Purification of human skin tyrosinase and its protein inhibitor: Properties of the enzyme and the mechanism of inhibition by protein

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 217. No. 2, September, pp. 73%747,1982

Purification of Human Skin Tyrosinase and Its Protein Inhibitor: Properties of the Enzyme and the Mechanism of Inhibition by Protein’ ELIMBAN

VIJAYAN,* AND

*Department

of Biochemistry

INTISAR HUSAIN,* ABBURI NARESH C. MADANt

and ~Lkpartment of Surgery All India New Lklhi-110029, India Received

May

Institute

RAMAIAH,*#’ of Medic&

sciences,

3, 1982

Tyrosinase from normal human skin was purified to high specific activity; 228 nmol of dopa formed/min/mg protein. The properties of the purified enzyme differ from those of the same enzyme in crude homogenates. The activity of the purified enzyme is not affected by dopa. It is not inhibited by excess tyrosine and exhibits no lag in its rate at 4 mM concentration of ascorbic acid. This preparation is free of peroxidase and yet will catalyze both hydroxylation of tyrosine to dopa and its further oxidation to dopa quinone with fourfold more activity with dopa as substrate suggesting that mammalian tyrosinase catalyzes both reactions rather than dopa oxidation alone as suggested by M. Okun, L. Edelstein, R. Patel, and B. Donnellan (1973, Yale J. Biol. Med 46, 535-540). A protein present in the cytosol and melanosomes that constitutes 30% of soluble epidermal proteins was purified and found to inhibit tyrosinase competitively with tyrosine. Its molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 66,000. In order to study the biochemical basis of depigmentation or vitiligo in human beings, which affects about 1% of world population (l), some properties of tyrosinase (Monophenol Dopa: Oz-oxidoreductase, EC 1.14.18.1) in the crude homogenate obtained from epidermis of normal and vitiligo skin of human beings were studied (2). Tyrosinase has been purified from mushroom (3), Neurospora (4, 5), Vibrio tyrosinut~ (6), epidermis of black mice (‘7), and from several melanomas (‘7-14). These procedures either involve several steps or the yields are low and therefore were unsuitable for purification of this enzyme from human skin, which cannot be obtained in large quantities. 1 Supported by the grants from the Department Science and Technology, India (HCS/DST/971/80) and the Indian Council of Medical Research. ‘Author to whom all correspondence should addressed. 0003-9861/82/100738-10$02.00/O Copyright All rights

0 1987, by Academic Press, Inc. of reproduction in any form reserved.

Mushroom tyrosinase was purified to homogenity by affinity chromatography using 4-aminobenzoic acid coupled to 6aminohexanoic acid Sepharose-4B (15). It was also partially purified (16) when 3-iodotyrosine was used in place of 4-aminobenzoic acid. However, this method was not successful for purifying tyrosinase from B16 melanoma, possibly because 3iodotyrosine may not be an inhibitor of mammalian tyrosinase (17). In this paper purification methods for human skin tyrosinase and a protein that inhibits tyrosinase are described, using 4aminobenzoic acid coupled to 6-aminohexanoic acid Sepharose-4B. The failure to purify human skin tyrosinase by eluting the enzyme with increase in pH (15) from the affinity column led to the discovery of the existence of tyrosinase inhibitor protein. Enzyme was therefore specifically eluted by tyrosine while tyrosinase inhib-

of

be

738

PROPERTIES

OF HUMAN

itor was eluted by increasing the pH of eluting buffer to 8.0. Some properties of the enzyme and the mechanism of inhibition of tyrosinase by epidermal protein also shown to be located in melanosomes are presented. EXPERIMENTAL

PROCEDURES

Materials L-Dopa,3 L-tyrosine, mushroom tyrosinase grade III, L-ascorbic acid, a-nitroso-@-naphthol, PPO, POPOP, cycloheximide, sodium nitrite, horseradish peroxidase type I RZ 0.73, 1-phenyl-Z-thiourea, bovine serum albumin, paminobenzoic acid sodium salt grade I, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (ECDI), 6-aminohexanoie acid Sepharose-4B (CH Sepharose-4B), phydroxyphenylpyruvic acid, phydroxyphenylacetic acid, Coomassie blue, acrylamide, Nfl-methylene-bis-acrylamide, SDS, ovalbumin, pepsin, and bovine plasma albumin were obtained from Sigma Chemical Company, St. Louis, Missouri. Dialysis tubing was purchased from Arthur H. Thomas Co., Philadelphia, Pennsylvania 19105. 0-Phenylenediamine was a product of Kochlight Laboratories Ltd., Colnbrook Bucks, England. Uniformly labeled L-[‘4C]tyrosine of specific activity 513 Ci/mol was obtained from Amersham International Limited, Amersham, England. Millipore filters of 0.45-pm pore size were purchased from Millipore Corporation, Bedford, Massachusetts. Igepal Co-630 from GAF Corporation, New York, was a gift from Professor S. H. Pomerantz, Department of Biological Chemistry, University of Maryland. All other chemicals were of analytic reagent grade. Human cadaver skin was taken from accident cases which were kept in cold. The skin was also obtained during plastic surgery operations.

Methods Processi~ of human cadaver skin. Connective tissue, fat, and hair were removed and the skin was washed several times with 20 mM sodium phosphate buffer, pH 6.8, and blotted. It was stretched on an 3 Abbreviations used: Dopa, L-@-3,4-dihydroxyphenylalanine; PPO, 2,5-diphenyloxazole; POPOP, 1,4-bis-2(5-phenyloxazolyl)benzene; ECDI, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide HCI; CH Sepharose-4B, 6-aminohexanoic acid Sepharose-4B, PHPP, phydroxyphenylpyruvic acid; PHPA, phydroxyphenylacetic acid; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid, BSA, bovine serum albumin.

SKIN TYROSINASE

739

enamel tray which was kept on ice-salt mixture (approximately -1O“C). The epidermis was scraped with a scalpel after the dermis portion was frozen. Preparation of melumcm& -ian. A weighed amount of epidermal scrapings (1 g/7 ml of 0.25~ sucrose) was homogenized in a tube kept in ice with polytron homogenizer for 1 min (20s each three times). The resulting homogenate was centrifuged at 7OOg for 10 min in Sorvoll R C-2B. The precipitate was discarded and the supernatant was further centrifuged at 10,OOOgfor 15 min. The pellet containing melanosomes was suspended in 20 mM sodium phosphate buffer, pH 6.8, containing 1% Igepal, a nonionic detergent that ruptures melanosomes (0.3 ml/g epidermis) and kept at 0-4°C overnight with occasional stirring with a glass rod. It was then centrifuged at 10,000g for 15 min. The supernatant solution which contained most of the tyrosinase was dialyzed against 2 liters of 5 mM phosphate buffer, pH 6.8, at 0-4°C with a change of 2 liters of buffer once over a period of 3 h. Preparation of epiderm& h.om.ogenate. A weighed amount of the epidermal scrapings was ground at O4°C in a precooled mortar and pestle in 20 mM sodium phosphate buffer, pH 6.8, containing 1% Igepal and Pyrex glass powder as abrasive for 15-30 min depending on the amount of tissue (25 mg tissue/O.5 ml of buffer). The homogenate was centrifuged at 7OOg for 10 min in a Sorvall RCZB centrifuge. The supernatant was further centrifuged at 10,000g for 15 min. The pellet was discarded and the supernatant thus obtained had most of the tyrosinase activity. It was dialyzed as described above for the melanosomal fraction. The dialysate was concentrated fivefold with respect to its original volume by lyophilization. Preparation of &nity column. CH Sepharose-4B coupled with paminobenzoic acid was employed for the affinity chromatography of human skin tyrosinase. It was prepared as recommended by the supplier, as was CH Sepharose-4B. The pH throughout the coupling procedure was kept at 5.0. Five grams of CH Sepharose-4B in 60 ml was coupled with 0.1 M paminobenzoic acid (20 times the theoretically required amount) in the presence of 0.1 M ECDI. The coupled gel was stored in 0.1 M sodium acetate buffer, pH 4.0. containing 1 M sodium chloride at 0-4°C. A portion of the coupled gel was thoroughly washed with double-distilled water and a column of 8 ml bed volume (1 cm diameter) was made. If the amount of protein loaded on the column was less than 20 mg, a smaller column of 4 ml bed volume (1 cm diameter) was used. Estimation of tgrosine. Tyrosine was estimated by a fluorometric method as described in Sigma Technical Bulletin 6OF/70F which was a modification of the procedure of Waalker and Undenfriend (18). Protein estimation. Protein was estimated by the method of Lowry et al. (19) except in the case of pu-

740

VIJAYAN

rified enzyme where the microassay method of Peterson (20) was employed. Estimation of tyrosinme a&v&. Tyrosinase a&vity was estimated by a fluorometric method (21) as modified by Husain et al. (2). The reaction was conducted at 3’7°C in rimless tubes of 12 X lOO-mm size, in a total volume of 55 ~1. The reaction mixture contained 40 rnM phosphate buffer, pH 6.8,0.75 mM ascorbic acid, 0.02% bovine serum albumin, tyrosine or/ and Dopa at concentrations as described in legends to figures and tables. After varying periods of incubation, 0.9 ml of 10 mM phosphate buffer, pH 6.5, containing 0.0025% zinc sulfate was added, followed by 0.1 ml of 0.25% potassium ferricyanide. After exactly 2 min the reaction was stopped by addition of 0.1 ml of a freshly made mixture of 5 N sodium hydroxide and 2% ascorbic acid (91 v/v). After 5 min the fluorescence of the sample was measured at 360nm excitation and 490-nm emission wavelengths, using a manual spectrofluorometer, Farrand Optical Company, New York. Quinine sulfate in 0.1 N sulfuric acid was used to fix the instrument to constant sensitivity during the assay and freshly made Dopa was used as standard. Estimutim of pemxikse activity. Peroxidase activity was estimated by a spectrophotometric method as described by Wolters et al. (22) except that hydrogen peroxide (0.012%) was used in place of urea peroxide. The reaction was terminated after 30 min and the product of enzymatic reaction was estimated at 492 nm. ~[U-“CjT~rn&e inmrpora tion into melanin. The incorporation of L-[U-“Cltyrosine into melanin was determined as described by Chen and Chavin (23). The incubation mixture contained the following components in a total volume of 57 ~1: L-Tyrosine, 0.75 mM (4 X lo6 cpm); cycloheximide, 500 wg/ml; sodium phosphate buffer, 40 mM (pH 6.8); and 16.83 units of enzyme. The reaction was carried out in air, in rimless tubes of 12 X lOO-mm dimensions at 37°C in a shaking water bath for a period of 30 min. Dopa and cycloheximide solutions were freshly prepared. Cycloheximide was added to inhibit any incorporation of tyrosine into protein. The reaction was terminated by adding 57 ~1 of 10% TCA containing carrier tyrosine (1.1 mM). Boiled enzyme (kept in boiling water for 10 min) and also containing phenylthiourea (1 mM), which inhibits any tyrosinase activity remaining even after boiling, were used as controls. All assays were run at least in duplicate and the average was taken. Maximum variation among duplicates was not more than 10%. The TCAtreated samples were processed according to the procedure described by Chen and Chavin (23). In brief, they were filtered through 0.45-am Millipore filters, washed with 36 ml 5% TCA, then with 16 ml water and 8 ml 0.1 N HCl, respectively, and finally with 25

ET AL.

ml water. The Millipore filters were then placed in vials and dried at 37°C. Of the scintillation fluid, 10 ml containing 4 g of PPO and 0.5 g POPOP per liter of toluene was added to the vials and the vials were counted in a Packard scintillation spectrometer. The counting efficiency was 87%. No quenching corrections were done. Results were expressed as picomoles of L-tyrosine incorporated into melanin per minute per milligram of protein. Estimation of Dopa oxiduse a&iv@. Dopa oxidase activity of the purified tyrosinase was measured by following the decrease in absorbance at 265 nm due to the oxidation of ascorbate by Dopa quinone formed by tyrosinaae (11). Ekctrop~~. Electrophoresis of tyrosinase inhibitor protein was done using the method of Davis (24). Lktemhdion of mobmular weight of @rosinme inhibitor protein. The molecular weight of the tyrosinase inhibitor protein was determined by SDS-polyacrylamide gel electrophoresis by the method of Weber and Osborn (25) using bovine plasma albumin, ovalbumin, and pepsin as marker proteins. RESULTS

Purification of Tyrosinase from Melanosomal Fraction The melanosomal fraction prepared as described under Methods was adjusted to pH 4.7 with 0.1 M citric acid. The enzyme under these conditions is stable at O-4% for at least 4 h. It was loaded on to the affinity column which was equilibrated with citrate phosphate buffer, pH 4.7 (50 mM citrate, 100 mM phosphate). The column was then washed with the same buffer (25 times the bed volume) until no more protein was eluted as judged by absorption at 280 nm. Tyrosine 5 mM in Citrate phosphate buffer of pH 4.7, was used to elute the enzyme. One-milliliter fractions were collected and fractions 4 to 20 were pooled and dialyzed against 1 liter of 5 mM sodium phosphate buffer, pH 6.8, for 3 h. The dialysate was lyophilized to 0.1 ml/g of original epidermis. The extent of purification and the yield of enzyme in a typical purification procedure are described in Table I. The yield of melanosomes from the epidermis was low because of incomplete breakage of cells by the polytron homogenizer. Homogenization of skin by grinding with abrasive and detergent was found

PROPERTIES

OF HUMAN

SKIN

TYROSINASE

741

to be more effective. Therefore homogenate obtained by grinding the skin with Igepal and sand was used as the source of enzyme for its purification. Putijcatim Epidermal

of Tyrosinuse from Homogenate

Tyrosinase was purified from the epidermal homogenate exactly as described for the enzyme from the melanosomal fraction. The extent of purification and the yield of enzyme in a typical purification procedure are described in Table I. The specific activity of the enzyme obtained from melanosomal fraction was about 1.5 times higher than that obtained from the epidermis homogenized with sand and Igepal, possibly because the homogenate may contain isozymes of tyrosinase (8-11, 14) which have different specific activities. In addition, the specific activity of tyrosinase in the melanosomal fraction is six times higher than in the homogenate. The specific activity of tyrosinase from the melanosomal preparation is comparable to the specific activity of tyrosinase from the skin of black mice (7) which was shown to be pure after taking into account the difference in the method of estimation of tyrosinase (26) which yield sixfold lower values for enzyme activity as compared to the method described here (2). It is planned to employ the microelectrophoretic method for determining the purity of our preparation. The enzyme was stored in 0.02% BSA at -20°C and was found to be stable. Properties

of Tyrosinase

Absence of lag in tyrosinase activity in the presence of 4 W&M asmbic acid. Tyrosinase purified from the homogenate was used as the enzyme source since the enzyme from the melanosomal fraction was not obtained in sufficient quantity. Bovine serum albumin (0.02% ) was routinely used in enzyme assays in view of the small amount of enzyme protein present. Tyrosinase activity was proportional to the enzyme concentration. The activity of tyrosinase was determined for varying intervals of time in the presence and absence of added Dopa and

742

VIJAYAN

15 TIME

OF

30 INCUBATION

45

60

U4INUTE.5)

FIG. 1. Lag in tyrosinase activity in presence and absence of Dopa. The reaction mixture contained at 3’7’C the following components at final concentration in a total volume of 55 ~1: L-tyrosine, 0.75 mM, ascorbic acid, 0.75 mM, sodium phosphate buffer (pH 6.8), 40 mrd, bovine serum albumin, 0.02%; 21.32 units of enzyme was used for reaction. Tyrosine was estimated in this preparation as described under Methods and it was found to contain 0.2 mM. This carryover was taken into account in estimating the final concentration of tyrosine in the assay mixture. No Dopa (o), 0.005 mM Dopa (A).

0.75 mM ascorbic acid, which was routinely used in the assay of enzyme activity. The results are presented in Fig. 1. The 0.75 InM tyrosine was employed in these studies since other studies of Dopa requirement were carried out with this concentration of tyrosine (2,21). The enzyme activity was linear up to 30 min with an initial lag of 5 min while with crude homogenates the reaction was linear up to at least 6 h with a lag of 1 h in presence of 5 pM Dopa and 2?4 h in absence of any added Dopa (2). In the purified fraction 5 pM Dopa neither abolishes the lag nor significantly alters the rate of reaction, in contrast to its effect on decreasing the lag and increasing the velocity by more than sixfold in the crude homogenate (2). Dopa had no effect on rate of reaction even at 20 I.IM (data not shown). This is in contrast to the absolute requirement of Dopa as a cofactor for purified tyrosinase of mouse skin (7). The 5min lag was entirely lost when the concentration of ascorbic acid was raised to 4 mM (Fig. 2). The hydrogen donor requirement under these conditions for the

ET AL.

hydroxylation reaction must have been satisfied by ascorbic acid. Absence of peroxidase activity and the ability of the purified enzyme preparation to incorpwate un$brmly labeled [‘4cJt~rosine into melanin. In view of the controversy over whether peroxidase, rather than tyrosinase, is the enzyme in the melanocytes which catalyzes the hydroxylation of tyrosine to Dopa (27-29), it was thought necessary to check whether the enzyme that was purified was peroxidase. No peroxidase activity could be detected in this preparation when estimated by the method of Wolters et al. (22). In addition phenylthiourea, a specific inhibitor of tyrosinase at 1 mM inhibited Dopa formation by 93% (data not shown). Moreover, we could demonstrate the incorporation of L-[U-14C]tyrosine into melanin by this enzyme preparation. The rate of L-tyrosine incorporation into melanin is about 1% of the tyrosine hydroxylation rate which is similar to the rate of tyrosine incorporation into melanin by the crude homogenate under similar conditions (2). It is, therefore, reasonable to conclude that tyrosinase indeed catalyzes both hydroxylation of tyrosine and oxidation of Dopa to Dopa-quinone as originally postulated (14, 30-33). The ratio of Dopa-oxidase to tyrosine hydroxylase activity of this enzyme is 4.11 when Dope-oxidase 270 I-

TIME

OF

INCUBATION

IMIN)

FIG. 2. The effect of excess ascorbic acid on lag in tyrosinase activity. The assay conditions were as described in Fig. 1 (a), except that ascorbic acid concentration was 4 mM and 19.5 units of enzyme was used for reaction.

PROPERTIES

OF HUMAN

SKIN

743

TYROSINASE

Elutkm of Other Protein(s) the Afinit2/ Column

FIG. 3. Effect of varying concentration of tyrosine on tyrosinase activity. The assay conditions were as described in Fig. 1 (O), except that tyrosine concentration was varied, 17.2 units of enzyme was used, and the incubation period was for 15 min.

activity was determined as described by Pomerantz (11) with 0.625 mM Dopa. Efect of varying concentrations of tyrosine on tyrosinase activity. The enzyme activity was determined at varying concentrations of tyrosine as shown in Fig. 3. Tyrosine was not inhibitory even up to 1.6 mM while in the crude homogenate 0.75 mM tyrosine inhibited tyrosinase activity by 70-80s (2). TABLE

Adsorbed on

Tyrosinase constituted only a small fraction of the protein bound to the eolumn. This suggested that there are protein(s) in the epidermis which have affinity for benzoic acid. It was therefore of interest to find the effect of these proteins on tyrosinase activity. For this purpose the affinity column after the elution of tyrosinase with 5 mM tyrosine was washed with citrate-phosphate buffer, pH 4.7, without tyrosine (five times the bed volume of column). The protein(s) on the column were then eluted by increasing the pH of the elution buffer to 8.0 (10 mM sodium phosphate buffer, nH 8.0). One-milliliter fractions were collected and fractions giving absorbance of 0.07 and above at 280 nm were pooled and dialyzed against 1 liter of 5 mM sodium phosphate buffer, pH 6.3, for 3 h at 0-4°C. The dialysate was concentrated 30-fold by lyophilization. The amount of protein eluted in relation to the epidermis and protein that was applied are presented in Table II. The epidermis was derived from whatever portion of skin was provided by the surgeon. It is not clear II

THE Abfouwr OF TYROSINASE INHIBITOR PROTEIN FROM EPIDERMIS AND ITS APPROXIMATE INTRACELLULAR CONCENTRATION

Experiment 1 2 3 4 5 6 I 8 9 10

Source

Amount of epidermis w

Cadaver Cadaver Cadaver Cadaver Normal Normal Normal Normal Normal Normal

3.08 4.33 4.30 0.22 2.75 0.38 0.14 0.28 0.16 0.88

Total protein applied (mrr) 18.85 42.60 71.50 4.66 9.75 5.00 2.35 3.90 3.45 3.60

Protein eluted from the affinity column by pH 8.V (me) 2.70 11.00 16.10 1.82 4.00 1.41 0.66 1.10 1.59 1.20

“The column wag first washed with 56 m?d citrate-100 mrd phosphate buffer, 5 rn~ tyrosine. The column thus freed of tyrosinase was washed further with times bed volume) to free tyrosine. The protein was then eluted with 10 mbf Results. b It is calculated on the assumption that water constitutes 75% of cellular

Percentage or applied protein eluted at pH 8.0

Intracellular concentration of protein elukd at pH 8.0 (mg/ml)

14.3 25.8 20.8 39.0 41.0 28.2 23.0 28.0 46.0 33.0

1.17 3.39 4.99 11.03 1.94 4.95 6.23 5.24 13.25 1.82

pH 4.7, followed by the same buffer containing 50 mM citrate-100 mM phosphate buffer (five phosphate buffer, pH 8.0, aa discussed under mass of epidermis.

744

VIJAYAN

ET AL. 50 t &wine Inhibitor Protein 1 5

Plasma 166.000l

Albumin

Ovalbumin K5.0001

4.5

E

Pepsin

--\

13L.7001

LO 1 L

I

L1

6

1 10

6 MOBILITY

FIG. 4. Polyacrylamide gel (7.5%) electrophoresis of the protein(s) eluted from the affinity column by increase in pH of elution buffer to 8.0 by the method of Davis (24). 26 pg protein was applied. Some protein remained at the top of the gel.

why variable amounts of protein were obtained per unit amount of epidermis. Purity of Protein(s) Eluted from the Ajinity Column by Iwea.Gng the pH to 8.0 The protein fraction eluted from the column with 10 mM phosphate buffer, pH 8.0, was electrophoresed under conditions described under Methods and found to be fairly pure (Fig. 4). Electrophoresis of this protein in SDS (25) along with bovine plasma albumin, ovalbumin, and pepsin standards gave a single band corresponding to a molecular weight of 66,000 (Fig. 5). The mobility of this protein in absence of SDS was similar to albumin suggesting that it may be monomeric. Inhibition of Tyrosinase Activity a Protein Purified frm the EpkbrmalHcm~ogenates

km1

FIG. 5. Determination of the molecular weight of protein eluted from the afbnity column by increase in pH of the elution buffer by SDS-polyacrylamide gel electrophoresis on 5% gel by the method of Weber and Osborn (2.5). 25 pg of each protein was applied on each gel for reference and 10 a when applied together.

tivity by about 80%. The inhibitory effect was completely abolished by heating for 10 min at 100°C. This protein had no effect on the method of estimation of tyrosinase as judged by the stability of Dopa in its presence. Eflect of Protein on Its Activity

Inhibitor

of Tyrosinase

with Time

It was thought possible that like the lag observed with mushroom tyrosinase caused by its protein inhibitor (34), the lag in ty-

bp

Tyrosinase activity at 0.75 mM tyrosine was tested with varying concentrations of protein purified from homogenates (Fig. 6). At about 2.6 pM (170 pg/ml) concentration, this protein inhibited tyrosinase ac-

ki7kkF /~g INHIBITOR OF REACTION

200 PROTEIN/ml MIXTURE

FIG. 6. Percentage inhibition of tyrosinase by varying concentrations of its protein inhibitor. The assay conditions were as described in Fig. 1 (O), except that the incubation period was for 15 min.

PROPERTIES TABLE

OF HUMAN

III

LACK OF EFFECT OF PROTEIN INHIBITOR OF TYROSINASE ON THE LAG IN ITS ACTIVITY Time of incubation (min)

Tyrosinase activity - Inhibitor

+ Inhibitor

Percentage inhibition

Picomoles of Dopa formed 10

71.29 188.0

15

346.0

5

25.50

61.00 118.0

64 67 66

Note. The assay conditions were as described in Fig. 1. The protein inhibitor concentration was 4.4 PM.

rosinase activity with the crude homogenate of human skin was due to the inhibition of tyrosinase by this inhibitor protein. If so, one would expect an increase in the lag with the highly purified enzyme on addition of the inhibitor. However, when the tyrosinase activity was tested with a fixed amount of the inhibitor protein at various intervals of time the extent of inhibition of tyrosinase by this protein was the same at all time intervals as shown in Table III. The protein inhibitor of tyrosinase from human skin had no effect on mushroom tyrosinase. Reversal of Inhibit&m of Tyrosinase by Protein Inhibitor with Tvrosine The enzyme activity was determined at a fixed concentration of protein inhibitor by varying the concentration of tyrosine and the data were plotted according to Lineweaver-Burk plot and presented in Fig. 7. The lines were fitted by visual inspection. It appears that tyrosine competitively reverses the inhibition of tyrosinase by the protein inhibitor. In the presence of 2.6 j&M (170 pg/ml) concentration of this protein in reaction mixture, the Km for tyrosine was increased from 0.5 to about 2 mM. DISCUSSION

Tyrosinase from melanosomes and from epidermal homogenates of human skin

SKIN

745

TYROSINASE

was purified to high specific activity with a yield of 36% by a single step using an affinity column and elution with tyrosine. Recovery might be improved by employing a larger column since in the present purification procedure about 40% of the enzyme was not adsorbed. Tyrosinase prepared from the human skin with a specific activity of 0.2 pmol of Dopa formed/mini mg protein and 0.82 pmol of Dopa oxidized/min/mg protein, which is comparable to the specific activity of pure enzyme from mouse skin, is only l/15 of the specific activity of tyrosinase purified from human melanoma (14). Tyrosinase in the homogenate from different sources (21, 34, 35) or partially purified enzyme (36, 37) exhibits a characteristic lag which was explained either as due to the dialyzable factors that inhibit tyrosinase (35) or due to a protein inhibitor of tyrosinase that binds to it with high affinity (34). The lag was abolished by Dopa or ascorbic acid (36). In the case of human skin tyrosinase, no lag was observed with 4 mM ascorbic acid. The enzyme showed no requirement for Dopa in contrast to absolute requirement of Dopa for pure mouse skin tyrosinase (7). Ascorbic acid appears to act as an efficient hydrogen donor for human skin tyrosinase. The addition of the protein inhibitor did not induce a lag in tyrosinase activity. 03% 03002s0.20-

[TYROSINE ,m”,j-’

FIG. 7. Lineweaver-Burk plot of tyrosinase activity in the presence (0) and absence (m) of the protein inhibitor. The assay conditions were as described in Fig. 3. The concentration of protein inhibitor was 170 ag/ml (2.6 PM) of reaction mixture.

746

VIJAYAN

The reason for the inhibition of tyrosinase by excess tyrosine when the dialyzed homogenate of human skin is assayed remains unclear. This inhibition is still present with the enzyme not adsorbed to the affinity column. Hydroxylamine markedly decreased the inhibition of tyrosinase by tyrosine in the homogenate. PHPP and PHPA were shown to inhibit tyrosinase both in the homogenate and pure enzyme and it appears possible that the inhibition of tyrosinase by tyrosine may be related to the metabolites of tyrosine that accumulate during the reaction. That tyrosinase inhibitor protein may bind to the enzyme at its catalytic site and thus compete with tyrosine rather than bind tyrosine directly is deduced from the following lines of evidence.

(i) Mushroom

tyrosinase, which has a of about 0.5 mM, similar to that of human skin tyrosinase, was not inhibited by this protein. (ii) The concentration of this protein is only 2 PM (132 pg/ml) in the assay mixture and thus it could not effectively reduce the tyrosine concentration, which is in millimolar concentration. Km for tyrosine

The inhibition of tyrosinase by the protein inhibitor and its apparent competitive reversal by tyrosine may have physiological significance in the melanogenesis in the human skin. The protein that inhibits tyrosinase constitutes 30% of the epiderma1 proteins and its concentration in the intracellular water is 5.4 mg/ml or 82 pM on average. Its concentration would be even higher in the melanosomal fraction where it is also present at 30% of total proteins of melanosomes. This concentration is about 23 times higher than that which inhibits tyrosinase by more than 80% at 0.75 mM tyrosine (Fig. 6). The intracellular concentration of tyrosine is in the range of 0.4 to 2 mM (38). Under these conditions the tyrosinase would be severely inhibited. The fact that some enzyme activity was measured in the assay with 0.1 mM tyrosine indicated that there

ET AL.

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