STUDIES 0? EH! fiUlTiPLE FORKS OF TYROSMASE EN “EUROSPGRA CRASSi ARE! Tfiiéfi RELATii‘NSHEP T0 GENETEC DiFFERENCES 5N YHERMOSTABRITY THESIS FOR THE DEGREE OF PH. D. fiMIMfl SYAYE UNWERSWY M08703! 5. FUCHS 1966 LIBRARY fl“ Mldiigan Sm: University This is to certify that the thesis entitled Studies of The Multiple Forms of Tyrosinase in Neurospora crassa and Their Relationship to Genetic Differences in Thermostability presented by Morton S. Fuchs has been secepted towards fulfillment of the requirements for Eh.D. degree in Watry .//11J7£ WVMajor proer sor Date February 27} 1967 0-169 ABSTRACT STUDIES OF THE MULTIPLE FORMS OF TYROSINASE IN NEUROSPORA CRASSA AND THEIR RELATIONSHIP TO GENETIC DIFFERENCES IN THERMOSTABILITY by Morton S. Fuchs Two strains of NeurOSpora Crassa, differing with respect to a pair of alleles (TS and TL), produce thermostable and thermo- labile tyrosinase respectively. Fractionation of the enZyme from each into three components has been accomplished by means of continuous-flow paper electrOphoresis and ion-exchange chroma- tography. A conversion phenomenon among these components is observed in extracts of both strains. Thermal inactivation studies show that the difference in thermostability of the enzyme from the two strains is not attribut- able to differences among and conversion of the three components in each. Gel—filtration and sucrose gradient centrifugation show that the three components produced by each strain do not differ signifi- cantly in molecular weight, and that the tyrosinase of the two strains also does not differ in molecular weight. This is interpreted to mean that differences in aggregation are not responsible for the differences among the three components in each strain, and that the difference between strains in heat stability cannot be attributable to differences in molecular weight. It seems most probable that the three tyronsinase components produced by each strain differ in tertiary configuration. The structural basis of the difference between strains remains uneluci- dated. None of this evidence excludes a difference in tertiary structure, but differences in primary structure await verification. STUDIES OF THE MULTIPLE FORMS OF TYROSINASE IN NEUROSPORA CRASSA AND THEIR RELATIONSHIP TO GENETIC DIFFERENCES IN THERMOSTABILITY by Morton S. Fuchs A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOC TOR OF PHILOSOPHY Department of Biochemistry 1967 .J I 1 ,Kv/ I K I / (/x _-._/ '\- .,./' — ‘ C / ACKNOW LEDGMENTS Much more than mere appreciation is extended by the author to his major professor, Dr. Allen S. Fox for his guidance in this work. It has been a privilege to work for and with him. His friendship will always be cherished. Thanks are extended to Mr. Sheldon D. Parzen for his interest and encouragement and to all other members of the "Fox group" for creating a stimulating environment. ii Dedicated to my wife Mary arm and to my wonderful Mother and Father TAB LE OF CONTENTS List of Figures . . . . ........ List of Tables ....... INTRODUCTION ....... . ...... . . ...... MATERIALS AND METHODS RESULTS A. Assay Methods . . . . . . . ............. B. Analysis by Continuous-flow ElectrOphoresis . . . C. Ion Exchange Column Chromatography D. Thermal Inactivation . . . . . . . . . . . ..... E. Sucrose Gradient Centrifugation . . . . ..... DISCUSSION AND CONCLUSIONS SUMMARY...... ............ ..... REFERENCES ....... iv 15 21 21 24 34 46 53 59 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 1B 5A 5B 6A 6B 7A 7B 8A LIST OF FIGURES Typical assay of T5 at various dilutions before dialysis or passage over G-ZS Sephadex. Typical assay of TS at various dilutions after passage over G425 Sephadex. Analysis of fractions collected from bottom of continuous-flow electrophoresis sheet. Analysis of fractions collected from bottom of continuous-flow electr0phoresis sheet. Analysis of fractions collected from two separate continuous-flow electrophoresis sheets. Analysis of fractions collected after gel filtration chromatography of undialyzed, crude TL extract on G-lOO Sephadex. Analysis of fractions collected after gel filtration chromatography of dialyzed, crude TL extract on G—lOO Sephadex. Analysis of fractions collected from bottom of continuous-flow electrophoresis sheet. Analysis of fractions collected from two separate continuous-flow electrophoresis sheets. Ion-exchange column chromatography of undialyzed crude TL extract on DEAE cellulose. Ion-exchange column chromatography of undialyzed crude TS extract on DEAE cellulose. Ion—exchange column chromatography of crude, TL extract on DEAE cellulose after passage over G-25 Sephadex. Page 23 23 26 28 31 33 33 36 39 39 41 Figure Figure Figure Figure Figure Figure Figure Figure 8B 10 ll 12 l3 14 15 Ion-exchange column chromatography of crude, TS extract on DEAE cellulose after passage over G-25 Sephadex. Ion-exchange column chromatography on DEAE cellulose of TL extract treated with 6M urea. Ion-exchange column chromatography on DEAE cellulose of continuous-flow electrOphoresis fractions from TL. See Figure 2. Thermal inactivation of TLA and TSA at three temperatures. Ordinate: residual activity plotted on a logarithmic scale. Abscissae: time of exposure to the indicated temperature. Arrhenius plot of the rate constants of thermal inactivation of TLA and TSA. k = rate con- stant; T = absolute temperature. Hinact = activation energy of inactivation in calories per mole. Thermal inactivation of TLB and TSB at 50° C. Ordinate: residual activity plotted on a logarithmic scale. Abscissae: time of exposure to indicated temperature. t 1/2 = half-life. Sedimentation patterns of crude, dialyzed and undialyzed TS extract and crystallized bovine hemoglobin in a linear 5 to 20 percent sucrose gradient. Sedimentation patterns of crude TL and TS extract in a linear 5 to 20 percent sucrose gradient. vi 41 44 47 50 52 54 56 58 Table Table Table Table Table Table Table LIS T OF TAB LES Page Distribution of Multiple forms of tyrosinase 42 before and after passage over G-25 Sephadex as determined by ion—exchange on DEAE cellulose for both TL and TS. Changes in tyrosinase during incubation of TS 42 extract after passage over G-25 Sephadex as determined by ion-exchange chromatography on DEAE cellulose. Affect of extraction in 6M urea on the distribution 45 of tyrosinases (TL) as determined by ion-exchange column chromatography on DEAE cellulose. Thermal half-lives of TLA and TLB —> TLA in 48 minutes. Determined in 0. 1M sodium phosphate buffer, pH 6.0. Rate constants of thermal inactivation (k) of TLA 48 and TLB -> TLA from half-lives shown in Table 4. l-linact, = activation energy of thermal inactivation in calories per mole. Thermal half-lives of tyrosinase TSA in sodium 49 phosphate buffer, pH 6.0 and T53 in barbital buffer, pH 8.6, in minutes. Rate constants of thermal inactivation (k) of TSA 49 and T83 from half-lives shown in Table 6. Hinact. = activation energy of thermal inactivation in calories per mole. vii INTRODUCTION One of the fundamental concepts of modern biology has been the so-called "central dogma" (l), which may be stated as follows: There is a DNA code which specifies an RNA code which in turn determines the amino acid sequence of a particular protein (2). This concept can be diagrammed in the following way: Etazlserzetlps, RNA -Tlr_a9§1stl<2n., protein DNA (enzyme) ----- > products (3). In the above, the arrows are presumed to indicate a flow of information (transcription and trans- lation), and the last arrow is meant to signify the products of the enzyme activity. Implicit in this concept is the prediction that the information contained within the amino acid sequence of a polypeptide chain is sufficient to cause it to fold into the precise configuration required for its particular function (4). According to this hypothesis, the polypeptide chain will take the three-dimensional configuration with the lowest configurational free energy, and hence under any specific set of conditions will be the one that is thermodynamically the most stable (5). As pointed out by Anfinsen et a1 (5), this 'thermodynamic' hypothesis has the great virtue (besides its innate simplicity) of being amenable to test. A model experiment would be to convert a native protein into a. linear polypeptide chain devoid of disulfide l ¥ bonds and of secondary and tertiary structure and then to restore this unfolded and reduced ("denatured") protein to its original configuration without the introduction of additional "inform ation" (5). Studies of just this sort have been carried out on a number of different proteins, some of which contain disulfide bonds and others that do not. Of the disulfide bond containing proteins studied, bovine pancreatic RNase (6, 7), egg white lysozyme (8, 9), tak- amylase A (10), bovine trypsin (11, 12), Escherichia coli alkaline phosphatase (l3), swine pepsinogen (5), human serum albumin (14), and soybean trypsin inhibitor (15) all show reversible denaturation (i. e. unfolding followed by proper refolding). Successful experi- ments of this type have also been performed with such diverse proteins as bacterial a -amy1ase (16), yeast enolase (l7) and bacteriOphage lysozyme (18, 19) all of which do not contain disulfide bonds. The criteria used to determine the ‘nativeness' of the renatured proteins mentioned has included the following comparisons with native proteins: specific activity, optical rotation, viscosity, ultra-violet spectrum, antigenic identity, peptide maps, chroma- tography, crystal form, sedimentation, and electrOphoretic prOperties. These criteria were used in various combinations for the different proteins mentioned. In all cases where estimates could be made for random yields, i. e. the probability of purely random sulfhydryl pairings to form the same disulfide bonds that are present in the native protein (5, 20), the actual recovery of activity (using combinations of the above criteria) was always con- siderably above that of the calculated random recovery. In spite of the preponderance of indirect evidence supporting Crick's statement that "folding is simply a function of the order of the amino acids" (1) unequivocable evidence exists for only one protein, RNase. This was produced by White (6) when he showed that the X-ray diffraction pattern of reoxidized RNase was indis- tinguishable from the native enzyme. Indeed, the generality of the phenomenon of Spontaneous folding may be questioned since as pointed out by Helinski and Yanofsky (21), "there have been many unsuccessful attempts to restore the tertiary structure or activity of other proteins denatured under relatively mild conditions ". A serious challenge to the "thermodynamic" hypothesis arises from the work of Berson and Yalow (22, 23). They found that certain antisera can distinguish between Sperm whale and pork insulins, proteins identical in amino acid sequence. They concluded, "it would seem that, in addition to amino acid sequence, the precise configuration of folding of the protein molecule is determined by the genetic appartus". Conversely, the tertiary structures of the a - and fi-chains of horse hemoglobins (24) and Sperm whale myoglobin 3 (25) have been worked out, and there exists a close similarity between these two proteins in their tertiary structures, despite marked differences in their amino acid compositions. In addition, H. C. Watson and Kendrew compared amino acid sequences of human hemoglobin and sperm whale myoglobin and found remarkably few correspondences, again despite their similar tertiary structures (26). Another complicating feature of the "thermodynamic" argu- ment of spontaneous folding, is the discovery and purification by Anfinsen and coworkers (27) of an enzyme isolated from beef liver which catalyzes the 'renaturation' of RNase, egg white lysozyme, and soybean trypsin inhibitor. Previous work on the reactivation of reduced RNase (7) had shown that air oxidation of the polypeptide chain of the reduced enzyme results in a protein with native con- formation and full activity. Such oxidation occurs spontaneously, but relatively slowly. By choosing the lowest protein concentration technically feasible (O. 01 mg/ml), a relatively high pH (8. 2), and the Optimal temperature (24°C) the lowest half-time reactivation achieved by air oxidation was 20 minutes (28). Reactivation in the presence of the enzyme, however, proceeds rapidly under more physiological conditions, i. e. pH 7. 0 to 7.4 and 37°C. With partially purified enzyme under these conditions half-maximal reactivation occured in less than 3 minutes (29) and in the purest 4 preparations obtained, ”the rate is in the range that would be expected for an enzyme which catalyzes the prOper folding of a newly synthesized polypeptide chain judged by estimates for the rate of protein synthesis in mammalian cells" (27). It is clear from the above studies that spontaneous folding does not constitute a major mechanism for the determination of tertiary structure of RNase, egg white lysozyme or soybean trypsin inhibitor (17, 29). In recent years it has become evident that a great number of proteins are composed of subunits which are able to associate and dissociate under proper experimental conditions (30). In fact, Changeux (31) points out that all known regulatory enzymes are multimers. The ubiquitousness of protein subunit structure indicates an important biological function, and indeed this subject has recently been given extensive discussion (32, 33). Two major ideas, not mutually exclusive, have emerged. Monod et a1 (34-36) propose that specific regulatory inter- actions of enzymes generally involve tOpographically distinct stero- specific sites and are mediated through a conformational alteration (allosteric effects). An allosteric enzyme is defined as one which can combine (with a substance sterically unrelated to the substrate at a site different from the active site and, as a result, undergo a conformational change or allosteric transition. According to Changeux (35) "the ability to mediate allosteric interactions appears 5 to be directly dependent on the presence of a quaternary structure". The second idea concerning the biological importance of subunit interactions derives from the extensive work done on tryptophan synthetase of Escherichia coli by Yanofsky and his group (37). The finding that the B protein subunit of the E. gg_li tryptOphan synthetase has serine deaminase activity and that this activity disappeared when the 5 protein was combined with the a protein subunit (38), led D. M. Bonner to postulate that protein subunit interactions have great evolutionary significance. Thus, Bonner's evolutionary scheme suggested (as stated by T. Crighton, 39), "that the combination of two enzyme molecules to form an enzyme complex which can efficiently combine two previously independent enzymatic activities may be the basis for the subunit structure of some enzymes". The fact that "hybrid" enzymes appear in hetero- zygotes, as in the cases of the esterases in maize (40), the acid phosphatases in Tetrahymena (41), and the alkaline phOSphatases in DrosoPhila (42) among others, caused Fincham (33) to speculate along similar lines. He points out that there is no evidence that the hybrid enzymes thus formed are functionally superior to 'pure' protein types but states: "It seems, nevertheless, reasonable to suggest that hybrid proteins are sometimes of advantage to the organism carrying them". If so, according to Fincham, this would form part of the explanation of heterosis and give the hybrid a 6 4‘ distinct selective advantage which through evolution would bring about a state of 'permanent hybridity'. Whatever the importance of the multimeric state of many proteins the question of the mechanism of their genetic control becomes pertinent. The original 'dogma' concerned itself with primary and tertiary structure. The more modern version incor- porates the idea of subunit structure and as expressed by Helinski and Yanofsky states (21): "The specific and unique association of the subunits of an enzyme can also be considered to be directed by amino acid sequences and thus by the conformation of the polypep- tide chain, rather than by the control or direction of a specific cyto- plasmic structure". The evidence for spontaneous formation of quaternary structure comes from many sources and has been demonstrated in both in vivo and in vitro systems (43-50). An outstanding case in point is lactic dehydrogenase of birds and mammals. In a wide range of species this enzyme has been shown to occur in five isozymic forms. Kaplan and his group (51) have shown that the active form of the enzyme is a tetramer of two interchangeable electrOphoretically distinct kinds of polypeptide chains. One kind, H, predominates in heart muscle and the other, M, in skeletal muscle. The pure tetramers are designated H4 or M4 as the case may be, and the heterotetramers as H3M, HZMZ, and HM3 respectively. A binomial distribution of the five forms 7 is found when the two types of enzymes are synthesized by one cell type; the distribution depends on the number of H and M subunits present. Evidence of the sort presented above coupled with the fact that efforts to demonstrate specific binding mutations involving enzyme subunits have failed, argue very strongly in favor of the Spontaneous synthesis of quaternary structure. However, as in the case of tertiary structure, there are complications. Thus, Zipser and Perrin (52) observed that the aggregation of B-galactosidase from subunits to make active enzyme proceeds at a much faster rate in the presence of ribosomes. Other types of protein-prote in inter- actions other than enzyme subunits exist and are thought to be under genetic control. Edgar and Wood (53) have implicated direct genetic control of prote in-protein binding in their complementation studies with phage amber mutants. Green and Hechter (54) in a paper con- cerned with the assembly of membrane subunits in mitochondria conclude with this statement: "Our consideration of the assembly in membranes has served to remind us that the heredity apparatus contains coded information, not only for the sequences of amino acids in proteins, but also for the sequences of proteins in macro- molecular assemblies". The argument that tertiary and quatermry structures of enzymes are products of the primary structure of their subunit 8 #‘ components is an attractive one, but not necessarily correct in all cases. There are enough indications in the literature to warrant further investigations into the possibilities that other mechanisms controlling tertiary and/or quatermry structure do indeed exist. The enzyme tyrosinase (EC 1.10. 3.1) in Neurospora crassa promised to be an ideal system for the uncovering of additional genetic control of tertiary and/or quatermry structure, superimposed upon the known mechanisms controlling primary structure. Exten- sive studies of Neurospora tyrosinase have been carried out over the last fifteen years. Most of the data accumulated over this period have come from the respective laboratories of A.S. Fox and N.H. Horowitz. Differences in results obtained by these two workers have led each to contradictory interpretations of the mechanisms of genetic control involved in this System. Genetic studies and detailed analyses by Horowitz and co—workers have revealed the following: Working with wild strain 4A of Neurospora crassa, Horowitz and Fling reported that two alleles at a locus designated T determine different forms of tyrosinase. The two enzymes are distinguished by a striking difference in stability at 59°C. and are therefore designated thermostable and thermolabile. The thermal difference was found to be simply inherited, the stable, S, form of the enzyme being determined by a gene TS and the labile, L, form by its allele TL, located on the mating type chromosome (55, 56). 9 Only one form, S or L is found in a homocaryon, depending on the T allele present (55). In heterocaryons possessing both the TS and TL genes both forms of the enzyme are demonstrable (57). Crude preparations of the L enzyme exhibit a half-life at 59° of 3-4 minutes while the S form at the same temperature had a half-life of 30 minutes. Crystalized L tyrosinase has a half-life of 5 minutes as opposed to the crystalline S form which exhibits a half—life at 59° of 65 minutes. The kinetics of inactivation is first order for both (58). The energies of activation for the thermal degradation of the enzyme are also different. Sueoka (59) reports L tyrosinase has an energy of activation of heat inactivation of 84, 000 calories per mole between 53°-59° and 59, 000 calories per mole between 49°-53° . For S tyrosinase, H = 90,000 calories per mole between 6l°-67° and 61, 000 calories per mole between 57°-6lo . Both forms of the enzyme exhibited considerable variability in their stability at a variety of temperatures both in crude and purified preparations. The two enzymes can also be distinguished by their different electrophoretic mobilities by paper strip ionOphoresis (60). At pH 6. 0 both forms migrate toward the cathode with the L form being fastest (61). A detailed study by Sussman (61) of thirty-three substrates, which included mono-, di-, tri-, and conjugated phenols, revealed no difference in Specificity between L tyrosinase and S tyrosinase. 10 Michaelis-Menten constants for the two forms of enzyme acting on L—tyrosine, and L- and D-dihydroxyphenylalanine (dOpa) were also found to be similar, as were their pH Optima and their response to several inhibitors. On the other hand, the activity of the thermola- bile enzyme was lost more rapidly than that of the thermostable one after incubation in high concentrations of urea or formamide. Sussman interprets this data to indicate that the difference between the two forms of tyrosinase reside in their secondary and tertiary structures. Crystalline L and S forms show no difference in their amino acid compositions, turnover number or "fingerprint‘l patterns (58). It is interesting to note that neither tyrosinase contains cys— tine and therefore neither forms disulfide bonds (58). Two other forms of tyrosinase were found in Neurospora by Horowitz et a1 (61). These were designated Puerto Rico-15 (or PR-15) and Singa- pore—2 (or Sing—2). Both the PR-15 and Sing-2 tyrosinases segregate with either L or S. They are distinguishable from one another on the basis of heat stability and/or electrOphoretic mobility. Horowitz and coworkers (62) also reported the presence of two other loci, ty-l and ty—2 not linked to each other which are epistatic to the T locus. The presence of genes ty-l and/or ty—2 (as opposed to ty-l+ and ty-2+) results in the cessation of tyrosinase production. The effect of the ty genes can be overcome by starving the mycelia or by adding certain D-aromatic amino acids to the growth medium. 11 The form of the induced enzyme is dependent upon which allele of the T locus (TS, TL, TPR‘15 or TSing‘Z) is present. Horowitz has concluded that the T locus is a structural one and that the ty-l and ty—2 loci are regulatory. He attributes the different forms of tyrosinase to genetic alteration at the T locus resulting in a change in primary structure thereby accounting for the differences in heat stability and electrophoretic mobility between the tyrosinases (63). Early data obtained by Fox's group (64, 65) working with the albino-2 mutant 15300, indicated that tyrosinase concentration and Specificity in Neurospora are controlled by two different genetic systems. Later Fox et a1 (66, 67) reported the existence of differ- ent forms of the enzyme utilizing a continuour flow paper electro- phoresis technique. The crude tyrosinase extracts separated into three components T1, T2, and T3, two or three of which were present in any one thallus. In order to rule out heterocaryosis as the reason for the simultaneous appearance of the multiple forms, hyphal tip and ascospore isolates were examined for their tyrosinase content. . The results clearly indicated that homocaryotic cultures are capable of producing two or three forms of the enzyme simul- taneously (68). Further analysis of T1, T2, and T3 led to the following observations (66-68): The activation energy for the thermal degradation of T1’ 95, 800 calories per mole, was similar to Horo- witz's L form (84, 000 calories per mole) and for T2 and T3, 12 H =-- 60, 900 and 57, 300 calories per mole reSpectively, virtually identical to the S form (64, 000 calories per mole). There were no differences in the Michaelis constants of the three enzymes T1, T2 and T3 with L—tyrosine and L-dOpa as substrates, nor were there any differences with regard to their dopa/tyrosine activity ratios. In material that contained all three forms, T1, T2, and T3 con- stituted 5. 9, 12.8 and 81. 3 percent respectively of the total tyro- sinase in fresh extracts. After incubation at 5° C. for six days of an aliquot of the same fresh extract T3 had increased to 91. 6 per— cent of the total. The gain in T3 was exactly accounted for by concomittant loss of T1 and T2, with no change in overall activity of the incubated aliquot. A linear scheme of interconversion was suggested when an electrOphoretic analysis of 33 ascospore isolates showed that T1 is found only in conjunction with T2 and T3, while T2 and T3 may appear together but only T2 appears alone. Based on his own observations, that of Horowitz's group and the fact that a tyrosinaseless mutant which segregates with the T locus has never been found, Fox suggested that the T locus controls the equilibrium in the interconversion of the alternate forms of the enzyme, and that the difference between the forms is not in their primary structure but in their tertiary structure (67, 68). Thus, this inter- pretation would mean the T locus determines the tertiary structure of tyrosine and that another, as yet unidentified, locus determines l3 primary structure. The purpose of the present work is to determine which of these two interpretations is correct and/or if a reconciliation of the two points of View is possible. Since both groups had been using different methods of growth and extraction as well as different stocks the first obvious step was to minimize these differences. To this end all the reported data heretofore was obtained with Horowitz's L S T and T stocks, and his methods of growth and extraction were adhered to closely. l4 MATERIALS AND METHODS Strains of Neurospora crassa producing either the S or the L form of tyrosinase were used throughout this study. The TS strain (No. 69-1ll3a) was kindly supplied by Dr. Norman H. Horo- witz of the California Institute of Technology, and the TL strain (No. 320) was obtained from the Fungal Genetic Center located at Dartmouth College in New Hampshire. They were maintained on solid Westergaard and Mitchell minimal medium (69). In order to obtain as much enzyme as possible three methods of growth and induction were used, duplicating the procedures reported by Horowitz et a1 (70, 58, 71). The first consisted of growing cultures in 125 ml. erlenmeyer flasks containing 20 ml. of Vogels N liquid minimal medium (72) at 25° C. After two days of growth the medium was decanted and the mycelial mat was washed twice with 20 m1. of sterile distilled water. The washing consisted of gently swirling the mycelium around and then decanting the water. After the second water wash, 5 ml. of sterile sodium phosphate buffer, 0.02 M, pH 6. 0, was added to each flask. The flasks were incubated in the dark at 25° C for 48 hours. At the end of this time the mycelial mats were collected by filtration, washed thoroughly and quickly with cold water, squeezed dry and stored at -25° C. until needed. In the second method N. grass}: was 15 grown in carboys. A two gallon carboy containing Six liters of Vogels N liquid medium was innoculated with a condial suSpension and allowed to grow with aeration in the dark at 25° C. for 3-4 days. The medium was aseptically replaced with sterile 0.05M, pH 5. 5 sodium phOSphate buffer, placed in the dark, and aerated for an additional 4-5 days at 25° C. before it was harvested. After har- vesting the mycelium was washed as before and frozen until needed. The last procedure, and the one that gave the highest yield of enzyme activity, consisted of growing the fungus in 6 liters of half strength Vogel's medium N containing 1/2 percent sucrose in the dark, at 25° C. , with aeration for 72 hours. At the end of this time one gram of D-phenylalanine was added to the growing culture and the carboy was allowed to sit in the incubator without changing conditions for another 4-5 days before harvesting. As before, the mycelium was thoroughly washed in cold water and stored at -25° C. until needed. This technique yielded 30-60 grams wet weight of mycelium per carboy. The frozen mycelium was ground into small pieces with a mortar and pestle. Ice cold buffer was added to a concentration of 2 ml. buffer to 1 gram of mycelium. The resulting suspension was then homogenized in an all—glass Duall tissue grinding apparatus in an ice bath for five minutes at 500 rpm. When more than 20 ml. of buffer was needed the suspension was homogenized in a pre—cooled l6 Waring blendor operated at 4° C. at high speed for one minute. The homogenate was centrifuged at 37, 500 xg for 20 minutes at 4° C. and the precipitate was discarded. The clear supernatant constituted the crude extract. In some experiments an aliquot of the homogen- ate was used immediately while the rest was placed in the cold with stirring for varying periods of time before centrifugation. Enzyme activity was determined by spectrophotometric measurement, at 475 mu, of the rate of dopachrome formation resulting from the oxidation of Ir-dihydrOXyphenylalanine (L—dOpa) (73). Reactions were carried out in spectrophotometric cells at a controlled temperature of 25° C. , regulated by a Haake circulating water bath. Reactions mixtures consisted of 1.0 ml. of apprOpriate enzyme dilution and 2. 5 m1. of 0.1 M sodium phosphate buffer, pH 6.0, containing 3.6 mg. of L—dopa. Absorbancy measurements were automatically recorded at 3 second intervals with a model 2000 Gilford recording spectrophotometer. In spite of the recommendation of the Enzyme Commission of the International Union of Biochemistry that an enzyme unit be defined as the amount of enzyme which catalyzes the transformation of one mole of substrate per minute under defined conditions (74) the tyrosinase unit reported here will be the one used by Fox and Burnett (67). Their unit is defined as that amount of enzyme which results in an initial rate of change of absorbance at 475 mu of 17 10'3/min./cm. light path. Taking into consideration that this assay measures the rate of dopachrome appearance rather than the disappearance of dopa and that 2 moles of dOpa are ozidized for every mole of dOpachrome produced (58), and given the molar extinction coefficient of dOpachrome at 475 mg of 3600 (75), then in a total volume of 3. 5 ml. with L—dOpa as substrate 514 of these units equal one Enzyme Commission Unit. Since our spectrOphoto- metric assay gives us a resolution of 0. 001 optical density (O. D.) per minute the unit used should be sensitive enough to reflect this resolution. The Enzyme Commission Unit in this context would obscure real differences in enzyme activity by compressing the numerical range of activities actually found in this study. Electrophoretic fractionation was accomplished by continuous- flow paper electrophoresis on Whatman No. 31ET filter paper, 35. 5 x 37. 5 cm. , in a Spinco Model-CP cell. Details of this pro- cedure have previously been published by Fox and Burnett (67). For the determination of thermostabilities the pH of the enzyme fractions were adjusted to pH 6. 0 in O. l M sodium phos- phate buffer either by dialysis or by passage through a G—25 Sephadex column. At the beginning of each experiment, the sample being tested was brought rapidly from room temperature to that of a constant-temperature water bath (i 0. 250 ). At predetermined intervals, aliquots were pipetted into tubes immersed in an ice 18 bath. These were then brought up to 25° C. and assayed for enzyme activity. Protein concentration was determined by the method of Lowry et a1 (76), using bovine serum albumin as a standard or by the absorbance obtained at 280 and 260 mp respectively using Kalckar's formula (77). The experimental procedures outlined in detail by Martin and Ames (78) for the handling of sucrose gradients were followed closely. Only linear gradients were used and were of 5 to 20 per- cent sucrose made up in various buffers as indicated, to a volume of 5. 3 ml. The volume of enzyme preparation applied to the gradient was 0. 2 m1. A standard was always included in a separate tube in every experiment. The standard used was 0. 2 ml. (2mg.) of twice crystallized bovine hemoglobin (sigma Chemical Company) and was assayed by absorbancy at 410 mp . After centrifugation for 20-24 hours at 34, 000 rpm and 4°C. in a SW-39 rotor in a refrigerated model L—Z Spinco centrifuge, 10 drop fractions were collected by a device similar to that described by Martin and Ames (78). Each tube yielded an average of 400 drops or approximately 0. 14 ml. per fraction. Of this amount, 0. 10 ml. was routinely assayed for enzyme activity. Ion exchange chromatography was performed using the anion exchanger diethylaminoethylcellulose (DEAE-Cellulose), 19 obtained from Carl Schleicher and Scheull Company, New Hampshire. Cleaned (79) DEAE-cellulose was repeatedly sifted through a N0. 100 standard sieve (149 microns) while suspended in distilled water, to remove the fines. After drying, the DEAE—cellulose was resuspended in O. 01 M, pH 7. 2 sodium phOSphate buffer and the slurry was poured into a column. At least three volumes of this buffer was passed through the column before a sample was applied. The enzyme preparation to be fractionated was in 0. 01 M, pH 8. 0-8. 2 sodium phOSphate buffer and elution of the enzyme was accomplished using 0. 01 M concentration of this buffer with increas- ing amounts of sodium chloride, either in step-wise fashion or with a linear gradient (80). The size of the columns used was 12 x 400 mm or 25 x 900 mm and the flow rates obtained with gravity were between 0. 5 ml. per minute to 1. 5 ml. per minute. All fraction- ation was carried out at 4° C. 20 RESULTS A. ASSAY METHODS Figure 1A shows the results of a typical assay using undialyzed extract. The lag periods and the discrepancies in activities between the various dilutions were exhibited by every S or TL, regardless of the method of growth. When extract, T these extracts were dialyzed overnight against the appropriate buffer, or when buffer exchange was accomplished by gel filtration using G-25 Sephadex, the lags and the dilution discrepancies were eliminated (Figure 1B). These results suggest that a small molec- ular weight tyrosinase inhibitor was present in the extracts. Horo- witz and Shen (81) reported the presence of an inhibitor of tyrosinase in Neurospora'crassa which was lost after dialysis. They also demonstrated that when the fungus was grown on a low sulfate- containing medium (0. 00785 gm. of MgSO4‘ 7H 0 + 0. 4 MgClz' 6H 0 per liter, instead of 0. 5 gm. of MgSO4' 7H 0) the inhibitor could not be detected. Growth on low sulfate medium was tried but did not result in elimination of the apparent inhibitor in the extracts. L or TS extracts were incubated for long When undialyzed T periods in the cold there was a gradual loss of the inhibitor effect. However, this observation was not consistent and varied qualitatively and quantitatively from extract to extract. 21 Figure 1A. Typical assay of TS at various dilutions before dialysis or passage over G—25 Sephadex. Amount Observed Activity (Units) Activity/m1 (Units) Assayed lst Minute Maximum 1st Minute Maximum (ml) 1.0 110 220 110 - 220 O. 5 140 240 280 480 0. 1 35 75 350 750 Figure 1B. Typical assay of TS at various dilutions after passage over G-25 Sephadex. Amount Observed Activity (Units) Activity/m1 (Units) Assayed lst Minute Maximum lst Minute Maximum (m1) 1. 0 760 760 760 760 0. 5 325 325 750 750 0. 1 75 75 750 750 22 ABSORBANCY AT 475 mM ABSORBANCY AT 475 mp. FIGURE 1A. Typical assay of TS at various dilutions before dialysis or gel-filtration through G-25 Sephadex. Absorbancy calculated for l. 0 ml abstract in each case. Amount Assayed 2.400“ e 1.0ml / x 0.5ml o 0.1m1 1.800— 1.200—1 0.600 —- TIME IN MINUTES FIGURE 1B. Typical assay of TS at various dilutions after passage over G-25 Sephadex. 2.000 ‘1 1.500 “ 1.0m1 1.000 - 0.5ml 0.500 - 0.1 ml I I T 1 I I 30 60 90 120 150 180 TIME IN SECONDS 23 In order to determine the true activity of any extract, dilutions were made until a constant activity per ml was obtained. To ascertain if this was the true activity the extract was dialyzed and then assayed again. In all extracts tested this way, the activity per ml obtained by dilution equaled the activity per m1 of the same dialyzed extract. The reliability of the dilution method to obtain the true activity was verified in over 100 different extracts. No trace of the inhibitor effect was apparent in any of the fractions of tyrosinase obtained after continuous-flow electr0phoresis, ion-exchange column chromatography on DEAE cellulose, or sucrose gradient centrifugation. B. ANALYSIS BY CONTINUOUS-FLOW ELECTROPHORESIS When crude, undialyzed TL and TS extracts are subjected to continuous—flow electr0phoresis and the samples collected at the bottom sheet are analyzed for enzyme content, it is evident that more than one tyrosinase component exists for each. Moreover it is clear that their electrophoretic patterns are different (Figures 2 and 3). Adopting Fox and Burnett's nomenclature (67), the most cathodic fraction, tubes 3-9, will be called TL3 or T53; tubes 10—15 will be called TLZ or TS 2; and tubes 16—25 will be referred to as TLl or Tsl. The TL extracts showed much more variability in their 24 k “\n i'ua FIGURE 2. Analysis of fractions collected from bottom of continuous-flow electrophoresis sheet. Sample: Crude, undialyzed TL extract. Sample Volume: 75 ml Total Activity: 52, 500 units. Activity Recovered: 25, 426 units (48. 4° /0) Conditions of ElectrOphoresis: Time: 60 hours Buffer: sodium barbital, pH 8. 6, I = 0.02 Constant voltage (960 volts) with current of 40—50 milliamperes. 25 ‘. 0 EN ZYME UNITS FIGURE 2. Analysis of fractions collected from bottom of continuous-flow electrophoresis sheet; TL. ('1 5400 “‘ 4800 r 4200 '7 3600 .. 3000 '3 2400 — 1800 - 1200 600 11 15 19 23 27 FRACTION NUMBER (Numbered From Cathode to Anode) 26 FIGURE 3. Analysis of fractions collected from bottom of continuous-flow electrophoresis sheet. Sample: Crude, undialyzed TS extract. Sample Volume: 20 ml Total Activity: 100, 000 units Activity Recovered: 49, 655 units (49. 7°/o) Conditions of Electrophoresis: Time: 18 hours Buffer: sodium barbital, pH 8.6, I = 0.02 Constant voltage (960 volts) with current of 40-50 milliampe res . 27 o ENZYME UNITS FIGURE 3. Analysis of fractions collected from bottom of continuous-flow electrophoresis sheet; TS. 9000 J 8000 r 7000 4 6000‘ _ . 5000. 4000‘ 3000‘ 2000 - 1000‘1 . ' oc 3 7 11 15 19 23 27 FRACTION NUMBER (Numbered From Cathode to Anode) 28 electrophoretic patterns (compare Figures 2, 4, and 6A) than TS. S3. However, the ratio of TL2zTL3 was always greater than TSZ:T Dialysis of crude TL or TS extracts against pH 8.6 barbital buffer, and subsequent continuous-flow electrophoresis, reveals a shift in the direction of T2 or T3. Figure 4 shows one such experi- ment. The TL extract used in this case was almost all TLZ before dialysis. After dialysis it was converted almost entirely to TL3. These results parallel those obtained by Fox and Burnett (67) using their l3la-15300 strain of Neurospora crassa. In order to test the possibility that the conversion of TLZ to TL3 was due to change in molecular weight, gel filtration column chromatography on G-100 Sephadex was performed. Dialyzed and undialyzed TL extracts were chromatographed separately and the fractions collected were analyzed for enzyme content. The extracts were similar, exhibiting a single species of identical chromatographic mobility, and presumably of identical molecular weight. The con- version of TLZ to TL3, therefore, is not attributable to changes in molecular weight caused by dialysis. In contrast, the electrophoretic components do not exhibit conversion after isolation. In an experiment to determine the stability of the electrophoretic components, TL3 and TLZ (tubes 38 and 10-15, respectively, from Figure 6A) were collected. They were concentrated by dialysis against 25 percent Ficoll (Pharmacia 29 m L ”‘3' FIGURE 4. Analysis of fractions collected from two separate continuous-flow electrophoresis sheets. Sample: Crude undialyzed TL extract Crude dialyzed TL extract Sample Volume: Undialyzed = 18 m1 Dialyzed = 30 m1 Total Activity: Undialyzed = 7, 200 units Dialyzed = 8, 700 units Activity Recovered: Undialyzed = 6, 208 units (86.2%) Dialyzed = 1, 824 units (20. 9%) Time of Electrophoresis: Undialyzed = 20 hours Dialyzed = 25 hours 30 o ENZYME UNITS FIGURE 4. Analysis of fractions collected from bottom of continuous-flow electrophoresis sheet; TL 2700—] 2400‘“ 2100—« 1800- 1500‘ 1200 _. 900 A 600 —~ 300 -« Extract not dialyzed A n A U U \f Extract dialyzed. if. t Y T Y 7 T‘ I 7 11 I Y 15 19 23 FR AC TION NUMBER (Numbered from Cathode to Anode) 31 L 450 - 400 L 350 “ 300 *" 250 _ 200 r—150 - 100 0 EN ZYME UNITS FIGURE 5A. Analysis of fractions collected after gel filtration chromatography of undialyzed, crude TL extract on G-lOO Sephadex. Dimensions of Column Bed: 1.8 cm by 22. 5 cm Equilibrated and eluted with sodium barbital buffer, pH 8. 6, I : O. 02. Sample: 5 ml of undialyzed, crude TL extract, in barbital buffer. Total Activity Applied to Column :2 3, 250 units Total Activity Recovered From Column 2 3, 250 (100%) Fraction Size = 5 ml. FIGURE 5B. Analysis of fractions collected after gel filtration chromatography of dialyzed, crude TL extract on G-100 Sephadex. Dimensions of Column Bed: 1.8 cm by 22. 5 cm. Equilibrated and eluted with sodium barbital buffer, pH 8. 6, I = 0. 02. Sample: 5 m1 of dialyzed, crude TL extract. Dialysis was carried out in cold overnight against barbital buffer. Total Activity Applied to Column 2 2, 750 units Total Activity Recovered From Column = 2, 700 units (98. 2%) Fraction Size = 5 ml. 32 ENZYME UNITS ENZYME UNITS FIGURE 5A. Gel filtration column chromatography of undialyzed crude TL extract in G-lOO Sephadex. 1200 1050 900 750 600 450 300 —— 150 -‘ 2 3 4 5 6 7 8 9 10 ll 12 FRACTION NUMBER FIGURE 5B. Gel filtration column chromatography of dialyzed crude TL extract in G-lOO Sephadex. 1200 l 1050 l 900 ~ 750 "l 600 __ 450 - 300 ~ 150 - H» ‘l .4 i 456789101112 Fine Chemicals, Inc.) in pH 8. 6 barbital buffer and subjected to continuous-flow electrophoresis on two fresh sheets. Figure 6B demonstrates that the TL3 isolated from the first electrophoretic separation remains most cathodic in its electrophoretic mobility while TLZ remains slightly anodic. C. ION EXCHANGE COLUMN CHROMATOGRAPHY Column chromatography on DEAE cellulose of undialyzed TL or TS extracts in a sodium barbital buffer system (pH 8. 6, I = 0. 02; the same buffer used for continuous-flow electrOphoresis) did not result in fractionation. All of the enzyme in both extracts was eluted completely in the break-through volume. However, resolution of the multiple forms of both TL or T8 is revealed on DEAE cellulose in a sodium phosphate buffer system (see Materials L and TS are both fractionated into three compon- and Methods). T ents, but with different quantitative distributions. Figures 7A and 7B depict typical patterns. The elution patterns were the same whether the salt concentration was increased in a step-wise fashion or by means of a linear gradient. No additional enzyme was eluted in NaCl concentrations exceeding 0. 5 M: Enzyme eluted in the absence of NaCl will be called TLA or TSA; that eluted in 0.1M NaCl, TLB or T58; and the 0.5M NaCl fraction, TLC or TSC. TL extracts, as was the case with 34 FIGURE 6A. Analysis of fractions collected from bottom of continuous-flow electrophoresis Sheet. Sample: Crude undialyzed TL extract Sample Volume: 150 m1 Total Activity: 90, 000 units Activity Recovered: 32, 160 (35. 7%) Time of Electrophoresis: 84 hours FIGURE 6B. Analysis of fractions collected from two separate continuous-flow electrophoresis sheets. Sample: TL3; tubes (3-8) from Figure 4A TLZ; tubes (10—15) from Figure 4A Sample Volume: TL3 = 32 ml TLZ = 20 ml Total Activity: TL3 = 7,680 units TLZ = 3,800 units Activity Recovered: TL3 = Z,445(31.8%) TLZ = 681(17.9%) Time of Electrophoresis: TL3 = 27 hours TLZ = 24 hours 35 o ENZYME UNITS o ENZYME UNITS FIGURE 6A. Continuous-flow electrophoresis analysis, TL 7200 6400 5600 4800 4000 3200 2400 1600 800 l 1 1 1 l l IIYTTVfT'TITI’rl 791113151719212325 FRACTION NUMBER (Numbered From Cathode to Anode) FIGURE 6B. Continuous-flow electrophoresis analysis of 990 880 770 660 550 440 330 220 110 fractions indicated, obtained from above ex eriment. (3-8) U0"! ‘1 Ulsh ‘1 (10-15) 91113151719212.3255 FRACTION NUMBER (Numbered From Cathode to Anode) 36 s126 r112 * 98 - 84 '70 s56 —42 o ENZYME UNITS r28 p14 continuous-flow electrophoresis, showed much more variability in the quantitative distribution of the three components than did the TS extracts. However the ratio of TLBzTLA was always greater than TSBzTSA. After buffer exchange by dialysis or gel filtration a shift to TA is evident in both extracts (compare Figures 7A and 8A, 7B and 8B, and Table 1). It was observed that with either extract a greater percentage of TA was present when the extract was dialyzed in a bag overnight than when subjected to gel filtration. Since the only difference in principle between these two methods is time (20 minutes on Sephadex versus 24 hours in a bag) it was postulated that a spontaneous con- version to TA takes place. In order to test this possibility an extract was prepared, immediately passed through a G—25 Sephadex column, and an aliquot was chromatographed on DEAE cellulose. The rest of the extract was allowed to incubate in the cold and aliquots were chromatographed on DEAE cellulose after 24 and 48 hours. Table 2 illustrates the results of such an experiment using aTS extract, and clearly shows that incubation of the filtered extract results in an increase of the TSA component and losses of both TSB and TSC. These results are comparable to those obtained by Fox and Burnett (67), who showed a quantitative conversion of T1 and T2 to T3 by continuous-flow electrophoresis with their 131a-153OO strain of Neurospora grassa. 37 FIGURE 7A. Ion—exchange column chromatography of undialyzed crude TL extract on DEAE cellulose. Dimensions of Column Bed 2 1. 2 cm by 40. 0 cm Sample Size = 10 m1 Tyrosinase Activity Applied = 4, 200 units Tyrosinase Activity Recovered = 4, 095 (97.1%) Fraction Size = 9. 0 ml Method of Elution = Step-wise; 0. 0M NaCl, 0. 1M NaCl, and 0. 5M NaCl FIGURE 7B. Ion-exchange column chromatography of undialyzed crude Ts extract on DEAE cellulose. Dimensions of Column Bed 2 1. 2 cm by 40. 0 cm Sample Size = 10 m1 Tyrosinase Activity Applied = 3, 500 units Tyrosinase Activity Recovered = 3, 438 units (98. 2%) Fraction Size = 9. 0 m1 Method of Elution = Step-wise; 0. 0M NaCl, 0.1M NaCl, and 0. 5M NaCl 38 FIGURE 7A. Ion-exchange column chromatography of undialyzed, ENZ YME UNITS EN Z YME UNITS crude TL extract on DEAE cellulose. 1800 "_ 0.0MNaC1 ( 0.1M NaCIl 0.5MNaC1 1600 " 1400 " 1200 - 1000 -* 800 -— 600 —- 400 ~— 200 -- Tilll[[l ‘11IITIIIYIIII 24681021416181)2224% FRACTION NUMBER FIGURE 7B. Ion-exchange column chromatography of undialyzed, crude TS extract on DEAE cellulose. 1800 *0. OM NaCl +0.1M NaC‘H‘” 0. 5M NaCl *- 1600- 1400 —- 1200 - 1000 .._. 800 “ 600 .. 400 ... 200 --+ IT 1'I'YIII'UtlIIIvu 24 6310121416182022’2426 FRACTION NUMBER 39 FIGURE 8A. Ion-exchange column chromatography of crude, TL extract on DEAE cellulose after passage over G—2 5 Sephadex. Dimensions of Column Bed = 1. 2 cm by 40. 0 cm Sample Size = 10.0 ml Tyrosinase Activity Applied = 4, 200 units Tyrosinase Activity Recovered = 3, 636 units (86. 6%) Fraction Size = 9. 0 ml Method of Elution = Step- Wise; 0. 0M NaCl, 0.1M NaCl, and 0. 5M NaCl FIGURE 8B. Ion-exchange column chromatography of crude, TS extract on DEAE cellulose after passage over G-2 5 Sephadex. Dimension of Column Bed 2 l. 2 cm by 40. 0 cm Sample Size = 7. 5 ml Tyrosinase Activity Applied = 2, 600 units Tyrosinase Activity Recovered = 2, 520 units (96. 9%) Fraction Size = 9. 0 m1 Method of Elution = Step-wise; 0. OM NaCl, 0. 1M NaCl, and 0. 5M NaCl 40 DI crude, isage FIGURE 8A. Ion-exchange column chromatography of crude T extract on DEAE cellulose, after passage over G-25 Sephadex. 1800 *‘ 0.0M NaCl ‘TU. 1M NaCl-AT 0.5M NaCl Moo a 1400 e TLA 1200 i 1000 — 800 - TLB 600 r ENZYME UNITS 400 __ 200 «d TLC - /\ I I ? 1 y ‘ y r i T ‘f T I l l l I T 8 10 12 14 16 18 Z) 22 24 FRACTION NUMBER R» 5 FIGURE 8B. Ion-exchange column chromatography of crude TS extract on DEAE cellulose, after passage over G-25 Sephadex. ""0.0MNaCl ""l“‘0.lMNaC‘I'F‘ 0.5MNaC1 - 1800 1 1600 ~ 1400 — 1200 a TsA 1000 a 800 _. 600 ~ ENZYME UNITS 400 ~ 200 .. TSB TSC I 7 I 1 l I , 1‘ 4 6 8 10 12 14 16 18 20 22 24 26 FRACTION NUMBER 41 TABLE 1. Distribution of Multiple forms of tyrosinase before and after passage over G—25 Sephadex as determined by ion-exchange on DEAE cellulose for both TL and TS. TLA TLB TLC Before gel filtration 14. 7 75. 8 9. 5 (percent ot total) After gel filtration 73. 0 23. 5 3. 5 (percent of total) TSA TSB TSC Before gel filtration 68 . 8 23. 3 7. 9 (percent of total) After gel filtration 85.1 11. 4 3. 5 TABLE 2. Changes in tyrosinase during incubation of TS extract after passage over G-25 Sephadex as determined by ion- exchange chromatography on DEAE cellulose. Length of Incubation TSA TSB TSC Total at 5° C. , . . . Units Percent Units Percent Units Percent Units (hours) 0 1314 81.6 207 12.8 90 5.6 1611 24 1467 93.5 51 3.2 51 3.2 1569 48 1719 95.0 45 2.5 45 2.5 1809 42 Six molar urea did not change the DEAE cellulose elution patterns of either TL or TS extracts (Figure 9 and Table 3), nor was any change observable in the presence of various concentrations of cupric nitrate, cuprous nitrate, or ethylenedinitrilotetraacetic acid (EDTA). Crude NeurOSpora extracts usually contained between 4-5 mg of protein per m1. This protein was eluted in a ratio of 30:50:20 in the three fractions eluted from the DEAE cellulose columns used. Recovery of total protein from these columns was between 70 and 80 percent. Total tyrosinase recovery was generally 90-100 percent. TA, TB, and TC tyrosinase components were purified approximately 18-fold, lZ-fold, and 16-fold respectively. A second passage over DEAE cellulose increased the purity to approximately 25 fold. Rechromatography of either TLB and TSB on DEAE cellulose after dialysis or gel filtration to remove the NaCl always resulted in conversion, so that at least 98 percent of the original TB com- ponent was recovered as TA. In addition, direct cross identification between the enzyme components obtained electrOphoretically and those obtained chromatographically proved impossible because of the necessity to change buffers by dialysis or gel filtration. Thus when TL3, TLZ, and TLl (fractions 5-9, 10, and 11-22, respectively, from Figure 2) were individually chromatographed on DEAE cellu- lose after dialysis or gel filtration against sodium phOSphate buffer 43 ENZYME UNITS FIGURE 9. Ion—exchange column chromatography on DEAE cellulose of TL extract treated with 6M urea. 900 *‘0.0M NaCl -+— 0.1M NaCl +0.5M NaCl 800 ‘ _ Control - no urea 70° treatment 600 4 . _®__0_0__ 500 -‘ Extracted in 6M 400 — urea ——~A—A—A— 300 ‘ 200 ‘ 100 ‘ T Ifi I 3468101214161820222426 FRACTION NUMBER 44 TABLE 3. Affect of extraction in 6M urea on the distribution of tyrosinases (TL) as determined by ion-exchange column chromatography on DEAE cellulose. Fraction Control Urea Extracted Percent of Total Percent of Total TLA 58. 7 50. 0 TLB 37.0 44. 0 TLC 4 3 o 0 45 (pH 8, 0.11M), they were all recovered predominately as TLA (Figure 10). Similarly, TLB exhibited the electrophoretic prOper- ties of TL3 after dialysis or gel filtration against sodium barbital buffer (pH 8. 6, I = O. 02). There is sufficient indirect evidence, however, to say with some assurance that TA is equivalent to T3, that TB is equivalent to T2, and the TC is equivalent to T3, and that these difficulties are attributable to conversion (see Discussion). D. THERMAL INACTIVATION Thermal inactivation of TLA and TSA has been examined. In all cases inactivation exhibited first order kinetics. Inactivation curves observed at three temperatures are illustrated in Figure 11. Tables 4 and 6 give the thermal half-lives of TLA and TSA calcu- lated by means of the least-square method from data such as is shown in Figure 11. The rate constants of inactivation are given in Tables 5 and 7. A typical Arrhenius plot of these data is shown in Figure 12. The conversion of TLB to TLA upon dialysis or gel filtration prevented an unambiguous determination of its thermal stability. However, it was possible to compare authentic TLA with TLA obtained by the conversion of TLB (referred to as TLB -* TLA). L extract was chromato- TLA To accomplish this comparison, crude T graphed on DEAE cellulose and TLA and TLB were isolated. 46 ENZYME UNITS FIGURE 10. 1350 1200 1050 900 750 600 450 300 150 ._—-d —C O O cellulose of continuous flow electrophoresis fractions from TL. See Figure 2. Ion-exchange column chromatography on DEAE *‘ 0.0M NaCl *1‘0. 1M NaCTI'" 0.5M NaCl continuous flow electrophoresis fractions (59). C) .. J continuous flow electrophoresis fractions ( 10 ). continuous flow electrOphoresis fractions (ll—22). ‘4 A A A o -‘ A ‘3 ‘ 9 c. . A; la. ‘A-c 246810111214161820222426 FRACTION NUMBER 47 TABLE 4. Thermal half-lives of TLA and TLB —- TLA in minutes. Determined in 0. 1M sodium phosphate buffer, pH 6. 0. Temperature Tyrosinase 50 ° 55 ° 59° TLA 215.0 42.4 10.4 TLB —> TLA 200.1 30.1 9.5 TAB LE 5. Rate constants of thermal inactivation (k) of TLA and TLB -* TLA from half-lives shown in Table 4. ”inact. = act1vat10n energy of thermal 1nact1vat10n in calories per mole. k X 102 minute"1 Temperature Tyrosinase 50° 55° 59° “inact. TLA 0. 32 1.63 6.68 76,440 i 3, 621 L L T B —> T A 0.35 2.26 7.30 75,799i9,911 48 TABLE 6. Thermal half-lives of tyrosinase TSA in sodium phosphate buffer, pH 6. O and T83 in barbital buffer, pH 8. 6, in minutes. Temperature Tyrosinase 48° 55° 59° 'Tszx 301.00 100.30 75.25 T53 2.18 1.31 0.85 TABLE 7. Rate constants of thermal inactivation (k) of TSA and T53 from half-lives shown in Table 6. Hinact. = activation energy of thermal inactivation in calories per mole. k X 103 minute-1 Temperature Tyrosinase 48° 55° 59° Hinact. TSA 2.3 6.9 9.2 27,113 T53 318.0 529.0 812.0 21,755 49 liar] It -5. l., 1. €11||/.||}||III|I(II\\1\\III|IIII. [if LOG PERCENT INITIAL ACTIVITY Thermal inactivation of TLA and TSA at three FIGURE 11. temperatures. Ordinate: residual activity plotted on a logarithmic scale. Abscissae: time of exposure to the indicated temperature. 1004' 59° 55° 50° 90 .. 80 ‘i 70 “ 60 “1 50 ‘7 40 r 30 —- TLA TSA TLA TSA TLA TSA I r l 1 l I r I 1 4O 80 120 50 100 120 125 250 375 TIME, MINUTES 50 was passed through a G-25 Sephadex column previously equilibrated with 0.1M sodium phosphate buffer, pH 6. O. TLB was passed over G-25 Sephadex, but in a column equilibrated with 0. 1M sodium phosphate buffer pH 8. 1. The enzyme was then rechromatographed on a new DEAE cellulose and was recovered as TLA; the TLB -’ TLA conversion had occurred. This was passed through a G-25 Sephadex column equilibrated with 0. 1M sodium phosphate buffer, pH 6. 0, in order to provide ionic conditions similar to those of authentic TLA. Thermal half-lives of the two preparations were determined at three different temperatures and their respective activation energies of inactivation were calculated (Tables 4 and 5). There was no significant difference, and the values agree reasonably well with those reported for TL by Sueoka (59). The thermal half—lives at three temperatures of TSA in 0. 1M sodium phosphate buffer, pH 6. 0, were determined and are given in Table 6. The half-life of TSA at 59°C. (75 minutes) is consistent with Sueoka's value (65 minutes) for both crude and partially purified TS (59). T53 obtained from a continuous-flow electrOphoresis experiment was also tested for thermal inactivation. The determination was carried out in pH 8. 6 barbital buffer (Table 6). In spite of the differences in their thermal half-lives at the temperatures tested, the activation energy of inactivation for TSA and T53 were the same (Table 7). This value, however, is very 51 LOG k. MINUTES” FIGURE 12. I F—l I -2_ -2_ Arrhenius plot of the rate constants of thermal inactivation of TLA and TSA. k = rate constant; T = absolute temperature. Hinact = activation energy of inactivation in calories per mole. ”inact. : 27’ 000 TLA = 71, 000 cal/mole Hinact. TSA cal/mole 3. 01 I T“ _ 7 r I 3.03 3.05 3.07 3.09 3.11 l/T x 10‘3 52 different from the one reported by Sueoka (59). A fuller discussion of this point will be found in the next section. The thermal inactivation data clearly illustrate that TLA and TSA are different (Figure 12). Figure 13 also clearly shows that TLB and TSB are different. Thermal inactivation at 59° C. of TLB and TSB was studied immediately after collection from separate DEAE cellulose columns. The fractions were not dialyzed or treated in any way, so that both determinations were carried out in the same buffer with the same NaCl concentration. TSA and TSB, respectively, are more heat stable than TLA and TLB. E. SUCROSE GRADIENT CENTRIFUGATION Sucrose gradient centrifugation of undialyzed and dialyzed TS extracts at pH 8. O in 0. 1M sodium phosphate buffer, shows a single symmetrical peak of tyrosinase activity for each, with the same S value (Figure 14). From the position of the bovine hemoglobin standard the molecular weight is estimated to be about 67, 500, which agrees with Horowitz's determination (58). Figure 15 compares undialyzed TL and TS extracts at pH 8. O in 0. 1M sodium phosphate buffer and again reveals a single peak of activity for each in the same place. The sucrose gradient centrifugation data was confirmed by gel filtration with G-lOO and G-200 Sephadex. 53 FIGURE 13. Thermal inactivation of TLB and T513 at 59° c. Ordinate: residual activity plotted on a logarithmic scale. Abscissae: time of exposure to indicated temperature. t 1/2 = half-life. LOG PERCENT INITIAL ACTIVITY 100 T513; t 1/2 = 210 seconds. 90.. \ \ \TLB; t 1/2 = 85 seconds. 80~ \ 4 70... 60 1 T I 15 3O 45 TIME, SECONDS 54 . 1 .. ’ :1 "Z 'A -l'd." Taufi'il if— .1 ' ".II 1"- 1 FIGURE 14. Sedimentation patterns of crude, dialyzed and undialyzed TS extract and crystallized bovine hemoglobin in a linear 5 to 20 percent sucrose gradient. Sample Sizes: Dialyzed TS = O. 2 ml Undialyzed TS = 0.2 ml Hemoglobin Standard 2 O. 2 ml (2. 0 mg) Centrifuged for 24 hours in a Spinco SW 39 rotor at 34, 000 rpm at 5 C, in sodium phosphate buffer, 0.1M, pH 8.0. Total volume for all tubes was 5. 3 ml 55 1 . .. SEC CFOEE O A ENZYME UNITS FIGURE 14. Sedimentation patterns of crude dialyzed and 180* , s _ 0’ Dialyzed T extract —A—-A——A— 1604 ' 0‘ Undialyzed TS extract. l40~r —. . . ,. 0' Bovine hemoglobin 120, ’ 0- —o o o 1001 P 0‘ 80 n ” O. 60 d - O. _ O, _ o. ’\‘A: . _ . . . . 12 8 4 TOP FRACTION NUMBER BOTTOM undialyzed TS extract and crystallized bovine hemoglobin in a linear 5 to 20 percent sucrose gradient. 56 o ABSORBANCY AT 410 mu "PCI' FIGURE 15. Sedimentation patterns of crude TL and TS extract in a linear 5 to 20 percent sucrose gradient. 1‘ Sample Sizes: TL = 0.2 ml TS 0.2 ml ll Hemoglobin Standard (Not shown) = O. 2 ml (2. 0 mg) ‘ Centrifuged for 24 hours in a Spinco SW 39 rotor at 34, 000 rpm at 5° C, in sodium phOSphate buffer, 0.1M, pH 8.0. Total volume for all tubes was 5. 3 ml 57 O EN ZYME UNITS FIGURE 15. Sedimentation patterns of crude TL and TS extract in a linear 5 to 20 percent sucrose gradient. 135* “45 . TL extract. 120* ~40 A A A 105” TS extract. *7 35 o c o 90~ ( 7‘ 30 U) E-1 H 25.5 1 L, 2 ' i; 20 60 _. T" 2 Lil 4 45 __ "' 15 r 30 .. - 10 15* " 5 ------ IIIITUII'III'T "'--_"‘_'-‘---_r'r 36 32 28 24 20 16 12 8 4 TOP BOTTOM FRACTION NUMBER 58 DISCUSS ION AND CONC LUSIONS The evidence presented in this study clearly indicate the presence of multiple forms of tyrosinase in strains TS and TL, and their convertibility. The phenomena originally reported by Fox and Burnett (67) in a different strain of Neuospora crassa have been confirmed. Interconversion of different tyrosinases is not restricted to Neurospora. Jolley and Mason (82) reported a similar phenomenon among the multiple forms of mushroom tyrosinases. Unlike horse heart cytochrome c (83), the conversion of Neurospora tyrosinase is not due to protein aggregation and dis 5 ociation. Study of the multiple forms of tyrosinase in NeurOSpora was made extremely difficult by their instability. The most pronounced S L difference between T and T in their quantitative distribution was observed in crude extracts. Gel filtration and dialysis promoted conversion in both extracts to the same electrOphoretic and chromatographic form even though this component differed with regard to heat stability. Dialysis also relieved an apparent inhibition of tyrosinase found in all extracts. Indeed, it may be suggested that the multiple forms themselves are artifactual and due to differential association with a small molecule or ion acting as an inhibitor. In fact, however, TL3 and TLZ do not exhibit 59 __ L1. -9! .F-hl .. 111 apparent inhibition and when they were subjected to re—electrOphoresis under conditions which did not change their environment after the original fractionation (i. e. after dialysis against Ficoll), they remained stable and did not change their electrophoretic positions. The inhibitor therefore must be ruled out as the cause of the multiple LA and TSB to TSA forms. In addition, conversion of TLB to T proceeded even though the inhibitor effect was removed by previous dialysis or gel filtration. The considerations presented here do not rule out the possibility of the association of tyrosinase with another small molecule or even another protein. The important point, however, is that the multiple forms exist regardless of the uncertainty about the basis of their differences. The significance of this statement is strengthened by the fact that the distributions of the multiple forms is different in TL and TS, two strains with a known genetic difference. It is probable that the three tyrosinases produced by each strain differ in tertiary configuration. The data strongly suggest that T3: TA, T2=TB, and T1=TC, even though a direct demonstration was not possible. The prOportions and order of appearance of TA, TB, and TC upon elution from DEAE cellulose are what one would predict from the distribution of T1, T2, and T3 after continuous-flow electrophoresis. ElectrOphoresis reveals TLZ>TL3>TL1 and TS3> TSZ> T51, while DEAE cellulose chromatography shows TLB> TLA> TLC and TSA> TSB>TSC. With 60 regard to charge, T1 is more negative than T2 and T2 is more nega- tive than T3, while TC is more negative than TB and TB is more negative than TC. T2 and T1 are converted to T3, while TB and TC are converted to TA. Moreover the l31a-153OO strain of Neurospora crassa produces a tyrosinase indistinguishable from the TS tyrosinase. Fox and Burnett (67) reported that the prOpor— tions of T1, T2, and T3 in this strain were 5. 9, 12.8, and 81. 3%, respectively, of the total tyrosinase content in fresh extracts as determined by continuous-flow electrOphoresis. They also showed that the conversion, after incubation, of T1 and T2 to T3 was quantitative. In fresh extracts TC, TB, and TA constitute 5. 6, 12.8, and 81.6% of the total tyrosinase, respectively. The con- version of TB and TC to TA, after incubation was shown in this study also to be quantitative. The thermal inactivation data of tyrosinase obtained with the TL strain agrees reasonably well with that reported by Horowitz's group. A major discrepancy exists with regard to the TS strain. The activation energy of inactivation for TS has been given by Horowitz and Fling (56) and Sueoka (59) as 60, 000 calories per mole. The value obtained in this study was 27, 000 calories per mole. Using the thermal half-lives for TS at three different temper- atures given by Horowitz in an early paper (55), the activation energy of inactivation was calculated to be 25, 000 calories per mole, a 61 figure in agreement with that reported here. The reason for these discrepancies is unknown. Based on the data obtained herein, Fox's suggestion that the T locus controls the equilibrium in the interconversion of the alternate forms of tyrosinase (67, 68) must be abandoned. It can also be concluded that the multiple forms found in both TL and TS are not due to the presence of different size protein aggregates. In addition differences in molecular weights do not form the basis of an explanation for the differences in heat stability between TL and T5. The reason for the heat stability difference is still unknown. A difference in tertiary structure between the L form of tyrosinase and the S form is not excluded by anything reported here or else— where. Horowitz's suggestion that the difference is attributable to differences in primary structure must await a complete sequence analysis for ve rification. 62 SUMMARY 1. Multiple forms of tyrosinase were found in extracts of two strains of Neurospora crassa (TS and TL). 2. Three forms of tyrosinase were observed in each of the strains by continuous-flow electrOphoresis. The quantitative dis- tribution of the three forms was different in the two strains. The forms were designated T1, T2, and T3. 3. In both strains, conversion of T1 and T2 to T3 occurred after dialysis or gel filtration. 4. Ion-exchange column chromatography also reveals three. forms in both strains. These were designated TA, TB, and TC, and the quantitative distribution paralleled that found by continuous- flow electrophoresis . 5. Dialysis or gel filtration of the extracts before chromatog- raphy results in a conversion of both TB and TC to TA in both strains. 6. In TS extract the conversion is quantitative. 7. Direct attempts to cross identify the electrophoretic and chromatographic forms were frustrated, but a variety of indirect 63 evidences indicates that TA=T3, TB=T2, and TC=T1. 8. Gel-filtration with G-lOO and G—ZOO Sephadex and sucrose gradient centrifugation of dialyzed and undialyzed TL and TS extracts show only one molecular species with a molecular weight of approximately 67, 000—68, 000. This result was interpreted to L and TS are mean that the multiple forms of tyrosinase found in T not a result of differential protein aggregation. In addition, the difference in heat stability between TL and TS cannot be explained by differences in molecular weight. 0 9. Thermal inactivation studies of some of the multiple forms of TL and TS tyrosinases indicate that the multiple forms them- selves do not account for the heat stability differences between TL and TS . 64 ‘3‘ REFERENCES Crick, F.H.C., 1958. On Protein Synthesis. Symp. Soc. Exper. Biol., 12: 138-163. Spiegelman, S. , 1960. Factors Modulating the Biochemical Expression of Genetic Systems, in GENETICS, TRANS- ACTION OF THE FIRST CONFERENCE ed. by H. Eldon Sutton, p. 177, Josiah Macy, Jr. Foundation, New York. 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