w: 25¢ per day per item RETURNING LIBRARY MATERIAL§: Place in book return to ram charge from circulation recon IDENTIFICATION OF A LACCASE PRODUCED DURING ENCYSTMENT OF ACANTHAMOEBA CASTELLANII BY Donald Edward Sykes A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1980 ABSTRACT IDENTIFICATION OF A LACCASE PRODUCED DURING ENCYSTMENT OF ACANTHAMOEBA CASTELLANII BY Donald Edward Sykes Acanthamoeba castellanii contains an enzyme, identified as a laccase, which is capable of oxidizing phenolic compounds. This enzyme was found in the outer wall of the mature cyst, in the cyto- plasm of precyst amoebae, and in the medium where amoebae have encysted. Although laccase activity was found in the mature cyst wall, this insoluble form of the enzyme could not be used for puri- fication and characterization of the enzyme. The enzyme was secreted into the encystment medium in soluble form. Established encystment procedures were modified to promote an increase in the production of soluble enzyme. The final procedure consisted of placing slow-growing organisms (amoebae that showed fewer than two doublings in five days) into an inorganic saline with high osmotic pressure at a concentration of 3-4 X 106 amoebae/ml. After 36-48 hr of incubation, encysting amoebae were removed from the medium. The medium was found to contain soluble laccase in a quantity 20-30 fold more than that realized when using published encystment procedures. With these new encystment pro- cedures, a large quantity of soluble laccase was produced and the enzyme was purified and characterized biochemically. Acanthamoeba Donald Edward Sykes laccase has a molecular weight of 160,000. The pH optimum is 6.0, and the Km value is 0.21 mM with dihydroxyphenylalanine. It does not use tyrosine as a substrate, and it is inhibited by chloride but not by inhibitors of peroxidase. Although the function of laccase for these amoebae is unknown, two lines of evidence suggest that the enzyme is necessary for normal encystment: known inhibitors of laccase activity were found to inhibit cyst formation without inhibiting vegetative growth; and laccase activity and cyst formation appeared concomitantly. It is hypothesized that Acanthamoeba laccase may have a role in making the cyst resistant to breakdown by mechanical and chemical agents. This suspected role for laccase in Acanthamoeba is similar to that proposed for the melanized mycelium of fungi. Due to the melanin content of these fungi, they have a greater resistance to bacterial lysis and breakdown. The restricted occurrence of laccase in the plant and animal kingdoms, and its presence in most fungal groups, suggest either a relationship of Acanthamoeba to the fungi, or convergent evolution among soil-dwelling microorganisms. Laccase was also found in the cysts of other soil-dwelling microorganisms, Naegleria gruberi and Acanthamoeba palestinensis, but was absent from the cysts of the parasite Entamoeba invadens. To my wife Barbara and daughter Kristina ii ACKNOWLEDGMENTS This research was possible through the generous support of Dr. R. Neal Band. His cooperation, guidance and encouragement were necessary for the successful completion of this thesis research. Thank you Dr. Band for everything. I would also like to thank Drs. Edward Cantino, Ralph Costilow, Hironobu Ozaki and Ralph Pax for their cooperation and helpful suggestions during this research. iii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . LIST OF FIGURES . . . . . . . . ABBREVIATIONS . . . . . . . . . INTRODUCTION . . . . . . . . . Description of Acanthamoeba . . . . Acanthamoeba as a Cytodifferentiation Model Enzyme Description . . . . . . . Laccase Function . . . . . . . Enzyme Purification . . . . . . Enzyme Properties . . . . . . . MATERIALS AND METHODS . . . . . . Cell Cultures . . . . . . . . Encystment Procedure . . . . . . Enzyme Preparation . . . . . . Enzyme Assays . . . . . . . . Substrate Specificity . . . . . . Enzyme Location in Cysts . . . . . Laccase Inhibitor Studies . . . . . Laccase Production During Encystment . . Inhibition of Laccase by High Salt Ions . Extraction of Laccase from Cysts . . . Altered Encystment Procedures . . . . iv vii ix xi 10 14 14 14 15 15 16 17 17 18 20 20 21 Protein Secretion by Cysts . . . . . . Variables Involved in Soluble Enzyme Production . Growth rate of vegetative amoebae . . . . Cell density during encystment . . . Enzyme Purification . . . . . . . . Large batch preparation . . . . . . Enzyme concentration and acetone fractionation Column separation . . . . . . . . Enzyme Purity . . . . . . . . . . Absorbency spectrum of Acanthamoeba laccase . Gel electrophoresis . . . . . . . Molecular Weight Determinations . . . . . Enzyme Properties . . . . . . . . . Comparison of Cyst Wall Enzyme and Soluble Enzyme . Materials . . . . . . . . . . . RESULTS . . . . . . . . . . . . Laccase Activity from Encysting Amoebae . . . Identification of the Enzyme . . . . . . Substrate Specificity . . . . . . . . Enzyme Location in Cysts . . . . . . . Laccase Involvement in Encystment: Inhibitor Studies Laccase Production During Encystment . . . . Ion Effect on Laccase Activity . . . . . Extraction of Laccase from Cysts . . . . . Altered Encystment Procedures . . . . . . Protein Secretion During Encystment . . . . V 21 21 21 23 23 23 23 24 25 25 25 25 26 26 26 28 28 28 32 32 35 39 43 45 45 45 Variables Involved in Soluble Enzyme Production . . . . 49 Growth rate of vegetative cells . . . . . . . 49 Cell density during encystment . . . . . . . 52 Harvest time for the enzyme . . . . . . . . 52 Enzyme Purification . . . . . . . . . . . 54 Large batch preparation . . . . . . . . . 54 Concentration and acetone fractionation of laccase . . 56 Column separation . . . . . . . . . . . 56 Enzyme Purity . . . . . . . . . . . . . 61 Molecular Weight Determination . . . . . . . . 66 Properties of the Enzyme . . . . . . . . . . 68 Optimal pH . . . . . . . . . . . . 68 Optimal temperature . . . . . . . . . . 68 Enzyme kinetics . . . . . . . . . . . 68 Enzyme concentration . . . . . . . . . . 71 Laccase Assays . . . . . . . . . . . . 71 Cyst and Purified Soluble Laccase Compared . . . . . 77 DISCUSSION . . . . . . . . . . . . . . 79 Enzyme Identification . . . . . . . . . . . 80 Laccase of Cysts . . . . . . . . . . . . .81 Laccase Function in Acanthamoeba . . . . . . . . 82 Laccase in Other Amoebae . . . . . . . . . . 87 Enzyme Secretion in Acanthamoeba . . . . . . . . 88 Enzyme Purification . . . . . . . . . . . 93 SUMMARY . . . . . . . . . . . . . . . 97 LIST OF REFERENCES . . . . . . . . . . . . 100 TABLE 10 11 12 13 14 15 LIST OF TABLES Description of active and slow growing cell cultures. (Cells/ml) . . . . . . . . . . . . Laccase activity yields from the encystment medium of different cell suspensions. . . . . . Identification of Acanthamoeba enzyme as a laccase and not a peroxidase. . . . . . . . . . Identification of Acanthamoeba enzyme as a laccase and not a tyrosinase. . . . . . . . . . Substrate specificity of Acanthamoeba laccase. . . . Localization of laccase within cysts. . . . . . The effect of laccase inhibitors on Acanthamoeba laccase activity. . . . . . . . . . . . . Growth of vegetative amoebae in the presence of laccase inhibitors. . . . . . . . . . . . . Inhibition of cyst formation in the presence of laccase inhibitors. . . . . . . . . . . . . Laccase activity in the presence of various ions. . . Attempts to extract laccase from Acanthamoeba cysts. . The effect of alterations in the encystment procedure on the secretion of soluble laccase. . . . . . Soluble laccase activity from amoebae that were starved compared to laccase activity from amoebae that were not starved. . . . . . . . . . . . . Total protein secretion by encysting amoebae. . . . Laccase activity yields from the crude soluble enzyme preparations of one liter suspensions. . . . . . vii PAGE 22 29 3O 31 33 34 36 37 38 44 46 47 48 50 55 TABLE PAGE 16 Summary of the purification of laccase from Acanthamoeba. 57 17 Properties of particulate (cyst wall) laccase compared to soluble laccase. . . . . . . . . . . 78 viii FIGURE LIST OF FIGURES PAGE Diagram of the procedure for measuring laccase production and cyst formation. Parentheses indicate preparations that were assayed. . . . . . . . . . . l9 Laccase activity in the CSEP of encysting amoebae as a function of time. The points shown in all of the figures in this thesis represent the average of duplicate deter- minations, unless stated otherwise. The vertical bars show the range between the duplicate values. . . . 40 Laccase activity measured from total cells and from the homogenate of precysts as a function of time. . . . 41 Laccase activity measured in the whole encystment sus- pension and cyst number as a function of time. The line with points marked with Xs ( X X ) indicates cyst number. The line with points marked with dots ( o c ) indicates laccase activity in the whole encystment suspension. . . . . . . . . . 42 A comparison of laccase yields from encysting cells derived from active and slow growing cultures. The values in this figure represent the average of duplicate determinations on two independent cell cultures. The difference in value between these duplicate determinations ranged from 0.2 to 1.8 units. . . . . . . . 51 Laccase yields from the CSEP of encysting amoebae as a function of cell number. The broken line (---) indicated assays after 24 hr of incubation of amoebae in HS. The solid line (-——9 indicates assays after 48 hr of incubation of amoebae in HS. . . . . . . . . . . 53 Acanthamoeba laccase elution profile from a Whatman CM-52 column. Fractions 15-50 were pooled. . . . . . 58 Acanthamoeba laccase elution profile from a Whatman DEAE-52 column. Fractions 178-183 were pooled. . . . . 6O Ultraviolet absorption spectra of purified Acanthamoeba laccase. . . . . . . . . . . . . 62 ix FIGURE 10 ll 12 13 14 15 16 17 18 19 20 Visible absorption spectra of purified Acanthamoeba laccase. . . . . . . . . . . . Gel electrophoresis pattern of Acanthamoeba laccase. Stained for laccase. . . . . . . . . . Gel electrophoresis pattern of Acanthamoeba laccase. Stained for protein. . . . . . . . . Relative mobility of Acanthamoeba laccase and marker proteins with sodium dodecyl sulfate polyacrylamide gel electrophoresis. The marker proteins and their molecular weights were: ceruloplasm, 150,000; tyro- sinase. 128,000; tranferrin, 85,000 and albumin, 67,000. . . . . . . . . . . . Optimal pH for Acanthamoeba laccase. Whole cyst laccase is indicated by the broken line (---). Purified soluble laccase is indicated by the solid line (-——9. . . . . . . . . . . . Optimal temperature for Acanthamoeba laccase. Whole cyst laccase is represented by the broken line (---). Purified soluble laccase is represented by the solid line (-——0. . . . . . . . . . . . Michaelis-Menten kinetics of Acanthamoeba laccase. . Linewever-Burk plot of Acanthamoeba purified soluble laccase. . . . . . . . . . . . . Linewever-Burk plot of Acanthamoeba Whole cyst laccase. Proportionality of purified laccase units to the quan- tity of enzyme assayed. . . . . . . . . Absorbency change at 475 nm as a function of time, produced by purified Acanthamoeba laccase acting on DOPA. . . . . . . . . . . . . . PAGE 63 64 65 67 69 7O 72 73 74 75 76 ABBREVIATIONS Abbreviations used are: CSEP, crude soluble enzyme preparation; KNP, potassium and sodium phosphate buffer; DOPA, L-B-3,4-dihydroxy- phenylalanine; HS, (hypertonic) high salts; LS, low salts. xi INTRODUCTION Understanding the mechanism involved in cytodifferentiation is one of the most interesting challenges facing the embryologist today. Spemann's (1924) demonstration that the eye lens is formed from ectoderm after mesodermal induction has brought about a flood of experiments seeking to provide a better understanding of the process of differentia- tion. In more recent experiments, embryologists are concerned with the nature of the "trigger," or chemical inducer, that initiates a series of events which eventually leads to the differentiation of cell type. For a developmental system to be conducive to experimental manipulation, it needs to be simple. The differentiated cell type must be distinguished from the undifferentiated cell by the presence or absence of some organic molecule. Thus, the presence of a well- defined macromolecule in the differentiated state is needed to serve as a marker which can be followed as experimental manipulations are performed on the undifferentiated cell. Unfortunately, most developmental systems studied are very complex, and cellular interactions complicate interpretation of the results. A simple system for the study of differentiation is that of the cellular slime mold, Dictyostelium, and valuable information has been gained from scrutiny of this differentiation system (Bonner 1959; Ashworth 1976; Sussman 1965). However, this system is also complicated l 2 by the interdependence of the cells in the multicellular stages. An ideal system for studying cytodifferentiation would be a single-celled organism that is not dependent on other cells, yet has a distinct differentiated state. For this reason, Acanthamoeba is favored as a cytodifferentiation model (Neff and Neff 1969; Byers 1979; Edwards and Lloyd 1977). Description of Acanthamoeba Acanthamoeba is an obligate aerobe which is free-living in the soil (Band 1959). The vegetative amoeba (trophozoite) feeds on bac- teria and other soil microorganisms (Band 1963; Bowers 1977). In times of environmental stress, this amoeba differentiates into a dormant cyst. Environmental stress conditions thought to be involved in the initiation of cyst formation are starvation (Marzzoco and Colli 1975; Neff, Ray, Benton and Wilborn 1964), drying (Band 1963), and oxygen depletion (Byers, Rudick and Rudick 1969). Encystment is considered to be a differentiation process. The Acanthamoeba cyst is, however, not a reproductive stage. The excystment of Acanthamoeba is also considered to be a differentiation process (Neff and Neff 1969). The Acanthamoeba cyst is morphologically and physiologically very different from the trophozoite. The cytoplasm of the cyst is very dense (Vickerman 1962) and the net negative surface charge of the cyst is greater than that of the trophozoite (Band and Irving 1965). The mitochondria decrease in size and number (Neff and Neff 1969), an occurrence consistent with the fact that oxygen is not taken up by cysts (Neff and Neff 1969; Band and Mohrlok 1969). The cyst is smaller than the trophozoite, and has a thick and wrinkled outer 3 cyst wall composed largely of protein (Neff and Neff 1969). Between the outer cyst wall and the plasma membrane there is a second mass of cell-wall material consisting largely of cellulose (Neff and Neff 1969; Bowers and Korn 1969). Acanthamoeba castellanii has been cultured axenically in various laboratories for many years. Methods have been developed that cause the trophozoite to differentiate into the dormant, resistant cyst in a synchronous manner. Encystment has been achieved under laboratory conditions: actively growing cells are placed in a starvation medium (low carbon source) of one type or another, such as a buffered in- organic saline solution (Neff, Ray, Benton and Wilborn 1964), or a culture medium containing ten-fold less glucose (Band 1963). In the latter case, the cells are placed secondarily in an inorganic medium of high osmotic pressure. Within 4-6 hrs after being placed in the inorganic saline solution, vegetative amoebae round up and a thick outer cyst wall is laid down first and later a second wall of cellu- lose fibers is laid down between the outer cyst wall and the plasma membrane (Neff and Neff 1969; Bowers and Korn 1969). Acanthamoeba as a Cytodifferentiation Model In spite of its obvious advantage of simplicity, Acanthamoeba has not been used extensively for cytodifferentiation studies. This is due to the lack of a well defined biochemical marker for the differentiated cell. Although the mechanisms of cellulose synthesis and its initiation have been examined (weisman 1976; Verma and Raizada 1975), little understanding of the control mechanism of encystment has been gained. 4 As was mentioned earlier, one of the most prominent features of the cyst is its outer wall. If not the most important feature for mature cyst survival, the outer cyst wall is surely a major, gross morphological feature. However, due to its resistance to chemical or physical analysis (Neff and Neff 1969; Barrett and Alexander 1977), little is known of its organic composition or how it is organized. The outer cyst wall is thought to be composed largely of protein (Neff and Neff 1969; Weisman 1976). For a single differentiating amoeba these proteins are incorporated as cyst wall components very rapidly (Neff, Ray, Benton and Wilborn 1964). Since little is known about these proteins or how they are organized in the outer cyst wall, there are no well defined biochemical markers for experimental studies. However, it seems reasonable to assume that there might be an enzyme involved in the synthesis of the outer cyst wall that could be iden- tified and used as a marker of the differentiated cell. A group of enzymes called the phenoloxidases are known to par- ticipate in the oxidation of phenolic compounds. Following this initial oxidation step, the phenolic intermediates polymerize and crosslink the existing proteins by autooxidation (Prota and Thomson 1976). The final product is very resistant to chemical and physical breakdown (Das, Abramson and Katzman 1976; Foerder and Shapiro 1977; Kuo and Alexander 1967). Chemically resistant proteinaceous materials found in several different phyla consist of crosslinked inter-protein chains. The sea urchin fertilization membrane is composed of dityrosine-linked proteins (Foerder and Shapiro 1977). In many fungi, protein crosslinking and melanin formation is considered to be the basis for resistance to lysis 5 and breakdown by soil bacteria (Kuo and Alexander 1967; Wheeler, Tolmsoff and Meola 1976). The resistant wall of the Eimeria oocyte is formed from quinone tanned proteins (Sleigh 1973). In various arthro- pods, tumor-like structures are formed around parasites by a melan- ization process (Soderhall and Unestam 1979; Hall, Andreadis, Flanagan and Kaczor 1975). Finally, the melanized cuticle of various insects is formed by a tanning process (Schlein 1975; Hepburn and Roberts 1975). Thus, it seems plausible that a similar enzyme mechanism might be involved in the formation of the outer cyst wall of Acanthamoeba. The appearance of this enzyme (e.g., phenoloxidase) in early encyst- ment might then be used as a marker for cytodifferentiation studies. Acanthamoeba was, therefore, examined for the presence of phenol- oxidase, and was found to contain an enzyme that would oxidize dihydroxyphenylalanine (DOPA) and p-phenylenediamine. Enzyme Description The enzymes commonly referred to as phenoloxidases are found in most animals (Molitoris and Esser 1971), plants, and microorganisms (Mayer and Harel 1979) so far examined. The Enzyme Commission has renamed this group of enzymes as EC l.l4.18.l monophenol monooxygenase (Mayer and Harel 1979), and included in this group catechol oxidase and laccase (Mayer and Harel 1979; Hoffman and Esser 1977). There is a significant difference between these two enzymes which is not implied in the collective name EC 1.14.18.l monophenol monooxygenase. Further, the function of phenoloxidase can be per- formed by peroxidases as well as by phenoloxidases, and methods for 6 distinguishing between these two are necessary. In general the phenoloxidases act on a variety of phenolic compounds to withdraw hydrogen and, in the presence of oxygen, to form water. However, peroxidases are also able to remove hydrogen from certain compounds and to reduce hydrogen peroxide to form water. The literature describes several diagnostic features which can serve as bases for distinguishing between laccase, catechol oxidase and peroxidase. Catechol oxidases oxidize, among other substrates, tyrosine (a monophenol) and o-diphenols, but not p-diphenols (Mayer and Harel 1979; Keilin and Mann 1939). In the presence of ferrocyanide, this enzyme will not catalyze the uptake of oxygen (Leonard 1971), and its catalytic activity is inhibited by carbon monoxide (Keilin and Mann 1939) and phenylhydrazine (Lerner, Harel, Lehman and Mayer 1971). In contrast, laccase usually will not oxidize tyrosine, but can oxidize o- and p-diphenols (Mayer and Harel 1979; Leonard 1971). Laccase will also catalyze the uptake of oxygen in the presence of ferrocyanide, and is not inhibited by carbon monoxide or phenyl- hydrazine. Peroxidases can be distinguished from the above two enzymes in several ways (Mayer and Harel 1979), due largely to the presence of an iron-prosthetic group rather than a copper-prosthetic group. Thus, phenanthroline, an iron chelator, inhibits peroxidase activity (Leonard 1971). Catalase reduces enzyme activity if the enzyme is a peroxidase, and hydrogen peroxide stimulates peroxidase activity (Mayer and Harel 1979). The enzyme in Acanthamoeba castellanii that catalyzes the oxidation of DOPA will be shown, in this thesis, to be properly 7 identified as a laccase (EC 1.14.18.1). Therefore, for convenience and uniformity, this enzyme will be referred to as laccase hereafter. Since the enzyme of concern in this thesis is laccase, further information concerning this enzyme is now presented. In contrast to catechol oxidase, which occurs in most organisms, laccase has a more- restricted occurrence in living organisms (Mayer and Harel 1979). It is found in all major groups of fungi and 511 the group of green plants with secretory ducts (Anacardiaceae) (Mayer and Harel 1979). The lacquer tree, a member of Anacardiaceae, is the source of the first laccase prep- aration, and is the basis of the enzyme's name (Yoshida 1883). Laccase may also be present in some gymnospenms (Cambie and Books 1966). The enzyme is not found in procaryotes, nor in most animals (Mayer and Harel 1979). The reaction for this enzyme is as follows: OH O OH 0 or LACCASE 3' H 02 or + H20 OH 0 8 The oxidation of p-diphenols shown in the first equation is considered to be diagnostic for this enzyme, since catechol oxidase does not catalyze the oxidation of p-diphenols (Mayer and Harel 1979). In addition to the o- and p-diphenols, laccase can oxidize m-diphenols, ascorbic acid, and p-phenylenediamine (Mayer and Harel 1979). Laccase Function In general, the function of the phenoloxidases remains unknown. Several theories have been advanced on a function for these enzymes. There is some basis for the theories regarding the function of catechol oxidases (e.g., tyrosinase). In mammals, tyrosinase has been studied extensively, and it clearly functions in melanin formation within the melanosome (Prota and Thomson 1976). In fungi, as was mentioned earlier, tyrosinase is thought to be involved in melanin formation (Katan, Arnon and Galun 1975; Potgieter and Alexander 1966), providing protection against destructive breakdown by microorganisms (Kuo and Alexander 1967; Potgieter and Alexander 1966; Martin and Haider 1979). Furthermore, it is believed that the quinone or polymerized quinone may be toxic, serving as a deterrent to potential pathogens (Kuo and Alexander 1967; Mayer and Harel 1979). While there are no adequate demonstrations of function for the laccase enzyme, some of the functions assigned to catechol oxidase might also apply to laccase, since the end product is likely the same for both enzymes. In addition, by pursuing two indirect lines of evidence, Leonard (1971) established a functional role for laccase in the forma- tion of fruiting bodies in the fungi Schizophyllum commune. First, after adding known inhibitors of laccase to developing mycelia, retarda- tion of fruiting body development was observed. Secondly, Leonard 9 showed that the level of laccase increased in fungi during the formation of fruiting bodies. It was concluded that the laccase enzyme was necessary for the production of fertile fruiting bodies in these fungi (Leonard 1971). Laccase is found in the white rot fungus, but is absent in soft rot fungus (Mayer and Harel 1979). In the white rot fungus, laccase is thought to function in the oxidation of lignin (Mayer and Harel 1979). In this role, the level of lignin oxidation products in the medium may regulate the formation of other enzymes involved in the breakdown of lignin and polysaccharides (Ander and Eriksson 1976). The function of laccase may be different in different fungi. In the lacquer tree, the enzyme is secreted only by ducts, and it may have a role in the hardening of the secretion for wound healing (Mayer and Harel 1979). Laccase of the lacquer tree may also have a role in defense: the production of toxic quinones or polymerized quinones would serve as a deterrent to invasive pathogens (Mayer and Harel 1979). Enzyme Purification Although it is stated by Mayer and Harel (1979) that laccase is easily purified, its behavior during purification depends considerably upon various characteristics of laccase. Mayer and Harel (1979) also state that the laccase enzyme is best considered as a "family of enzymes, not a single enzyme which underwent minor modification during evolution." For this reason, the literature on behavior during purifica- tion and characterization of the enzyme appears confusing. Purification of the lacquer tree enzyme was achieved by 10 Reinhammer in 1970. Purification was accomplished by acetone precip- itation, CM-Sephadex C-50 and DEAE-Sephadex A-SO chromatography. Reinhammer (1970) states that the lacquer tree enzyme is incompletely precipitated by ammonium sulfate, and thus, acetone precipitation is widely used in the first step of purification for this laccase. The laccase from fungi is usually precipitated and fraction- ated by ammonium sulfate. Fahraeus and Reinhammer (1967) purified the laccase of Polyporus versicolor by precipitation with saturated ammonium sulfate, followed by Sephadex G 25 and DEAE-Sephadex A-50 chomatography. DEAE cellulose chromatography was also used in the final step of purification of the laccase from Botrytis cinerea (Dubernet et a1. 1977). In contrast to Rhus (lacquer tree) laccase, the fungal laccases are retained by anion exchange columns. Isoelectric focusing columns have been used for the purification of the laccase of the fungus Polyporus versicolor (Mayer and Harel 1979). Enzyme Properties Reports indicate that, regardless of the techniques used to purify the laccase enzyme, considerable heterogeneity of laccase exists (Thomas, Delincee and Diehl 1978). Gel electrophoresis of an unpurified and a purified preparation of laccase from the grape rot fungus, Botrytis cinerea, shows two bands (Dubernet et a1. 1977). Six electro- phoretically distinct gel bands were obtained from a crude laccase prep- aration from the fungus Pleurotus ostreatus (Leonowicz and Trojanowski 1975a). One of the laccase bands was shown to be indicible by ferulic acid in this white-rot fungus, and a purified preparation of this laccase, when subjected to sodium dodecyl sulfate polyacrylamide gel ll electrophoresis, was shown to contain six distinct molecular weight species (Leonowicz and Trojanowski 1975b). These different laccase bands may represent isozymes, or the microheterogeneity that has been observed also with other enzymes. The band of laccase from Pleurotus ostreatus that resulted from ferulic acid induction has been traced to the induction of a specific laccase m-RNA (Leonowicz and Trojanowski 1978). This suggests that an isozymic form of the enzyme is present. Finally, Esser and Minuth (1970) report that there are three distinct molecular weight species in the fungus Podospora anserina. Minuth, Klischies and Esser (1978) concluded that, after amino acid analysis, one smaller molecular weight species is a distinct enzyme, not a subunit of the large molecular weight laccase. Other researchers are not so final in their assignment of isozyme status to the electro- phoretically distinct laccase components. These differences between species and within the same species probably arise from differences in the length of the attached carbo- hydrate chain. Laccase is a glycoprotein with 15-41% of the molecule composed of carbohydrate (Mayer and Harel 1979). Such glycoproteins have a basic structural unit consisting of between 50,000 and 70,000 molecular weight, which can aggregate to form larger enzyme units (Mayer and Harel 1979). The following variable molecular weights have been reported for the fungus Podospora anserina: laccase I, 390,000; laccase II, 71,000; laccase III, 78,000 (Minuth, Klischies and Esser 1978). The major component of Polyporus versicolor laccase has a molecular weight of 60-65,000 (Fahreus and Reinhammar 1967). The pH optima for the various laccases are likewise varied, 12 depending on the conditions of testing and the species from which the enzyme is derived. For the lacquer tree species, the optimum is between 6.4 and 7.5 (Mayer and Harel 1979). For the fungi, the optimum is between 4.0 and 6.0 (Mayer and Harel 1970). It is known that laccase is a blue, copper-containing enzyme. A monomer of laccase, with a molecular weight of 50,000-70,000, contains four atoms of cooper (Mayer and Harel 1979). The objective of this research, after discovery of the phenol— oxidase enzyme of Acanthamoeba castellanii, was to obtain information about the enzyme. The identification of this phenoloxidase as a laccase rather than a catechol oxidase or a peroxidase was established by the use of inhibitors and specific substrate molecules and by biochemical char- acterization. Since the enzyme was found in the wall of the mature cyst, the possible involvement of this enzyme in the formation of the outer wall of the cyst was considered. To establish a functional role for laccase in cyst-wall formation, two indirect lines of evidence were examined. First, an effort was made to determine whether there is a correlation between the production of laccase and the onset of cyst formation. Second, known inhibitors of laccase were used to examine the potential inhibition of encystment. To characterize the enzyme and further establish its import- ance, it was necessary to purify the enzyme. The cysts produced by the established encystment procedures were observed to have good enzyme activity, but not in a soluble form. To obtain a large quantity of the enzyme in soluble form, efforts were directed toward the portion of the enzyme that was secreted into the encystment medium. Thus, 13 alterations in the encystment procedure were made, and experiments were performed to define the parameters for encystment that would result in maximal secretion of the enzyme into the inorganic saline. With these altered encystment procedures, a large quantity of the enzyme was pre- pared and then purified. This purified preparation was used to study some of the biochemical properties of the enzyme. MATERIALS AND METHODS Cell Cultures Acanthamoeba castellanii, strain Ifii (Hartmannella rhysodes Singh), was obtained from R. N. Band. The vegetative amoebae were cultured in Proteose Peptone (Difco) glucose medium by the method of Band (1959). Routinely, Proteose Peptone-glucose medium was inoculated with an initial cell density of 2-4X105/ml and 200 m1 of culture were placed in a one-liter, silicone-coated flask. The cell cultures were incubated at 291:10C on a rotary shaker at 100 revolutions per minute. Amoebae grown under these conditions reach a maximum cell density of 2-4X106/ml in about four days. Encystment Procedure In experiments where healthy, mature cysts were desired, cysts were produce by the method of Band (1963). This method involves amoeba starvation, followed by placement of the cells in a medium with in- creased osmotic strength. Starvation medium was derived from Proteose Peptone-glucose medium by reducing the amount of glucose ten-fold, and was then filtered through a 0.45 um.Mi11ipore filter (Millipore Corp., Bedford, Mass.). After 48 hrs in the starvation medium, amoebae were washed in a low salt solution (LS): 50 mM NaCl, 4.6 mM MgSO and 0.36 4' mM CaC12. After 48-72 hrs in HS medium, nearly 100% of the amoebae encysted. 14 15 Enzyme Preparation During the course of this research, it was observed that laccase was secreted, in soluble form, into the HS encystment medium. The amount of secreted enzyme could be increased about 20-30 fold if the starvation period was omitted. The following procedure was adopted to obtain a soluble enzyme preparation. The vegetative cells were harvested at a cell density of 1-3X106/m1, washed in LS, and resuspended in HS. After 24-48 hrs of incubation, the soluble enzyme was collected by cen- trifuging or filtering (Whatman #1) the cysts from the HS encystment medium. The enzyme preparation was further clarified by filtration using a 0.22 um membrane filter (Millipore Corp.), to give a crude soluble enzyme preparation (CSEP). For some experiments, the enzyme preparation was then dialyzed against distilled water or 20 mM potassium and sodium phosphate (0.349 g NaZHPO4 and 2.387 g KH2P04/liter) buffer, pH 6.0 (KNP). . O . These preparations could be stored at 4 C for several weeks Without significant loss of enzyme activity. Enzyme Assays The conditions for the laccase assay were modified from those of Leonard and Phillips (1973). The assay mixture was buffered at pH 6.0 with 20 mM KNP. A 15 mM stock solution of the substrate, L-B-3,4- dihydroxyphenylalanine (DOPA), was made in the same buffer. In those experiments in which the enzyme was not dialyzed against 20 mM KNP buffer, 15 mM DOPA was made up in 60 mM buffer. The reaction was initi- ated by adding 1 m1 of the DOPA solution to 2 m1 of the enzyme prepara- tion. Since the reaction rate is not linear during the first 3-5 min after initiation, the change in absorbency at 475 nm was determined 16 after five minutes of incubation after initiation. A unit was arbitrarily defined as that amount which would cause an absorbency change of 0.001/min. The values presented as data (points on a graph or numerical values) in this thesis represent the average from duplicate determinations, unless stated otherwise. In experiments in which particulate enzyme preparations were used or where turbid solutions were involved, the assay mix was incubated for 5-8 min and was then clar- ified by centrifugation (3,000 g for l min). The absorbency change was determined by subtracting the absorbency reading of a similarly treated sample to which substrate had not been added. For most experiments where relative enzyme activity was the only major concern, the assays were performed at room temperature (23-250C) using a Spectronic 20 colorimeter. For the determination of proportion- ality of enzyme quantity to absorbency change, the temperature of the cuvette was maintained at 25 i 0.1°C and the change in absorbency was determined from the linear portion of a line traced by the chart re- corder of a Gilford 2400-2 spectrophotometer (Gilford Instrument Lab- oratory, Inc., Oberlin, Ohio). All other assay conditions were as de- scribed above. Substrate Specificity Substrate specificity of the enzyme was studied by measuring oxygen uptake with a Clark-type oxygen electrode (Yellow Springs In- strument Co., Yellow Springs, Ohio). The temperature was maintained at 25 i 0.10C. Oxygen uptake was determined from the linear portion of a line produced by a Sargent chart recorder (E. H. Sargent and Co., Chicago, Illinois). The values reported in Table 5 represent the 17 percentage of the available oxygen consumed in 1 min. The enzyme source for these experiments was a suspension of whole cysts at a concentration of 1X107 cysts/ml of KNP buffer. The substrate solutions listed in Table 5 were made up in 20 mM KNP buffer. The reaction was initiated by adding 1 ml of the substrate solution to 2 ml of cysts. Enzyme Location in Cysts Mature cysts, prepared by using the starvation method of Band (1963), were broken open by use of a French press (Fred S. Carver, Inc., Summit, New Jersey). This was performed at 4°C using a piston pressure of 20,000 lbs/sq. inch. Cysts were suspended in buffer at a concentra- tion of 1X107/m1 and were passed through the press six times. Micro- scopic examination showed that more than 98% of the cysts were broken open by this procedure. A portion of the homogenate was solubilized in 1 percent Triton x-1oo. Both the Triton X-100 treated and the untreated homogenates (including pieces of cyst wall) were centrifuged at 8009;for 5 min at 4°C and the pellet was washed four times in buffer. The super- natants were subjected to increasing centrifugal force (as given in Table 6) and each supernatant and the pellets were assayed for laccase activity. Laccase Inhibitor Studies The effect of several known inhibitors of laccase (Leonard 1971) was studied using a crude soluble enzyme preparation. The inhibitors and their final concentrations in the assay mixture are given in Table 7. The effect of these known inhibitors of laccase on vegetative growth and encystment was also examined. For these experiments, 10 ml 18 of culture were incubated in 50-ml silicone-coated flasks. These experiments were done in duplicate. For these experiments, the inhib- itor was first dissolved in 1 m1 of the incubation medium. Since the pH of p-aminobenzoic acid in the medium was about 5.0, the pH was ad- justed to 6.2 by adding sodium hydroxide. The inhibitor solutions were sterilized by filtration, using a 0.45 um Millipore filter. After the cell number was determined by duplicate hemacytometer counts, vegetative amoebae were incubated in Proteose Peptone-glucose medium containing the inhibitors listed in Table 8. After three days of incubation, the growth was compared with growth in cultures which had no inhibitor. The effect of these inhibitors on encystment was studied using the methods described above. Thus, inhibitors (see Table 9) were added to amoebae during both starvation and HS incubation in some experiments and during only HS incubation in other trials. The percentage of cysts was determined by examining at least 100 cells in several microscopic fields. The cultures with inhibitor were compared to the cultures without inhibitor. Laccase Production During Encystment Vegetative amoebae were washed in LS and resuspended in H8 at a concentration of 3.4X106 amoebae/ml. In 100 m1 quantities, these suspensions were incubated in 500—ml flasks on the rotary shaker, as described earlier. At a given interval, the culture from a flask was processed as shown in Figure 1, and the different preparations (indicated in parentheses) were assayed for laccase activity. Dialysis of the var- ious preparations was performed at 4°C. Less than 10 m1 of each enzyme solution was dialyzed against two, 2-liter changes of distilled water 19 (Whole culture) Centrifuge 1000 g 5 min at room temperature Pellet Supernatant washed 3X in 20 mM KNP buffer; filtered 0.22 um resuspended in buffer at a Millipore cell density of 1x107/m1 crude soluble (washed total cells) enzyme preparation (CSEP) homogenized 3X in Logeman dialyzed against Mulsifier water homogenate of vegetative d' 1 .d EP cells and cysts ( 1a yzp CS ) centrifuged 100 g 5 min supernatant pellet dialyzed against discarded distilled water (dialyzed homogenate) Figure 1. Diagram of the procedure for measuring laccase production and cyst formation. Parentheses indicate preparations that were assayed. 20 over a 24 hr period. Washing and harvesting of cells and cysts was performed by centrifugation at 1,000 g for 5 min at room temperature. Homogenization was performed at room temperature using a Logeman Mul- sifier (Arthur H. Thomas Co.). Inhibition of Laccase by High Salt Ions The inhibitory effect of ions, contained in HS, on laccase activity was studied by adding ions to a crude soluble enzyme preparation that was dialyzed against water for three days. Each 20 m1 volume of the enzyme solution was dialyzed against six changes, of 4 liters each, of distilled water. The ion or chelator stock solutions were prepared in water at a concentration 10X greater than is given in Table 10, and 0.2 ml of the stock solution was added to 1.8 m1 of the enzyme prepara- tion. After mixing and adding buffered DOPA, the absorbency change/min was recorded. Extraction of Laccase from Cysts Mature cysts, prepared by the starvation method of Band (1963), were harvested, washed in buffer by low-speed centrifugation at room temperature, and resuspended in one of several extraction solutions at a concentration of 1X107 cysts/ml. After a 20 min incubation in the extraction solution, the cysts were washed in buffer and assayed for residual laccase activity. In some experiments, the extracting solution (supernatant from cysts) was dialyzed against water and assayed for soluble enzyme. 21 Altered Encystment Procedures Various additions or deletions were made in the encystment pro- cedure in an attempt to promote the secretion of soluble enzyme into the HS medium. In those experiments where the composition of HS was changed, the medium after encystment was dialyzed against water before assays were performed. The activity levels in each case were compared with the levels in control cultures. Protein Secretion by Cysts The method of Lowry, Rosenbrough, Farr and Randall (1951) was used to determine the protein concentration in crude soluble enzyme preparations (CSEP). In particular, the protein concentration of CSEP from encysting cells prepared by the method of Band (1963) was compared with the protein concentration of CSEP prepared by placing vegetative cells directly in HS. Thus, protein and laccase activities were deter- mined in the two CSEPs and expressed as mg protein/106 cells and as units/mg protein. Variables Involved in Soluble Enzyme Production Growth rate of vegetative amoebae In the study of the effect of the growth rate of vegetative cells on laccase production, active and slow growth were arbitrarily de- fined as shown in Table 1. After culturing of the vegetative amoebae under the two different growth conditions, the amoebae were induced to encyst in HS. The two cultures were harvested, washed in LS, and re- suspended in HS to a final density of 6.6, 4.4, 3.3 and 1.7X106 cells/ml. Crude soluble enzyme preparations (CSEP) were prepared, in duplicate, and 22 Table 1. Description of active and slow growing cell cultures.(Cells/ml) Initial Cell density Cell density Doublings cell density after three days after five days in five days ACtlve 0.1x1o6 1.3x1o6 4.4x106 >5 growth 6 6 Sigzth 2.0x106 4.8X10 6.6X10 <2 23 assayed after 24 and 48 hrs of incubation. Amoebae were grown and encystment was achieved in 500-ml flasks (100 m1 of cells per flask). Cell density during encystment The effect on laccase production of cell density during encyst- ment was studied. Vegetative cells, derived from a culture with a density of 4X106 cells/ml, were harvested, washed in LS, and resuspended in HS at densities of 1x104 to 1X107 cells/ml. Duplicate 10 ml suspen- sions were incubated in SO-ml flasks. After 24 and 48 hrs of incubation, the CSEPs were prepared and assayed for enzyme activity. Enzyme Purification Large batch preparation Cells were cultured in four-liter silicone-coated flasks with one liter of culture in each flask. After four days, under conditions of slow growth (see Table 1), cells were harvested under sterile con- ditions, washed in LS, resuspended in HS (3.5-4.0x106/m1), and incubated on the shaker for 40 hrs. Cells were removed from the HS medium by filtration (Whatman #1) and the filtrate was further clarified by filter- ing it through a 0.22 pm membrane filter. Enzyme concentration and acetone fractionation From a total of ten flasks, 9,250 m1 of crude soluble enzyme were obtained. This enzyme preparation was concentrated ten-fold by filtration using eight Millipore immersible CX probes (Millipore Corp., Bedford, Mass.). To each liter of the concentrated enzyme preparation, 1.45 liters of acetone (60% acetone) was added with continuous stirring 24 for 2 hrs at 40C. A11 further purification steps were performed in a cold room at 40C. The precipitate was collected by centrifugation for 20 min at 27,000 g. The pellets were resuspended in 100% acetone, and the precip- itates were collected by centrifugation as given above. After decanting of the supernatants, the pellets were allowed to air dry overnight. The dried pellets (which had a slight greenish-blue color) were resuspended in 10 mM KNP buffer and dialyzed against four liters of the same buffer (two changes). Column separation The methods of column separation were modified from those of Reinhammar (1970). The acetone-precipitated enzyme, dissolved in 10 mM buffer, was placed on a Whatman CM-52 cation exchanger (2.SX40 cm) equil- ibrated with 10 mM KNP buffer. The column was eluted with 360 m1 of the same buffer at a flow rate of 80 ml/hr, and 6.5 ml fractions were col- lected. A 500 ml linear gradient of 10-200 mM KNP buffer was then ap- plied. The greenish—blue fractions containing the laccase activity were pooled and placed on a Whatman DEAE-52 anion exchanger (2.5x40 cm) equilibrated with 10 mM KNP buffer, and 6.5 ml fractions were collected. A greenish-blue band formed at the top of the column and was not eluted with 250 m1 of 10 mM KNP buffer at a flow rate of 80 ml/hr. A 500 m1 continuous gradient of 10-200 mM KNP buffer was then applied, and some elution of laccase was achieved. Elution of the remaining laccase activity was achieved with 500 mM KNP buffer. The fractions that were eluted with 500 mM KNP buffer were pooled and stored at 4°C. 25 Enzyme Purity Absorbency spectrum of Acanthamoeba laccase Using fraction number 181 from the anion exchanger, both UV and visible absorbency spectra were determined by a Gilford 24,000-2 record- ing spectrophotometer. The blank cuvett contained 500 mM KNP buffer. Gel electrophoresis Purity of the major fractions from the anion exchanger was assessed by electrophoresis on 7% polyacrylamide gels by the method of Davis (1964). A 0.1 ml volume of the enzyme solution (45 ug protein) was analyzed on each gel. After electrophoresis, the gels were removed, and parellel gels were stained for protein and laccase activity. Proteins were stained using the method of Fairbanks, Steck and Wallach (1971). Laccase activity was visualized using a modification of the method of Leonowicz and Trojanowski (1975a). The gels were soaked in 5 mM DOPA, in 20 mM KNP buffer. A black, melanin-like material was deposited at the site of laccase activity after 30 min of incubation at 35°C. Densitometric traces of stained gels were made with a Gilford 2400-2 gel scanner. A wavelength of 550 nm was used for both laccase and protein. Molecular Weight Determinations The molecular weight of Acanthamoeba laccase was estimated by sodium dodecyl sulfate gel electrophoresis using the method of Weber and Osborn (1969). The following proteins were used to calibrate the gels for molecular weight determinations: albumin, transferrin, tyrosinase 26 and ceruloplasm. After the gels were stained for protein by the method of Fairbanks, Steck and Wallach (1971), the relative mobility of each marker protein was determined. Mobility was then plotted against the known molecular weights of these marker proteins. Enzyme Properties The effects of pH, temperature, enzyme concentration, and sub- strate concentration were assessed using the enzyme preparation eluted from the anion exchange column. The enzyme preparation was dialyzed against water or against 20 mM KNP buffer before analysis. Phthalate buffer (20 mM) was used for the pH range of 4.0 to 6.0. KNP buffer was used for the pH range above 6.0. Comparison of Cyst Wall Enzyme and Soluble Enzyme In studies totally independent of the soluble enzyme prepara- tion tests, cysts were used as the enzyme source for enzyme characteri- zation. These cysts were prepared by the starvation method of Band (1963). The laccase activity of these cysts was determined by adding 1 m1 of DOPA to 2 ml of buffered cysts. The reaction was terminated after 20 min by centrifuging out the cysts (1,000 g for 2 min at room temperature). The absorbence at 475 nm was then determined. Activity was calculated by dividing by the number of min of incubation. The cyst density for these experiments varied between 1-5><106 cysts/m1, but the density was the same for a particular variable that was examined. Materials The following chemicals were obtained from the Sigma Chemical Co. (Saint Louis, Missouri): phenylhydrazine, phenanthroline, phenyl- thiourea, thioglycollic acid, p-aminobenzoic acid, diethyldithiocarbomate, 27 the substrates listed in Table 5, and the reagents and protein markers for gel electrophoresis. Catalase was obtained from Worthington Bio- chemical Corp. (Freehold, New Jersey). All other chemicals were analyt- ical reagents. Distilled water was used in all preparations. RESULTS Laccase Activity from Encysting Amoebae The laccase activity produced by several different suspensions of encysting amoebae, after crude soluble enzyme preparations were prepared, is given in Tables 2 and 15. The encystment conditions were similar in all experiments, and did not involve starvation. These results demon- strate that the laccase enzyme can be recovered from the HS medium of encysting cells. The quantities produced (units/ml) vary considerably from one culture to another. Identification of the Enzyme The DOPA oxidizing enzyme from Acanthamoeba was identified as a laccase, and not a catechol oxidase or a peroxidase. The enzyme from Acanthamoeba was not inhibited by phenanthroline (Table 3). Inhibition by phenanthroline is considered diagnostic of per- oxidases (Leonard 1971). Further, a peroxidase would show augmented activity if hydrogen peroxide was added, and the addition of catalase would cause a decrease in activity (Mayer and Harel 1979). In the presence of hydrogen peroxide or catalase, there was no significant change in activity for Acanthamoeba laccase (Table 3). Therefore, the enzyme from Acanthamoeba is not a peroxidase. Catechol oxidases are able to catalyze the oxidation of tyrosine and other monophenolic substrates, and are inhibited by phenylhydrazine 28 29 Table 2. Laccase activity yields from the encystment medium of different cell suspensions. Trial Laccase units Standard laccase units Standard after 24 hrs deViation after 48 hrs* deViation 1 6.2 .14 4.0 .35 2 2.3 .07 5.2 .21 3 1.9 .14 2.6 .14 4 2.0 .14 1.6 .65 5 1.7 .21 3.1 .08 6 3.8 .35 4.4 .28 7 4.6 .14 2.3 .07 8 2.9 .21 7.0 .21 9 5.5 .00 10 3.3 .35 11 6.4 .00 Average 3.2 (8) ' 1.59 4.1 (11) 1.74 NOTE: Values are expressed as units/2 m1 of the crude soluble enzyme preparation. The values are averages from duplicate assays. *The values for the 24 and 48 hr measurements were not deter- minations from the same encystment medium. 30 Table 3. Identification of Acanthamoeba enzyme as a laccase and not a peroxidase.* Additions to the ' Units of laccase activity 5 mM L-DOPA reaction mix NOne 6.9 Hydrogen peroxide (0.3 mM) 6.9 Catalase (150 us) 6.8 Phenanthroline (1 mM) . 6.4 w *Unless stated otherwise, the numerical values in all tables of this thesis represent averages of duplicate determinations. 31 Table 4. Identification of Acanthamoeba enzyme as a laccase and not a tyrosinase.* Reaction mix Z of the available oxygen utilized/min KNP buffer 0.4 L-DOPA 6.4 L-DOPA plus 0.1 mM 6.8 phenylhydrazine 5 mM potassium 7.3 ferrocyanide L-DOPA plus 1 mM 0.0 potassium cyanide L-DOPA plus S'mM 0.0 sodium azide *Whole cysts were the enzyme source and oxygen uptake was measured.‘ 32 (Mayer and Harel 1979). The enzyme from Acanthamoeba was found incapable of oxidizing tyrosinase and other monophenolic compounds (Table 5) and was not inhibited by phenylhydrazine (Table 4). The enzyme was com- pletely inhibited by 1 mM potassium cyanide and 5 mM sodium azide (Table 4). Oxygen uptake was observed when potassium ferrocyanide was added (Table 4). This is considered typical behavior for a laccase, but not for a tyrosinase (Leonard 1971). In fact, tyrosinase from the mushroom (Sigma preparation) did not cause the uptake of oxygen in the presence of potassium ferrocyanide. The enzyme from Acanthamoeba did catalyze the oxidation of several o— and p-diphenols as well as phenylenediamine (Table 5). The catalysis of p-diphenols (e.g., hydroquinone) is considered diagnostic for the laccase enzyme (Mayer and Harel 1979). The enzyme from Acanth- amoeba cannot be a catechol oxidase. Substrate Specificity Table 5 shows the relative rates of oxygen uptake with washed cysts (1x107 cysts/ml) in the presence of different substrates. Sig- nificant oxygen uptake, comparable to that of L-DOPA, was noted for all substrates except tyrosine and other monophenols. These results show that the enzyme of Acanthamoeba acts specifically on o- and p-diphenols and does not act on monophenols. Enzyme Location in Cysts When mature cysts were ruptured in a French press and the cyst components were separated by centrifugation, the low-speed particulate fraction (800 g for 5 min) was found to contain all of the significant laccase activity (Table 6). Homogenate fractions, with or without 33 Table 5. Substrate Specificity of Acanthamoeba laccase.* Substrate(5 mM final concentration) 1 of the available oxygen __7 utilized/min Water 0.8 KNP buffer 0.7 L-dihydroxyphenylalanine (DOPA) 5.3 D-DOPA 5.3 L-DOPA methyl ester ‘ 5.5 OH-tyramine 5.3 Epinephrin 6.2 P-phenylenediamine 7.4 Catachol (no enzyme added) 0.2 Catachol 6.5 Hydroquinone (no enzyme added) 0.2 Hydroquinone 9.5 Pyrogallanol 10.3 Ferulic acid 6.1 Tyrosine methyl ester 0.7 Tyroamine 0.9 m-cresol 0.8 B-phenylethylamine 1.3 *Whole cysts at a concentration of l x 107/ml were used as the enzyme preparation. 34 Table 6. Localization of laccase within cysts. Cyst frac ion laccase units laccase units (1.5 X 10 / ml) (with Triton X-100) Washed particulate 50.4 65.4 material Supernatant of 1.1 0.7 washed particulate material (lg,lhr) Supernatant of 0.3 0.4 homogenate (800g, 5 min) Supernatant of 0.3 0.2 homogenate (7,000g, 20 min Supernatant of 0.1 0.2 homogenate (10c,0005, 2 hr) CSEP from cysts 1.4 - CSEP from cysts 1.5 - (100,0003, 2 hr) None (buffer) 0.05 0.03 35 Triton X-100, that did not contain cyst walls or pieces of cyst walls did not have laccase activity. Table 6 also shows that the enzyme of a CSEP does not sediment at 100,000 9. Thus, although the enzyme of a CSEP may or may not be totally soluble, this enzyme is more soluble than the enzyme of the cyst wall. Laccase Involvement in Encystment: Inhibitor Studies Two areas of functional importance of laccase in Acanthamoeba were studied: the effect of laccase inhibitors on cyst formation, and a correlation between cyst formation and enzyme production. The effects of several known inhibitors of the enzyme laccase on Acanthamoeba laccase are shown in Table 7. In the presence of inhibitor, 21-100% of the laccase activity of Acanthamoeba was lost. When these same inhibitors were added to amoebae in growth medium, vegetative growth was not sig- nificantly suppressed, except in the case of thiourea (Table 8). When these inhibitors were added to encysting cells at the same concentration as that which permitted vegetative growth, the fraction of amoebae that formed cysts was reduced by as much as 50% (Table 9). Note particularly the results with diethyldithiocarbamate. Table 7 shows that the enzyme of Acanthamoeba is completely inhibited by this known laccase inhibitor (Dubernet et a1. 1977). This inhibitor also caused a reduction in the percenthfcysts formed (Table 9) without inhibiting vegetative growth (Table 8). Although inhibition was not complete with any inhibitor, a sig- nificant reduction in the percent of cysts formed was observed (Table 9). In these particular experiments, the percentage of cysts in the 36 Table 7. The effect of laccase inhibitors on Acanthamoeba laccase activity.* Inhibitor I Final Laccase 1 inhibition concentration units .__. --- _—_ .i__i-r- E .- __l__ None (buffer) s - 6.9 I - Phenylthiourea l 0.1 ms 2.0 I 70.8 Thioglycollic acid ' 0.5 mM l 0.0 100 p-aminobenzoic acid . 0.5 mM i 5.4. 21.2 p-aminobenzoic acid ‘ 5.0 mM 3 1.7 75.2 Diethyldithiocarbomate 0.3 mM 0.0 100 Sodium azide 5.0 mM 0.0 100 Potassium cyanide 1.0 mM 0.0 100 *Enzyme preparation was a CSEP. 37 Table 8. Growth of vegetative amoebae in the presence of laccase inhibitors.* Cell number/ml after three days incubation in culture medium plus Final concentration of inhibitor -- . ... ‘dm-_~.“--‘ --—— inhibitor.* 1 mM phenylthiourea 2.0 x 105 ** 5 mM Thioglycollic acid 8.8 X 105 5 mM p-aminobenzoic acid T 6.3 X 105 0.3 mM.diethyldithiocarbamate : 8.8‘x 105 None (buffer) 8.1 x 105 *Initial cell density was 2.2 ><105/ml. ** values in this table represent the averages of duplicate determinations on two independent cultures. 38 Table 9. Inhibition of cyst formation in the presence of laccase inhibitors.* Final concentration: of inhibitor Inhibitor added to starvation and to HS encystment medium. Inhibitor added to H3 encystment medium only. Cell number] [I cysts ml times 105*l 5 mM Thio- i glycollic acid 5 mM p-amino- benzoic acid 1 mM p-amino- benzoic acid 0.5 HIM p- 8101110" benzoic acid 0.3 mM diethyl- dithiocarbomate 1 mM phenyl- thiourea None (buffer) 14.0 *** cytolysed 10.0 5.9 10.0 ,64 48 44 93 *Initial cell density was 11 x 105/ ** Initial cell density was 15 x 10 *** Values in this table represent m '31 ' teel'averages of duplicate determinations on two independent cultures. ”— cell number/ 1 cysts ml times 105** 16.0 46 cytolysed - 14.0 41 16.0 50 14.0 36 15.0 40 15.0 90 39 control was less than 100% but much greater than in the experimental cultures. For one reason or another, 100% encystment is not always realized for Acanthamoeba. That there was an inhibition of encystment by laccase inhibitors suggests a role for laccase in Acanthamoeba cyst formation. Laccase Production During Encystment Soluble enzyme production from amoeba suspensions in HS encyst- ment medium, measured as a function of time, is shown in Figure 2. These results show that laccase was first detected in the harvested medium after 8 hrs of incubation, and that the amount detected increased with further incubation up to 42 hrs. When samples of the harvested enzyme were dialyzed overnight, against water, the enzyme activity was increased six-fold. In the next section of this paper, this six-fold increase in activity after dialysis is shown to be due to the removal of chloride ions. Laccase activity of the total cells in suspension also increased after an 8-hr lag period (Figure 3). When the activity of homogenized, non-cyst cells was measured after overnight dialysis against distilled water, an increase followed by a decrease was observed (Figure 3). The activity from cells and encystment medium reflects the activity measured in the whole encystment suspension (Figure 4). Parallel to the profile of laccase activity shown in these graphs is the profile of cyst formation shown in Figure 4. Cyst for- mation as a function of time is shown in Figure 4 as a line whose points are marked with Xs ( X X——*). This cyst formation profile shows that, following a lag period, enzyme activity increases concomitantly 40 .mosHo> oumoflaano on» :mo3uos moose ocu 305m mama Hmofluum> och .omw3uoauo ooumum manna: .usoauosusuouoo ounuuuano mo owouo>u on» ucououoou mucosa «us» ca nouswwu may no age so csozu mucaoo och .oawu mo scuuocsu o no caboose wcuuusoco mo mumo ecu cu huu>wuoo unsound u: we A: an Jn w~ ON 0H Nu m a ‘II ammo Aoon>amflov ammo .N ouawum o o l.n I 9 O D 13 n s m 7.. 3 U l.n~ [I z u I I.o~ IInN Inca 41 .ofifiu mo cofiuocam a mu summoned mo oumcomoEo: osu Scum can maamo HouOu Scum consumes huu>uuos omsuoma u; we we on on «N on ma Na w a I _ b _ p _ _ I, Aoonwacwov III|\\ mummuoua mo . ouacowoaom lame..ac adaoo asuOH .n ounwwm o o s 3 o e s a loam .l. 3. 8 II 7v m I: ma 1. 1: ON T: mm [I on 42 .cOwnsoousm ucoauuhoco egos: ecu cw muu>auuu unsound mousuwocu AnIIOIIIoIIIv muoo nous eczema oucwoa so“: mafia one .uobssc ammo moumowoc« Asllxullxlllv ax so“: ooxuce eunuoa buds cows 05H .oaau uo noduosam s as Hopes: uuho one cowusoouao usoauuhoso muons any :« couscous huu>auom unsound 5. 3 3 on o... .a S 3 S m r _ a _ p e . ‘ 1d.o a K B 3 I¢.m u m. a N m rIN.N T. ) X P I 0 (w. ld.n 1m.n l¢.e .e shaman o o 1IH Tl T~n a m 1.. .4 8 In, 2 m I Ina Inn 1:0 43 with cyst formation up to 42 hrs of incubation. The decline in laccase activity after 42 hrs of incubation, observed in these experiments, is not easily explained but does represent unusual behavior. In other experiments, a decline in laccase activity after 42 hrs was not observed. That cyst formation begins only after laccase is produced suggests a functional role for laccase in cyst formation. Ion Effect on Laccase Activity Since dialysis of the CSEP consistently caused about a six-fold increase in laccase activity, the inhibitory effect of ions contained in HS was studied. Various ions or chelators were added to a dialyzed CSEP. The results are shown in Table 10. When the ions of HS were added to a dialyzed CSEP, the activity was reduced from 13.8 to 2.5 units. A sim- ilar reduction in activity was observed when potassium chloride was sub- stituted for sodium chloride in the HS, and when sodium or potassium chloride alone was added. When the anion consisted of sulfate or acetate, no reduction in activity was observed. Thus it was concluded that the chloride ion is inhibitory to this enzyme. Others have reported the inhibition of phenoloxidases by halogens (Brander, Malmstrom and Vanngard 1971; Ben- Shalom, Kahn, Harel and Mayer 1977). The effect of the calcium and magnesium ions of HS was also studied. Table 10 shows that there is no significant change in laccase activity when these ions are added to CSEP at the concentration found in HS. Magnesium sulfate at a higher concentration (17 mM) did cause some inhibition. EDTA was observed to cause a reduction in laccase activity (Table 10). 44 Table 10. Laccase activity in the presence of various ions.* Substance added and the concentration Laccase units None (water) 13.8 NaCl, 250 mM ) MgSO4, 4.6 mM I 118 2.5 CaClz, 0.36 mM KCl, 250 mM Mg804, 4.6 mM } Potassium HS (K-HS) 2.8 CaClZ, 0.36 mM NaCl, 250 mM 2.8 KCl, 250 mM 3.1 Sucrose, 250 mM 14.0 Sodium acetate, quantity of sodium same as as f 13.8 Potassium acetate, quantity of potassium same as K-HS g 14.0 Sodium sulfate, quantity of sodium same as HS ! 14.1 Potassium sulfate, quantity of potassium same as K-HS 14.3 MgSO4, 17 mM 9.3 MgS04, 1.7 an 12.8 MgSOa, 0.17 mM 13.5 M3304, 4.6 mM 13.1 CaClz, 0.36 mM 13.8 CaClz, 0.5 mM 15.2 EDTA, 0.1 mM 9.5 EDTA, 1 mM 9.0 *The enzyme preparation was a CSEP that was dialyzed against water. The reaction mix consisted of 1.8 ml of the enzyme preparation and 0.2 ml of a 10x solution of the substance. The molar concentrations are based on two ml of enzyme prior to the addition of one m1 of the DOPA substrate. 45 Extraction of Laccase from Cysts Cysts produced by starvation prior to incubation in HS were ob- served to have significantly high laccase activity and several attempts were made to solubilize the enzyme from the cysts. Table 11 shows sev- eral methods employed to solubilize the enzyme from mature cysts. None proved successful in removing active enzyme. Lithium chloride (6-8M), considered to be a chaotropic agent (Hills, Phillips, Gay and Roberts 1975), did cause loss of cyst laccase activity, as did 20% TCA. Attempts to regain the lost activity by dialysis were unsuccessful. Altered Encystment Procedures Since attempts to solubilize the enzyme from mature cysts failed, a series of experiments was performed to alter encystment conditions such that more soluble enzyme would be secreted into the medium. Those varia- tions that did not cause a significant increase in the level of soluble laccase are outlined in Table 12. The procedure that did increase the level of laccase activity secreted to the medium was simply to omit the starvation period and place the vegetative cells directly into HS. The laccase activity yields from CSEPs prepared with and without starvation are shown in Table 13. The quantity of enzyme secreted into the encystment medium was effectively increased from 20- to 30-fold when the starvation period was omitted. As emphasized earlier, these data show that dialysis increases the laccase activity of a given preparation by about six-fold. Protein Secretion During Encystment The basis for this increased soluble laccase activity in the en- cystment medium was considered. It was observed, with omission of the 46 Table 11. Attempts to extract laccase from Acanthamoeba cysts.* Treatment Laccase activity of cysts after treatment for 20 min** None (control) ‘+ Autoclave for 30 min (control) 30% hydrogen peroxide 20% TCA 10 M LiCl 8 M LiCl 6 M LiCl 2% SDS 52 n-butanol 10 mm EDTA 4 M NaCl 4 H.KCI +++++++ 101 Roccal *Cysts at a concentration of l X 107/ml of extraction substance were incubated in the presence of the extracting substance for 20 min. After the 20 min treatment, the cysts were washed and DOPA was added. Activity was determined by visual inspection of the pink color development. **+ indicates activity nearly the same as the control. - indicates that all activity was lost. 47 Table 12. The effect of alterations in the encystment procedure on the secretion of soluble laccase.* ———.~_———-——..- Treatment Conditions Laccase units/2 ml None (control) Control (dialyzed Ferulic acid Ferulic acid. (dialyzed) Sodium azide (d ialyzed) Sodium chloride Deep culture (shaker) -———- Deep culture (stationary); 1 DMSO (dialyzed) Incubation at 4°C Deep culture plus cysteine (dialyzed) PH 13 I See Methods See Methods ' 0.1 an in HS for 48 hr 0.1 mM in NS for 48 hr 5 mM in as for 48 hr 250 mM NaCl for 48 hr 200 ml in 300 ml flask 200 ml in 300 ml flask ; 0.1 ml in 10 ml of LS Stationary, in HS for 48 hr ! 11 cysteine in HS for 48 hr NaOH added to HS 0.10-0.16 0.61-0.88 0.03 0.19 0.11 0.53 0.05 0.04 0.20 0.04 0.30 0.02 48 Table 13. Soluble laccase activity from amoebae that were starved compared to laccase activity from amoebae that were not starved .* Encystment Laccase units/2 m1 CSEP Laccase units/2 ml procedure dialized CSEP With starvation 0.1-0.3 0.5-1.5 (3 determinations) Without starvation 2-7 10-40 (3 determinations) *The encystment medium from amoebae prepared by the starvation method of Band (1963), compared to the encystment medium prepared by incubating amoebae directly in HS. Comparable cell numbers were used in all determinations. 49 starvation period, that only 60-90% of the amoebae produced cysts, and the cysts were notably heterogenous in size and morphology. Of those that did form cysts, 10-20% did not exclude dye (dead). The possibility of precyst lysis due to stressed encystment procedure was examined by comparing the amounts of protein in the medium and by comparing the specific activity of laccase. If lysis was occur- ing in encysting cells, induced to encyst by placing them directly into HS without prior starvation, an increase in total protein secretion would be expected. As shown in Table 14, on a per-cell basis, there was actually less protein secreted by cells placed directly into HS than was secreted by cells starved prior to being placed in HS. Since encyst- ing cells induced by starvation secrete less laccase to the medium, the specific activity (units/mg protein) from these cells was also less than that observed from cells placed directly into HS (Table 14). Thus, the basis for the increased activity from cells placed directly into HS remains unknown. However, these data suggest that the increased laccase activity is not due to the lysis of precysts. Variables Involved in Soluble Enzyme Production Growth rate of vegetative cells As is evident from Table 2, there is considerable variation in the quantity of laccase produced by different cultures. The following results are from experiments designed to obtain reproducible culture conditions for Optimal production of soluble laccase. Figure 5 demonstrates that the source of cells used for encyst- ment will affect the quantity of laccase secreted to the encystment medium. The laccase produced from slow-growing cells (see conditions in Table 14. —-~ --~—.~-—_-—-— —- .. Encystment* procedure 50 Total protein secretion by encysting amoebae. ‘pg protein/loécells With starvation (3 determinations) Without starvation (4 determinations) 58.1 :2.6 30.9 3.7 ........__-- __._. ....e-.__.--..> .-- v Laccase units/mg protein +. 2.2 0.4 15.5‘+5.6 *With and without starvation described in Table 13. oofim> ca oocoHoMMfio ens oumofiamso mo oumuo>m on» ucomoumon ounmflw wasp :H mosHm> one .muacn m.H on «.0 scum oomcmu ma0fiumswshouoo oumowHQDo omen» cooauon .mousufino HHoo ucoocomoocw 03» co mnofiuscfifiuouoo .mousuflsu mcfizoum 30am one o>wuom Eoum oo>wuoo mHHoo mcaumhbco scum moaofim ommoooH mo comwummsoo d .m shaman menu! as we no unsound mo oauu mason wcuuuhoso o>uuum sous o>uuus 30am mo ounce eusoum .81 5 Sins xv .rw Mm ewaoo NH mm as we “H1mm as oo 5H mm as we Hones: Hgou .\\. \x . o \\\\ \\~ \\.. \ II N I a "M I! Q m 8 a n 3 3 / Z Il.w m I O .4. S a. .1 II Na fl SH 52 Table 1), regardless of the cell density during encystment, was about three-fold greater than the laccase activity from actively growing cells (Figure 5). Cell density during encystment As can be observed in Figure 5, cell density is an important variable for maximal yield of laccase from encysting cells. The laccase yield appears to be proportional to the cell number below a cell density of 3.3X106 cells/m1, but is not when the cell density is above this number. This nonproportionality is probably due, among other causes, to poor encystment in the heavy-density cultures. The percentage of cysts was much lower in the high-density cultures. The critical cell density for maximal laccase yield is shown in Figure 6 to be 5X106 cells/ml. Cell densities above 5x106 cells/m1 gave poor laccase yields. It was observed that, at high cell densities, encystment was retarded and thus laccase secretion to the medium was minimal. This retarded encystment at high cell densities is shown dra- matically if the cell density is le07 cells/ml (Figure 6). At this density, encystment is nearly completely inhibited and secretion of laccase is likewise near zero. Harvest time for the enzyme The time of harvest of the HS medium is an important variable that affects the final yield of laccase. It was observed that maximal enzyme production was reached from 24 to 48 hrs after incubation in HS. (See Figures 5 and 6.) In most cases there was no decrease in enzyme activity from 24-48 hrs. In fact, the activity level usually increased with additional incubation time (Figures 5 and 6). There may only be a .m: s« caboose mo noduupsosw mo an we nouns masons mousouosu Alllvvoswa page. one .m: nu caboose mo soundness“ no u: «N nouns «humus nousowocq Acucv saws coxoub any .uobssc Hams mo combossw m as caboose wcwumzoco mo ammo onu scum apnea» unsound .0 shaman gassed N caboose mo season coca con OOH on ad m H snrun 1m z/Astarsos aseoost go 54 minimal increase in laccase activity between 24 and 48 hrs of incubation, as is the case shown in Figure 5. From these results, it was concluded that optimal laccase yields could be obtained by harvesting CSEP after 36 to 48 hrs of incubation. A summary of the optimal encystment procedures for the production of laccase by Acanthamoeba is as follows. Slow-growing amoebae that had fewer than two doublings in 5 days were placed into inorganic saline at a concentration of 3-4X106 cells/ml. After the amoebae had incubated in the inorganic saline for 36-48 hr, the encystment medium containing soluble laccase was harvested. These conditions for good enzyme produc- tion are reliable and repeatable, as demonstrated by the production of a large batch of cysts and enzyme from them (Table 15). Although consider- able variation is still evident among cultures, a yield of 20-30 units/m1 (after dialysis) can be realized. Enzyme Purification Large batch preparation From ten liters of cells grown in ten four-liter flasks, cells were harvested, washed in LS, and resuspended in ten liters of HS. The cell density was 3.5-4.0X106 cells/ml, and incubation was performed in four-liter flasks, one liter of HS/flask. After 40 hrs of incubation, CSEPs were prepared and assayed for laccase activity. The laccase yields from these suspensions are shown in Table 15. These results show that these methods are useful for producing about 20,000 units of soluble laccase from each liter of encystment medium (Table 16). 55 Table 15. Laccase activity yields from the crude soluble enzyme preparations of one liter suspensions.* Flask Actual counts of Laccase ‘pg protein] laccase number cells prior to units/ml ml unitsflng harvest (times 106) protein 1 3.9 3.9 103 37.9 2 3.3 3.1 101 30.7 3 4.1 3.6 87 41.4 4 3.6 2.1 102 20.6 5 3.7 2.8 77 36.4 6 3.4 2.9 100 29.0 7 3.9 3.5 88 39.8 8 3.3 1.9 98 19.4 9 2.1 64 32.8 10 1.5 71 21.1 Mean 3.7 2.7 89.1 30.3 Pooled 3.3 88 37.5 56 Concentration and acetone fractionation of laccase One of the major problems with attempts to purify laccase from Acanthamoeba encystment medium was the very dilute nature of the solution containing the enzyme. Attempts to concentrate the CSEP of Acanthamoeba through ammonium sulfate precipitation resulted in incomplete precip- itation, as was found by Reinhammar (1970). Preliminary attempts at precipitation in 50% acetone solution also resulted in significant loss of activity. With both methods, the volume was considerable, and cen- trifugation was cumbersome. An effective means for concentrating the enzyme preparation was found in the use of a Millipore CX probe (Millipore Corp., Bedford, Mass.). The volume of CSEP was reduced ten-fold without any significant loss of activity (Table 16). Thus, 9,250 ml of CSEP were reduced to 925 ml by using 8 probes over a 48-hr period. This ten-fold concentration of CSEP was made 60% in acetone, and the solution became cloudy. After being stirred for 2 hrs, the solution was centrifuged and the supernatant, which was clear, was discarded. The pellet was resuspended in 100% acetone and again collected through cen- trifugation. The residual acetone was then allowed to evaporate over- night at 4°C. The small pellet of dried material, about one gram, was resuspended in a 100 m1 volume of 10 mM KNP buffer and dialyzed overnight against 4 liters of buffer (two changes). Column separation Figure 7 shows the elution profile from the Whatman CM-52 cation exchanger after application of 220 m1 of the dialyzed enzyme preparation. uuohdmuo he snow ocauoaso mo He>oEou nouns huaaquom unsound sq ousouosu oaow o s wswasso summmm;:sr- cease m:r -Jas_\m~mme -2 .-...iwamoma.;zztz-:smmuwmri sauce «nauseous 58 laccase units .ooaooa one: owing ocowuosym .csnaoo Nnuzu usages: s sown adduced souusao unsound snooaunusso< .s enough Hones: noduosum CON cog ONH co ea c _ a r _ c Suwauuoe 1|.o.o :88qu L e .Ii muconHOmnm m. m II. a. m. ml e 1:46 w z I 8 0 I m can! .Iuw.o 7 «all J i o m u. o nl.o~ 055E ,1 59 Most of the laccase activity was eluted unretarded from the column by 10 mM buffer. After applying a continuous gradient of KNP buffer (10-200 mM), two small peaks of laccase activity were eluted. These peaks accounted for less than 7% of the total activity, and were discarded. The unretarded laccase, contained in 35 fractions (6.5 ml/fraction), had a greenish-blue color. These fractions (15-50) were pooled and dialyzed overnight against 4 liters of 10 mM KNP buffer (two changes). The pooled fractions from this column, containing 108,200 units, had a specific activity of 895 units/mg protein (Table 16). The specific ac- tivity of this preparation was about the same as that of the preparation before the use of this column, indicating that no purification was achieved. A total of 280 ml of the dialyzed enzyme preparation was applied to a Whatman DEAE-52 anion exchanger. The elution profile is shown in Figure 8. A greenish-blue band formed at the top of the column, and neither it nor any laccase was eluted with 10 mM KNP buffer. While a continuous gradient from 1-200 mM KNP buffer eluted what appeared to be three separate peaks of laccase, the greenish-blue band, although less intense, was not eluted and remained at the top of the column. After an increase in the concentration of the buffer to 500 mM, elution was continued. The greenish-blue band then moved through the column in a tight band and was collected. Fraction numbers 178-183, containing the laccase activity, were light greenish-blue. These fractions were pooled. This enzyme preparation accounted for about 27% of the concentrated CSEP (Table 16). The most prominent peak accounted for more than 67% of all enzyme activity recovered from the anion exchange column. The other chromatographically distinct enzyme peaks were not considered further in 60 laccase units .ooaooa one: mwanwna usouuosuh .ceaaoo unuu_:. _ _ _ (illlll Isa ufi Hu EC owN #m . >oconuomnm «1 (To a. a. . a c_ll llie.o m K D. 1 a! Tad m nu m w I: t ll.w.o 3 Cal. A192" :\ on: I; III ON 05:5— ] on OONi1 61 this study. The major component from this column contained 47,000 units (Table 16). A major difference between the behavior of Acanthamoeba laccase and that of Rhus (lacquer tree) needs to be considered. Rein- hammar (1970) found that Rhus laccase was retained by a CM Sephadex cation exchanger, but Acanthamoeba laccase was not retained by a com- parable cation exchanger. This difference in behavior may reflect a difference in the laccases of these two organisms. Laccase from Polyporus versicolor behaves in a similar manner as that of Acanthamoeba laccase (Fahralus and Reinhammar 1967). Enzyme Purity A lO-fold purification over that of the CSEP, with specific activity of about 5,000 units/mg protein, was achieved (Table 16). An index of purity for this enzyme is its 280/615 nm ratio. Note that, indeed, Acanthamoeba enzyme has absorbency peaks at 280 and 615 nm (Figures 9 and 10). The 280/615 nm ratio was 19 for this purified Acanthamoeba enzyme. This can be compared to a ratio of 14.6 reported by Farver, Goldberg, Wherland and Pecht (1978) for lacquer tree laccase, and a 280/610 nm ratio of 14-17 reported by Brander, Malmstrom and Vanngard (1971) for fungal laccase. When the purity of this preparation was further analyzed by gel electrophoresis, there was good correspondence between DOPA-stained and protein-stained bands (Figures 11 and 12). All of the three major bands of protein were those of the enzyme laccase. A couple of additional minor bands may be present (Figures 11 and 12). The use of DOPA as substrate for staining laccase was better 62 1.0"‘ _---—.-v— 0.8'-‘ 0.6—4 absorbency c> ¢~ 0.2-‘ 1 l l 250 300 350 400 wavelength in nm Figure 9. Ultraviolet absorption spectra of purified Acanthamoeba laccase 63 con .oosoosu sauce—snags counts.— uo sun-code coauauoube oak—«sway .3 one»: a: a.“ games.— 263 one use coo can can one coo \ 1n8.o 1 loHoé Aoueqzosqe llnuoé llo~o.c IlnNo.o 64 0.5—i 0.4‘"‘ E C. O 0.3— L0 L0 U (‘0 > D C 0.) fi 0.2-—- O U) .Q (‘0 0.1'—‘ 0.00 I I I 1 1 1— 0 2 4 6 8 10 12 gel length (relative units) Figure 11. Gel electrophoresis pattern of Acanthamoeba laccase. Stained for laccase. 65 0.5‘-— 0.4-—— E 003— O 1 L0 L0 .LJ f0 6‘ c 0.2— Q) .0 H O U) .Q (U 0.1"‘ 0.0 l 1 I ‘ 0 1 4 6 8 10 14 gel length (relative units) Figure 12. Gel electrophoresis pattern of Acanthamoeba laccase. Stained for protein. 66 than the use of phenylenediamine. Phenylenediamine was used by Leonowicz and Trojanowski (1975a), and the use of this substrate was tried. This substrate gave a dark background due to autooxidation. With DOPA as a substrate, there was less non-specific staining. Although the pH of the staining solution rose to near 7.5 during incubation (the electrophoresis buffer was 8.3), melanin deposits were specifically associated with protein bands (Figures 11 and 12). Molecular Weight Determination The purified enzyme and several marker proteins were electro- phoresed in the presence of sodium dodecyl sulfate. The purified enzyme of Acanthamoeba showed three bands on these gels (Figure 13). The most prominent band corresponds to a molecular weight of 160,000. The two minor components have molecular weights of 40,000 and 80,000 (Figure 13). These different molecular weight proteins may indicate that there are three different subunits of the Acanthamoeba laccase. However, Minuth, Klischies and Esser (1978) report that the laccase II of Podospora anserina is not totally dissociated by 1% sodium dodecyl sulfate, although inactivation of the enzyme was observed. After removal of the copper from this enzyme, a new band in sodium dodecyl sul- fate gel electrophoresis was observed. These authors conclude that this suggests a subunit structure for laccase II. This subunit structure was not evident on sodium dodecyl sulfate gels by the method of Weber and Osborn (1969). Thus, sodium dodecyl sulfate gel electrophoresis may not completely dissociate the laccase enzyme into monomers. If this is true for the Acanthamoeba laccase, the more prominent band may be a tetramer of laccase, and the monomer would have a molecular 100‘" 80“ 60- 40— 30" molecular weight X 104 67 laccase ceruloplasm tyrosinase transferrin laccase Q albumin laccase Figure 13. l l l o 0.1 0.2 relative mobility Relative mobility of Acanthamoeba laccase and marker proteins with sodium dodecyl sulfate polyacrylamide gel electrophoresis. The marker proteins and their molecular weights were: ceruloplasm, 150,000; tyrosinase, 128,000; transferrin, 85,000 and albumin, 67,000. 68 weight of 40,000. Without additional studies, the molecular weights reported here are difficult to interpret in terms of subunit structure. Properties of the Enzyme Optimal pH The purified enzyme preparation was used to study the properties of Acanthamoeba laccase. The enzyme showed a single peak of optimal sctivity at pH 6.0 (Figure 14). An optimal pH of 6.0 was also found when the enzyme source was whole cysts (Figure 14). Optimal temperature The optimal temperature for the DOPA-dopachrome reaction with o Acanthamoeba laccase was found to be 40 C (Figure 15). A temperature optimum of 35°C was determined for the whole cyst enzyme, although this preparation was not assayed at 400C (Figure 15). There was a broad temperature optimum shown for the purified laccase, ranging from 40 to 55°C. In this temperature range, the conversion of dopachrome to other products was very rapid. The formation of melanin also complicated interpretations from assays in this temperature range. At 25°C, the dopachrome end product appeared to be stable for at least 30 min. The 010 for the 20-300C temperature range was calculated to be 2.1 for this enzyme. This 910 value would be expected for an enzyme . . O . reaction. When the enzyme was incubated at 65 C for 2 min, complete inactivation was observed. Enzyme kinetics When the enzyme reaction rate was considered as a function of substrate concentration (DOPA), a typical Mechaelis-Menton (1913) 69 12--i . /\ / \ / \ / \ / \ 10—4 / \ ' q \ \ \ 8— \ \ \ \ \ 6— \ \ 'E' \ N \ E \ g \ 9 \ o 4 _— \ g \ 33 b 2—4 0 I I l l 4.0 5.0 6.0 7.0 7.4 pH Figure 14. Optimal pH for Acanthamoeba laccase. Whole cyst laccase is indicated by the broken line (---). Purified soluble laccase is indicated by the solid line (--). 70 35-‘ 25-—- 20-~ 15"“ 10— ' \ laccase units/2 m1 ° I I F-l I 10 20 3O 40 50 60 7O assay temperature (degrees C) Figure 15. Optimal temperature for Acanthamoeba laccase. Whole cyst laccase is represented by the broken line (---). Purified soluble laccase is represented by the solid line (—). 71 relationship was found for Acanthamoeba laccase (Figure 16). A Linewever-Burk plot (1934) reveals a Km of 0.21 mM and a Vmax of 17.9 units/min (Figure 17). This compares with a Km of 0.24 mM and a Vmax of 16.7 units/min for whole-cyst preparation (Figure 18). These Km values are about 20x lower than the value reported by Leonard (1971) for the laccase of the fungus, Schizophyllum commune. Enzyme concentration The reaction rate for Acanthamoeba laccase, measured as a change in absorbency at 475 nm, was proportional to the quantity of enzyme from 3.2-320 ul (Figure 19). This corresponds to an activity range of 1-125 units (Figure 19). When the quantity of enzyme was such that the change in absorbency was less than 0.001/min (less than one unit), the reaction rate was not proportional to the quantity of enzyme in the assay. Since most experiments reported in this paper dealt with enzyme activities greater than one unit, valid comparisons between experiments were pos- sible. Laccase Assays The reaction rate for Acanthamoeba laccase was not linear for a 20-min reaction period. A typical reaction profile using purified laccase, but similar also to the profile observed with CSEP, is shown in Figure 20. It can be seen that there is an initial lag period of about 5 min, followed by a linear period from 5-10 min and then a decline (Figure 20). For most experiments in this report, absorbency readings were taken during this linear period of the reaction. 72 .ousooua snooasnusso< mo mouuosax sousoxuoanssAofix as ea souusuuooosoo Ao3ucwq Azev scuusuucoocou oumuumaom mo auuououoou «N 0N ed «a a _ _ _ b P _ _ _ _ _ — .na owswwm sarun assoost go Isooadroai 74 em .ommoomH ammo oaona mooosmnucmo< mo Dodo stmuuo>osocfi4 AZEV cowumuucoocoo museumbsm mo Hmoouafioou ca NH w a o _ a P _ _ P _ .ma suamam l 3 3 Te. 1 O 3 e I 13 I 9 3 3 I e s a n m S EUR. 75 .oozsmmm ceases mo augusosv can ou mugs: success oouuuwsm mo Suwassowuuoaowm cause no #1 owu _ _ IIIONH winced .ma shaman (sarun) Anrnrnas aseooet 0.4-—i 76 0.2-— 0.1-- absorbency at 475 nm 0.0 [ 0 4 8 12 16 20 time (min) Figure 20. Absorbency change at 475 nm as a function of time, produced by purified Acanthamoeba laccase acting on DOPA. I! 77 Cyst and Purified Soluble Laccase Compared A comparison of the enzyme properties of whole cysts and of the purified soluble enzyme preparation is shown in Table 17. Although the possibility did exist that the CSEP was a digest product from the wall of whole cysts, the data would suggest they are the same enzyme. It is not possible to determine if subtle differences exist--for example, in glycoprotein chain length. It is possible that the soluble enzyme may differ from the whole cyst enzyme in glycoprotein chain length. 78 Table 17. Properties of particulate (cyst wall) laccase compared to soluble laccase. Property Whole cysts Partially purified examined . CSEP pH optima : 6.0 6.0 i Temperature ! 335° 40° optima 1 Km 1 0.24 mm 0.21 1111! V 16.7 units/min 17.9 units/min max Linear range of enzyme concentration 0.3-3 x 106cells/m1 3.2-320 p1 DISCUSSION The objective of this research was to define and characterize a cyst-specific enzyme usable with Acanthamoeba as a marker for further differentiation studies. As a cytodifferentiation model, the Acanth- amoeba vegetative-cyst system has the obvious advantage of being simple. There has been much experimentation with the mechanism by which inactive genes of the vegetative cell become activated to produce proteins or other gene products unique to the cyst stage. More specifically, these studies have been directed toward the mechanism by which a change in the environment of the amoebae causes certain active genes to become inactive and other genes to be "turned on" during encystment. However, these studies have suffered from the lack of a well defined, cyst specific gene product. The outer cyst wall is composed largely of protein. Little is known about how protein synthesis is regulated during the encystment process, or about how proteins are transported through the membrane and assembled into the cyst wall. It is known that the protein material of the outer wall is particularly resistant to chemical and physical agents, suggesting that the bonding and cross-linking of this wall material is unusually stable or cryptic. To learn more about the organization pattern of the outer cyst wall, and the trigger for its formation, Acanthamoeba was examined for the presence of an enzyme that might be involved in the formation of 79 80 resistant protein material. This research demonstrates that an enzyme capable of oxidizing phenolic compounds is present in Acanthamoeba (Table 5). The enzyme was found in the mature cyst wall, in the cyto- plasm of precyst vegetative cells, and in the medium in which encystment occurred (Table 6 and Figures 2-4). Enzyme Identification Since phenolic compounds can be oxidized by several different types of enzymes, it was necessary to identify the type of enzyme found in Acanthamoeba. This can be achieved by the use of inhibitors and of specific substrate molecules. Inhibitors of peroxidase (phenanthroline) and of catechol oxidase (phenylhydrazine) were added to the enzyme from Acanthamoeba. The lack of inhibition by these compounds, combined with the enzyme's failure to use hydrogen peroxide, tyrosine or other mono- phenols as substrate, suggest that the enzyme from Acanthamoeba is not a peroxidase or a catechol oxidase. The catalysis of o- and p-diphenols by the Acanthamoeba enzyme indicates that the enzyme is a laccase. This identification is further supported by the biochemical data reported in this thesis. Moreover, the biochemical characterization of the enzyme shows that the laccase of Acanthamoeba is comparable to the laccase found in the lacquer tree and in fungi. An optimum pH of 6.0 was found for both the purified preparation and a whole-cyst preparation. A comparable optimum of 6.5 has been reported for the fungal laccase of Schizophyllum commune (Leonard 1971). A pH optimum of 6.0 has been found for the fungus Leptosphaerulina briosiana (Simon, Bishop and Hooper 1979). The substrate affinity constant for Acanthamoeba laccase was found to be 0.21 mM with DOPA. This Km value compares well with that reported for 81 fungi in general (Mayer and Harel 1979), although DOPA was not used as a substrate in these determinations. Dubernet et a1. (1977) report a K.m value of 0.19 mM for quinol by the grape rot fungus, Botrytis cinerea. Leonard (1971) reports a higher Km value of 4.2 mM with DOPA for the fungus Schizophyllum commune. The absorbency spectrum of Acanthamoeba laccase also compares well with those reported for the fungal and the lacquer tree laccases. Thus the 280 and 615 nm absorbency peaks found for Acanthamoeba are the same as those found by Farver, Goldberg, Wherland and Pecht (1978) for the lacquer tree laccase. An absorbency ratio of 280/610 was used by Branden, Malmstrom and Vanngard (1971) as a purity index for their fungal laccase preparation. As mentioned earlier, Acanthamoeba laccase and the laccase from the fungi Botrytis cinerea (Dubernet et al. 1977) and Polyporus versi- color were retained by a DEAE column, while the enzyme from the lacquer tree was not retained by this column (Reinhammar 1970). Taken together, these comparable characteristics and behavior of the Acanthamoeba enzyme to those of the lacquer tree and fungi laccases constitute additional evidence that the Acanthamoeba enzyme is a laccase. The similarities between the fungal and Acanthamoeba laccase are partic- ularly interesting, and will be discussed later in this discussion section. Laccase of Cysts After demonstration that the phenoloxidase enzyme of Acanthamoeba is a laccase, its usefulness as a unique marker of the cyst stage was studied. Figures 2, 3, and 4 show that, indeed, this enzyme is unique to the cyst stage of this amoeba. In all fractions of an encysting amoeba 82 suspension, no laccase activity was detected prior to the formation of cysts. The line that is marked with Xs ( X X ) in Figure 4 repre- sents the cyst number in an encysting suspension and shows that the vege- tative cells, as a population, do not contain the enzyme. Thus, from the start of incubation until 81uuslater, no cysts were observed and no lac- case was detected in the homogenate of amoebae or in the suspension medium. Laccase was detected in precyst amoebae (broken open by a Logeman Mulsifier) only after an induction period of at least 8 hrs (Figure 3). These data suggest that laccase is synthesized in amoebae that have been "induced" in HS for longer than 8 hrs. However, it is also possible that 8 hrs of incubation in inorganic saline brings about increased enzyme ac- tivity in ways that do not involve de_ngza synthesis of laccase. The pro- duction of enzyme activity by the amoebae after 8 hrs of incubation may also be achieved by the activation of a proenzyme, or by the removal of competing substrate or inhibitor. Additional research is needed for a better understanding of the absence of laccase in vegetative amoebae, and of its presence in cells and in the medium only after cyst induction. This pattern does, however, provide a unique enzyme marker for the cyst stage of this developmental system. Laccase Function in Acanthamoeba After establishment of the presence of laccase in encysting amoebae, it was of interest to determine if this enzyme had a functional role in cyst formation or was present during encystment only coincidental- ly. The presence of the enzyme during cyst formation does suggest that it 83 may function in some way during Acanthamoeba cyst formation. Since the enzyme was associated with the outer cyst wall of mature cysts (Table 6), its possible role in the formation of the outer cyst wall was considered. Attempts to release the enzyme by washing and more drastic methods were not successful, suggesting that the enzyme becomes incorporated into the outer cyst wall protein during the formation of the wall. In order to establish a functional role for the enzyme in outer cyst wall formation, two types of experiments were performed. First, known inhibitors of laccase were added to encysting amoebae and the ef- fect on cyst formation was observed. Second, the time needed for cyst formation and its dependence on the presence of laccase were studied. When known inhibitors of laccase were added to encysting amoeba suspensions, inhibition of encystment was noted (Table 9). This suggests a possible role for the enzyme in Acanthamoeba outer cyst wall formation. However, there could be other explanations for the observed inhibition of encystment. The inhibitors may affect some metabolic process other than the enzymatic oxidation of phenolic compounds, or they may affect encyst- ment only by preventing polymerization or autooxidation of the wall com- ponents. Complete inhibition of encystment in the presence of these in- hibitors was not observed in these experiments, and incomplete inhibition has also been observed by Leonard (1971), using the same inhibitors to affect fungus fruiting body development. As was mentioned earlier, there is a good correlation between en- cystment and the appearance of laccase (Figures 2-4). Although this does not prove that laccase has a function in cyst-wall formation, it is highly suggestive. An increase in laccase with the appearance of fruit- ing bodies in Schizophyllum commune was considered by Leonard (1971) to 84 suggest a functional role for laccase in development of fruiting struc- tures. Inhibition of encystment with laccase inhibitors, the demonstrat- ed association of the enzyme with the outer cyst wall, and the presence of laccase only in encysting amoebae strongly suggest a role for laccase in Acanthamoeba outer cyst wall formation. If this enzyme is involved in the formation of the outer cyst wall, its function may be to catalyze the oxidation of phenolic substan- ces or proteins that are produced during encystment. Thus, proteins (containing phenolic amino acids) or hydroxylated phenolic compounds may be secreted through the membrane along with the laccase enzyme during encystment. The enzyme then may catalyze their oxidation. The further autooxidation of these phenolic substances could then cause the proteins to become cross-linked, forming the thick outer cell wall material. The nature of this substrate is not known, but it is possible that o- and p- diphenols are produced and secreted during encystment. The Acanthamoeba enzyme was shown to cause the oxidation of these molecules (Table 5). Furthermore, it was observed that the resulting autooxidation of DOPA and epinephrine produced melanin-like polymers. The autooxidation and poly- merization of other compounds may result in formation of other types of polymers. Thus, the yellowish-brown color of cysts may be due to the cross-linking and polymerization of proteins by one or several phenolic substrates. Several observations with Acanthamoeba show effects consistent with this mechanism of cyst-wall formation. First, debris-like material has been observed to be trapped in the outer cyst wall. This debris could be extruded material that becomes trapped in the wall during polymerization of substrate molecules, including proteins. 85 Second, Band (1963) has shown that oxygen is required for encystment. This may partially be a result of oxygen being a co-sub- strate for laccase. Band and Mohrlok (1969) found an increase in the uptake of oxygen by encysting amoebae that had been incubated in HS for 3-4 hrs. Interestingly, this 3-4 hr incubation time corresponds to the induction phase, which is followed by outer cyst wall formation. These authors concluded that the increase in oxygen uptake may be due to the functioning of a peroxisomal, non-energy-yielding pathway used by the amoebae to dump hydrogen. Although theirsiesstill a valid hypothesis, the requirements for oxygen in laccase oxidation is also a possibility. Finally, the resistance of the outer cyst wall to breakdown by microorganisms and chemical and physical agents (Barrett and Alexander 1977; Neff and Neff 1969) is consistent with formation of the outer cyst wall by melanized cross-linking of proteins. It has been shown that as- comycetes mycelia that are melanized have a greater resistance to lysis and microbial breakdown than those without melanin (Potgieter and Alex- ander 1966; Kuo and Alexander 1967). Although the mycelium of ascomy- cetes is melanized by the enzyme catechol oxidase (tyrosinase), laccase is also able to catalyze the formation of melanin. Thus, resistance by melanized or melanized-like protein in the outer cyst wall of Acanth- amoeba may serve to protect the cyst from breakdown by soil micro- organisms. This resistance, in the form of a physical barrier, is not abso- lute. It has been demonstrated that a natural population of Alternaria fungi were able to degrade Acanthamoeba cysts (Verma, Raizada, Shukla and Murti 1974). Barrett and Alexander (1977) demonstrated that bacteria can also participate in cyst degradation. 86 Although the exact function of laccase is not known, even for organisms which produce large quantities (fungi and lacquer tree), sev- eral theories have been offered. In addition to the idea that laccase is involved in the formation of a resistant physical and chemical barrier, two other theories have been offered that may apply equally well to Acanthamoeba. The toxicity of quinones and quinone-tanned proteins has been suggested as a deterrent to invasive predators (Mayer and Harel 1979; Kuo and Alexander 1967). This would be an important defense for a dormant, resistant cyst that lives in the soil among potential invasive microbes (Barrett and Alexander 1977). In the white-rot fungus, laccase is thought to function in the oxidation of lignin and other potentially resistant soil components, using them as an energy source (Ander and Eriksson 1976). It has been demon- strated that ferulic acid, a degradation product of lignin, serves to induce laccase in the fungus Pleurotus ostreatus (Leonowicz and Trojanow- ski 1975a). For these fungi, laccase may serve not in a protective cap- acity, but in a nutrient-providing capacity. It has been shown that, for these fungi, an adequate carbon source suppresses laccase production and starvation causes an increase in laccase production (Grabbe, Koenig and Haider 1968). For Acanthamoeba, the possibility exists that a pool of oxidized lignin and other nutrients is produced by the laccase of the outer cyst wall prior to excystment. This pool of nutrients may, in fact, be the signal that causes the amoeba to excyst; excystment has been shown to occur when nutrients are present in the medium (Neff and Neff 1969). 87 Laccase induction in Acanthamoeba by the addition of ferulic acid to the encystment medium did not cause an increase in laccase production (Table 12), but the timing of addition of ferulic acid and other variables were not fully studied. Thus, it is possible that ferulic acid induction might be shown for Acanthamoeba. Although these theorized functions for laccase in Acanthamoeba are speculative, they should serve as a basis for further research. Laccase in Other Amoebae Since laccase is present in soil-dwelling fungi and in Acanth- amoeba, it is possible that this enzyme may be present in other soil dwelling amoebae that form cysts. Thus, Acanthamoeba palestinensis, also a soil amoeba, was assayed for laccase activity. A very low level of lac- case activity was found in the encystment medium of this amoeba. The major basis for such low activity is probably that effective encystment procedures for this amoeba have not been established. Band's (1963) method for Acanthamoeba castellanii encystment yielded very poor encyst- ment with Acanthamoeba palestinensis, but those amoeba that did encyst showed some laccase activity in the medium and in the cyst walls. The cysts of Naegleria gruberi were also able to convert DOPA to melanin. The parasite Entamoeba invadens did not have this ability. These observations suggest that the occurrence of laccase in encysting soil amoebae may be a widespread phenomenon. The wide occurrence of laccase in fungi has been well established: laccase has been found in all fungi so far examined (Mayer and Harel 1979). Band (1962) and Adams (1959) suggest that a relationship might exist between algae and Acanthamoeba, since both groups have the same 88 vitamin requirements. A relationship between the fungi and Acanthamoeba is also a possibility. The restricted occurrence of laccase in the plant and animal kingdoms, its presence in most fungal groups, and its possible function in the production of a durable structure for both Acanthamoeba and fungi suggest the possibility of a relationship between these two organismic groups. Furthermore, the biochemical similarities between the fungal and Acanthamoeba laccases (described earlier) also support this idea. It could be theorized that amoebae were derived from fungi in a retrograde evolutionary process--that is, that Acanthamoeba is a spore stage of a fungus that has not reentered its normal, multi-cellular, vegetative state. If the laccase of Acanthamoeba is shown to function in lignin oxidation, the suggestion of an evolutionary relationship between fungus and Acanthamoeba could be further qualified to that of the white- rot fungus. The presence of laccase in these two organismic groups may, however, be the result of convergent evolution among soil-dwelling micro- organisms. Enzyme Secretion in Acanthamoeba Another question of interest is the mechanism by which the laccase enzyme is produced in the cell and is secreted through the mem- brane. The enzyme in the mature cyst wall is firmly bound and is thus particulate in nature. In several attempts, I was unable to extract, or to cause the release of, the enzyme from the cyst wall (Table 11). Entrapment or anchorage in the cell wall was also reported for the fungus Leptosphaerulina briosiana. In this case, laccase activity was found only in the cell wall (Simon, Bishop and HOOper 1979). Through use 89 of a histochemical stain and transmission electron microscope tech- niques, it was concluded that the enzyme becomes activated as it is po- sitioned in the cell wall. The rapid polymerization of protein and entrapment of catechol oxidase is thought to occur upon isolation of this enzyme, leading to the false conclusion that catechol oxidase is particulate (Mayer and Harel 1979). In melanosomes of mammals, the tyrosinase forms the nucleating point for melanin granule formation (Prota and Thomson 1976). Although laccases in general are considered to be soluble cyto- plasmic enzymes (Mayer and Harel 1979), the mechanism of secretion is not known for these enzymes. Phillips and Leonard (1976) report that, in the fungus Schizophyllum commune, there is an intracellular (cytoplasmic) laccase and an extracellular laccase. Again, the mechanism for the secretion of this extracellular enzyme is not known. In contrast to the conclusion reached by Simon, Bishop and Hooper (1979) that the enzyme becomes activated after it is positioned in the wall, these data (Figure 3) show that the enzyme is in an active form prior to that time. Specifically, it was found that the homogenate of precysts does contain some laccase activity. For Acanthamoeba the enzyme must be produced in the cytoplasm in soluble form, and then secreted through the membrane. One can only speculate as to why more laccase ac- tivity was not found in the cytoplasm. The cytoplasm of mature cysts, vegetative cells and early precysts does not show significant laccase ac- tivity (Figures 2 and 3). In several other experiments, no laccase ac- tivity was detected in precyst cytoplasm. Since preliminary experiments with Triton X-100, added to the ho- mogenate of precysts to give a 1% solution, failed to increase the laccase 90 activity level, it was concluded that the laccase of Acanthamoeba was not vesicle bound. This is consistent with the fact that there have been no reports of laccase being found in a subcellular organelle (Mayer and Harel 1979). Any effort to explain the mechanism of this secretion would be highly speculative at this time. The results shown in Table 14 indicate that secretion is not achieved by cell lysis. The demonstration that the laccase enzyme is found in the outer cyst wall of the mature cyst suggests that the enzyme must pass through the membrane during encystment. (The outer cyst wall is external to the cell membrane.) Although the mechanism of secretion remains unknown, methods that caused an increase in the amount of enzyme secreted were studied as part of this research. Since increased activity was secreted to the medium in soluble form, it is likely that amoebae lack control over the normal secre- tion process when these altered encystment procedures are used. Cysts produced by the altered encystment procedures were heterogenous in size and in outer-wall appearance. This suggests that the altered encystment procedures were stressful to cells. It is interesting to note that the final encystment procedure was such that, on a total suspension basis, more laccase was recovered from the HS medium than was found in the cytoplasm of precysts yielding maximal activity. In Figure 3, it can be seen that the homogenate of precysts yields less than ten units after 30 hrs of incubation. Since this homogen- ate was concentrated about 3-fold, this yield would be about 3.0 units/2 m1 on a comparable volume basis of CSEP. The yield from CSEP after 30 hrs is about 8 units/2 ml (Figure 2). After 42 hrs of incubation the difference is more dramatic. The 91 22 units/2 ml yield from dialyzed CSEP represents a much greater quantity than that recovered from precyst cytoplasm. The total yield from washed whole cells (including cysts added to precyst-cytoplasm activity) repre- sents about one-third of the quantity of laccase recovered in CSEP after 42 hrs of incubation. This illustrates that the encystment medium is the best source of the enzyme. These results also emphasize that these cells are under stress and lack control over secretion. For the enzyme to be useful to the cell, it would be expected that more activity would be associated with the cyst wall and the cytoplasm of cells than is lost to the medium. Normal encysting cells produced by the method of Band (1963) did not secrete significant laccase to the encystment medium, but cysts them— selves contained good activity. How each major change in the encystment procedure causes a change in the normal mechanism of release can only be speculated upon. The placement of cells directly into HS without prior starvation caused a 20- 30 fold increase in secreted enzyme activity. This drastic change in the ionic composition of the culture compared to the encystment medium could cause an increase in secretion rate, or a loss of association of the en- zyme with the outer wall after secretion. Since normal encystment by the method of Band (1963) also employs HS, the release of the enzyme from the cell wall is probably not the basis of this increased secretion. Without prior starvation, however, the stress on the membrane may be greater when the cells are placed directly into HS. An increase in activity by slow-growing cells compared to active- ly growing cells is not surprising, since one would expect slow-growing cells to be in the process of "gearing up" for encystment. These cells 92 are likely to be experiencing starvation prior to being harvested from growth medium. (I.e., the nutrients have been depleted.) It is known that the secretion of laccase in fungi increases when vegetative growth ceases (Mayer and Harel 1979) and is retarded if an adequate carbon source is present (Grabbe, Koenig and Haider 1968). Molitoris and Esser (1971) demonstrated that Podospora anserina mycelial extracts have max- imal phenoloxidase activity when they reach stationary growth. Active, growing amoebae encyst slower than slow growing cells, consistent with the earlier demonstrations that activity increases as the number of cysts produced in a culture increases (Figures 2, 3 and 4). When laccase activity secreted to the medium was studied as a function of cell number, a critical cell density was found. The pro- portionality of cell number to laccase activity at low cell densities (Figure 6) is not surprising. The lack of secretion by higher cell den- sities (1x107 cells/ml) is consistent with the poor level of encystment observed. Poor encystment may result from increased oxygen demand by heavy cell densities, since encystment requires oxygen. Finally, when the timing of harvest is considered, 36-48 hrs of incubation appears to be optimal for maximal laccase yields. Although normally there was no decline in laccase activity after 48 hrs, the in- crease was minimal from 36 to 48 hrs of incubation. This could be because encystment brings about a decrease in metabolic activity, thus preventing further production and secretion of the enzyme. Another possibility is that the enzyme becomes increasingly associated with the outer cyst wall during additional incubation time. Yet another explanation may be that the proteolytic enzymes in the medium begin to degrade the soluble enzyme that is secreted. 93 The enzyme secreted by these encystment methods is the same enzyme as that found in the outer cyst wall, as demonstrated by comparing their similar biochemical characteristics (Table 17). It can therefore be con- cluded that, for biochemical studies and large-scale production of the enzyme, the secreted soluble enzyme is the same enzyme as that found in normally encysting amoebae. Enzyme Purification In addition to being a simple system for cytodifferentiation studies, Acanthamoeba has the advantage of being conducive to biochemical studies. Large quantities of these amoebae can easily be grown in axenic culture in either Proteose Peptone-glucose or in a completely defined medium (Band 1962). Thus, in this research, a large quantity of the laccase enzyme was produced and purification was achieved. This and other biochemical studies with this laccase should help to establish this enzyme as a well defined biochemical marker of the cyst stage. Although four-liter flasks are rather cumbersome, use of these large flasks was considered the best method for the production of ten liters of soluble enzyme preparation. Whether due to the geometry of the four-liter flask (compared to the one-liter flask), or just to an individ- ual difference in this particular batch, the yield in units/ml was not as great as in some individual preparations from smaller flasks. (Compare Table 2 to Table 15.) However, as mentioned earlier, the average yield from the suspensions in Table 2, 4.1 units/2 m1 (2 units/ml), was lower than that shown for suspensions in Table 15, about 3 units/m1. This in- dicates that the use of the altered encystment procedure established by this research, and summarized earlier, provides a method for producing 94 predictable and reproducible laccase yields. The laccase yields in Table 2 are those from cells placed in HS without regard for the optimal cell density, or phase of growth of the amoebae. The yields in Table 15 are from cells whose encystment conditions were optimal for maximal laccase production. Thus the range for laccase yields produced by these encyst- ments methods was 20-30 units/ml, after dialysis (Table 15). Whatman #1 filter paper was used to separate the cysts from their encystment medium rather than centrifugation because it was quicker. Clarification by filtering through a Millipore 0.22 pm filter was effec- tive even though the filter membranes required changing after each 300 ml of medium, and required about one hr for filtering the ten liters. Millipore C-X immersible probes were effective for producing a ten-fold concentrated preparation. About 48 hrs were required to concen- trate this ten-liter preparation ten-fold. Experimental tests indicated that there usually was no significant loss of activity during a 48-hr period if the filtration was performed at 4°C. The use of more probes could shorten the time required for concentration considerably. As an alternative for concentration, precipitation of the enzyme by acetone or ammonium sulfate was found to be incomplete and cumbersome. In preliminary experiments with unconcentrated CSEP, 50% and 60% acetone fractionation was tried. When 60% acetone was used, nearly 90% of the activity was recovered, compared with less than 50% recovery with 50% acetone. Therefore, this concentrated enzyme preparation was fractionated with 60% acetone, and washed and dried in 100% acetone. There was a loss of 24% of the activity by acetone fractionation in this experiment. The acetone-dried pellet dissolved in KNP buffer only after considerable stirring. 95 Since the behavior of Acanthamoeba laccase in column chromatog- raphy was not known, a cation exchanger was used first as was done by Reinhammar (1970). This column did not retain any significant laccase activity, and the laccase was eluted unretarded. There would appear to be little reason to use this column in future attempts, since little purification is realized. Almost all of the protein corresponded to laccase (Figure 7). All of the laccase activity was retained by the Whatman DEAE-52 anion exchanger, and was not eluted with the 10 mM KNP buffer (Figure 8). A continuous gradient of 10—200 mM KNP buffer eluted some activity in three peaks. However, a greenish-blue band at the top of the column accounting for about 67% of the total activity recovered from the anion exchanger was eluted neither by this gradient nor by 200 mM KNP buffer. When 500 mM KNP buffer was applied, the greenish-blue band moved through the column. In future attempts, a gradient of 200-500 mM KNP buffer would be useful for elution of the anion exchange column. A molecular exclusion column would also be worth consideration, rather than the use of the cation exchanger. Again, it should be noted that the Acanthamoeba laccase behaved differently than did laccase from the lacquer tree with respect to the DEAE cellulose column. Lacquer tree laccase was not retained by this column (Reinhammar 1970), while Acanthamoeba laccase was in fact bound quite firmly by this column. The fungal laccase of Polyporus versicolor is also retained by anion exchange columns (Fahralus and Reinhammar 1967). The purity of laccase after DEAE cellulose chromatography was about ten times that of the starting material. The ten-fold purification 15”.:- 96 achieved does represent a comparatively pure preparation. Acanthamoeba CSEP may be nearly all laccase to begin with, as is the case for fungi, which secrete laccase to the medium in nearly pure form (Fahralus, Tullander and Ljunggren 1958; Fahralus and Reinhammar 1967). As an index of laccase purity, a 280/615 nm ratio was determined, and found to be 19. This value compares well to a ratio of 14.6 reported by Farver, Goldberg, Wherland and Pecht (1978) for Rhus laccase, and a 280/610 nm ratio of 14-17 reported by Brander, Malstrom and Vanngard (1971) for fungal laccase. The purity was also checked through use of polyacrylamide gel electrophoresis; nearly all protein bands corresponded to bands of laccase activity (Figures 11 and 12). Although there were several minor peaks of laccase eluted from the anion and cation exchangers, these were not considered further in this study. These activity peaks may represent genuine isozymic forms of laccase or simply represent microheterogeneity that has been reported for the fungal laccase preparation of Podospora anserina (Minuth, Keischies and Esser 1978). The basis for these other activity peaks will require further study with this Acanthamoeba enzyme. S UMMARY The free-living soil amoeba Acanthamoeba castellanii was found to contain an enzyme capable of oxidizing various ortho and para substituted phenolic compounds. This enzyme did not oxidize monophenols including tyrosine. After analyzing the behavior of the enzyme from Acanthamoeba with several different substrates and inhibitors, it was concluded that the enzyme is a laccase and not a tyrosinase nor a peroxidase. This laccase was specifically found in the mature cyst wall, in the cytoplasm of precyst cells and in the medium where encystment occurred. The soluble form, secreted into the medium, was demonstrated to be identical to that found in the mature cyst wall. Since the soluble form of laccase secreted into the medium was not quantitatively signif- icant when published encystment procedures were used, an alternative en- cystment procedure was developed which resulted in a 20-30-fold increase in the secreted enzyme. Briefly summarized, this procedure employed slow growing vegetative amoebae (amoebae that have had fewer than two doublings in five days) that were suspended in an inorganic saline of high osmotic pressure at a concen- tration of 3-4x106 cells/ml. After incubation from 36 to 48 hrs, the medium containing soluble laccase was harvested. The population of cysts produced by this method may be considered other than "normal." This altered encystment procedure was used to prepare a large quantity of the soluble enzyme which was then purified and characterized 97 98 biochemically. Acanthamoeba laccase was found to have a molecular weight of 160,000 for its major component, a pH optimum of 6.0, a 18“ value of 0.21 mM with dihydroxyphenylalanine and the enzyme was inhibited by the chloride ion. A role for Acanthamoeba laccase in the formation of cysts was demonstrated in two ways. First, inhibitors that are considered specific for laccase were found to inhibit the formation of cysts by as much as 50%, without inhibiting the growth of vegetative amoebae. Second, the production of laccase by a suspension of amoebae was shown to be dependent on the appearance of cysts. How this enzyme functions in encystment was not determined, but one can speculate that the enzyme may have a function similar to that re- ported for other laccase-containing organisms. One possibility is that Acanthamoeba laccase is involved in the oxidation of phenolic compounds which subsequently oxidize further and cross-link protein molecules to form the outer cyst wall. This type of melanin-like linkage is consistent with the fact that amoeba cysts are very resistant to mechanical and chem- ical breakdown. Melanin formation is known to be the basis of resistance of fungal mycelium to lysis and breakdown by bacteria. Oxidized phenolic compounds are also thought to be toxic and may serve to protect the dor- mant cyst from invasive microorganisms. Finally, Acanthamoeba laccase may act at the cyst wall location to oxidize soil debris and render the oxidized products available as a food source. The production of a food source by cysts could then be the mech- anism by which dormant amoebae are induced to excyst. In light of these results, the phylogenetic position of Acanth- amoeba castellanii might be reexamined. Band (1962) and Adams (1959) -m_ . 99 suggest that a relationship might exist between the algae and Acanth- amoeba. The restricted occurrence of laccase in the plant and animal kingdoms and the presence of laccase in all fungi so far examined suggest a relationship between the fungi and Acanthamoeba. Thus, Acanthamoeba may be a reproductive cell of a fungus that evolved in a retrograde man- ner to become an independent, free-living amoeba. These data will, it is hoped, rekindle interest in Acanthamoeba castellanii as an important model system to use for cytodifferentiation studies. Since laccase is an enzyme unique to the cyst stage of Acanth- amoeba, the use of laccase as a marker might yield interesting information concerning the "trigger" for cyst formation. If it is demonstrated that laccase is an inducible enzyme, information concerning the environmental inducer, and the enzyme's natural substrate, could be obtained. LIST OF REFERENCES LIST OF REFERENCES Adams, K. M. G. 1959. 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