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Enzymes of Amino Acid Metabolism in Normal Human Skin

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Enzymes of Amino Acid Metabolism in Normal Human Skin CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector THE JOURNAL OF INVESTIGATIVE DERMATOLOGY vol. 45, No. 3 Copyright 1557 by The Williams & Wilkins Co. Printed in U.S.A. ENZYMES OF AMINO ACID METABOLISM IN NORMAL HUMAN SKIN II. ALANINE AND ASPARTATE TRANSAMINASES* KENJI ADACHI, Mill., Pu.D., CHARLES LEWIS, Ja., B.S.t AND FALLS B. HERSHEY, M.D. In the preceding paper(1) we described the MATERIALS AND METHODS partial characterizationandquantitative Alanine (or aspartate) transaminase activity bistoehemieal localizationof glutamate dehy-was assayed according to the steps shown in drogenase in normal human skin. In compari-Equation 1, with L-alanine (or L-aspartate) and son with glutamate dehydrogenase, which cat-a-ketoglutarate as substrates. Alanine (or aspar- tate) transaminase catalyzes the formation of alyzes reversible oxidative deamination, thepyruvate (or oxaloacetate) and L-glutamate. The transaminases catalyze transferof aminoresulting pyruvate (or oxaloacetate) is imme- groups, usually from an amino acid to a keto-diately converted to lactate (or malate) in the acid, without net loss of nitrogen. In otherpresence of NADH' and purified lactate (or malate) dehydrogenase. The NAD' produced was words, transamination represents the inter-measured fluorometrically (1,2). molecular exchange of amino nitrogen. Trans- The complete alanine transaminase substrate amination plays a significant role in metab-reagent consisted of 2.5 mM a-ketoglutarate, 100 olism by providing numerous alternativemM L-alanine (adjusted to pH 7.9), 1 mg% pyridoxal phosphate, 0.5% bovine plasma albumin, pathways between amino acids. Obviously, it20 mM nicotinamide, 1 mM NADH, and 5 g/ml is a useful process for tissues, by which ex-reagent mixture of crysta]line lactate dehydro- cessive amounts of certain amino acids cangenase in 100 mM Tris-HC1 buffer at pH 7.9. The complete aspartate transaminase substrate be removed rapidly without storage, andreagent consisted of 2.5 mM re-ketoglutarate, 20 the amino acids of body protein deaminatedmM aspartate, 1 mg% pyridoxal phosphate, 0.05% to serve as energy sources upon oxidationbovine plasma albumin, 20 mM nicotinamide, 2 via the citric acid cycle in ease of caloriemM NADH and 1.5 g/ml reagent mixture crystal- line malate dehydrogenase in 100 mM Tris-HC1 deprivation. buffer at pH 8.2. This paper characterizes human epidermal Human epidermal homogenate was prepared alanine and aspartate transaminases (E.C.as described in the preceding paper (1), and S of this homogenate (generally 1%, W/V) were 2.6.1.2, L-alanine: ex-oxoglutarate aminotrans-added to 50 el of the buffered substrate reagent. ferase and E.C.2.6.1.1, L-aspartate: a-oxoglu-The mixture was incubated at 37° C for an ap- tarate aminotransferase) and undertakes a quan-propriate period (generally 30 minutes). The titative histochemical study of these enzymes inresulting NAD was measured fluorometrically in the same way as for glutamate dehydrogenase normal skin and its appendages. assay (1). Frozen and dried sections (0.5 to 5.0 'eg) of Received for publication January 25, 1967. * From the Oregon Regional Primate Researchskin and its appendages were prepared in the Center, Beaverton, Oregon 97005 (K. Adachi) andmanner described by Hershey (3). Skin sections the Department of Surgery, Washington Univer-were placed on the bottoms of 2.5 x 50 mm test sity School of Medicine, St. Louis, Missouri (F. B.tubes, and S N1 of iced buffered substrate added Hershey). Publication No. 248 from the Oregonto each tube. After incubating 1 hour at 37° C, Regional Primate Research Center. This investi-the mixtures were returned to the ice bath and gation was initiated while the authors were at the Beatrice F. and Melville N. Rothschild Surgical3 N1 of 0.6 N HC1 added to each to arrest the Research Laboratories, Michael Reese Hospitalreaction. A 5 N1 aliquot was taken from each and Medical Center, Chicago, Illinois. The presenttube and the NAD formed determined fluoro- study was supported by funds from the Unitedmetrically as previously described. Standards con- States Public Health Service (GM 12874, FRtaining 1 and 5 mpmoles of NAD were carried 00163, AM 08445, AM 5512), the Beatrice F. andalong with the samples. Reagent blanks (reagent Melville N. Rothschild Memorial Foundation, and Revlon Research Center, Inc., New York. 'Abbreviations used: NAD and NADH = nie- t Present address: Central Research Division,otinamide adenine dinucleotide, oxidized and re- Monsanto Chemical Company, 800 N. Lindbergduced forms; tris-HC1 buffer = tris(hydroxy- Boulevard, St. Louis, Missouri. methyl)amino-methane-HC1 buffer. 240 ALANINE AND ASPARTATE TEANSAMINASES IN SKIN 241 coofl R C= 0 CHa -S H-C-NH;+ tjfl2 + Coo- coo- CH H_?_NH; Coo- Coo- L—aianine ÷ + ____s. Pyruvate + L—gtutamate or or glutarate (L—aspartate) (oxaloacetate) mixtureonly) and enzyme blanks (complete assay mixture without substrate) were also run simul- taneously. The lactate dehydrogenase was obtained from the Sigma Chemical Company, St. Louis, Missouri, and was substantially free of transaminase and pyruvate kinase activity. It had a specific activity of approximately 300 pmoles of pyruvate to lactate/mm/mg protein at pH 7.5 and 37° C. The malate dehydrogenase (Sigma, Type I) was ob- •— Asp.trans. tained as a dry preparation and reconstituted with £ Alatrani, water to make a 1.5 mg/ml suspension. The specific activity was approximately 125 imoles of oxalo- acetate to malate/min/mg protein at pH 7.5 and 25° C. •' Protein was measured according to the method 7 8 9 of Lowry et al. (4). pH RESULTS Fxo.1. Effect of pH on alaninc and aspartate transaminase activities. The assay conditions are Theeffects of pH on alanine and aspartatethe same as described in the text except for pH. transaminase activities are shown in Figure 1.The ordinate shows relative enzyme activities The optimum pH ranges for alanine and as- (maximum activity as 100). Homogenate used. partatc transaminases are 7.6—7.9 and 8.1—8.3 respectively. The pH curves for both enzymes9 x10respectively. Figure 3 shows the ef- show somewhat flat-topped peaks, and enzymefects of varied a-kctoglutaratc concentrations activities at physiological pH are approxi-on epidcrmal alaninc and aspartatc trans- mately 80% of those at optimum pH values.aminase activities. It should be noted that Figure 2 shows the effects of various alanineexcessive amounts of a-kctoglutaratc cause and aspartate concentrations on the respectivemarked inhibition. Optimum activities for enzyme activities. Optimum alaninc trans-both enzymes are obtained at 2.5 mM a- aminasc activity is obtained at L-alaninc con-ketoglutaratc. Alanine transaminase activity centrations above 100 mM and remain opti-is inhibited nearly 50% at an a-kctoglutarate mum with concentrations up to 200 mM, whileconcentration of 8.5 mM, while aspartate optimum substrate concentrations for aspar-transaminasc activity is depressed 40% at 30 tate transaminasc activity are 10 to 30 mM;mM. The Km values of alaninc and aspartate concentrations higher than 50 mM are in-transaminases for a-kctoglutaratc were 1 x10 hibitory (25% inhibition at SO mM). Theand 2 x10respectively. Michaclis constants (Km values) of alaninc Table I shows the effects of omitting each and aspartate transaminascs for their respec-constituent of the test medium. In the absence tive amino acid substrates were 1 >< 102 andof any one of the substrates, no transaminase 242 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY 10 mjzmoles of NAD production per reaction 100 vessel (55 1d volume) for alanine transaminase and up to 16 mtmoles NAID per tube (55 fd) for aspartate transaminase activity, respec- 50 tively. Both transaminase activities were di- TABLE I Effect of omitting one constituent from the reaction mixture 50 (asp. mM) 100 0 50 100 (ala.mM) 200 Constituent omitted Asp. trans. Ala. trans. Fm.2. Effect of L-alanine and L-aspartate con- centrations. The reaction systems are as describedNone* 100* 100* in the text except for varied substrate concentra- tions. Homogenate used. Aspartate (or alanine) 1.5 3.0 a-Ketoglutarate 2.5 2.1 NADH 0 0.5 Pyridoxal phosphate 64 77 Auxiliary enzyme 71 74 Bovine plasma albumin 83 90 Nicotinamide 100 105 * Thecomplete mixtures with 1.5% (W/V) epidermal homogenate as described in the text were incubated for 30 mm. at 37° C. This activity 0 2.5 50 75 was taken as 100%. Each figure is expressed as Oç—tstogiutarats mM % of the maximal activity. Fm. 3. Effect of a-ketoglutarate concentration. Test systems as in Fig. 1 at optimum p11's. Ho- NI An •— Asp.trans. mogenate used. s—-—— Alo.trans. activities could be assayed. L-alanine, L-aspar- tate, a-ketoglutarate, and NADH were essential for the reaction. On the other hand, omission of pyridoxal phosphate caused 25 to 35% re- duction of maximal activities. Omission of nieotinamide did not change activities under the assay conditions, while omission of bovine plasma albumin caused 10 to 20% reduction.00) of maximal activity. The latter finding indi- (0 a, cates protective action of the plasma albumin 0 against loss of transaminase activities. When E their respective auxiliary enzymes were omit- ted, both transaminase maintained 70—75% of optimum activities. Since malate and lactate dchydrogenases in skin have 10 to 40 times more activity than the transaminases and the respective Km's for oxaloaeetate and pyruvate are extremely low, the endogenous malate and lactate dehydrogenases in the homogenate are 0 30 60 nearly sufficient for the assay of transaminases. Mm. Figure 4 shows the time course of alanine Ftc.4. Reaction rate curves and the effects of and aspartate transaminase reactions and theenzyme concentrations. Enzyme concentrations as indicated in the figure. Other conditions are the effects of enzyme (epidermal homogenate) con-same as described under "methods." Homogenate centrations. A linear rate was observed up toused.
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