, 2. micromoles MgSO4, in 2 micromoles phosphate buffer, all at ph 7.. ** 4 hour incubation at 38 C. disease psoriasis have been found to have markedly increased arginase activity (18, 19). Perhaps the most unique function of epidermal tissue is its production of keratinous proteins Mercer (27) has proposed that two types of keratinous protein are formed by one cell line in the epidermis, though at different levels of differentiation. In the epidermally derived hair root, however, each protein is synthesized by cell lines that are morphologically separable, the one cell type producing the hair shaft proteins, whereas the other cell type produces the proteins of the internal root sheath. Since arginase activity has been found only in the epidermal sheath

6 292 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY portion of hair roots (28), this may be associated with the production of a specific keratinous protein common to the cells of the epidermis and the internal sheath, but not with the production of the proteins in the hair shaft; hence its presence in both epidermis and internal root sheath, hut not in that portion of the hair root which synthesized the hair shaft proper. SUMMARY The enzymatic steps in the Krebs-Henseleit urea cycle were evaluated in preparations of human epidermis. Neither ornithine transearbamylase nor arginine synthetase activities could be demonstrated. The implications of these negative findings are discussed in terms of a possible role of arginase in epidermal tissue. REFERENCES 1. KnEEs, H. AND HENSELEIT, K.: Untersuehungen uber die harnstoffbildung in tierkorpen. Z. Physiol. Chem., 21: 33, COHEN, P. P. AND HAYANO, M.: The conversion of citrulline to arginine (transamination), by tissue slices and homogenates. J. Biol. Chem., 166: 239, CoHEN, P. P. AND HAYANO, M.: Urea synthesis by liver homogenates. J. Biol. Chem., 166: 251, COHEN, P. P. AND GRISOLIA, S. J.: The role of carbamyl L-glutamic acid in the enzymatic synthesis of citrulline from ornithine. J. Biol. Chem., 182: 747, Gmsou.&, S. AND COHEN, P. P. Catalytic role of glutamate derivatives in citrulline biosynthesis. J. Biol. Chem., 24: 753, BuRNETT, G. H. AND COHEN, P. P.: Study of earbamyl phosphate-ornithine transcarbamylase. 3. Biol. Chem., 229: 337, MARSHALL, M., METZENBEEG, R. L. AND COHEN, P. P.: Purification of carbamyl phosphate synthetase from frog liver. J. Biol. Chem., 233: 12, S. RATNEE, S. ANn PAPPAS, A.: Biosynthesis of urea. I. Enzymatic mechanism of arginine synthesis from eitrulline. J. Biol. Chem., 179: 1183, RATNEE, S. AND PAPPAS, A.: Biosynthesis of urea. II. synthesis from eitrulline in liver homogenates. J. Biol. Chem., 179: 1199, RATNEE, S. AND PETEACK, B.: Biosynthesis of urea. IV. Further studies on condensation in arginine synthesis from citrulline. J. Biol. Chem., 2: 161, RATNER, S., PETEACK, B. AND RcHAYAN5KY,.: Biosynthesis of urea. V. Isolation and properties of argininosuccinie acid. J. Biol. Chem., 24: 95, RATNEE, S., AN5LOw, W. P. AND PETEACK, B.: Biosynthesis of urea. VI. Enzymatic cleavage of argininosuccinic acid to arginine and fumarie acid. J. Biol. Chem., 24: 115, GREENBERG, D. M.: in Sumner, J. B., and Myrback, K., (Editors), The Enzymes, Volume I Part II, p Academic Press Inc., New York, KossEL, A. AND DAKIN, H. D.: Uber die arginase. Z. Physiol. Chem., 41: 321, SHAW, J. C. AND PETERSEN, W. E.: Arginase in the mammary gland. Proc. Soc. Exp. Biol. Med., 38: 631, MIHAEA, S.: Beitrage zur kenntis der fermente der stierhoden. Z. Physiol. Chem., 75: 443, GREENSTEIN, J. P., JENEETTE, W. V., MIDEE, G. B. AND WHITE, J.: Chemical studies on the components of normal and neoplastie tissues. V. The relative arginase activity of certain tumors and normal control tissues. J. Nat. Cancer Inst., 1: 687, VAN SCOTT, E. J.: Arginase activity in human skin. Science, 113: 3943, MAEDESHEv, S. R. AND SEMINA, L. A.: Arginase in the skin. Biokhimiia, 13: 236, VAN SCOTT, E. J.: Studies on the arginase activity of the skin. J. Invest. Derm., 17: 21, ROTHBEEG, S. AND VAN SCOTT, E. J.: Evaluation of arginase activity in normal epidermal tissue and pathological stratum corneum. J. Invest. Derm., 31: 263, VAN SCOTT, E. J.: Mechanical separation of the epidermis from the corium. J. Invost. Derm., 18: 377, BEOWN, Jn., G. W. AND COHEN, P. P.: Comparative biochemistry of urea synthesis. I. Methods for the quantitative assay of urea cycle s in liver. 3. Biol. Chem., 234: 1769, ARCHIBALD, R. M.: Determination of citrulline and allantoin and demonstration of citrulline in blood plasma. J. Biol. Chem., 156: 121, RATNEE, S.: in S. P. Colowick and N.. Kaplan, (Editors) Methods in Enzymology, Vol. II, p Academic Press Inc., New York, ROTHBEEG, S.: Possible significance of elevated arginase activity in psoriasis scales. Ann. N. Y. Acad. Sci., 73: 14, BALDWIN, E.: Arginase. Biol. Rev., 11: 247, MALMGEEN, H. AND SYLYEN, B.: The histological distribution of arginase activity in solid mouse tumor transplants and a comparison with ascites tumors and normal tissue. Cancer Res., 19: 525, Mnncnn, E. H.: in W. Montagna and R. A. Ellis, (Editors), The Biology of Hair Growth, p. 17. Academic Press Inc., New York, CROUNSE, R. G. ANn ROTHBEHG, S.: Distribution of arginase in human hair roots. 3. Invest. Derm. 35: 17, 196.