MULTIPLE FORMS OF PLANT PHENOLOXIDASE Thasis for the Degree of Ph. D. MECHEGAR STATE EleVERSiTY Spéroa Minas Censtanténidas 196.6 TYIJBRARY ‘ Michigan State mme'r/fl/I/lmm Ill/W -._.. w“ i...__ This is to certify that the thesis entitled MULTIPLE FORMS OF PLANT PHENOLOXIDASE presented by Spiros Minas Constantinides has been accepted towards fulfillment of the requirements for _P_h_,_n_,_ degree in .Eond_S.c_ie.n c e Majofiéofessor Date JUly I9, 1966 0-169 University {- w ABSTRACT MULTIPLE FORMS OF PLANT PHENOLOXIDASE by Spiros Mines Constantinides The phenoloxidase (PO) system present in the tissues of mushrooms, potatoes and apples was shown to exhibit the phenomenon of multiple forms. Using an electrOphoretic method which employs polyacrylamide gel, distinct multiple forms of P0 were obtained. Cathodical and anodical enzyme forms were separated. These were specific for each species or variety studied. Elution, recovery and other tests ruled out the possibility of artifacts. Substrate specificity was clearly shown to exist. Two tyrosine specific multiple forms were separated. Other substrates showed distinct variations in the mode of reactions with each multiple form. The lag period for a given substrate was different for the different forms of PO. Room.temperature up to five hours had no effect on the mul- tiple molecular form pattern, but higher temperatures of up to 60 C caused fragmentation of the dihydroxyphenylalanine (DOPA) specific forms. A group of closely related DOPA specific multiple forms had the unique ability to withstand the temperatures of 70 C for sixty minutes. Sulfite incubated with the enzyme extract caused inactivation of certain forms. Spiros Minas Constantinides The tyrosine specific multiple forms were little affected by relatively high concentrations of sulfite. Short periods of incubation of the multiple forms with sulfite on the gels followed by washing with water showed no permanent inactiva- tion of the multiple forms. High doses of 2 x 106 rads of X’-irradiation caused very slight inactivation, the pattern of multiple forms being similar to those held for seven hours at 22 C. Urea (4.0 M) inactivated completely the tyrosine specific forms while the DOPA specific forms were gradually inactivated as the urea concentration was increased. Ethylene- diamine tetra acetic acid (EDTA) at low concentrations had no effect on the DOPA specific multiple forms, while at ex- tremely low concentration of 0.05% the tyrosine specific bands were inactivated. Trypsin degradation inactivated most of the forms and also gave rise to a new form. Mercapto- ethanol and cysteine-H01 completely destroyed all forms. Ascorbic acid had no effect on the multiple forms. Incuba- tion with excess of DOPA caused inactivation of all the multiple forms except for one group. Drastic purification treatments tended to inactivate some of the multiple forms. Fewer multiple forms were separated from commercial mush- room P0 than from the Agaricus campestris preparations used in this study. The P0 system seems to be installed in the plant tissues at the very early stages of development. No tissue Spires Mines Constantinides specificity of multiple forms was apparent. The intra- cellular distribution of P0 seemed to be specific for each species. In potatoes most of the activity was present in the microsomal and the final supernatant fractions, while in mushrooms and apples most of the activity was in the mitochondrial fractions. Intracellular compartmentalization of the multiple forms of P0 was also shown'to exist. In mushrooms the tyrosine specific form existed only in the final supernatant, and not in the mitochondria, while the mitochondria exhibited two DOPA specific bands only. Also in apples the pattern of the mitochondrial fraction was different from that of the supernatant. MULTIPLE FORMS OF PLANT PHENOLOXIDASE By Spiros Mines Constantinides A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1966 DEDICATION To my Mother and my Wife ii ACIG‘IOWLEDC‘JMNTS The author wishes to express his sincere appreci- ation to Professor Clifford L. Bedford for his guidance and continuous encouragement throughout the course of this work and during the preparation of this manuscript. The author is also deeply indebted to Professor P. Markakis for his beneficial suggestions on the multiple forms of enzymes, and to Professor J. R. Brunner who pro- vided laboratory facilities and contributed valuable dis- cussions. ‘ Thanks are also extended to the members of the guidance committee, Dr. Geo. Borgstrom, Dr. W. Deal, Dr. L. G. Harmon, Dr. P. Markakis and Dr. S.Schanderl. Sincere gratitude is expressed to Mr. G. Birk for his assistance in obtaining specimens of mushrooms through the invaluable c00peration of the Great Lakes mushroom 000p., Warren, Michigan. The excellent photographic job performed by Mr. P. Coleman of the Experimental Station Photo Laboratory of Michigan State University is greatly appreciated. The author most gratefully acknowledges the financial assistance for the scholarship granted for the initial part of his studies by the Greek Foundation of State Scholarships iii (I.K.Y) and for the financial support by the Michigan State Agricultural Experiment Station. The patience, understanding and encouragement of the author's wife, Niovi, throughout the course of this graduate program are gratefully acknowledged. iv ACKNOWLEDGMENTS . . . LIST OF TABLES . . . LIST OF EIBURES . . . INTRODUCTION . . . . REVIEW OF LITERATURE TABLE OF CONTENTS MATERIALS AND METHODS . . . . Enzyme source Separation of multiple forms Intracellular distribution of Extraction of mitochondrial phenoloxidase Protein determination Enzyme activity determination RESULTS AND DISCUSSION phenoloxidase Multiple forms of phenoloxidase Substrate specificity of the multiple forms Effects of various treatments on the multiple form pattern of mushroom phenoloxidase Intracellular distribution of phenoloxidase Intracellular compartmentalization of the multiple forms of phenoloxidase CON C LUSIONS O O O O O O O O O LITERATIJRE CITED 0 O O O O O O O O O O O O O O Page iii vi vi 21 29 A6 50 Table I. Figure 1. 2. 3. 4. 5. 7. 8. 9. 10. ll. 12. LIST OF TABLES Substrate specificity of multiple forms of mUShroomSoooooeoooeoeecoco. LIST OF FIGURES Differential centrifugation scheme . . . . . . Effect of concentration of gel on the resolution of multiple forms of phenoloxidase (P0) in mUShI'OomSoooeoooooooooeooo P0 active multiple forms and inactive protein in mUShroomSooooooooooooooooo Multiple forms of P0 from different genera . . Cathodical (pH 8.3) and anodical (pH 4.3) mul- tiple forms of P0 in mushrooms . . . . . . . Two-phase polyacrylamide electrophoresis. Sep- aration of the multiple forms of P0 in mush- roomSooooooooeooooooooeo Effect of current on the separation of multiple forms Of P0 in mushrooms 0 o o o e o o o o 0 Recovery of multiple form of P0 in mushrooms following elution from'the gels . . . . . . DL dihydroxyphenylalanint(DOPA) and L-Tyrosine specificity of multiple forms of P0 in mUShroomScoco-00000000000. Substrate specificity of multiple forms of P0 inmUShroomSoooooooeoeoecoo. Species and genus specificity of multiple forms OfPOinmUShroomSooeoooeoococo Varietal specificity of multiple forms of P0 inapples................. vi Page 33 Page 60 60 60 60 61 61 61 61 62 62 62 63 Figure Page 13. Effect of acetone on multiple forms of P0 in mushrooms . . . . . . . . . . . . . . . . . 63 14. Multiple forms of commercial mushroom P0 from four different companies . . . . . . . . . 63 15. Effect of 5 C with time on the multiple form of POinmUShrOOHlSooooeoooooocoo 63 16. Effect of room temperature (22 C) with time on the multiple forms of P0 in mushrooms . . . 64 17. Effect of 40 C and 50 C with time on the mul- tiple forms of P0 in mushrooms . . . . . . 64 18. Effect of 60 C, 70 C and 100 C on the multiple forms of P0 in mushrooms . . . . . . . . . 64 19. Effect of sulfite on the multiple forms of P0 in mushrooms. Incubation on the gel. Sub- Strate:lXoeooooooooeoeeoo 65 20. Effect of sulfite on the multiple forms of P0 in mushrooms. Incubation on the gel. Sub-' Strate:2Xoooooooooooooeo 65 21. Effect of 1000 ppm sulfite with different sub- strate concentrations on the multiple forms OfPOinmUShrooms 000000000000 65 22. Effect of sulfite on the multiple forms of P0 in mushrooms following washing off of the sulfite. Incubation on the gel.. . . . . . 65 23. Effect of incubation of sulfite (6 hrs.) on the multiple forms of P0 in mushrooms. Sub- StrateDL'mPAooooooeooooooo 66 24. Effect of incubation (6 hrs.) of sulfite on the multiple forms of P0 in mushrooms. Sub- Strate: L-TYI‘OSine o o o o o o o o o o o o 66 25. Effect of Ethylene diamine tetracetic acid (EDTA) on the multiple forms of P0 in mush— rooms. Substrate: L-Tyrosine . . . . . . 66 26. Effect of EDTA on the multiple forms of P0 in mushrooms. Substrate: DL—DOPA . . . . 66 vii Figure Page 27. Effect of urea on the multiple form of P0 in mUShrOOmSo SUbStrate: DL’DOPA e o o e e o o 67 28. Effect of urea on the multiple forms of P0 in mushrooms. Substrate: L-Tyrosine . . . . . 67 29. Intracellular distribution of total P0 activity 67 30. Intracellular compartmentalization of the mul- tiple forms of P0 in mushrooms . . . . . . . 67 viii INTRODUCTION Although the first observation of enzyme multiplicity was made with lactic dehydrogenase, it is now known to be a wide- spread, perhaps general phenomenon. More than one hundred en- zymes have been shown to exist in unicellular and multicellular species of plants and animals (Furness 1961). Studies over the past seven years have shown that specific types of enzy- matic activity may be associated with more than one protein. Differences were found between comparable enzymes in differ- ent tissues of the same organism. In addition, a single tissue may yield several enzymes catalyzing the same reaction but having differences in their physical, chemical and kinetic preperties (Markert and Moller 1959). At the present time there is no unanimity on the method of identification of a particular form. The multiple molecular forms have been dis— tinguished from one another by electrOphoresis, chromatogra- phy, salt fractionation ultracentrifugation, immunoelectro— phoresis and reaction kinetics. The electrOphoretic method is the most commonly employed, resolving macromolecules on the basis of both charge and size (Whipple 1964). The following terms have been used by different au- thors for the multiple forms: isoenzymes, isozymes, iso (enzyme's name) as isoamylases, electrOphoretic variants, 1 multiple forms, polymorphs, electrOphoretic components, and multimolecular forms. Webb (1964) recommended that multiple molecular forms of an enzyme in a single species should be known as isoenzymes, although this recommendation is not to be interpreted as excluding the use of isozyme if any individual author prefers it. We do not know how or why enzymatic heterogeneity exists, what advantage it offers to the cell, or how the mul— tiple forms differ chemically or structurally from each other. Multiple enzyme forms could be products of single genes, by modification in the tertiary structure of a gene product or by alterations of a basic protein structure, depending on the site of attachment within the cell. The relationship of this phenomenon to the one—gene one-polypeptide theory and to the problem of cellular differ- entiation poses important biological questions which remain to be solved. Their study promises to expand our knowledge in a variety of fields ranging from embryology and the studies of evolution, to physiology and pathology. The study of them has already proved that it has clinical diagnostic applica- tion (Kaplan et a1. 1960). Phenoloxidase (PO), classified as an o-diphenol: 02- oxidoreductase EC 1.10.3.1, by the Commission on Enzymes of the International Union of Biochemistry (1961), and referred to as tyrosinase, polyphenoloxidase, phenoloxidase, phenolase, catechol oxidase, laccase and cresolase, is widely distributed in plants, invertebrates and higher animals. Although phenoloxidases are widely distributed through- out the plant kingdom, their role in plant metabolism is not clearly understood. It has been preposed that plant P0 acts as a hydroxylating enzyme in vivo, catalyzing hydroxylations which are believed to occur during the biosynthesis of o- diphenolic compounds in the potato tuber (Patil and Zucker 1965). P0 is characterized by two different catalytic activ- ities, both involving molecular oxygen. The oxidation of mono- hydric phenols such as tyrosine, phenol or p-cresol involving the insertion of a hydroxy group ortho to the one already present is referred to as cresolase activity. The removal of two hydrogen atoms from a dihydric phenol as catechol to yield the corresponding o-quinone is referred to as catecholase activity. .Mallegg and Dawson (1949) and Mason 1955a, b) have indicated the reaction as follows: monOphenol + 2e + 02 3 o-diphenol + 0= 2 o-diphenol +02 > 2 o—quinone + 2 H20 However Dressler and Dawson (1960a, b) have indicated that oxidation of monOphenol does not proceed via an o-dihydric phen- ol. In the past few years data have been presented indi- cating that plants contain several different phenoloxidases that exhibit unique catalytic and physical preperties. How- ever the information is very confusing and the results are contradictory. The present study was therefore undertaken in an attempt to elucidate the nature and properties of the phenoloxidase system from plant tissues and from within the cell structures. REVIEW OF LITERATURE In recent years molecular heterogeneity of proteins has often been observed, particularly for enzymes, where the existence of multiple molecular forms has been extensively documented. Markert and Muller (1959) introduced the concept of an isoenzyme, which they defined as one of the molecular forms in which protein may exist with the same enzymatic specificity within a single organism. This term has gained wide popularity and has been used in an operational sense to cover any series of enzymes with roughly similar prOperties regardless of genetic relationship. In nearly all cases the genetic relationships are not known. A review of the work up to 1961 on multiple forms of different enzymes is presented by Furness (1961). The first electrophoretic method used to demonstrate the multiplicity of enzyme forms was starch gel electro- phoresis (Furness 1961). Later other gel electrOphoretic methods, especially polyacrylamide gel (disk), were used (Whipple 1964). The heterogeneity of lactic dehydrogenase was first tested by Wieland and Pfleiderer (1957). Markert and Appella (1961) investigated the physicochemical preperties of two purified isozymes from beef heart lactate dehyrogenases (LDH) and found a remarkable similarity between the two. Markert 5 (1963) later found that LDH from beef tissue may be resolved electrophoretically into five isozymes, each being a tetramer. These tetramers can be dissociated into 2 monomers by freez- ing in l M.NaCl, namely LDH-l and LDH-5, that are differenti- ated by charge and aminoacid composition. 0n thawing, reasso— ciation into functional tetramers occurs. A mixture of equal quantities of the two isozymes after dissociation and reasso- ciation produces all five isozymes in the expected proportions of l:4:6:4:1. Kaplan and Ciotti (1961) suggested that there has been considerable evolutionary change in the lactic and malic dehydrogenases and that this enzyme change is not with- out significance and is of importance in the survival and perpetuation of new species. Laufer (1961) found the existence of isozymes among the hydrolytic dehydrogenating enzymes that occur in cell free blood preparations. He also preposed that the insect hormones act on specific sites of the chromosomes, the isozymes being a particular expression of these developmental interactions. Higgins and Kiser (1964) discovered that urine from tract inflamations gave a different isozyme pattern than normal. Withy- combe and Wilkinson (1964) found that spermatozoal extract ex- hibited greater dehydrogenase activity with 2-oxobutyrate as substrate than with pyruvate as the substrate. Mahy and Rowson (1965) reported that of the five electrOphoretically distinct forms normally present, only the slowest migrating LDH-5 is increased in amount during Riley virus infection. Brody (1964) reported that the individual isozymes of LDH differs not only in electrophoretic mobility but also in chemical composition, Michaelis constant, activity against substrate analogues, reaction rate with coenzyme analogues, immunolog- ical characteristics, thermal stability and susceptibility to inactivation by excess substrates and by certain chemical inhibitors. Nine forms of LDH have been reported to be pres- ent in various tissues of the brook trout (Goldberg 1965). This was an indication that three polypeptide subunits take part in the lactate dehydrogenase composition of this species. Goldberg also observed that the sperm specific LDH contains polypeptide subunits that differ from those in LDH 1, 2, 3, 4 and 5. The recognition of the molecular heterogeneity of enzymes in organisms has offered a new dimension for studying the genetic control of enzyme synthesis (Vesell 1965). In the case of lactate dehydrogenase five distinct types can be identified in most mammalian and avian tissues by the method of starch electrOphoresis. Katz and Kalow (1965) reported several isozymes of lactate, malate and isocitrate dehydrogenase in human skeletal muscle, heart and liver. The multiple forms of acid phOSphatase found in animal organs are distinct molecular forms. There is probably a genetic basis for the multiple forms of the enzyme since they appear to be species specific and not organ specific (Moore and Angelletti 1961). Alcohol dehydrogenases in liver were studied by McKinlethcKee and Moss (1965). They found that horse liver alcohol dehydrogenases are heterogeneous on starch gel electro— phoresis and that this heterogeneity is due to coenzyme- enzyme complexes. Carter et a1. (1961) separated five forms of ribon- uclease from crystallyzed bovine pancreatic ribonuclease. Three electrophoretic variants of catalase were found in maize endosperm and these were under genetic control (Scan- dalios 1965 and Beckman et al. 1964b). Catalases purified from human and rat livers showed significant differences when compared to purified erythrocyte catalases of these species by immunoelectrophoresis (Nishimura et a1. 1964). Starch block zone electrOphoresis was used to isolate five electrophoretically distinct active cellulolytic com- ponents in the crude extracellular cellulase system of Streptomyces antibiotigus (Enger and Sleeper 1965). Starch gel electrOphoresis showed three distinct mole- cular forms of enolase in different species on Salmonidae (Tsuyuki and Weld 1964). Augustinsson (1961) studied the multiple forms of esterases in vertebrate blood plasma and found each animal species had its own typical set of plasma esterases. The pattern of protein and of esterases found in Streptococcus faecalis differed from those in Streptococcus durans. Other investigators studied the substrate specificity and inhibit- ing effects of esterases (Allen 1961 and Ecobichon 1965). Secchi and Dioguardi (1965) found four esterase fractions in serum and six in the hepatic tissue of man. Two multiple forms of fumarase isolated from Candida utilis showed marked differences in kinetic properties from that of pork heart fumarase (Hayman and Alberty 1961). The presence of multiple forms of,B-galactosidase were reported by Furth and Robinson (1965) in rat tissues, and in Egcherichia coli by Appel and Alpers (1965). In ‘Nggrgspora crassa twof9-galactosidases were found by salt and column fractionation, and they differed markedly in their pH Optima, heat susceptibility and affinity for substrates (Lester and Byers 1965). Kinetic and electrOphoretic evidence has indicated the presence of only two forms in human cell cultures of glucose adenosine triphosphate phosfotransferase. In rat liver four distinct electrOphoretic forms were found (Katzen et a1. 1965). Three major components of glutamate-aspartate trans- aminase were found in the heart of pig when analyzed with starch gel (Martinez-Carrion et a1. 1965). Yeast hexokinase has been separated into several 10 fractions by diethylaminocellulose column chromatography (DEAE) (Kaji et al. 1961). In rat tissues at least four types of hexokinases were distinguished by starch gel electro- phoresis and chromatography on DEAE cellulose or calcium hydrozylapatite. The liver contained all four types while the kidney contained three, epidydimal fat pad, skeletal muscle, heart and brain, each contained two. Each type of this family of hexokinases was shown to be different from the other (Katzen and Schimke 1965). Schulze et al. (1965) found hexokinases to be susceptible to proteases. Tissue specific variants of leucine aminopeptidase have been shown to exist in maize (Scandalios 1964). Four different molecular forms of leucine aminopeptidase were found in maize endosperm by means of starch gel electro- phoresis (Beckman et al. 1964a). Malate dehydrogenase activity is a preperty of a number of different proteins present in tissues of sea urchins and star fish. These proteins differ in electro— phoretic mobility, in rates of reaction with pyridine nucle- otide analogues and d-malate, in thermal inactivation, and solubility in ammonium sulfate. 0f the five l-malate dehy— drogenases present in Arbacia eggs only three are found in very young embryos and four after 12 hours of development (Moore and Villas 1963). Kitto et al. (1966) reported five com- ponents of the mitochondrial malate dehydrogenase obtained from chicken heart tissue. 11 Evidence was presented based on differential extrac- tion procedures, pH dependence studies, activation or inac- tivation by sodium dodecyl sulfate, and differential temper— ature inactivation, that the pulp of the banana contains at least three molecular forms of pectinesterase (Hultin and Levine 1963). McCune (1961) obtained six peroxidase active frac- tions from corn leaf sheath preparations by starch gel electrophoresis. The four major bands differed in their substrate specificity. Paul and Fottrell (1961) demonstrated the existence of differences in the isozyme pattern between species, while Klapper and Hackett (1964) showed that commer- cial horseradish peroxidase contains multiple components. Yu and Hampton (1964) reported that pathogens can influence the peroxidase isozyme content of plant tissues. Three pro- tein components with peroxidase activity were separated from Eigg§_glabrata latex by chromatography on DEAE cellulose at pH 7.0. The peroxidase differed slightly from that of horse- radish peroxidase (Kon and Whitaker 1965). Multiple Forms of Phenoloxidase In the past few years experiments showed that crude phenoloxidase preparations actually are mixtures of several P0 proteins each exhibiting unique catalytic and physical properties. 12 Mushrooms: Mallette and Dawson (1949) first reported the presence of multiple forms of mushroom tyrosinase. Smith and Krueger (1962) using a column of hydroxylapatite, chro- matographically separated the crude extract of mushrooms into a series of purified fractions, that included the classically catecholase and cresolase enzymes as well as other enzyme types. Bouchilloux et al. (1963) obtained four active pro- teins using a column of hydroxylapatite. Jolley and Mason (1965) concluded that the existence of two unlike subunits, one largely cresolase active and the other catecholase active, combining in several proportions may explain the dif- ferences observed among the multiple forms of tyrosinase toward mono and diphenols. They also found that the mushroom isozymes were to a certain degree interconvertible, depending on pH, ionic strength and protein concentration. Neurospora crassa: Horowitz and Fling (1953) showed the presence of several forms of tyrosinase in N. crassa. Initially they were distinguished on the basis of their heat resistance and later they were Shown to differ in their electrOphoretic prOperties (Horowitz et al. 1961). They also suggested that the different forms of tyrosinase were alike functionally but differed in structure since the prep- arations appeared to have similar substrate specificity, Km values and pH optima. Sussman (1961) found that a thermostable and thermolabile form of tyrosinase existed. Fox and Burnett 13 (1962) were able to separate the enzyme into three components by continuous flow paper electrOphoresis. These components differed in their electrOphoretic and immunochemical properties as well as thermostabilities, and interconversion of the three forms was observed. Fling et al. (1963) isolated two forms by diatomaceous earth and found that a rapid association- dissociation between molecular species (monomer and tetramer) of tyrosinase occurred. Mammalian: Brown and Ward (1958) and Shimao (1962) indicated the presence of multiple forms of tyrosinase. Pomerantz (1963) separated and partially purified two tyrosin- ases from hamster melanoma. They were distinguished by DEAE cellulose chromatography and starch gel electrOphoresis. Potatoes: The P0 system of Kennebec potatoes had two components Patil et al. (1963). Uritani (1963) using starch gel electrophoresis, separated crude extracts of sweet potatoes into three distinct bands, each showing a capacity to catalyze the oxidation of chlorogenic acid. Broad beans: Robb et a1. (1965) showed the presence of four multiple forms of tyrosinase. He was not able to distinguish their heterogeneity by their copper content or substrate specificity. Tobacco: Sisler and Evans (1958) reported that chlorogenic acid is a better substrate for the crude P0 from tobacco than for a comparable preparation from mushrooms. 14 Apples: walker (1964) found apple P0 to be associated with the mitochondria. Harel et al. (1965) found the enzyme to exist also in the chlorOplasts. Other sources: Two forms of laccase have been puri- fied from the culture medium of Polyporus versicolor using ammonium sulfate precipitation, chromatography on hydroxylap- atite and DEAE chromatography (Mosbach 1963). The P0 system of DrOSOphila melanegaster was shown to come from at least four protein components. Two components had tyrosinase ac- tivity while the other two had dihydroxyphenylalanine activity (Mitchell and Weber 1965). Phenoloxidase: Preparation and General Properties The phenoloxidase system of enzymes is widely distrib- uted in nature, and highly purified P0 from microbial, plant and mammalian sources have been Studied (Kubowitz 1937, Keilin and Mann 1938, Dawson and Magee 1955, Sussman 1949, Mallette 1950, Joslyn and Ponting 1951, Schwimmer 1953, Mason 1955a and b, Bonner 1957, Kassab 1961, Hayaishi 1962, and Mason 1965). Yasunobu (1959) tested the substrate specificity of a number of P0 from various sources, both plant and animal. He concluded that these enzymes catalyze the oxidation of a wide variety of substrates, but that each individual enzyme tends to catalyze the oxidation of one particular phenol or a par- ticular type of phenolic compound more readily than others. 15 He also suggested that true tyrosinases are these enzymes which catalyze the oxidation of both mono and diphenols. Different molecular weights of phenoloxidases have been reported. Dawson and Magee (1955) in their review indi- cate that as the enzyme occurs in nature it is likely to have a molecular weight of 200,000 to 400,000 and that purifica- tion results in fragmentation. Other different molecular weights have been reported by'Mallette and Dawson (1949), Yasunobu (1959), Frieden and Ottesen (1959), Krueger (1959) and Kertesz and Zito (1957). The tyrosinase of N; crassa was found to be a crys- tallizable COpper containing enzyme with a molecular weight of 32,000 - 34,000. It is normally produced during the sexual phase of the life cycle, but not during vegetative growth ex- cept when growth is inhibited by starvation or by aminoacid analogues or d-aromatic amino acids (Horowitz et al. 1964). Tyrosinase of N; crassa is synthesized under conditions which are unfavorable for growth and general protein synthesis, i.e. after exhaustion of culture medium (Fox et a1. 1963). This implies some difference between the mechanism of tyro- sinase synthesis and that of general protein synthesis and it seemed likely that the steps involved are those concerned with the synthesis of the polypeptide chain of the enzyme. The polypeptide as originally released from the sites of syn- thesis lacks enzymatic activity. It therefore constitutes a l6 kind of proenzyme (protyrosinase) which requires activation. The presence of one or more additional macromolecular "acti- vating" factors, not tyrosinase itself, apparently is re- quired for protyrosinase activation. Rearrangement of ter- tiary structures seemed to be responsible for activation, either with or without the splitting off of a terminal peptide segment. Dressler and Dawson (1960a, b) suggested that the enzymatic sites for phenol and catechol are different, also that the oxidation of a monohydric phenol does not proceed via an o-dihydric phenol. Aerts and Vercauteren (1964) also reported that the oxidation of a monohydric phenol does proceed via the corres- ponding o-dihydric phenol and that phenoloxidase bears two types of active centers, one for the cresoloxidase activity and one for the catecholoxidase activity. Kertesz and Zito (1962) reported that the oxidation of a monohydric phenol by P0 in the presence of hydroquinone is preceded by an induction period. The induction period is increased rapidly by increasing the hydroquinone concentra- tion and decreased by the addition of small amounts of cate- chol. When the phenol or enzyme concentration was increased the induction period decreased and the rate of the oxygen consumption increased. Osaki (1963) stated that prolonged induction periods are to be expected when the concentration 17 of the substrate such as tyrosine is high. The induction period can be decreased by increasing the enzyme concentra- tion. Pomerantz (1964, 1966) found that the rate of tritium release as H20 in the tyrosinase l-Tyrosine-3, 5 T reaction was directly prOportional to the rate of hydroxylation. Dihydroxyphenylalanime (DOPA) was the most efficient hydrogen donor for hydroxylation. Tyrosine was found to exhibit an apparent substrate inhibition. Pomerantz suggested that re- action was prevented by the combination of excess tyrosine at the DOPA site and excess DOPA at the tyrosine site. Kean (1964) indicated that monophenols could inhibit the oxidation of o-diphenols by occupying sites on the enzyme that would be available to oxygen or to o-diphenols. Nelson and Dawson (1944) found that phenol inhibited the action of tyrosinase on catechol. Kendal (1949) also re- ported that phenol competitively inhibits the oxidation of catechol. Karkhanis and Frieden (1961) found a protein in- hibitor in the crude mushroom tyrosinase preparations. Lerner (1953) has shown that tyrosinase was inhibited by compounds which complex with cepper, by analogues which competitively inhibit its action and by metals that compete with copper. Mayberry and.Mallette (1962) reported that excess catechol inhibits its own oxidation by a competitive process, thus accounting for an observed optimum in substrate concentration. 18 Added phenol, although itself a substrate, inhibits the enzymatic oxidation of catechol by a mixture of two processes, competitive and non-competitive. Walker (1964) reported that diethylthiocarbamate, and dimercatoethanol were powerful inhibitors of the chlorogenic acid oxidation. He also reported that caffeic acid was formed when apple P0 was incubated with p-coumaric acid for long periods of time (4 hours). The thermal inactivation of apple P0 was investigated by Walker (1964) who found that inactiva- tion first became marked at 70 C and that activity was de- stroyed at 80 C. Sulfur dioxide is known to inhibit the browning caused by P0 (Ponting 1960). Sulfite was shown to prevent browning in the systems by combining with the enzymatically produced o-quinones and stOpping their condensation to melanins (Embs and Markakis 1965). Lyr and Luthardt (1965) found that by adding metabolic inhibitors to the culture fluid of fungi a strong induction could be obtained and the synthesis of a new tyrosinase pro- tein occurred. This mode of induction of tyrosinase iS' probably not restricted to fungi but may also occur in higher plants where tyrosine activity often increases in the region of sublethal injuries as a part of a defense mechanism. Intracellular Distribution of Enzymes and Their Multiple Forms Much has been written about the intracellular distribution 19 of enzymes and their isolation. Some of the early classical papers are those of Hogeboom et a1. (1947), Schneider and Hogeboom (1950), deDuve et al. (1955), Schneider and Hogeboom (1956), and Novikoff and Podber (1957). Many reviews have also been published. Allen (1964) found two groups of six isozymes in the esterases of Tetrahymena. One isozyme appeared to be local— ized in the microsomes, another in somewhat larger particles, while the remaining isozymes appeared to be localized in frac- tions that sediment with low centrifugal force. Hsu and Tappel (1964) found six intracellular hydrolases in the rat intestinal mucosa that were associated with lysosomes. Met— zger et al. (1965) reported that hepatic glucose dehydro- genase exists predominantly in the heavy microsomal fraction of homogenates. Kun and Volfin (1966) reported kinetic dif- ferences between malate dehydrogenase activities of cytoplasmic and mitochondrial extracts of the same tissue, and marked differences in catalytic activities between homologous enzyme preparations obtained from different tissues. Very little work has been done on the intracellular distribution of P0 or its multiple forms within the cell particles. Goldfish tyrosinase was found to exist both in the particulate and in the soluble fraction of skin homgenate (Kim and Chen 1962). Catechol oxidases were shown to exist in several subcellular fractions of apples (Harel et al. 20 1965). Starch gel electrophoresis separated three components from the cholorOplasts of apples and one component from the mitochondria. In the ink gland of the squid, tyrosinase was found to be present in the particulate form within the mitochondria (Vogel and McGregor 1964). It was suggested that this enzyme is synthesized or assembled in some measure in this site, remains latent, and is transmitted through the mitochondrial membrane into the cytoplasm to participate in melanogenesis. Chloroplasts of sugar beets have P0 activity (Mayer 1965). MATERIALS AND METHODS Enzyme Source The following plant materials were used to deter- mine enzyme activity, the presence of multiple forms and the intracellular distribution of the enzyme system and its multiple forms. Apples: (Pyrus malus). The following varieties at harvest maturity were used: Cortland, Jonathan, Northern Spy, Red and Golden Delicious, and Rhode Island Greening. In addition representative samples of Northern Spy at 30, 46, 63, 87, and 130 days after full bloom were used to de- termine the effect of maturity on the multiple form pattern. Mushrooms: Agaricus compestris, Agaricus placomyces, Amanita rubenscens, Coprinus comatus, qurinus micaceus, Suillus revillei, and Tricholoma venenata. Potatoes: (Solanum tuberosum). The following varieties were used: Aranac, Katahdin, Kennebec, Ontario, Rural Russett and Sebago. Representative samples used for enzyme preparations and the determination of multiple forms were packaged in moisture vapor proof polyethylene bags, frozen and stored at -22 C until used. For the determination of the intracellular distribution of the enzyme, the samples were stored at 2 C. All samples were prepared for analysis in a 2 C room. 21 22 Separation of Multiple Forms The least drastic procedures were used to homo- genize the tissue and to retain the integrity of the dif- ferent multiple forms present in the plant tissues. One part by weight of plant material was homogenized by hand in an all glass tissue grinder (Kontes Glass Corp.) with three to six parts of 0.25M sucrose in 0.05 M phosfate buffer at pH 7.0. The homogenate was immediately centrifuged at 20,000 X g for 20 minutes in a superspeed Serval Angle Centrifuge 88-1, and the supernatant recentrifuged at 100,000 X g for 2 hours (Beckman, Ultracentrifuge, Prepara- tive, Model L-2). The final supernatant was used for elec— trophoresis. The method of Davis (1964) and Ornstein (1964) de- veloped for serum proteins using polyacrylamide gel was modi- fied for the separation of the multiple forms of phenol- oxidase. Stock solutions for cathodical proteins:* A. l N HCl 48 m1, TRIS (Tris hydroxy methyl aminomethane) 36.3 gr, TEMED (N, N, N, N1, Tetramethylenediamine) 0.23 ml, and H20 to make 100 m1 (pH 8.8 - 9.0). B. 1 N HCl 48 ml, TRIS 5.98 gr, TEMED 0.46 ml, and H20 to make 100 m1 (pH 6.6 — 6.8). *All regents used were Eastman Chemicals products, Rochester 3, N. Y. 23 C. Acrylamide 60.0 gr, BIS (N. NéMethylenebiscrylamide monomer) 0.4 gr, and H20 to make 135 m1. D. Acrylamide 10 gr, BIS 2.5 gr, and H20 to make 100 ml. E. Riboflavin 4.0 mg and H20 to make 100 ml. F. Catalyst: Ammonium persulfate 0.14 gr and H20 to make 100 ml. G. Buffer (dilute to 1/10): Tris 6.0 gr, Glycine 28.8 gr, and H20 to make 1 liter (pH 8.3). H. Protein stain: Aniline black 1 gr and 7% acetic acid to make 200 ml. I. Tracking dye: 0.005% bromphenol blue solution. Working solutions: Lower gel: 7% A= 1.0 part, C= 1.4 parts, and H20 = 2.1 parts. 8% A= 1.0 " C= 1.6 " H20 = 1.9 " . In order to form gel the lower gel is combined with the catalyst F 1:1. Upper gel: B= 1 part, D= 2 parts, E= 1 part, and H20 = 2 parts. Stock solutions for anodical proteins: (pH 4.3)* A. 1N KOH 48 m1, Glacial acetic acid 17.2 ml. TEMED 4.0 ml, and H20 to make 100 ml (pH 4.3). B. 1N KOH 48 ml, Glacial acetic acid 2.87 ml, Tmmn 0.46 ml and H 0 to make 100 m1 (pH 6.7). 2 *All reagents used were Eastman Chemicals products, Rochester 3, N. Y. 24 F. Catalyst: Ammonium persulfate 0.28 gr and H2) to make 100 ml. G. Buffer (dilute to 1/10): Beta alanine 31.2 gr. Glacial acetic acid 8 ml, and H20 to make 1000 ml (pH 5.0). The rest of the stock solutions and working solutions were identical to the cathodical proteins stocks solutions. The enzyme preparation was not incorporated in the sample gel, but was layered directly on the top of the al- ready polymerized upper gel. About 0.4 ml of enzyme prepara- tion was used with 2% sucrose added to increase the specific gravity to facilitate layering and to prevent diffusion into the upper liquid. The gel tubes used were 3" x 0.5 mm o. d. From bottom to t0p, 2 inches of lower gel was introduced, 3/8 inches upper gel, and 6/8 inch length of tube was left for the sample to be introduced. The current used for the inactive protein separation was 5 milliamperes (MA) per tube. For the separation of the mushroom multiple forms 2 1/4 MA per tube was found to be optimum, for the potatoes 4 MA and for the apples 3 MA per tube were used. Higher currents resulted in band distortions. With mushroom and apple multiple forms the front was allowed to migrate 1 inch toward the cathode, but with potatoes the migration was extended to 1 3/4 inch. Total time of the "run" was 1 1/2 hours. 25 Preliminary studies were made to determine the Opti- mum concentration of substrate for color develOpment of the multiple forms. A concentration of 1.5 x 10"3 leor all sub- strates used gave good resolution. Higher concentrations resulted in a dark background. The gels were introduced in the substrate solution and left there until multiple form color bands develOped. Ethyl alcohol facilitated the develOpment of the bands. The gels were finally stored in 30% ethyl alcohol. Inactive proteins were stained with stock solution H, according to Davis (1964). The gel was destained by re- peated washings with 7% acetic acid. Electrophoresis was carried out at room temperature (22 - 24 C). Some of the preparations such as those from mushrooms seemed to have slow moving components as well as very fast components. To get the whole pattern within the limits of the gel tube with highest possible resolution and sharpness and to be able to separate some components according to size only, "two-phase or multiphase" gel. electrOphoresis was introduced. The gel tube was divided into two or more portions, each portion having different concentrations of acrylamide. Usually the concentration ran from low to high (5% - 10%) anode to cathode, from t0p to bottom of the gel tube. The length of each gel concentration was based on the size of the molecules to be separated. 26 For recovery studies, the portion of the gel incor- porating the multiple forms or form to be recovered was cut off from the rest of the gel with a sharp razor blade and cut into pieces in a small beaker containing 0.5 to 1.0 ml 0.05 M phosphate buffer pH 7.0. After allowing the gel to stand for 30 minutes at room temperature it was frozen. When the gel was needed it was thawed and the drip with 2% sucrose added was pipetted on to the polymerized upper gel of the electrophoretic tube and electrophoresis was started. Studies made using the method of Fling et al. (1963) involving acetone precipitation and column chromatography were found unsatisfactory because of destruction of enzyme activity and the failure to resolve the enzyme into its possible multiple forms. Paper electrOphoresis using the Spinco Durrum Type cell, continuous flow paper electro- phoresis (Model CP Spinco) and starch gell electrOphoresis did not give good separations of the multiple forms of P0. Intracellular Distribution of Phenoloxidase One part of weight of the plant sample was homogenized in an all glass tissue grinder with three to six parts of 0.25M sucrose. The homogenate was centrifuged at 300 x g in a clinical centrifuge for ten minutes, the supernatant re- moved, the precipitate resuspended twice in 0.25M sucrose and recentrifuged. The supernatants were combined and centrifuged at 300 x g for ten minutes to remove the cell debris. The 27 supernatant was centrifuged at 10,000 X g for 20 minutes to obtain the mitochondrial fraction. The mitochondrial frac- tions were centrifuged twice in 0.25 M sucrose. The combined mitochondrial supernatants were centrifuged at 100,000 X g for 2 hours to obtain the microsomal fraction and the final supernatant (Fig. 1). Enzyme activity and protein of the fractions was determined immediately. Extraction of Mitochondrial Phenoloxidase The mitochondrial fraction was suspended in a solu- tion of 0.1% Triton X - 100 (Rohm and Haas), in 0.25M phosphate buffer Pb 7.0. The suspension was blended in a waring blender for 10 minutes, allowed to stand for 30 minutes and centrifuged at 100,000 X g for 2 hours. Protein Determination The Lowry method was used (Lowry et al. 1951) with crystalline bovine serum albumin used as the standard. Enzyme Activity Determinations Phenoloxidase activity was determined by measuring the rate of oxidation of D L DOPA to d0pachrome at 475 m¢t Reactions were carried out in Beckman DU spectrOphotometric cells 1 cm diameter at room temperature. The following were introduced in the cell: 3 m1 of buffered substrate (0 .1 M phosphate at pH 6.0), 0.00 - 0.15 ml H20, and 0.05 - 0.20 ml 28 enzyme solution. The mixture (3.2 ml) consisted afterchlution of 1.41 x 10'2M DOPA. The enzyme solution was added with plastic plunger. Absorbancy readings were made at 30 second intervals for 2 minutes after introduction of the enzyme. Under these conditions the initial rate of change of absorb- ancy is an adequate measure of enzyme concentration. Enzyme unit = Sgggigggcy x 10‘3. Range of absorbency units used were from 20 x 10'"3 to 80 x 10’3. Specific activity was expressed as units of enzyme per milligram of protein. RESULTS AND DISCUSSION Multiple Forms of Phenoloxidase Polyacrylamide gel electrOphoresis was first suc- cessfully used by Davis and Ornstein for the separation of serum proteins. These gels are thermostable, transparent, strong, relatively inert chemically, and non-ionic. They can be prepared with a large range of average pore size as indicated by Davis (1964) and Ornstein (1964). The concentration of the polyacrylamide gel played a very important role in the resolution. Different concen- trations of gel were used ranging from 5% up to 10%. With 7 mushrooms, gel concentrations of 6%, 7%, 8%, and 9% were used (Fig. 2). The multiple form pattern of the mushroom PO consisted of three major groups from the anode (top) to the cathode (bottom) of the gel tube. These will be referred to as A, B, and C groups, respectively. With the 6% gel A=2 bands, B=3 and C=1; with 7% gel A=2, B=3, and C=2; with 8% gel A=2, B=3, and c=4; with 9% gel A=2, B=2 and c=3. The inactive protein showed a different separation pattern depending on the gel concentration (Fig. 3). Apple and potato P0 was best resolved using 8% gel (Fig. A). 29 30 Anodical multiple forms of PO were also found. Four weak bands of activity were evident migrating toward the anode at pH 4.3 (Fig- 5). Using the two-phase electrophoresis with 7% and 8% gel a new pattern was obtained where the single band of group C (last band toward the cathode) could be split into four bands, indicating the molecular sieving effect of the gel (Fig. 6). Elution of the four bands from the 8% gel and re- running on 7% and 8% gels, resulted in the appearance of a single band on the 7% and the same four bands on the 8% gel. The results of these studies showed that using dif- ferent gel strengths and obtaining different pore sizes, per- mitted the separation of protein molecules that differed in size and charge, including the multiple forms of enzymes. The current played an important role in obtaining good band separations. A current of 1.25 MA per tube was found to be Optimum for mushroom PO (Fig. 7). The different multiple forms could not be easily. liberated from within the pores of the gel. Only 65% of the total activity could be eluted. Repeated separation and elution showed no change in the multiple form pattern or position (Fig. 8). This would indicate that the multiple forms separated by polyacrylamide electrOphoresis cannot be considered artifacts of preparation. To eliminate the possibility of enzyme adsorption on 31 inactive proteins, protein from potato or apple was mixed with the mushroom enzyme extract. The multiple forms were developed. The pattern obtained was identical to the con- trol. Inactive proteins or other substances present did not bring rise to pseudo-multiple forms. The pattern of multiple molecular forms was the same whether the enzyme extract was sonicated before electrOphore- sis, frozen and thawed rapidly and slowly many times, or run on polyacrylamide repeatedly. Examination of several fruit tissues showed that all tissues have the same pattern of PO multiplicity. The skin, the flesh and the core of apples were tested. The cap and the stalk of mushrooms were examined for multiple forms. In all cases no difference was evident. In the apple the core seemed to have the highest concentration of activity. During the development of the apple and mushroom the PO multiple form pattern was common throughout all the stages of development. Apples at different stages of development ranging from thirty days after full bloom to one hundred and thirty days were used. The P0 system of enzymes seems to be installed in all the fruit tissues at the very early stages of fruit setting. Substrate Specificy of the Multiple Forms The mushroom PO system (Ap_campestris) clearly exhibited substrate specificity. With 7% gel, DOPA, catechol, phenol, 32 p—cresol, catechine, dOpamine, chlorogenic acid, caffeic acid, and p—coumaric acid reacted with all seven bands, while only two bands showed tyrosine activity (Fig. 9 and 10). The color develOpment was characteristic for each substrate and was the same for each reacting band. The intensity of color develOp- ment showed considerable differences between the various bands (Table I). Different lag periods of activity for each multiple form were observed. These lag periods depended on the sub- strate used, the species, and the particular multiple form. For Ap_campestris, catechol gave the fastest reaction for almost all the bands. The DOPA specific multiple forms of A:,p1acomyces developed faster than the DOPA specific forms of 2;,venenata. In the case of g, campestris and using DOPA as a substrate band No. 2 of group B, developed first, then the following bands appeared in order: band 2 of group A, band 1 of group B, band 3 of group B, band 1 of group A and finally after twenty minutes band 1 of group C. The existence of different lag periods showed that the different multiple forms have different affinity for the substrate, or were present at different concentrations. The periodical initiation of color development could bring about irregular reaction rates over a period of time, so initial velocities would not be enough to determine the true activity of all the PO system. Reaction kinetics of each multiple 33 30HHoh noopw csonn pstH noSHm omnmmo esownanmm HomHm when 90Hoo omnosz omCmpGH omnoan panH econ omnman H 0 peace pamaa scene a neon a a 2 2 8 2 3 2 m panH mmcoch oncoan a omcoch a N anmm chmm pstH omnoan econ omnman H m = = = = a z N pmeH omcoch oncoan panH econ oncouQH H < UH0< UHo¢ manoopmo Honompmo ochowha anon nnmm msoho oHnowoAOHmo onmmo .Eoopnmde mo mahom oHaHpHSE Ho thoHMHoodm opmnpmnsmun.H oHnme 34 form should be studied individually. The same could be true for inhibition studies. The mushroom genera and species showed their own in- dividual pattern of separation and in all instances the forms were also substrate specific (Fig. 11). In potatoes differ~ ences in the enzyme multiplicity pattern developed with DOPA were evident between the varieties of Aranac, Kemebec, Rural Russett, Sebago, Katahdim and Ontario. The apple varieties (Fig. 12) showed a common major migrating band toward the cathode and two to four other slow moving bands. The multiple form pattern was different for each variety, based on color develOpment with DOPA and Catechol. Sugarbeets and Freestone peaches both had at least three bands of DOPA activity. No detailed study was made on them. The electrOphoretic pattern of the inactive protein was different for each variety, species or genus studied in all the above plant sources. These results led to the conclusion that each mul- tiple form was an entity of its own, with specific character- istics concerning affinity and general behavior toward the substrate. Effects of Various Treatments on the Multiple Form Pattern of Mushroom Phenoloxidase Acetone The classical acetone powder technique has been used 35 for the study of many proteins and the preparations of enzymes. As acetone is capable of dissolving certain proteins and of denaturing others, although to what extent has not been deter— mined (Keller and Block 1960), it would seem desirable to determine whether acetone purification of the enzyme would alter the pattern of the multiple forms. An acetone powder preparation seemed to destroy the activity of bands 1 of group A and bands 1 and 2 of group B (Fig. 13). The preparation of commercial mushroom PO (prpgp- pestris) probably involving long and exhausting steps of purification, apparently resulted in the destruction or loss of some of the multiple forms (Fig. 14). Temperature ElectrOphoretic "rune" on mushroom samples stored for 2, 8, 20, and 72 hours at 5 0, showed no change in the poly- morphic pattern as indicated in Fig. 15. Samples stored at room temperature Q2 0) showed significant changes after 8 hours (Fig. 16). All forms withstood 40 C for 3 minutes as shown in Fig. 17, but after 60 minutes some of the forms were destroyed and others were broken into smaller distinct frag- ments having DOPA activity as seen in Fig. 17. Similar re- sults were obtained at 50 C and 60 0 (Figs. 17, 18). Tyro- sine activity showed no fragmentation, but its activity de- creased at temperatures of 50 and 60 C. Group 0 bands were stable for 60 minutes at 70 C (Fig. 18). All forms were 36 inactivated after 1 minute at 100 C. The fragmentation occurring at high temperature could lead to the assumption that multiple forms have more than one site for activity, or that one site of activity exists, but the non-active moiety is.fragmented causing different sized macromolecules to appear. The unique property of band C to withstand the temperature of 70 C for 60 minutes suggests ex— treme stability of the active site or sites of this molecule and probably a different configuration. Sulfite The gels, after the separation of the multiple forms, were removed from the tubes and incubated for 10 minutes in sodium bisulfite solutions containing 20, 100, 250, 500, 1000 ppm 802. The gels were then removed, allowed to drain for 20 seconds, and placed into a 1.5 x 10‘3M DOPA solution. Con- centrations of 20 and 100 ppm SO2 had very little effect on the multiple form pattern. There was a progressive decrease in color develOpment with increased concentration of SO2 and at 1000 ppm only slight activity was observed in group C (Fig. 19). Doubling the DOPA concentration showed activity in all except two bands (Fig. 20). Thus studying the effect of SO2 on enzyme activity, it is essential that the substrate con- centration be considered as it plays an important role in show- ing the presence or absence of enzyme activity (Fig. 21). 37 Gels were also thoroughly rinsed with fresh water for at least twenty minutes after treatment with 250 and 500 ppm 302 solution to remove free 802. They were then treated with DOPA. The washed gels showed enzyme activity while the un- washed gels showed little or no activity (Fig. 22). The P0 enzyme system was not inactivated when the original enzyme extract was incubated with concentrations of 802 in the range of 20 to 500 ppm for 5 hours. However, at 1000 and 2000 ppm the DOPA active bands 1 and 2 of group B were inactivated and at 4000 ppm only band 3 of group B was still active (Fig. 23). The tyrosine specific band was still active after exposure to 1000 ppm (Fig. 24). These results indicated that sulfite apparently interfered with the DOPA specific site of the enzyme but did not interfere strongly with the tyrosine specific site. The degree of color formation depended upon the amount of substrate present. These results supported the theory of Embs and.Markakis (1965) that no color or melanin formation occurred when sufficient sulfite was present to react with the non-colored intermediate products of o-diphenols (pyrocate- chol, chlorogenic acid and caffeic acid). Higher concentrations of 302 are necessary to inacti- vate the multiple forms when the enzyme system is in the ex- tract form than in the separated form on the gel. Foreign materials in the extract may bind with the 802 so that the 38 total free 302 decreases. Bedford and Mayak (1965) have shown that a certain percentage of the SO2 is actually not recovered from the cherry extract to which it was added. Irradiation Irradiation at 250 kilorads from a source of 10,000 curies of Cobalt 60, resulted in the inactivation of the multiple forms of group A. Two thousand kilorads were needed to inactivate band 1 of group B. The other multiple forms were not inactivated, however there was an indication of a slight reduction in their activity as the amount of irradi- ation was increased. The effect of irradiation was similar to that obtained during the storage of the enzyme at 22 C (Fig. 16). Ethylene-Diamine Tetra Acetic Acid (EDTA) EDTA apparently acts exclusively on the tyrosine bands as seen in Fig. 25. A concentration of 0.05% EDTA in- hibited tyrosine activity, whereas 4.0% EDTA did not inactivate the same band with DOPA activity. Increasing the EDTA con- centration from 0.01% up to 4.0% caused the DOPA specific mul- tiple forms to be inactivated differentially (Fig. 26). COpper could be the very important moiety for the enzyme active site function. It may affect the tertiary structure of the multiple forms of the enzyme as far as the stereochemistry of the active sites of the molecule is con- cerned, and the removal or binding of c0pper may cause a 39 change in tertiary structure which no longer favors enzyme activity. The cOpper may also bind the substrate forming a complex. The DOPA specific forms were not inactivated by con- centration of less than 4.0% EDTA. It could be postulated that the copper is deep within the three dimensional con— figuration of the protein or is tightly bound to the protein of the DOPA specific multiple forms. The absence of compe- tition between the chelating agent and DOPA could also imply that the metal is not primarily engaged in binding any of the components to the enzyme. gpeg The enzyme extract was incubated for six hours with urea 1.5 M, 4.0 M, 6.0 M and 8.0 M all at pH 7.0. After development of the multiple forms with DOPA, total inactiva- tion of most of the bands occurred at 1.5 M urea except for band 3 of group B. Complete inactivation of this band occurred at 8.0 M urea (Fig. 27). However 4.0 M urea inactivated com— pletely the tyrosine specific bands as may be seen in Fig. 28. The fact that urea affected the multiple forms prefer- entially may suggest that the active site of the enzyme is different in each form. Unfolding the molecule had a greater effect on the tyrosine specific forms than on the DOPA specific forms of group B. The tertiary structure may contribute ex- clusively to the tyrosine specific site on the molecule. 40 The hypothesis that the bands are distinct species and not artifacts arising one from the other is strengthened by the fact that when treated with denaturing agents no inter- conversion occurred. This finding contradicts the results of Jolley and Mason (1965) where interconversion of P0 was ob— served in mushrooms. Sodium Chloride Different concentrations of NaCL, 0.1%, 0.5%, 1.0% and 2.0% were incubated with the enzyme extract for six hours. At the concentration of 1.0% or higher all forms were in- activated except for band No. 3 of group B, and also the same band with tyrosine specificity remained active. Effect of DOPA on the Tyrosine Specificity and Vice Verse The gels after multiple form separation were incubated for 15 minutes with DOPA or Tyrosine. The gels were then re- moved end allowed to drain for 5 seconds. The gel treated with DOPA was then treated with tyrosine and the gel treated with tyrosine was then treated with DOPA. There was no effect of one substrate on the multiple form pattern of the other. Active sites apparently are not affected by the presence of different substrates. Incubation with Excess DOPA The enzyme was incubated with DOPA for 5 hours. The multiple form pattern showed that the group C band remained 41 very active while 2 bands of group B still had weak activity. It is assumed that all products formed must competitively inhibit the enzymes that produce them. Monod and Jacob (1961) stated that, "the products of an enzyme necessarily are an- alogues of the substrate, and competitive inhibition is ex- pected in any case, whether it is physiologically significant or not depends on the specific construction of the enzyme site." The resitence of the fast moving multiple form group C to the inhibitory effects of excess products due to the reaction with excess DOPA may indicate that one of the bio- logical roles of these multiple forms is to maintain activity in the presence of excess of products, a kind of defense mechanism against product inhibition. What role this may play in cell metabolism is still to be determined. Trypsin Digestion Trypsin (2 X crystallized, Nutritional Biochemicals), 1:50 w/v at pH 8.0 was incubated for 10 hours with the enzyme extract. Electrophoresis showed that only band 3 of group B still had DOPA activity. However a new slow moving band appeared with DOPA activity at a new position. Arginine and lysine seem to be indispensable for the active sites of most of the P0 multiple forms. They may take part directly in the active site or indirectly as maintaining the tertiary structure of the protein on which the active site may depend. 42 Other Treatments The inactivation of all DOPA and tyrosine bands was complete at even extremely low concentrations of 0.001 M mercaptoethenol. Sdfur may be involved directly or in- directly in the formation of the active sites. The multiple form pattern was not affected by 2% concentration of ascorbic acid. Intracellular Distribution of Phenoloxidase The differential centrifugation scheme used to de- termine the possibility of intracellular localization.of P0 activity is shown in Fig. 1. The first indication of intracellular localization of the PO enzyme suggested that most of the enzyme activity was found in the mitrochondria. and microsomal fractions as well as in the supernatant of all species and varieties (Fig. 29). In potatoes, the microsomal fraction and the final supernatant contained 40% and 46%, respectively, of the total activity. The specific activity of the microsomal fraction was about 15 times that of the supernatant. From a balance scheme of activity and inactive protein the recovery of both was 90-95%. In mushrooms the mitochondrial fraction contained 37% and the supernatant 50% of the total enzyme activity, with the mitochondrial fractions having the highest specific ac- tivity, about four times that of the supernatant. Most of the 43 total activity was found in the mitochondrial fractions of the apples. The specific activity was ten times that of the supernatant, which only had 15% of the total activity. All varieties of apples and potatoes tested had the same intra- cellular distribution pattern. Intracellular Compartmentalization of the Multiple Forms of Phenoloxidase Multiple forms of phenoloxidase have been found to exist in different parts of the cell having different char- acteristics. All members of the same species or varieties exhibited the same characteristics. Mushrooms: Fig. 30 depicts the difference in patterns of multiple forms between the final supernatant, mitochondrial solubilized enzyme. Tyrosine specificity seems to belong ex- clusively to the final supernatant. The soluble enzyme of the mitochondria has two DOPA specific band of Group B and two other week bands. Apples: The final supernatant of the apple (Red Delicious) showed three bands, while the mitochondrial soluble enzyme fraction had four bands. ‘ One approach that will lead to understanding the function of an enzyme is to determine precisely the location in the living system in which the enzyme Operates. Literature resulting from this work has provided us with information about the topographical distribution of P0 in some plant tissues, but remarkably little concerning their function. 44 The distribution pattern of phenoloxidase activity within the cell was found to be a varietal characteristic. All apple varieties tested had the same distribution pattern; the same was true with potatoes and mushrooms. Also the dis- tribution Of the multiple molecular forms was a characteristic belonging to the species or variety. The enzyme was shown to be a particulate as well as a supernatant enzyme. Various multiple forms were situated at different sites within the cell. Such topographical heterogeneity might be expected to have important implications in terms of the activity of the enzymes within the cell. A different situation could result in the exposure Of an enzyme to different microenvironmental influences that would be expected to affect its function. Also specific localization of the enzyme could result in channeling biochemical reaction sequences. Indeed a great deal of present day cytological research demonstrates the restrictions of enzymatic capacities to this or that cell structure. The specific situation of macromolecules and the importance of this, for the completion of a biochemical re— action sequence (electron transport and oxidative phosphor- ylation) is very important. The mitochondria of all plant tissues tested did contain large amounts of P0. The intra— cellular studies reported here indicate that one of the sites of activity, specifically that of tyrosinase, is restricted to a specific cellular region, in this case the final super— natant. Restriction of a particular member or members to a 45 specific cellular region could lead to the physiological im- portance of multiplicity of forms. It is possible that many of the multiple forms of other enzymes separable by electro- phoresis may represent enzymes with Specific cellular local- izations. The multiple forms found to be located in different parts of the cell should function under different physical or chemical conditions and they should be serving different functions in vivo. This suggests that multiple forms of these cases represent components of alternative metabolic pathways some of which are always functioning and others operative only during specific active phases of develOpment. They could be subject to different feedback control which would have the effect of maintaining useful concentration of enzymatic activity to provide products for two quite dis- tinct metabolic pathways. Recently various multiple forms have been demonstrated to be subject to feedback control by compounds in divergent metabolic pathways (Stedtmen et a1. 1961). The multiple forms associated with the microsomes may represent the molecular forms Of the newly synthesized P0 and other multiple forms may be derived from this form. The phenomenon of enzyme compartmentalization is evident. Enzymes cannot exist in a disorganized manner within the cell. Since they have a role to perform, they should exist at the right place and at the right time to help coordinate the complex system of life within the cell. CONCLUSIONS The excellent resolution of disk or polyacrylamide electrophoresis was employed for the first time in the in- vestigations of the phenoloxidase system for multiple forms. The fourteen multiple forms of PO found in the common mush- room, the eleven found in the potato variety Rural Russett and the three forms in the Golden Delicious apple may not be the only forms present. The stability and reproducibil— ity of the enzyme pattern of PO suggested that this multi— plicity is not the result of random changes or experimental manipulations, but represents the intracellular state of the enzyme system. The present study shows that the phenomenon of multiplicity of forms is of considerable value in phylo- genetic, taxonomic and genetic studies. Comparative enzyme structure also appears to be of importance in classification studies and in evaluation of changes associated with evolu- tion. Future studies on some phenoloxidases may help us ascertain which part of the enzyme molecule undergoes change during evolution. The multiple forms of P0 from mushrooms exhibited the phenomenon of substrate specificity. 0f the fourteen DOPA specific bands only two of these were active toward tyrosine, while the rest of the bands showed different re- actions toward different phenolic substrates. 46 47 The different multiple forms showed different degrees of tolerance toward temperature, sulfite,“ - irradiation, urea, EDTA, NaCL, and trypsin degradation. A temperature of 60 0 caused fragmentation of the DOPA specific forms while the tyrosine specific forms re- mained intact, with some loss of activity. Two different molecules, one DOPA specific and the other tyrosine specific are postulated, although both have similar electrOphoretic prOperties. The DOPA specific molecule may have many active sites, or it may have only one active site and high temper— atures may tend to break this molecule at different points thus changing the original electrOphoretic properties of the molecule. The high degree of heat tolerance which one group Of multiple forms showed points out the importance of blanch— ing to accomplish enzyme inactivation in the area of food processing. The inhibition by sulfite on the multiple forms was different for each form, and the tyrosine specific multiple forms withstood sulfite inhibition while DOPA specific forms did not. Sulfite probably blocks the active sites of the forms thus causing inactivation. It is speculated that sulfite may be tied up by different substances present in the enzyme extract thereby decreasing its effect on the enzyme. Unfolding of the molecule by urea had a greater ad- verse effect on the tyrosine specific form than on the DOPA 48 specific form. The tertiary structure may contribute to the tyrosine specific site of the molecule. EDTA inactivated the tyrosine specific forms at ex- tremely low concentrations while much higher concentrations were needed to inactivate the DOPA specific forms. COpper may affect the tertiary structure of the molecule, in so far as the stereochemistry of the active sites is concerned, so tyrosine specificity which may depend on the tertiary struc- ture is inactivated when the copper is removed. The little competition between the chelating agent and DOPA could also imply that the metal is not primarily engaged in binding any of the components to the enzyme. Contrary to the hypothesis of Pomerantz (1966) that excess tyrosine combines at the DOPA site thus preventing DOPA from acting and vise versa, it was found that the effect of excess tyrosine or DOPA did not affect the active sites for DOPA and tyrosine activity. Products formed when excess DOPA was incubated with the enzyme extract failed to inhibit one group of multiple forms, while all other DOPA specific forms were inactivated. A defense mechanism against product inhibition is postulated. Trypsin degradation partially inactivated most of the multiple forms, indicating the significance of arginine and lysine as contributing directly or indirectly to the active site formation. Mercaptoethanol and cysteine-HCL caused 49 complete inactivation of all forms, while ascorbic acid had no effect on the multiple form pattern. The P0 system seems to be installed in the plant tissues at the very early stages of development, and no tissue spe- cificity of multiple forms is apparent. The intracellular distribution pattern of the PO system is specific for each species. Intracellular compartmentalization of the multiple forms of P0 was shown to exist. In mushroom the tyrosine specific form exists only in the final supernatant, while the mitochondrial fraction has only two DOPA specific bands. The phenomenon of enzyme compartmentalization and multiple form compartmentalization is evident. Enzymes cannot exist in a disorganized manner within the cell. 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Nachweis der Heterogen- itat von Milchsauredehydrogenosen verschiedenen Ursprungs durch TragerelktrOphorese. Biochem Z. 329:112. Withycombe, W. A., and J. H. Wilkinson. 1964. Properties Of human spermatozoan lactic dehydrogenases isozymes. Biochem. J. 93:11p. Yasanobu, K. T. 1959. Mode of action of tyrosinase. Pigment Cell Biology, pp. 583-607. ed. M. Gordon. Acad. Press, N. Y. Yu, M. L., and R. E. Hampton. 1964. Biochemical changes in tobacco infected with Colletotricum destructivum. II. Peroxidases. Phytochem. 3:499. Figure 1 *ufio ‘— Differential centrifugation scheme. Figure 2 Effect of concentration of gel on the resolution of the multi- ple forms of phenoloxidase (P0) in mushrooms. Left to right: ‘op, 7%, z, 9% gel. Substrate: DL—DOPA Figure P0 active multiple forms and inactive protein in mushrooms. Left: Multiple molecular forms of phenoloxidase in the common mushroom (Agaricus campestris) separated with 7% and 8% polyacrylamide gel. Sample applied 0.4 m1. Substrate: DL—DOPA. Right: The corresponding protein pattern, separated in 9% gel. Sample applied 0.1 m1. Developed with 1% Amide Black in 7% acetic acid. Fi. re Multiple forms of PO from different genera. Left: Common Mushroom (Agaricus campestris) Middle: Potato (Russett var.) Right: Apple (Yellow delicious var.) Substrate: DL-DOPA. 60 Homog, 1 i I A t . i i n" can» . R Q . T . . . 3 Mitochond. .5 d I ~ .I .I u MicrosOmes. O .‘ n O s""ERNArANr . PREC|p|TATE A . ’ Si 4 5 I fig. ! ‘ l . I300’X98’IO xsllo x9 '0 20’ . IZO Fig. 1 Fig. 2 Ii! pt L., Fig. 3 Figure 5 Cathodical (pH 8.3) and anodical (pH 4.3) multiple forms of P0 in mushrooms. Left: Cathodical multiple forms. Right: Anodical multiple forms. Substrate: DL-DOPA Figure 6 Two—phase polyacrylamide electrOphoresis. Separation of the multiple form of P0 in mushrooms. Left: 7% Hal. Right: 7% tOp half of column, 8% bottom half of column. Substrate: DL—DOPA. Figure 2 Effect of current on separation of multiple forms of P0 in mushrooms. Left to Right: 1 MA per tube, 2 m, 2 1/4 rm, 3 MA. Substrate: DL-DOPA. Flgpre 8 Recovery of multiple forms of P0 in mushrooms following elution from the gells. Left: Recovery of the one band of group C. fiddle: Control Right: Recovery of the two bands of group B. Substrate: DL-DOPA. Figure ~ DL dihydrooxyphenylalamine and L-Tyrosine specificity of mul- tiple forms of P0 in mushrooms. Left: DL-DOPA specific multiple forms. Right: L—Tyrosine specific multiple forms. Figure 10 Substrate specificity of multiple forms of P0 in mushrooms. Left to Right: DL-DOPA specific multiple forms, L-Tyrosine, catechol, catechine, cafeic acid, cholorogenic acid. (The fast band is the front). Figure 11 Species and genus specificity of multiple forms Of P0 in mush- rooms. Left to Right: Agaricus campestris Substrate: Aggricus placomyges " " Tricholoma venenata " fl Suillus_gpeville; " DL-DOPA a)DL-DOPA b)L-Tyrosine a)DL-DOPA b)L—Tyrosine a)DL-DOPA b)L—Tyrosine 62 Figure 12 Varietal specificity of multiple forms of P0 in apples. Left to Right: Spy, Red Delicious, Golden Delicious, Cort- land, Jonathan, Grimes Golden. Substrate: DL—DOPA + Catechol. Figure 13 Effect of acetone on multiple forms of P0 in mushrooms. Left: Control. Right: Acetone treated (Acetone powder). Substrate: DL—DOPA. Figure 14 Multiple forms of commercial mushroom PO from four differ- ent companies. Left to Right: A, B, C, D. Substrate: DL—DOPA. Figure 15 Effect of 5 C with time on the multiple forms of P0 in mush- rooms. Left to Right: Hours: 2, 8, 20, 72. Substrate: DL—DOPA. lily-r- Figure 16 Effect of room temperature (22 C) with time on the multiple forms of P0 in mushrooms. Left to Right: Hours: 2, 8, 20, 72. Substrate: DL-DOPA. Figure 17 Effect of 40 C and 50 C with time on the multiple forms of P0 in mushrooms. Left to Right: Control: 40 C 3 min.; 40 C 60 min.; 50 C 3 'min.; 60 C 60 min. Figure 18 Effect of 60 C, 70 C and 100 C on the multiple forms Of P0 in mushrooms. Left to Right: 60 0 minutes: 3, 15, 60. 700 ,. : 3, 50. lOOC " : 2. 61+ Figure 19 Effect of sulfite on the multiple forms of P0 in mushrooms. Incubation on the gel, 10 minutes. Right to Left: 802 ppm 20, 100, 250, 500, 1000. Substrate: 1.5 x 10‘3m DL-DOPA RgmneZO Effect of sulfite on the multiple forms of P0 in mushrooms. Incubation on the gel, 10 minutes. Left to Right: 302 p.p.m. 250, 500, 1000. Substrate: 3.0 x lO'3M DL-DOPA. Figure 21 Effect of 1000 p.p.m. 802 with different substrate concentra- tions on the multiple forms of P0 in musarooms. Incubation on the gel, 10 minutes. Right to Left: DL-DOFA 0.5 x 10'3M, 3.0 x 10’3M, 1.2 x 10'3M. Figure 22 Effect of sulfite on the multiple forms of P0 in mushrooms, following washing off of the sulfite. Incubation on the gel, 30 minutes. Left to Right: 1) 302 p.p.m. 250. Substrate: 3.0 x 10‘3M DL-DOPA Gel not washed. 2) n n n n n n u n n washed. 3) 802 p.p.m. 500. Substrate: " " " " " not washed. 1+ ) n n n . n n n n n n wa she d . 65 Figure 23 Effect of incubation of sulfite (6 hrs.) on the multiple forms of P0 in mushrooms. Left to Right: Control: 802 p.p.m. 1000 2000, 4000, 8000. Substrate: DL-DOPA 1.5 X 10‘3M. Figure 24 Effect of incubation (6 hrs.) of 802 on the multiple forms of P0 in mushrooms. Left to Right: Control: 802 p.p.m. 1000, 2000, 4000, 8000. Substrate: L—Tyrosine. Figure‘25 Effect of EDTA on the multiple forms of P0 in mushrooms. Right to Left: Control: EDTA 0.05%; 0.25%; 0.5%; h.0%. Substrate: L-Tyrosine. Figure 26 Effect of EDTA on the multiple forms of phenoloxidase in mush- rooms. Left to Right: 0.01% EDTA 0.5%; 0.25%; 0.50%; 4.0¢. Substrate: DL—DOPA. 66 : § 4‘; C 1 '. I 4' ti r 1 , Q 1' -' . a l L- ! ~ .'. , .2 . _ J u/ L _—— — ViL—fl‘ Figure 27 Effect of Urea on the multiple forms of P0 in mushrooms. Right to Left: Control: Urea 1.5M; 4. K; 6. M; 8.0M. Substrate: DL—DOPA. O) re 2 if )8 H H: H: (D O (.1. of Urea on the multiple forms of P0 in mushrooms. wight: Control: Urea 1.5M; b.0fi; 6.0K; 8.0T. t“ (u H: c+ d O U) C‘ O“ 0) cf "3‘ Q.) d (b ’: L-Tyrosine. Figure 2Q Intracellular distribution of total P0 activity. Figuro‘3Q Intracellular compartmentalization of multiple forms of P0 in mushrooms. ' 0 Right to Left: a) Final Supernatant. Substrata: DL-DOPA. b) " " " : L-Tyrosine. c) Mitochondrial elution. Substrate: DL-DOPA. d) " " " : L—Tyrosine. 67 Fig. 27 60” ®Mitochondric@ Final Supern, 30F % ACTIVITY POTATO MUSHROOM APPLE Fig. 29 ®Cell Debris @ Microsomos v " l "‘mm1111111ES