Mammalian melanogenesis: Tyrosinase versus peroxidase involvement, and activation mechanisms

ARCHIVES

OF

BIOCHEMISTRY

Mammalian

AND

168,

BIOPHYSICS

72S725

Melanogenesis: Involvement,

Tyrosinase

and

Activation

VINCENT Dermatology

Branch,

National

Cancer

(1973)

Institute,

Versus

Peroxidase

Mechanisms

J. HEARING National

Received

July

Institutes

of Health,

Bethesda,

Maryland

20014

10, 1973

A study of the enzymes functioning in murine melanogenesis was carried out on tissue homogenates of the black mouse. Several major points were resolved: (a) while the enzyme peroxidase is capable of converting tyrosine to melanin in vitro, it is not responsible for observed melanogenesis in the mouse, (b) a proteolytic activation system for tyrosinase, such as that described for amphibian skin, does not seem to function in mammalian tyrosinase activation, and (c) tyrosinase activity in normal murine tissues can be stimulated with a variety of treatments.

Recently there has been much controversy surrounding the roles of peroxidase (EC 1.11.1.7) and tyrosinase (EC 1.10.3.1) in melanogenesis. While the oxidation of tyrosine to dihydroxyphenylalanine (DOPA) and thence to melanin has been traditionally regarded as a function of the enzyme tyrosinase (l-3), the ability of peroxidase to catalyze such a reaction has been known for some time (4). In light of recent suggestions that (a) peroxidase was responsible for observed melanin synthesis in the melanocyte, and (b) that tyrosinase was incapable of the tyrosine to DOPA conversion (5, S), and in view of the importance of these implications to the study of enzymes and control mechanisms in melanogenesis, a study of enzymes functioning in mammalian melanogenesis seemed necessary. In addition, since it has been shown that in a variety of melanogenic tissues treatment with either proteases or detergents results in increased melanin synthesis (7, 8) we have investigated the status of murine tyrosinase activity after these types of treatments. EXPERIMENTAL

PROCEDURES

Black (C57B1/6N) mice, homozygous gouti, were obtained originally from Laboratories. They were sacrificed by phyxiation, the tissue was immediately

RESULTS

nonaJackson CO, asdis-

Table I reports the amount of melanin synthesizing activity in eye tissue homogenates from black mice at various ages post720

Copyright All rights

@ 1973 by Academic Press, of reproduction in nny form

Inc. raerved.

sected into 0.1 M phosphate buffer (pH 6.8) at 4% and ground in a Tenbriieck (glass:glass) tissue grinder. Treatment of the resulting homogenate varied for different experiments and is detailed in the table legends. Melanin synthesis was measured by the radioassay technique originally described by Kim and Tchen (9), which was modified according to Achazi and Yamada (10) to include a final concentration of 1.5 pg/ml cycloheximide, and 40 units/ml penicillin G. Onehundred microcuries of L-[U-Wltyrosine (sp act, 100 mCi/mmole) was added to each incubation mixture. Protein concentrations were determined by the method of Bramhall et al. (11). Polyacrylamide gel electrophoresis, utilizing System A (pH 9.45, 25%) (12) was run at 2 mA/tube for 2 hr. All gels were removed as the pyronin Y tracking dye reached the bottom of the tube; they were immediately rinsed for 15 min in 1 M phosphate buffer (pH 6.8), then stained. Coomassie blue staining was used for protein visualization (12); the method of Fling et al. (13) was used for tyrosinase localization; and the o-dianisidine staining technique of Lundquist and Joseffson (14) was utilized for peroxidase staining. Gels were fixed permanently after staining with 7.5% acetic acid. Control enzymes used were tyrosinase (Grade III, mushroom, Sigma) and peroxidase (Type III, horseradish, Sigma).

MAMMALIAN

TABLE MEL.\NOGENIC

ACTIVITY

IN BLACK

Age (days)

MOUSE

721

MEL.4NOGENESIS

EYE

I HOMOGENATES

.-

AT VARIOUS

Supernatant lpC cpm f

AGES Pellet

SE

Sp act

f 21 f 115 Lk 4 f 285 f 4

1881 1395 1053 995 828

Tt

cpm f

SE

Sp act

107 156 74 100 607

15657 8015 6087 6600 3760

__-___ 1 5 10 20 30

2220 2093 3059 4476 3892

POSTPARTUM”

7359 10420 12478 12541 11659

LzE;ye tissue homogenates were prepared as described in Methods; 1OOOg X 10 min spin, and then centrifuged at 10,ooOg X 30 min. pellets resuspended in the original volume of phosphate buffer, described in Methods. Sp Act& cpm/mg protein.

partum. Approximately 20-30% of the activity is found in the supernatant of each tissue; this represents activity present in the microsomal and soIuble prot,ein fraction. The remainder of the activity is pelleted, and is present primarily in the premelanosomes. While the total amount’ of activity increases with age in both the supernatant and pellet fractions, the specific activity in both drops off rapidly after birth. It is interesting to note that there is much melanin synthesizing activity in the older mice, since melanogenesis in black mice is essentially completed shortly after birth, as seen ultrast,ructurally (15). The remainder of experiments reported were carried out on 5-day newborn mice; this age seemed to optimize the tot.al amount of activity, t.he specific activit,y and ease of dissect.ion. There are several criteria for distinguishing betwetan tyrosinase and peroxidase oxidation mechanisms (5) : (i) diethyldithiocarbamatc may act as a nonspecific ant,ioxidant in the mcubation medium, although when removed after preincubation before assay by thorough washing (or dialysis), peroxidaw activity (an iron-containing enzyme) is recovered, while tyrosinase (a copper-containing enzyme) activity remains bound by DDC and is not recovered, (ii) the removal of HZOZ (a substrate of peroxidase) inhibits peroxidase activity, but not tgrosinaw activity, which utilizes O2 as a substrate. In an effort to resolve the controXWlXg regarding peroxidase versus tyrosinasc irlvolvement in melanogenesis in the mouse, a variety of experiments were carried

+ f f f k

the homogenates The supernatants and both fractions

were cleared with a were removed, the were assayed as

out, the results of which are detailed in Table II and III. The characteristics of tho tyrosine to DOPA to melanin conversion bJ tyrosinase and by peroxidase as resolved bj this assay are presented in Table II. Five units of tyrosinase are capable of rather high activity (ca. 13,000 cpm), while 50 units of peroxidase are required to achiew similar activity (ca. 10,000 cpm). The addition of HzOz stimulates tyrosinase activity slightly, by about lo%, but is essential for peroxidase activity. Incubation wit,h diethyldithiocarbamate results in better than 95 % inhibition of both peroxidase and tyrosinase activity; however, pretreatment with DDC, followed by exhaustive dialysis, still results in inhibition of tyrosinase by bcttcr than 95 %, while about 65 % of peroxida.se’s melanogenic activity is recovered. When mouse Gssue homogenates wro examined for melanin synthesizing activity, the following data resulted, presented m Table III. The homogenate by itsrlf is capable of a high specific activity of incorporation (ea. 64,000 cpm/mg protein). However, treatment with diethylditr complet,e inhibition of activitv. In addi t~ion, treatment with cat.alasc (beef liver, Sigma, EC 1.11.1.61, which reduces the amount. of available Hz02 i11 the homogenate: (but incrcascs the free oxygen content), stimu-

722

HEARING TABLE

TABLE

II

CHARACTERIZATION OF MELANOGENIC ACTIVITY BY TYROSINASF, AND PIGR~XIDASE

No.

Components

IT cpm f SE

No. 1 2 3 4 5 6 7 8 9 10 11 12

Tyrosinase” Tyrosinase Tyrosinase Tyrosinase Tyrosinase Tyrosinase Peroxidasec Peroxidase Peroxidase Peroxidase Peroxidase Peroxidase

$ + + + +

Ha02 DDCd DDC + Hz02 DDCb DDC + HzO$

+ + + + +

H202 DDC DDC + Hz02 DDCb DDC + H20sb

13105 15143 554 727 659 973 675 9811 424 370 360 6364

f f f f f f f f f f f f

270 155 32 5 62 9 20 251 23 37 22 123

All enzyme solutions were prepared immediately before use. 0 All tyrosinase experiments contained a total of 5 units of activity. b Dialyzed against water (3 X 1000 vol, 30 min each). c All peroxidase experiments contained a total of 50 units activity. Final concentrations used throughout the experiments were: DDC-10 mM, HtOs-9.1 mM, catalasea.1 mg/ml, Triton X100-l%, trypsin-1 mg/ml, phospholipase C-l mg/ml. d DDC = diethyldithiocarbamate.

lates activity greater than 40%, and with HzOz added as well, stimulates activity by more than 160%. When intact eyes are preincubated with diethyldithiocarbamate and then washed, as is the procedure for histochemical examination, and subsequently homogenized and assayed, again 95 % inhibition is observed (Table III). Catalase by itself is unable to oxidize tyrosine to melanin, and thus has no contaminating peroxidase or tyrosinase. Figure 1 showsthe results of histochemical staining experiments for tyrosinase and peroxidase activity on polyacrylamide gels. Mushroom tyrosinase (Figs. la-c) has a very strongly DOPA-oxidized band (Fig. lb), which is negative for peroxidase activity (Fig. lc). Horseradish peroxidase (Figs. ldf), which migrates only marginally into the gel, not only is peroxidase positive (Fig. lf), but also is capable of a slight amount of DOPA oxidation (Fig. le) (as might be expected based on the evidence presented in

III

CHARACTERIZATION OF MELANOGENIC ACTIVITY BY TISSUE AND TISSUE HOMOGENATES OF THI BLACK MOUSE EYE

5 6 7 8 9 10 11 12

Components Homogenate Homogenate + HlOn Homogenate + DDCd Homogenate + DDC + H&a Homogenate + DDCb Homogenate + DDC + HaOzb Homogenate + catalase Homogenate f catalase -IH&z Whole eye” Whole eye + DDCc Catalase Catalase + HaOn

14Ccpm f SE* 63690 95700 790 720

f 1500 f 1650 f 90 32 130

820 f 290 2100 zk 75 90412 f 167690 f

1200 2100

78780 f 3990 f Of Of

2075 170 37 25

Tissues were prepared as described in Methods, then cleared by a spin at 1OOOgX 10 min, and the supernatant assayed. Total volume of assay mixture in the table was 150 rliters; normal volume for other assays was 125 pliters (in all a total of 100 rliters was placed on filters, washed, and counted). a cpm reported as cpm/mg protein. b Dialyzed against water (3 X 1000 vol, 30 min each). c Whole eye preparations were incubated with or without DDC, dialyzed, and then homogenized and assayed for activity. d DDC = diethyldithiocarbamate.

Table II). Finally, separation of proteins by gel electrophoresis of black mouse eye tissue homogenate (Figs. lg-i) reveals the presence of two DOPA-oxidized bands, one of which is very weak (Fig. lh) and both of these are peroxidase-negative (Fig. li). In addition, both of these bands are capable of converting [‘*Cl-tyrosine into [14C]-melanin, further verifying the ability of mammalian tyrosinase to utilize tyrosine as a substrate (data not presented). In view of the recent evidence presented that proteolytic digestion leads to increased tyrosinase activity in amphibians in both dorsal and ventral skin (7), we investigated the effects of trypsin on mammalian tyrosinase. As can be seen in Table IV, in both eye and skin tissue homogenates, trypsin

MAMMALIAN

b

723

MELANOGENESIS

F

.a.

FJG. 1. Histochemical demonst,ration of tyrosinase and peroxidase activity on proteins separated by polyacrylamide gel electrophoresis. (a) Mushroom tyrosinase, Coomassie blue staining; (b) mushroom tyrosinase, I,-DOPA; (c) mushroom t)yrosinase, o-dianisidine; (d) horseradish peroxidase, Coomassie blue staining; (e) horseradish peroxidase, L-DOPA; (f) horseradish peroxidase, o-dianisidine; (g) black mouse eye tissue homogenate, Coomassie blue st,aining; (h) eye tissue homogenate, I,-DOPA; (i) eye tissue homogenat,e, odianisidinc. TABLE EFFECTS

OF V.~RIOUS

AGENTS

-. NO.

-

IV

ON TYROSIN-\SE THK BJACK

Treatment

Black 14C cpm f

1 2 3 4 5 6 7

Control Trypsin Triton X-100 Phospholipase C 50°C for 10 min 1)ialysis vs water Iliffusate (10 X concn)

ACTIVITY Movsr~

62520 16550 268790 160676 99750 301190 67440

rt f f f f * *

‘1 Tissiies were prepared as described in Methods, det,ailed in the table, before radioactivity was added 6 Results are presented as cpm/mg protein. AB, Oct~, 4052, Biirki et al, 2 t,abs, 2 figs.

results in almost complete inhibition. When normal mclanogenic tissue from t’he mouse is treated with Triton X-100 or with phospholipasc C, there is a large increase in tyrosinase act,ivity. In addition, it can be shown t’hat, grntle heating and dialysis against, water also results in an increase in activity. Treatment of t,hr homogenate with a 10 X concrntrato of the diffusat’e caused cssen tially no change in activity.

IN Trssr~r:

HOMOGEN.~TI’:S

eye* SE

Black % Diff

OF

skin”

14C cpm f SE .__-~

2220 19680 f 740 1390 -74 920 f 290 23050 rt 1520 9230 330 7034 25i 3960 60 2250 380 1347 7 .__~. -_~ .- ~~~~ homogenates were t,hen pretreated for for assay.

‘%, Diff

- 95 IT

30 nun

as

1~)1SCUHS10N

The results presented in Table II are interpreted to indicatr that pcroxidase is capable of the tyrosine to melanin convwsion as has been suggested (5, 6). Further, preincubation with diethyldithiocarbamate, followed by thorough washing, seemsto bc a valid method of differentiating bet,n-ecn melanin synthesis mediatIed by peroxidase and/or tyrosinase. However, it should be

724

HEARING

noted that addition of Hz02 increases tyrosinase activity (Table II). This observed stimulation of activity could be due to the additional oxygen made available to the reaction by either the spontaneous breakdown of H202, or by the reaction of tyrosinasewith Hz02 (which yields free oxygen) as suggested by Jolley et al. (16). For this reason, addition of Hz02 is not a valid control for differentiating between tyrosinaseand peroxidase-dependent melanin synthesis. The data presented in Table III and Fig. 1 indicate convincingly that peroxidase activity is not responsiblefor observed melanogenesisin the mouse. While it is not possible to deny categorically that peroxidase is implicated in melanogenesis in all murine tissues (especially in light of its ability to utilize tyrosine and DOPA as substrates), it appears that the bulk of normal melanin synthesis in murine tissues is tyrosinasedependent. The inactivation of tyrosinase activity by trypsin (Table IV) seems to rule out the possibility that a tyrosinase activation mechanism, such as that described in amphibian epidermis, is present in this mammalian system. The stimulation of demonstrable melanogenic activity by treatment with Triton X-100, phospholipase C, dialysis and mild heat might suggest either: (1) the presence of a thermolabile, low molecular weight inhibitor of tyrosinase in the normal black mouse (tyrosinase inhibitors have been described in melanoma tissues (17, 18), although they are diffusable and heat-stable), (2) a restricted substrate permeability which is overcome by these treatments [substrate permeability has been suggested as a possible method of controlling tyrosinase activity in pigmented tissues (19, 20)], or (3) a conformational change in the enzyme induced by these treatments which results in a stimulation of activity (conformational changes in enzymes has been implicated in the control of their activity, and can be brought on by such treatments (21,22)). When thediffusate is added back to the homogenate, there is no inhibition of tyrosinase (Table IV), and for this reason, one of the latter two nossi-

bilities seems more likely. Work is currently in progress to further characterize the mechanism of this observed activation. In summary, several points have been resolved, (a) while the enzyme peroxidase is capable of converting tyrosine to melanin in vitro, it is not responsible for observed melanogenesisin the mouse, (b) a proteolytic activation system for tyrosinase, such as that described for amphibian skin, does not seem to function in mammalian tyrosinaseactivation, and (c) tyrosinase activity in normal murine tissues can be stimulated with a variety of treatments. ACKNOWLEDGMENTS The author is indebted to Mr. Tom Eke1 for his excellent technical support, and to Drs. Gary Peck and Marvin Lutzner for their helpful criticism of the manuscript. This work was in part supported by an NIH/NCI Postdoctoral Fellowship to the author (No. 1 F02 CA51551). REFERENCES 1. BROWN, F. C. AND WARD, D. N. (1957) J. Amer. Chem. Sot. 79, 2647-2648. K. (1962) Biochim. Biophys. Acta 2. SHIMAO, 62, 205-215.

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