0022 -2 0 2 X /8 ~ /8;J02 - 0 14;'$02.00/0 TH E .JOU RNAL OF INV ESTIGATI VE DERMATOI.O Gy.83:14 .'i - 14 9. 1984 Vo l. R:l. No. :! J by & Cop y right (0 984 The Willia ms Wilkins Co. j Jrinied in LI . , ~' . A . Dopachrome Oxidoreductase: A New Enzyme in the Pigment Pathway
JOAN 1. BARBER, PH.D. , DEWAYNE TOWN S END, PH.D. , DAVID P . OLDS, M .S ., AND RICHARD A. KING , M .D. , PH.D.
Department of M edicine, Medical School (JIB. DPO, RA I() and Division of Human and Oral nenetics. /J ental School (DT). Universityo( Minnes()t a. Minneap()lis. 11.:"".04 .
We report our preliminary characterization of a new us to beli eve t hat this activity is due to a new enzyme in the enzyme in the pigment pathway which we propose to pi gment pathway, which we propose to call dopachrome ox i call dopachrome oxidoreductase (DeOR). This enzyme, doreductase (DCOR), a nd our studies suggest t hat this enzy me prepared from mouse melanoma, catalyzed the conver acts as both the "dopac hrome co nversion" and t he "indole sion of dopachrome to 5,6-dihydroxyindole and also ap bl oc king" factors. peared to block the pigment pathway at this latter com pound in the absence of tyrosinase. DeOR was protease sensitive, heat-labile, and showed maximum stability in MATERJALS AND METHODS the range of pH 6-8. The molecular weight of DeOR Prepa ration ()( f)() pachrome Oxidoreductase from Tumor w-as estimated to be 34,000 by gel filtration. De OR had The preparation of crude DCOR from tumor was carried out at :; oC a subcellular distribution within the melanocyte which unless otherwi se spec ifi ed. Amelanotic a nd melanotic 8- 16 melanoma w-as similar to that of tyrosinase, but DeOR activity was tumors were coll ected from C57BL/6J mi ce, washed twice in 0.8:; 0;' found in melanocytes devoid of tyrosinase activity and saline, pell eted at :;00 g, and stored at -20°C. Amela notic tumors were w-as not inhibited by tyrosinase inhibitors. DeOR may lI sed in the cha racteri za t.i on studies t.o avoid confusion wi t h tyrosinase, prove to be an active regulatory enzyme in melanoge a fte r demonstrating DCOR activity in t hi s t issue. Thawed portions of tumor were homogeni zed wit.h a ground-glass tissue grinder in 10 nesis. volumes of 0.05 M sodium phosphate, pH 6.8, containing 1 % Triton X- 100, fo ll owed by incubation on ice for 30 min to release enzyme. The homogenate was centrifuged at 105,000 g for 15 min at. 5°C a nd the It has long been thought that the melanin pathway employed supernatant used as the crude DCOR. t he single enzyme tyrosinase for the oxidation of tyros ine ---+ In some experiments, Triton X -IOO was removed from the crude d opa ---+ dopaquinone and that subsequent. steps in melanin enzy me preparation wit.h BioBeads. The crude DCOR prepa ration was synthesis [l eukodopachrome ---+ dopachrome(D C) ---+ 5.' 6-dihy mixed with an equal volume of BioBeads 8 M2 (Bio-Rad) a nd gent ly dro xyindole(DHI) ---+ Il1dole -5 ,6-qull1one(lQ) ---+ melanll1J were rotated for 90 min at 4°C [71. The Triton X-lOO bound to the Bio wit hout enzyme requirement. However, recent studies have Beads and t he enzyme was removed in the supernatant. s u ggested that there may be pathway control distal to dopaqui Pr('parotion o( f) opachrome Oxidoreductase (rom Hairbulbs n o ne [1 - 6] . Logan and Weatherhead's studies on molting Si berian hamsters provided the first convincing evidence that Mouse hai:buLbs: All hai~ over t he dorsal trunk was removed by tyrosinase activity does not correlate directly with melani.n pluckIng to stllnulate new hall' growth. At 14 days, t he skin was removed and the ha ir follicles were scraped from the inner surface of one skin production. They found that t.yrosll1ase actIvIty Il1 the haIr in to 10 ml of co ld saline. This suspension was centrifuged at 105,000 g follicles of Siberi an hamsters was high during the spring and for 15 min, aft er which the fat layer and t he saline were removed. The a utumn molts but the hair produced following the latter molt. ent.ire pell et. was incuba t.ed on ice for 60 min for enzyme release in 500 wa s unpigmented [lJ. Subsequently, they showed t.hat. mela 1-' 1 of 0. 05 M sodium phosphate, pH 6.8, containing 0.001 M di ethyldi t onin or cG MP (guanosine-3',5' -cyclic phosphate) inhibited thioca rbamate (DOC), 0.001 M phenylt hi ourea (PTU), a nd 1% T ri t.o n m e lanin format ion in cultured hamster hair follicles without X- IO O. The sample was cen t. rifuged at 105,000 g for 20 min at 5°C a nd a ffecting tyrosinase activity 12J. At about. t.he same t.im e, Paw 2:;01-'1 of the supernata nt was used to test fo r DCOR activity. e le k , Korner, Bergstrom, and Bologna reported three melano Human hairbulbs: Fifty fresh human a nage n hairbulbs were incu genic regu latory factors that acted on the eumelanin pathway bated on ice in 250 m! of 0.05 M sodium phosphate, pH 6.8, containing after dopaquinone: "dopachrome conversion fa ctor" which pro 1% Trit.on X- IOO . for 1- 5 h to release enzyme. After incubation, t he hairbulbs were removed a nd the entire enzy me sample was used to test m otes the conversion of DC to DHI; "indole blocking fact.or" for DCOR activity. There was no significant. difference in DCO R w hich inhibits the conversion of DHI to IQ and "indole con act. ivity between the 1-, :3- or 5- h incubation for enzy me release. vers ion factor" which promotes the conversion of DHI to IQ [3,4]. Part of this latter regulatory factor activity is now thought Uopachrome Oxidareductase Assay to be the result of tyrosinase activity [5,6J. DC was produced by mixing cold dopa (0.5 mg L-dopa/ml in 0.05 M Our characterization of "dopachrome co nversion fa ctor" led sodium phosphate, pH 6.8) wi th silver oxide (6 mg Ag,O/ mg dopa) for :l min foll owed by filtrat.i on through a 0.22-/1m Millipore fil ter. T his M a nuscript received June 20, 1983: accepted for publicat ion March procedure resulted in a 77 % co nve rsion of dopa t.o DC. The sodium 20, 1984. phosphate buffer used in the prepa ra t.i on of DC was passed Ove r a S upported in part by N IH grants GM22 167 and AM32407. co lumn of Chelex- IOO chelating ion excha nge resin (Bio- Rad) for R eprint requests to: Dr. Ri cha rd A. King, Depart ment of Medicine. removal of t race metals before use. The sta ndard assay consist.ed of Box 485, University of Minnesota Hospital, Minneapoli s, Minnesota 125 fII crude DCOR (7 mg total protein), 250 fIl DC solution, and 0.05 5545.5. M sodium phosphate, pH 6.8, in a t.otal volume of I m l. Assays were A bbreviations: run with a buffer bl a nk at 25 °C in semi -micro disposable polystyrene DC: dopachrome cuvett es, and the value for t. he buffer blank was subt. ract.ed from the D COR: dopachrome oxidoreductase react ion va lu e for determinat ion of DCOR ac t.i vity. DCOR activit y was DOC: di ethyldithiocarbamate de t.ermined spectrophotometrically (Beckman Acta cIII ) by monit'oring DHI: dihydroxyindole the decrease in absorba nce at 475 nm as DC (red) was conve rt.ed to l Q: indole·5,G ·quinonc DH I (colorl ess), and was expressed in nmol per min (DC ext. inct ion PTU: phenylthiourea coeffi cient = :1700 at 475 nm) 181. All experiment s were run at least 2 T CA: trichl oroacetic acid tilll e~.
14 5 146 BARBER ET AL Vol. 83, No.2 Tyrosinase Assay and with 0.0001 M iodosobenzoate for 70 min at 25"C, after which t he residual activity of DCOR was determined. Crude DCOR preparation Tyrosin ase activity was determined using a t ri t iated tyrosi ne assay from amelanotic tumor was incubated wi t h a mi xture of 0.001 M DDC a~ described by King et al[91. The preparation to be assayed was mi xed and 0.001 M PTU on ice for 60 min, after which residual DCOR activity with an equal vo lume of 0.1 M sodiu m phosphate, pH 6.8, co ntaining was determined. Crude DCOR preparation from amelanotic tumor was 2% Triton X- I00 and in cubated on ice for 60 min for enzyme release, incubated with 1 or 2 X 10-' M 1,10-phenanthroline for 60 min, after after whi ch a sample was taken for assay. The assay incubation mixture which residual DCOR activi ty was determined. T yrosinase activity was co ntained 0.2 nmol L-[3,5-"Hjtyros ine (Amersham, 1 or 2 Ci/ mmol), determined on a number of DeOR preparations. Appropriate blank 0.1 nm ol L-dopa, 20 or 25 1'1 of the sample to be assayed and 0.1 M reactions were run with each experi ment. sodium phosphate, pH 6.8, in a total volume of 60 1'1. Incubation was Dialysis: Crude DCOR preparation from amelanotic tumor was dia at 37"C for 120 min. After incubation, 10 1'1 of the reaction mixture lysed against 0.01 M EDTA in 0.05 M sodium phosphate, pH 6.8, for 24 was pl aced on a Dowex 50W co lumn and the "HOH generated with the h at 4"C foll owed by dialysis against 0.05 M sodium phosphate, pH 6.8, ox id ation of tyrosine was recovered with a 0.1 M cit rate wash. The for 24 h at 4 "C. Cont rol DCOR was djalysed against 0.05 M sodium ty rosin e hydroxylase activity of tyrosinase was expressed as pmol of phosphate for 48 h at 4"C. After dialysis, residual DCOR activity was tyrosine ox idi zed per 120 min. determined.
Protein Determination Subcellular Fractionation Protein conce ntration was determined by t he Biuret method [10] . Melanotic B-16 melanoma was subjected to subce llular fr actionation Bovine se rum albumin was used in the preparation of standards. into soluble, mi crosomal, and melanosomal fractions by djfferential centrifugation, according to the procedure of Townsend et al [1 2]. Biochemical Characterization of DCOR Tumor was washed in 0.01 M cell -washing buffer co ntaining 0.007 M Molecular we ight: Melanotic B-16 melanoma was homoge nized in TES, 0.003 M Tris base, NaCI, and sucrose, pH 7.2, suspended in cold 0.01 M HEPES co ntaining 0. 3% Triton X-100 by the method described dj stilled water and placed on ice for 10 min. All further manipulations above. The crude preparation was ce nt rifuged at 105,000 g for 45 min were done at 4 "C. The tumor was then sonicated twice, dj[uted with and the supernatant passed through a 0.22- l'm Millipore filter. Two cell -washing buffer, and centrifuged at 500 g for 10 min. The superna mi lliliters of the filtered supernatant was placed on a 90 x 2.5 em tant was centrifuged at 11,000 g for 10 min and the 11 ,000 g pellet used AcA22 (LKB Ultragel) ge l fi ltration co lu mn equilibrated with 0.01 M as t he melanosomal fraction. The 11 ,000 g supernatant was centrifuged HEPES co ntaining 0.3% Triton X-100 and 3.5 ml fractions were at 105,000 g for 90 min; the 105,000 g supernatant was used as t he co ll ected at a fl ow rate of 12 ml / h. DCOR and tyrosinase activity we re soluble fraction and the 105,000 g pellet was used as the microsomal determined in each fraction by the methods described above. Molecular fraction. Tyrosinase and DCOR activit ies were determined on each weight was determined by camp,aring t he activity elution patterns to fraction. For DCOR activity, 125 1'1 of the fraction was mixed wit h 125 the elut ion pattern for a series of molecular weight standard proteins "I of 0.05 M sodium phosphate, pH 6.8, containing 2% Triton X -lOO as determined at 280 nm. The molecular weight standard proteins and incubated on ice for 60 min for enzyme release. The entire 250 1'1 (B ioRad ge l fi ltration standards) were bovine thyroglobulin (670,000), was used for the DCOR assay in a total volume of 1 ml, as described hovine gamma globulin (158,000), chicken ovalbumin (44,000), and above. Duplicate DCOR determinations were done for each fraction. equine myoglobin (17 ,000). For tyrosinase activity, 25 1'1 of the fraction was mixed with 25 1'1 of Heat inactivation: Crude DCOR preparation from amelanotic tumor 0.01 M sodium phosphate, pH 6.8, containing 1% Triton X-100 and was pl aced in plastic microfuge tubes and incubated at 25 ", 37.5", 45", incubated on ice for 60 min for enzyme release. A 20-1'1 sample of the 65", and 100"C fo r 15 min. After the incubation, the samples were preparation was used for the tyrosinase assay. Triplicate tyrosinase cooled on ice and ce ntrifuged at 12,000 g for 10 min at room tempera determinations were done for each fraction. Protein determinations ture. T he supernatant was used to determine DCOR activity. The were done on 100-1'1 samples of each fraction. co ntrol sample was incubated on ice for 60 min, and t he activity of this sample was taken as 100%. The thermal denaturation of DCOR at RESULTS 45"C was determined by incubating samples of crude DCOR prepara General Properties of DeOR tion at 45 "C fo r 5, 10, 15,30,45, and 60 min. After the incubation, the samples we re placed on ice, and centrifuged at 12,000 g for 10 min at DCOR had an (.stimated M , of 34,000 by gel filtration and 4 "C. The supernatant was t hen used to determine DCOR activity. The could be separated from tyrosinase which had an estimated Mr O"C sample was left on ice for 60 min and the activity of this sample 0[67,000 with t his procedure, as shown in Fig 1. DCOR activity was taken as 100%. The half-life of DCOR at 45 "C was determined was sensitive to temperature, with a decrease in activity above from the slope of the line for the change of activi ty with time [11]. 25'C (Fig 2A), and a loss of activity after 15 min at 100' C. pH S tability: Crude DCO R was prepared in 0.05 M sodium phosphate that co ntained 1.0% Triton X- I00 and had been made up with different Heating at 45' C caused a decrease in enzyme activity consistent pH values. The preparation was first incubated on ice for 1 h to release with protein thermal denaturation (Fig 2B). The first orde r enzyme and ce ntrifuged at 105,000 g for 20 min. The supernatant was rate constant for the inactivation of DCOR at 45'C was 0.0163 then incubated at 3"C for 18 h, the pH determined, and each sample min- I (half-life of 42.5 min). Crude DCOR was more stable at djalyzed against 0.05 M sodium phosphate, pH 6.8, co ntaining 1.0% 6"C than at -70'C.* T ri ton X-IOO for 2 h at 3"C. The di alysate was assayed for DCOR by DCOR had maximum pH stability in the near neutral range, standard procedures. with loss of stability at more basic and acidic pH, as shown in Protease digestion: Crude DCOR from amelanotic tumor was pre Table 1. pared and the Triton was removed. Trypsin IX, trypsin XI, protease D COR was sensitive to digestion by trypsin and protease 1lI, and protease XIV were dissolved in 0.05 M sodium phosphate, pH 6.8, and different amounts of each were incubated with 125 1'1 of t he (Table Il). Precipitation of crude DCOR with TCA destroyed crude DCO R preparation at 25"C for 30- 45 min. At the end of the enzym e activity. Free sulfhydryl groups did not appear to be in cubation, t he remaining DCOR activity was determined. The bl ank required at the active site as the activity was not affected by reaction co nsisted of buffer with trypsin or protease. The DCOR control iodoacetate, iodoacetamide, or iodosobenzoate (Table II). Ac co nsisted of DCO R without t rypsin or protease. The blank reaction tivity was not inhibited in the presence of the metal chelators and the DCOR co ntrol were incubated at 25"C for the same peri od of PTU a nd DDC. There was minimal inhibition with 1,1O-phen time as t he reaction. For.each trypsin experiment, a reaction consisting anthroline and with prolonged dialysis against EDT A (Table of crude DCOR, trypsi n, and soybean trypsin inhibit ion (amount II). Concomitant values for tyrosinase are a lso given in Table sufficient to inhibit approxim ately twice the amount of trypsin added) was run as a co nt rol of t he proteolytic action of the trypsin. II. Trichloroacetic acid (T CA) precipitation: Crude DCOR preparation was mixed with 50% TCA (0.5 ml TCA in 2.0 ml crude DCOR). The mixture was ce nt rifuged at 12,000 g for 10 min, after which the pH of the supernatant was adjusted to 6.3 and the residual DCOR activity • After 3 days, remaining activity was over 90% of initial activity in determined. preparations left at 6"C but only 61 % in preparations frozen at 70'C. Other reagents: Crude DCOR preparation from amelanotic tumor The lower stability of frozen vs cold samples may indicate a polymeric was incubated with 0.035 M iodoacetate or iodoacetamide for 30 min structure for DC OR. Aug. 1984 DOPACHROME OXIDOREDUCTASE 147 4 Molecular Weight ( x 10 ) A 30 10 54 3 2 100 e~ 120 t t i- t 24 e\ 80 • >- ':; 60 100 20 ""G p. 0 <[ 0 O· 0 0 40 1 : 9 ::v c : : ;;e .c: 80 16 »
TABLE II. Properties of dopa crh ome oxidoreductase DISCUSSION ,;;. R er.; idual activity Time in Recent studies on melanin synthesis have shown the melanin Age nt Amount minut.es DCOR Tyrosinase pathway to be more complex than originally thought. Korner, Pawelek, Murray, Hearing and their associates have described Trypsin IX 1685 units 45 56 94 three factors that act in the distal eumelanin pathway ND" Trypsin IX + inhihi- 1685 units 45 120 [3- 6,13). The activity of one of these factors, the "indole tion conversion factor," can be partially accounted for by tyrosinase Trypsin Xl 1072 units 45 71 ND Trypsin XI + inhibi- 1072 units 45 108 ND [5,6). We now present evidence that an enzyme, which we tion propose to call DCOR, is responsible for the activity of the Protease III (papaya .125 units 45 53 ND other 'two factors, "dopachrome conversion" and "indole block prolase) ing." We have partially characterized this new enzyme and find Protease III .75 units 45 28 88 that DCOR has an estimated M , of 34,000, and is heat-labile, Protease X IV (pronase .75 units 30 41 94 protease-sensitive, and not inhibited by the tyrosinase inhibi E) tors DDC and PTU. DCOR has a melanocyte subcellular dis 86 ND Iodoacetate 0.035 M 30 tribution similar to tyrosinase. 30 98 103 Iodoacetamide 0.035 M DCOR and tyrosinase were separated by gel filtration chro lodosobenzoate 0.0001 M 70 94 ND EDTA dialysis 0.01 M 1440 85 101 matography. The M, of the major tyrosinase peak with this DDC/PTU 0.001 M/ 60 100 0.8 procedure was estimated to be 67,000 (Fig 1) which is consistent 0.001 M with the previously reported M, for tyrosinase [14) . Korner and 1,10-Phenanthroline 0.0001 M 60 80 ND Pawelek found that dopachrome conversion activity cochro 0.0002 M 60 78 99.9 matographed with tyrosinase during the early stages of purifi " Not determined. cation, and their studies suggested a M, of less than 1000 [3). Their conversion factor underwent significant changes in phys ical properties, however, with further purification, suggesting TABLE III. Subcellular distribution of tyrosinase and DeOR in that the molecular weight studies had not identified the con melanotic B- 16 melanoma version factor directly. Hearing et al reported the association Percentage or total activity in each rraction of dopachrome conversion activity with tyrosinase isozyme T . Enzy me (membrane-bound tyrosinase), and a melanogenic blocking ac Soluble Microsmal Melanosomal tivity with purified isozyme T\ and To [1 3). It is not clear that DCOR 14 ± 1" 44 ± 8 43 ± 8 this latter blocking activity is the same as we have shown for Tyrosinase 18 ± 5 34 ± 7 49 ± 5 DCOR. With a M, of 34,000 DCOR should not be found in a 24 ± 2 25 ± 2 Protein 48 ± 3 sample of purified T :) or Th which have M ,s of 54,000 and " Mean ± SO. 72,000, respectively [14). We cannot resolve these differences at the present time. The gel filtration method used in these studies was similar to that used by Hearing et al to purify the De()R activity in mouse and human hairbulbs TABLE TV. tyrosinase isozymes. If DCOR has a polymeric structure, then Source DCO R activity it is possible that a dimer (with an estimated M, of 68,000) A. Mouse (C57BL/65) Coat color nmol DC/ could be found in a tyrosinase fraction of similar molecular min/ mouse skin weight. Purification of DCOR will be required to answer this 1. CJ /e-' White 52.97 ± 13.0 question. 2 2. C>"/C ' White 112.5 ± 19.9 Evidence tha'~ DCOR is a unique enzyme in the pigment 3. B/B Black 40.4 ± 11.5 pathway, not identical with tyrosinase, includes the following B. Human subject Hair color nmoI DC/min/50 (1) DCOR and tyrosinase chromatograph at different positions hairbulbs with gel filtration, (2) DCOR activity is present in albino mice l. Brown 2.02 ± 1.2 J that have a deletion at the c locus (c ) and in amelanotic B-16 2. Brown 1.98 ± 0.6 melanoma, both of which lack tyrosinase activity; and (3 ) DCOR activity is unaffected by the tyrosinase inhibitors DDC and PTU, and is more sensitive to proteolytic digestion. Al TABLE V. Action o[ DeOR and tyrosinase on distal eumelanin pathway though separate enzymes, it is likely that tyrosinase and DeOR have a coordinated physiologic function within the melano Tyrosinase DCOR Melanin ror · DDC/PTU some. Their distribution within the melanocyte is similar and activity activity motion both are solubilized by detergent, which suggests membrane Dopachrome plus: binding. A close membrane-bound approximation of these two Phosphate buffer +++ enzymes would allow pathway constituents to be more easily + ++ processed in the formation of melanin. Mushroom tyro- + +++ In the normal melanocyte, our studies suggest that DCOR sinase + ± ++ Melanotic 8 -16 + + +++ catalyzes the conversion of DC to DHI and tyrosinase oxidizes + ± + ± DHI to IQ which quickly forms melanin. When tyrosinase is Amelanotic B-16 + absent, DCOR reduces DC to DHI but further progress is Amelanotic 8 -16 + + +++ restricted, as seen by the fact that the red color of DC disap and mushroom pears and the reaction solution remains colorless. The biock in tyrosinase the pathway is the result of the lack of tyrosinase and the Note: Melanin formation was visually determined and graded as action of DCOR. DHI is not enzymatically converted to mela follows: - = solution colorless or red-tinged with no gray color; + = nin in the absence of tyrosinase and DHI does not auto-oxidize li ght gray color; ++ = moderate gray color, no €1occulation; +++ = to melanin in the presence of DCOR. One possible mechanism dark gray color with black flocculation; ++++ = black precipitation to explain this action of DCOR is suggested by the fact that with heavy flocculation. DC and IQ are both 5,6-quinones. DeOR may convert both DC and IQ to DHI. The slow spontaneous formation of IQ from ing activity and the slow formation of melanin in the heated DHI in the absence of tyrosinase is overcome by the rapid samples. conversion of IQ to DHI by DCOR, effectively blocking the Aug. 1984 DOPACHROME OXIDOREDUCTASE 149 pathway at this point. Thus, DCOR appears to express both 1980 4. Pawelek J , Korner A, Bergstrom A, Bologna J: New regulat.ors of "dopachrome conversion" and "indole blocking" activity. mela nin biosynthesis and t he autodestruction of melanoma cell s. The regulation of melal1ln formatIO n IS clearly more complex Nature 286:617- 619, 1980 than can be accounted for solely by the level of tyrosinase 5. Korner A, Pawelek J: Mammalian tyrosinase catalyzes three re activity. The high tyrosinase activity without accompanying actions in the biosynthes is of melanin. Science 217:1163- 116.5, pigment production described in molting Siberian hamsters 1982 6. Murray M, Pawelek JM, Lamoreux ML: New regulatory factors may represent a block in the pigment pathway at colorless DHI. fo r melanogenesIs: developmental change in neonatal mice of Tyrosinase-positive oculocutaneous .al.b inism in humans is also various ge notypes. Dev Bioi 100: 120-126, 1983 characterized by high tyrosll1ase actIvIty wIthout melal1lzatlOn. 7. Holloway P: A simple procedure for removal of Triton X-I00 from protein samples. Anal Biochem .53:304- 308, 1973 Whether DCOR may mediate the inhibition of pigment for 8. Mason HS: The chemistry of melanin III. Mechanism of the mation in t hese cases remains to be seen; however, it is likely oxidation of dihydroxyphenylalanine by tyrosinase. J Bioi Chern that DCOR wi ll prove to be an important regulatory enzyme in 172:83- 99, 1948 melanogenesis. 9. King RA , Olds DP, Witkop CJ Jr: Characterization of human hairbulb tyrosinase: properties of normal and albino enzyme. J We want to thank John Lipscomb for his helpful discussion of this Invest Dermatol 71:136- 139, 1978 10. Gornall AG, Bardwill CJ, David MM: Determination of serum work. proteins by means orthe Biuret reaction. J BioI Chern 177:751- REFERENCES 766, 1949 11. Moore WJ: Physical Chemistry, 3rd ed. Englewood Cliffs, NJ, 1. Logan A, Weatherhead B: Pelage color cycles and hair fo lli cle Prentice-Hall , 1962, pp 260-261 tyrosinase activity 111 the Sibenan hamster. J Invest Dermatol 12. Townsend D, Witkop CJ, Mattson J: Tyrosinase subcellular rus· 71:295- 298, 1978 tribution and kinetic parameters in wild type and ("- locus mutant 2. Weatherhead B, Logan A: Interaction of -melanocyte-stimulating C57BL/6J mi ce. J Exp Zool 216:113- 119, 1981 hormone, melatonin, cyclic AMP and cyclic GMP in the control 13. Hearing V, Korner A, Pawelek J: New regulators of melanoge nesis of melanogenesis in hair fo llicle melanocytes in vitro. J Endo are associated with purified tyrosinase isozymes. J Invest Der crinol 90:89- 96, 1981 matol 79:16- 18, 1982 3 . Korner A, Pawelek A: Dopachrome co nversion: a possible control 14 . Hearing V, Ekel T, Montague P: Mammalian tyrosinase: isozymi c point in mela nin biosynt hes is. J Invest Dermatol 75: 192-195, forms of the enzyme. Int J Biochem 13 :99- 103, 1981