OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. THE BIOCHEMICAL CHARACTERISTICS AND PHYSIOLOGICAL FUNCTION OF PHENOL OXIDASE IN SCHISTOSOMA MANSONI By John Lindon Seed A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology & Toxicology 1979 ABSTRACT THE BIOCHEMICAL CHARACTERISTICS AND PHYSIOLOGICAL FUNCTION OF PHENOL OXIDASE IN SCHISTOSOMA MANSONI by John Lindon Seed The schistosome egg appears to be primarily responsible for the development of most of the pathology associated with schistosomiasis. One component of the egg which has been demonstrated to be critical to the development of the pathology of the disease is the eggshell. It is the purpose of this thesis to establish the mechanism by which the eggshell is formed in Schistosoma mansoni and to evaluate the effective- ness of inhibiting this process in order to alleviate schistosomal pathology. Female §, mansoni were found to possess an enzyme which could be classified as phenol oxidase (E.C. 1.10.3.1). This enzyme was latent and could be activated by in_yi££g_incubation in a tissue culture medium or balanced salt solution. Enzyme activity increased from non-detectable levels up to .4li.02 umoles 02/min-mg protein. The enzyme was found 'exclusively in the female schistosome. Activation of the enzyme was not inhibited by 0.1 mM antimony potassium tartrate, a potent inhibitor of ATP synthesis. The enzyme sedimented in a 1,000 x g pellet and could not be solu- bilized by treatment with detergents, proteolytic and lipolytic enzymes, John Lindon Seed and freezing and thawing. The enzyme was found to have a pH optimum of 7.0 and was inhibited non-competitively by diethyldithiocarbamate (DDC) and allylthiourea. L-Dihydroxyphenylalanine (L-DOPA) was the best substrate (Km 8 0.5 mM; Vmax = .338 pmoles 02/min-mg protein). L-tyrosine and depamine were good substrates (Km = 0.5 mM and 2 mM; Vmax - .148 and .250 umoles 02/min-mg protein, respectively). Phenol oxidase was localized within the eggshell globules of vitelline cells by fluorescence histochemistry. The fluorescence of the product formed by L-tyrosine methyl ester in this assay was identical to the fluorescence of the product formed from the reaction of phenol oxidase with this substrate in_yi££g. The concentration of L-tyrosine in the female schistosome (252 ng/mg worm) was 3 times higher than the phenol oxidase Km for L-tyrosine while the concentrations of L-DOPA and dopamine (.954 and .790 ng/mg worm, respectively) were 100 and 500 times less than the Km for these substrates. L-DOPA was not actively accumulated by the female schisto- some. The peptide tri-L-tyrosine was oxidized at <32 of the rate of L- tyrosine methyl ester and a tyrosinezlysine peptide was not oxidized at a significant rate. Female g, mansoni did not incorporate L-tyrosine‘ preferentially into specific proteins and did not incorporate L-tyrosine into protein to a significantly greater extent than L-leucine. These results suggest that free L-tyrosine is the in yiyg_substrate for §3 mansoni phenol oxidase. Inactive analogs of phenol oxidase inhibitors, peroxidase inhibi- tors, autooxidation inhibitors and inhibitors of lipid peroxidation were incapable of inhibiting formation of the schistosome eggshell at 100 John Lindon Seed mg/kg in_zizg, This dose of a phenol oxidase inhibitor caused 100% inhibition of eggshell formation. 3H-Labeled proteins from female schistosomes were polymerized i§_yi3£g following incubation with S, mansoni phenol oxidase and excess 3H-labeled L-tyrosine. Fluorescent substances found in eggshell hydrolysates were similar to those formed from the reaction of phenol oxidase generated quinones with lysine. These observations support a concept of phenol oxidase catalyzed eggshell formation in which phenol oxidase reacts with tyrosine to form a quinone which subsequently reacts non-enzymatically with lysine residues in adjacent proteins to form a cross-linked protein which is one of the major constituents of the S, mansoni eggshell. DEDICATION To my wife, Ann, without whose support, patience and under- standing, this would not have been possible. ACKNOWLEDGEMENTS It is my pleasure to gratefully acknowledge the generous support, patience and guidance of my advisor, Dr. James L. Bennett, whose continuing efforts on my behalf have been of invaluable assistance in the completion of this thesis. Dr. Bennett's kind understanding and continuing support during the trials and tribula- tions of my graduate studies have contributed immensely to both my personal and scientific develOpment. His friendship and professional expertise has been and will continue to be a source of inspiration. I would also like to express my gratitude to those who have graciously contributed their time and knowledge in helping to complete these studies. They include Dr. Taie Akera, Dr. Kenneth E. Moore, Dr. Ralph Pax, Dr. Manly Pratt and Dr. Theodore M. Brody. My Special thanks to the graduate students and friends who have provided invaluable technical assistance and personal advice over the years, including Mike Boff, Clint Kilts, Jim Vrbanac, Mirdza Gramatins and many others. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS- ii LIST OF TABLES- vi LIST OF FIGURES ) 7 viii LIST OF ABBREVIATIONS- xi INTRODUCTION 1 A. Importance of the Schistosome Egg to the Development of Schistosomal Pathology B. Eggshell Formation l. Morphology 2. Biochemistry a. Reactions of quinones with amino acids and proteins 8 b. Requirements for the generation of quinones- 12 \lJ-‘bN 1) Enzymatic formation of quinones --------- 12 2) Non-enzymatic formation of quinones---- 15 c. Other mechanisms of protein hardening--—--- 16 d. Evidence for quinone—tanning in trematodes-- 20 C. Proposed Research 23 MATERIALS AND METHODS 25 A. Schistosomes 25 B. Activation of Phenol Oxidase in S, mansoni 25 C. Phenol Oxidase Assays 26 1. Standard phenol oxidase assay 26 2. Phenol oxidase assay for protein and peptide substrates 27 3. Mushroom phenol oxidase assay 29 D. Inhibitor Studies 29 E. Histochemistry 30 l. Catechol method 30 2. Fluorescent method 30 F. L-DOPA and Depamine Determinations 31 iii TABLE OF CONTENTS (continued) Tyrosine Determinations Identification of Crosslink Formation In Vitro-—-—----- Uptake Studies Tyrosine Utilization 1. Distribution of L-tyrosine into PCA-precipitable and non-precipitable fractions of S, mansoni homogenates 2. Tyrosine metabolism In Vitro Incubation Purification of Schistosome Eggshells Fluorescence Spectroscopy Fluorescence Characterization of the Endproducts of the Phenol Oxidase Reaction in S, mansoni Eggshells-—--—-- In Vivo Inhibition of Eggshell Formation ATP Assay Proteins Liquid Scintillation Counting Gel Electrophoresis Pathological Studies Statistical Methods Induction of Phenol Oxidase Activity Isolation and Histochemical Localization of Phenol Oxidase 1. Isolation of the enzyme 2. Histochemical localization Characterization of S, mansoni Phenol Oxidase 1. Specificity of the assay 2. Characteristics of the enzyme Identification of the In Vivo Substrate for S, mansoni Phenol Oxidase l. Soluble substrates a. L-Tyrosine concentrations b. L-DOPA and DA . Protein and peptide substrates Relative abundance of tyrosine in female proteins- LAN iv Page 37 38 39 40 4O 40 44 45 45 50 50 51 52 52 53 53 54 56 56 67 67 73 80 83 84 99 99 100 103 107 109 TABLE OF CONTENTS (continued) Page 4. Metabolism of tyrosine as it relates to eggshell formation 119 5. Soluble L-tyrosine and in vitro protein crosslink formation 123 E. Identification of the Link Between In Vitro Phenol Oxidase Inhibition and In Vivo Eggshell Formation---—- 130 F. Chemotherapy of Schistosomiasis Using Phenol Oxidase Inhibitors 142 DISCUSSI N 159 A. Induction of Phenol Oxidase 159 B. Isolation and Characterization of the Enzyme. 162 C. The Role of Phenol Oxidase in Eggshell Formation----- 170 D. Chemotherapy of Schistosomiasis Using Phenol Oxidase Inhibitors 176 SUMMARY AND CONCLUSIONS ‘ 178 BIBLIOGRAPHY 183 Table 10 11 LIST OF TABLES Page The effectiveness of various treatments which have been used to induce phenol oxidase activity The effects of temperature and type of incubation medium on the induction of phenol oxidase activity----- The effects of protein synthesis inhibitors on schistosome protein synthesis, ATP levels and phenol oxidase induction Oxygen consumption in pellets of female schistosome homogenates following successive administration of 1 mM succinate and 2 mM dopamine The effects of enzymes and detergents on phenol oxidase activity Protein levels in pellet of female schistosomes homo-' genized in either 5% Triton X-100 or 0.1 M phosphate buffer ' Effects of various treatments on relative intensity of fluorescent light from vitelline glands of adult female S, mansoni Effect of inhibitors on phenol oxidase activity in female S, mansoni Effects of substrates on phenol oxidase activity in homogenates of female S, mansoni Michaelis constants and maximal velocities for the sub- strates dopamine, L-DOPA methyl ester and L-tyrosine methyl ester L-DOPA and DA concentrations in male and female S. mansoni as determined by mass fragmentography (MF) and catechol-o-methyl transferase (COMT) radioenzymatic assay vi 58 61 63 69 71 72 79 87 91 92 104 LIST OF TABLES (continued) Table 12 13 14 15 16 17 18 19 Proteins and peptides as substrates for S, mansoni and mushroom phenol oxidase Incorporation of 3H-L-tyrosine and 3H-L-leucine into male and female S, mansoni PCA-precipitable proteins following 18 hrs in vitro incubation in egg producing (mod. RPMI 1640) and non-egg producing (BMEFC) media-- Male/female ratios for the incorporation of 3H-L- tyrosine and 3H-L-leucine into PCA pellets and superna- tants of S, mansoni pairs incubated 18 hrs in BMEFC or mod. RPMI 1640 Metabolism of 3H-L-tyrosine in female S, mansoni during 18 hrs in vitro incubation in BMEFC or mod. RPMI 1640-- Formation of insoluble protein polymers following in vitro incubation of H-female S, mansoni proteins with L-tyrosine plus S, mansoni phenol oxidase The effects of inhibitors of protein cross-link forma- tion on S, mansoni eggshell formation in vivo The effects of some inhibitors of protein cross-link formation on the DDC induced inhibition of eggshell formation in viva Number of eggs per mm2 of liver in mice chronically treated with disulfiram (0.3% in diet) vii Page 108 117 118 122 125 140 143 150 Figure 10 11 12 13 14 15 16 LIST OF FIGURES Anatomical features associated with egg production in female S, mansoni Reactions of benzoquinones with amines Two mechanisms of phenol oxidase catalyzed protein cross-link formation Electron impact mass spectrum of protonated L-DOPA---- Electron impact mass spectrum of d -DOPA 3 Migration of tyrosine and selected metabolites in 2- dimensional cellulose thin-layer chromatography---—-—-— Purified schistosome eggs Purified schistosome eggshells Activation of S, mansoni phenol oxidase by in vitro incubation s In vitro uptake of L-tyrosine by female S, mansoni---- Histochemical localization of phenol oxidase activity in female S, mansoni by catechol method Histochemical localization of phenol oxidase activity in female S, mansoni by fluorescent method Fluorescence spectrum of products extracted from female S, mansoni following incubation in 2 mM TME Specificity of the reaction between crude S, mansoni phenol oxidase and DA pH Optimum of S, mansoni phenol oxidase Stimulation of L-tyrosine oxidation by L-DOPA and DA—-- viii Page 18 33 35 42 46 48 59 65 74 76 81 85 89 93 LIST OF FIGURES (continued) Figure 17 18 19 20 21 22 23 24 25 26 27 28 29 3O 31 mansoni 122 days post-infection Noncompetitive inhibition of phenol oxidase by diethyl- dithiocarbamate Noncompetitive inhibition of phenol oxidase by allyl- thiourea L-Tyrosine concentrations in male and female S. mansoni as a function of age In vitro uptake of L-DOPA by female S. mansoni --------- In vitro incorporation of 3H-amino acids into male and female_S. mansoni PCA-precipitable proteins n vitro male/female ratios for the incorporation of H-amino acids into_S. mansoni PCA-precipitable proteins In vitro incorporation of 3H-amino acids into male and female S. mansoni PCA supernatants le/female ratios for the in vitro incorporation of H-amino acids into PCA supernatants Distribution of female S. mansoni protein in SDS-poly- acrylamide gel following incubation with S. mansoni phenol oxidase n Distribution of 3H—proteins from female-S. mansoni in a 10% polyacrylamide gel following incubation with S. mansoni phenol oxidase Fluorescence spectrum of acid hydrolyzed S, mansoni eggshells Appearance of normal S. mansoni egg in U.V. light---- Appearance of S. mansoni egg in U.V. light 1 hr follow- ing the adminigtrwation of 40 mg/kg diethyldithiocarba- mate Effects of disulfiram.on mortality of mice infected with S. mansoni Portal triad from liver of mouse infected with S. Page 95 97 101 105 110 112 115 120 126 128 131 135 137 145 147 LIST OF FIGURES (continued) Figure Page 32 S, mansoni eggshell globules in liver of disulfiram treated mouse 122 days post-infection 151 33 Eggshell globules in uterus of female S, mansoni ob- tained from disulfiram.treated mouse 153 34 Portal triad from liver of disulfiram treated mouse infected with S, mansoni,122 days post-infection------ 156 >4 ADP ATP BME BMEFC DA DOPA DOPAC DDC H & E HBSS HVA 3MT NE NM PAS PCA PFP PFPA PTTU S.E.M. SDS SbK TCA TCP TME TX-IOO UK-l4624 LIST OF ABBREVIATIONS adenosine-diphosphate adenosine-triphosphate Eagle's basal medium Eagles' basal medium containing 10% fetal calf serum and Earle's salt buffered with .02 M Tris, pH 7.4 dopamine dihydroxyphenylalanine dihydroxyphenylacetic acid sodium diethyldithiocarbamate hematoxylin and eosin Hank's balanced salt solution homovanillic acid 3-methoxytyramine norepinephrine normetanephrine periodic acid-shift perchloric acid pentafluorOprOpanol pentafluorOpropionic anhydride l-pheny1-3-(2-thiazoly1)-2-thiourea standard error of the mean sodium dodecyl sulfate antimony potassium tartrate trichloroacetic acid tranylcypromine tyrosine methyl ester Triton X-100 l-phenyl-3-(2-thiazolyl)-2-thiourea INTRODUCTION Schistosomiasis is a parasitic disease which has been estimated to infect between 117 and 200 million peOple throughout the world (Wright, 1968) and infects countless numbers of other animals in the vertebrate kingdom. This disease is particularly prevalent in developing nations where the lack of adequate sanitational facilties contributes to the maintenance of the life cycle of the schistosome. The life cycle of this parasite is complex, involving three morphologically distinct forms of the parasite and two hosts. ‘In brief, eggs excreted in the urine or feces of the vertebrate host release a free-swimming larval form known as a miracidium, when the eggs come in contact with fresh water. These miracidia then penetrate a snail host wherein they multi- ply and undergo transformation to an intermediate form known as cer- caria (Faust and Hoffman, 1934). The cercariae are subsequently released by the snail whereupon they penetrate a vertebrate host in the water and develop into mature schistosomes in the vertebrate host (Faust §£_§l,, 1934). The methods most commonly used in attempts to control the disease involve both destruction of the snail host and its habitat, and de- struction of the parasite within the host (WHO, 1973). However, these attempts have often met with rather limited success (WHO, 1973). The snail has proven to be highly resistant to the effects of molluscidal agents (Duke and Moore, 1976). The effectiveness of chemotherapy in 2 the host has been limited not only by the toxicity of most of the compounds currently in use (WHO, 1965; Katz, 1977; Bueding and Batzin- ger, 1977) but also by the predilection of the treated population for reinfection (WHO, 1965, 1973). In recent years, some interest has been expressed in the development of treatments which will either block the development of schistosomal pathology (Warren, 1968) or which will inhibit the process of egg production (Campbell and Cuckler, 1967). These two objectives are very similar inasmuch as they deal with the schistosome egg. A. Importance of the Schistosome Egg to the Development of Schisto- somal Pathology The deposition of schistosome eggs within host tissues is the primary cause of the pathology of schistosomiasis (Lichtenberg, 1955). The formation of a granuloma around each egg deposited in host tissues results in considerable necrosis and scarring of the affected organs. The primary organs involved include the bladder (S, hematobium), liver and intestine (S, mansoni, S, japonicum). The development of these granulomas is linked to the secretion of a number of specific, soluble, maturation-dependent antigens by intact schistosome eggs (Pelley g5 ‘31,, 1976; Hamburger §£_§l,, 1976). These antigens are produced by the developing miracidium within the egg and appear to diffuse through the eggshell into the surrounding tissues, providing the stimulus for granuloma formation (Hang §£_§;,, 1974). The proteinaceous shell which surrounds the miracidium does not possess antigenic activity and thus is not directly involved in granuloma formation (Boros and Warren, 1970; Lichtenberg and Raslavicious, 1967). However, the intact egg- shell has been shown to be critical to the development of the granuloma 3 since it protects the developing micracidium from immunolysis by the host immune system and permits the gradual release of schistosome antigens into host tissues (Boros and warren, 1970; Hang g£_§l,, 1974; Lichtenberg and Raslavicious, 1967). A number of investigators have attempted to alleviate the necrosis and scarring caused by granuloma formation through immunosuppression of the granuloma (Domingo and Warren, 1967, 1968; Mahmoud and Warren, 1974). However, it has recently been reported that in the absence of granuloma formation, freely diffusing schistosome egg antigens have a cytotoxic action on host tissues (Byram and Lichtenberg, 1977). An alternative approach to the alleviation of this pathology is the elimi- nation of the source of granuloma formation by suppression of egg production. Schistosomes which no longer produce eggs can no longer contribute to the miracidial pool which is essential to the maintenance of the life cycle (Duke and Moore, 1976). Thus, the suppression of egg production has the dual effect of alleviating the pathology associated with schistosomiasis and interrupting transmission of the disease. One aspect of egg production which has proven to be particularly susceptible to drug action has been the process of eggshell formation.~ In recent years, thiosinamine (Machado ggfl§1., 1970), dapsone (Katz, 1977), and disulfiram (Bennett and Gianutsos, 1978) have been noted to inhibit eggshell formation in S, mansoni. It has been suggested that these drugs inhibit eggshell formation by inhibiting an enzyme known as phenol oxidase. This enzyme is responsible for the hardening (sclero- tization) of proteins in a wide variety of invertebrate species (Pryor, 1962) and has previously been suggested to be responsible for the 4 process of eggshell formation in the trematodes (Stephenson, 1947; Smyth, 1954). However, at this time there is little evidence to support the concept of phenol oxidase catalyzed eggshell formation in schistosomes. It is the purpose of this thesis to establish the mechanism by which the eggshell is formed in S, mansoni and to evaluate the effective- ness of inhibiting this process in order to alleviate the pathology of schistosomiasis. B. Eggshell Formation The morphology and biochemistry of eggshell formation has been described in a number of trematodes, including S, mansoni (Stephenson, 1947; Gonnert, 1955; Rao, 1959; Smyth and Clegg, 1959; Nollen, 1968). The bulk of this work has been done with the trematode Fasciola hepatica (Stephenson, 1947; Rao, 1959; Smyth and Clegg, 1959). Thus, much of the following description is based on observations of eggshell formation in S, hepatica, although it appears that this process is quite similar in S, mansoni (Gonnert, 1955). 1. Morphology The anterior portion of the female schistosome (approximately one-fifth of its total length) contains the ovary, the ootype-~an enlargement of the oviductal canal in the vicinity of Mehlis' gland, the uterus and the uterine canal (Figure 1) (Gonnert, 1955). The posterior four-fifths of the schistosome is composed primarily of the vitelline glands and the associated ducts responsible for the transport of vitelline cells to the uterus. In the process of eggshell formation, vitelline cells are combined with the ovum (egg) in the ootype whereupon .Ammma .uumcaow Scum wouamtmv acomcme aw mamaom :H coauostomm wwo saga wmumfioommm momsumow HmoHEODms< .H muswflm H ouswwm .2... 2... n _ m «0 .... 8. n... ... .30; .0. n.. .wg.§.3~¢gg‘ ....m. G“ O... . . O . s ...G... ... téafiasi l ... ‘ 3:8 65.3: o 9 O o ...1 o ....z. .. . Benders. 7 they release from their cytoplasm numerous subcellular particles which are often referred to as vitelline droplets. These particles contain large quantities of membranes which histochemically react with stains for phenolic substances (Stephenson, 1947; Smyth and Clegg. 1959). Mehlis' gland, which empties into the ootype, may be responsible for the release of the vitelline droplets, but its function has not ade- quately been defined (Rao, 1959; Clegg, 1965; Clegg and Morgan, 1966). Following their release, the droplets coalesce to form a thin membrane _; around the surface of the uterus. At this time, formation of the eggshell appears to occur. The formation of the hardened shell is associated with the appearance of a dramatic yellow fluorescence which is characteristic of the eggshell (Kelley and Lichtenberg, 1970). Acid hydrolysis of S, hepatica eggshells has shown them to be composed almost entirely of amino acids (Rainsford, 1967). Thus, the eggshell appears to be composed primarily, of extensively cross-linked proteins to which some fluorescent chromagen may be attached. This cross-linked protein is highly resistant to immunolytic (Stenger gE_§;,, 1967) and chemical degradation (Stephenson, 1947). It is this resistance to immunolytic degradation which is responsible for the persistence of the egg within host tissues and which thus contributes significantly to the development of the granuloma. 2. Biochemistry The mechanism by which the schistosome eggshell is formed has been proposed to involve the process of quinone—tanning of eggshell precursor proteins (Smyth and Clegg, 1959; Stephenson, 1947). According to this theory, phenol oxidase catalyzes the oxidation of an ortho—phenol to an ortho—quinone. The ortho—quinone subsequently reacts with basic amino acids in adjacent proteins to form a cross- link. This concept of quinone-tanning in the process of shell-hardening was first introduced by Pryor (1940). In this study, phenol oxidase, phenolic substances and basic proteins were demonstrated to be inti- mately associated with the process of cuticular hardening in the cockroach Blatta Orientales. Mixing of the phenol (3,4-dihydroxybenzoic acid; Pryor ggwg1., 1946) with phenol oxidase produced a reddish color which is characteristic of the reaction of quinones with free amines (Pugh and Raper, 1927). The appearance of this reddish color was associated with the disappearance of histochemical staining character- istic of free amines (i.e., basic amino acids) in the cuticular proteins of Blatta, and was also associated with the onset of cuticular hardening. Considerable evidence has accumulated in the ensuing years which has strongly supported this concept of quinone-mediated protein cross-link formation. A brief review and discussion of this evidence follows. a. Reactions of quinones with amino acids and proteins The quinones are a group of highly reactive substances which readily react non-enzymatically with a number of nucleophilic moieties including primary and secondary amines and thiols (for a comprehensive review of this subject, see Mason, 1955). The reactions of the amines with quinones characteristically results in the genera- tion of numerous colored products which can be either amino— or imino- quinones (Figure 2) (Pugh and Raper, 1927; Hackman and Todd, 1953; Jackson and Kendall, 1949). The apparent ease with which one quinone molecule reacts with several nucleophilic ligands (Figure 2) makes the quinones ideal intermediates in the formation of protein cross-links. .mmoa .weoe paw assign: no new a .mmma .umamx tsm swam n< .mmcfiem :DHB mmsosfiavoucon mo mcoauommm .N muswfim :00 N: + . .o .\ P _ . ~12 are we .< 11 Following the reaction of a quinone with a nucleophilic side chain from one protein, the quinone reacts with a nucleophilic side chain from an adjacent protein in order to form a cross-linked protein. The types of nucleophiles which readily react with quinones (R-NH R-SH) are 2: commonly found in proteins (e.g., lysine, histidine and cysteine). In addition, proteins in solution (most notably insect cuticular proteins) develop color upon the addition of quinones (Mason and Peterson, 1965; Hackman, 1953). These quinoid proteins appear to have an absorption maximum around 480 nm which is characteristic of the products formed from the reactions of many amino acids with ortho-quinones (Mason and Peterson, 1965). In the absence of reactive nucleophiles, quinones often cross-react with other quinones to produce melanin, an insoluble, polymeric, acid-stable substance. However, not all reactive sites of the quinones are utilized in this polymer formation (Mason, 1955). Thus, the observation that melanin, 12.21229 is invariably isolated in a protein bound form is consistent with the known reactivity of qui- nones with nucleophilic amino acids (Piatelli 35 31,, 1962; Seiji 35_ 21,, 1963). In light of the multiple reactive sites of quinones and their abilities to react with amino acids and proteins, it is not surprising that it has been found that numerous proteins will form insoluble and non-reactive complexes in the presence of quinones generated enzymatically from the reaction of phenol oxidase with a phenolic substrate (Sizer and Brindley, 1951). With regard to the specific process of shell-hardening, Hackman (1953) has demonstrated that water soluble proteins extracted from unhardened insect cuticles will precipitate upon the addition of phenol oxidase and catechol. 12 Thus, it is apparent that quinone-tanning can be involved in the cross-linking of proteins and the hardening of insect cuticles. However, it is not certain that the presence of phenol oxidase is a necessary requirement for the generation of the quinones which are involved in the process of protein cross-linking. b. Requirements for the generation of_quinones 1) Enzymatic formation of quinones. The gggggr quinones, which appear to be capable of mediating protein cross-link formation, are formed from the oxidation of gfdihydroxyphenols. Sgtggfquinones can be generated enzymatically from grdihydroxyphenols by the enzyme phenol oxidase (also known as polyphenoloxidase, tyrosi- nase, gfdiphenolm2 oxidoreductase, E.C. 1.10.3.1). The chemistry of this well-known enzyme has been extensively reviewed elsewhere (Nelson and Dawson, 1944; Lerner 1953; Brooks and Dawson, 1966). In brief, phenol oxidase catalyzes the following reaction: OH OH OH H [E1 [E1 [02] [021 This enzyme is a copper containing enzyme and is inhibited by copper chelating agents such as diethyldithiocarbamate and thiourea and is also inhibited by c0pper complexing agents such as cyanide and various sulfhydryl containing compounds (Dubois ggnal., 1946; Flesch and Roth- man, 1948). Phenol oxidase is somewhat unique in that it can catalyze 13 both the hydroxylation of tyrosine and the oxidation of dihydroxyphenyl— alanine (DOPA). The only other enzyme which is capable of performing this dual function is peroxidase (Lerner, 1953). However, the phenol oxidase catalyzed hydroxylation of tyrosine is unique in that the end product of tyrosine hydroxylation is both a substrate for the second step of the reaction (DOPA oxidation) and a cofactor for the first step (tyrosine hydroxylation) (Hearing and Ekel, 1976; Pomerantz and Warner, 1967). In contrast, the peroxidative conversion of tyrosine to DOPA is not activated by DOPA and also requires the presence of peroxide. However, not all phenol oxidases possess the capability of converting tyrosine to DOPA. Karlson and Liebau (1961) were able to crystallize a soluble phenol oxidase obtained from the larvae of Calliphora which was copper containing and specific only for dihydroxyphenols. Rela- tively impure preparations of phenol oxidase from insect cuticles have also been found to oxidize only dihydroxyphenols while phenol oxidases purified from the haemolymph of these same organisms oxidizes both mono- and dihydroxyphenols (Mills g£_§;,, 1968; Ishaaya, 1972). In every case, if monophenol oxidase activity is present, it cannot be separated from diphenoloxidase activity. In those insects where monophenol oxidase activity has not been found, the onset of cuticular hardening has generally been associated with dramatic increases in the activity of enzymes such as tyrosine decarboxylase (HOpkins and Wirtz, 1976) and DOPArdecarboxylase (Wirtz and HOpkins, 1977). These enzymes are involved in the metabolism of tyrosine to a dihydroxyphenol which serves as a substrate for the phenol oxidase of the species in question (Periplaneta americana). The subject of tyrosine metabolism as it l4 relates to the process of cuticular hardening in insects has been extensively reviewed by Brunet (1963). In general, it appears that the metabolism of tyrosine within insects is optimized to produce specific phenolic substrates for diphenoloxidases during the process of cuticu— lar sclerotization. Those findings argue against a significant role for peroxidase (another enzyme capable of generating quinones) in the process of quinone-tanning in insects, since peroxidase can produce quinones from a wide variety of substrates (Mason, 1957). However, in cases where the major substrate appears to be tyrosine or DOPA, the situation is not as clear. Both of these compounds serve as substrates or are intermediates in other common metabolic processes such as pro- tein synthesis and/or neurotransmitter synthesis and thus changes in the metabolism of these compounds during sclerotization may be ob- scured by other biochemical changes not associated with sclerotization. Furthermore, both peroxidase and phenol oxidase can catalyze the formation of quinones from either tyrosine or DOPA. Okun ES 31. (1975) and Patel ggflgl. (1974) have obtained evidence which suggests that peroxidase may be primarily responsible for the generation of melanin from tyrosine in mouse melanomas. Thus, it is necessary to rule out this possibility in the elucidation of mechanisms of quinone- mediated protein cross-link formation in which tyrosine or DOPA are con- sidered to be the major ig_yizg precursors of the quinone. This task should not be difficult since phenol oxidase and peroxidase are readily distinguishable from each other. Peroxidase is an iron containing protein while phenol oxidase is cepper containing. Peroxidase re- quires the presence of hydrogen peroxide in order to catalyze the 15 hydroxylation of tyrosine and the oxidation of DOPA (Mason, 1957; Paul, 1959) while phenol oxidase requires molecular oxygen. Other enzymes or enzyme systems which have been demonstrated to be able to produce quinones or melanin from phenolic precursors include the cytochrome oxidase system (Hesselbach, 1951) and laccase (Whitehead g£_a1,, 1960). However, these enzymes have not been adequately demonstrated to play a significant role in the generation of gfquinones during protein hardening and thus their role in this process has not been given much credence (Pryor, 1962). 2) Non-enzymatic formation of quinones. The ggghgf quinones can be readily generated from the corresponding gfdihydroxy- phenols (catechols) by autooxidation.l The autooxidation of the cate- chols occurs rapidly in the presence of excess hydroxide ions, ferric ions, or any suitable hydrogen ion acceptor (Senoh g£_§l,, 1959: Harrison ggfial., 1968; Hawley ggugl., 1967). The monophenol tyrosine can also be converted to a quinone (DOPA-quinone) in the presence of L- DOPA and cupric or ferric ions (Foster, 1950). However, the autooxida- tive generation of quinones is also known to produce hydrogen peroxide (James and Weissberger, 1938), a feature which distinguishes it from the enzyme catalyzed formation of quinones. Since it has not been possible to correlate increases in autooxidation with the hardening of the cuticle in insects, the autooxidative formation of quinones is not thought to play a significant role in the quinone-tanning of insect cuticular proteins (Pryor, 1962). However, it has not always been possible to detect all of the components which are required for the process of protein hardening by quinone-tanning. The component which has been most 16 notably absent in some studies is the phenolic substrate which is necessary for the generation of a quinone (Brown, 1952; Blower, 1951). In such cases, one must consider several alternative hypotheses. c. Other mechanisms of_protein hardening Protein cross-links can be found in a number of struc- tural proteins including collagen, elastin and fibrin. However, these proteins do not have the same characteristics as the proteins involved in cuticular hardening. Many of these protein structures are somewhat elastic and contain a number of unique amino-acid sequences (Mahler and Cordes, 1971). In addition, the formation of cross links in these protein structures appears to be catalyzed by some very specific cross- linking enzymes such as lysyl oxidase (Siegal g; 31., 1970) or serum trans-glutaminase in fibrin (Matavic and Loewy, 1968). Thus, cross- links formed by these enzymes are readily distinguished from quinone- formed cross-links both in terms of the characteristics of the protein formed and the enzyme involved. On the other hand, Brown (1950) and Blower (1951) have proposed that, in the absence of free phenolic substrates, protein bound tyrosine or DOPA is converted to protein bound DOPA-quinone by phenol oxidase. This protein bound quinone subsequently cross reacts with e-amino groups from lysine molecules in adjacent proteins to form 'a hardened protein mesh. This mechanism eliminates the necessity of demonstrating free phenolic substrates for phenol oxidase and has been proposed in those cases where these substances have been difficult to identify, although most of the other components and characteristics of quinone-tanned proteins can be readily identified (Brown, 1952; Blower, 17 1951; Smyth and Clegg, 1959). Evidence to support this notion of protein tanning has come from studies by Hackman (1953) and Yasonobu 23.21; (1959) who have shown that tyrosine rich proteins and poly- peptides can serve as substrates for phenol oxidase in the absence of exogenously added phenols. However, although proteins and peptides can serve as substrates for phenol oxidase, they have not been conclusively demonstrated to form cross-links following exposure to phenol oxidase. Pryor (1962) has suggested that the above mechanism is not feasible since steric hindrance would not permit effective attack of the e-amino group at the reactive site of the protein bound quinone (Figure 3a). Furthermore, Gianutsos and Bennett (1977) have recently demonstrated the presence of high concentrations of the dihydroxyphenol, dapamine (DA), in the egg-producing regions of S, hepatica using a specific and sensitive radioenzymatic assay for this compound. Earlier workers had not been able to detect this substance histochemically (Smyth and Clegg, 1959) and had subsequently proposed a mechanism of auto-tanning similar to that proposed by Brown (1950). The recent findings of Gianutsos and Bennett (1977) make it unnecessary to postulate a mecha- nism of quinone-tanning which involves the action of phenol oxidase on a protein bound substrate. Thus, while it appears possible that phenol oxidase could be involved in the formation of direct protein cross- links, the bulk of the evidence strongly favors cross-link formation mediated by a quinone or quinone-derived polymer. An alternative mechanism of protein cross-link formation involves the enzyme peroxidase. Peroxidase has previously been men- tioned with regard to its ability to catalyze the formation of quinones 18 Imzumz A< .Aoqaav poaum mo Emwcmzomz Am .Aommav =30um mo Ema: .coHumEmow mxsaalmmomo :«muoua woN>Hmumo mmmmfixo Housed mo mamfiamzome 039 .m ouswam mmmmmmm 20 from dihydroxyphenols and tyrosine (Patel 25H51., 1974; Mason, 1957), and thus also catalyze the formation of quinone-linked protein polymers (Stahmann g; 31., 1977). This alternative scheme is based upon the ability of peroxidase to catalyze the formation of dityrosine (Gross and Sizer, 1953). Recent studies by Foerder and Shapiro (1977) and Garcia-Castineiras §£_§1, (1978) have demonstrated the presence of dityrosine residues in cross-linked proteins. Aeschbach g5 El: (1976) have recently shown that peroxidase can catalyze the formation of dityrosine from proteins rich in tyrosine residues. Foerder and Shapiro (1977) have shown that the release of peroxidase from sea urchin egg membranes is intimately associated with the production of dityrosine residues and the simultaneous hardening of the eggshell. Thus, it is clear that peroxidase might be involved in the process of protein hardening either in the production of quinones or in the formation of direct dityrosyl links. Resolution of the question of whether phenol oxidase or peroxidase is involved in the formation of the hardened eggshell in S, mansoni will depend upon the isolation and characterization of the enzyme or enzymes involved in this process. Lipid peroxidation has also been implicated in the process of protein cross-link formation (Roubal and Tappel, 1966). However, the mechanism by which free radicals form protein cross-links in a lipid peroxidizing system has not been extensively investigated. Thus, lipid peroxidation has an uncertain potential for being involved in the process of protein hardening. d. Evidence for gginone-tanning in trematodes The evidence for quinone-tanning in trematodes is based primarily on histochemical studies (Nollen, 1971; Erasmus, 1975; 21 Smyth and Clegg, 1959; Clegg and Smyth, 1968; Ramalingan, 1973) with only limited support from biochemical studies in this area (Mansour, 1958; Gianutsos and Bennett, 1977; Bennett and Gianutsos, 1978). While these studies provide a limited amount of information from which the mechanism.of eggshell formation might be inferred, the inherent lack of specificity of most histochemical stains does not provide sufficient information to definitively rule out any one mechanism of protein sclerotization. In S, hepatica, histochemical evidence has suggested the presence of phenol oxidase in the vitelline cells of S, hepatica (Smyth, 1954) and in freshly laid eggshells (Ramalingam, 1973). However, this histochemical evidence is based simply upon the oxidation of catechol to a red color and thus does not differentiate between phenol oxidase and other enzymes (e.g., peroxidase) which are capable of oxidizing catechol. 0n the other hand, Mansour (1958) was able to isolate a crude, membrane-bound enzyme in S, hepatica homogenates which appeared to possess many of the characteristics of phenol oxidase including some degree of selectivity in its affinity for different phenolic substrates, a characteristic which is not typical of peroxi- dases (Mason, 1957). In addition, catalase was not found to alter the kinetics of catechol oxidation, thus ruling out the possibility of a contaminating peroxidase. Thus, there appears to be fairly good evidence based on histochemical and biochemical studies which impli- cates phenol oxidase in the process of eggshell formation in E, hepatica. Unfortunately, Mansour (1958) did not recognize the signi- ficance of his findings with regard to role of phenol oxide in egg 22 production and hence did not attempt to correlate his ig'yiggg data with ig_yiyg studies on eggshell formation. Thus, it might still be argued that another enzyme such as peroxidase is involved in the process of eggshell formation (Ramalingham, 1973). Furthermore, although phenol oxidase has been identified in S, hepatica, free phenolic substrates for this enzyme have been difficult to identify. Smyth and Clegg (1959) were unable to histochemically identify any phenolic substances in S, hepatica vitelline glands except for tyrosyl peptides. In the absence of detectable phenolic substrates these investigators proposed a mechanism for eggshell formation in trematodes based on tanning of protein bound phenols (Figure 3a). However, the observation of Gianutsos and Bennett (1977) that the egg producing regions of Fasciola have elevated concentrations of DA, suggested a potential role for this compound in the classical process of quinone tanning described by Pryor (1940). With regard to the role of basic amino acids, Smyth (1954, 1956) and Stephenson (1947) have noted that the staining characteristic of basic amino acid residues gradually disappears as the eggshell becomes hardened in the uterus. Thus, there appears to be a fair amount of evidence which is suggestive of a role for quinone-tanning in the process of eggshell formation in S, S323: .EAEE- However, Ramalingam (1973) and Wilson (1967) have presented evidence which suggests that dityrosine may be present in hydrolysates of S, hepatica eggshells. These findings have created a controversy concerning the role of phenol oxidase and the possible role of peroxi- dase in eggshell formation in S, hepatica. In S, mansoni it has been reported that phenol oxidase may be present in the vitelline cells of the female (Clegg and Smyth, 23 1968). However, this data has not been published in detailed form. The only histochemical evidence for phenolic substances localized in the vitelline cells of female S, mansoni is based upon a positive diazo test (Johri and Smyth, 1956). Unfortunately, the test is positive for a number of other substances including the aromatic amines (Pearse, 1968) which may be present in the vitelline cells in the form of the basic amino acid histidine. Gianutsos and Bennett (1977) observed that DA was present in higher concentrations in the female than in the male. It was thus suggested that this difference might reflect the importance of this substance to the process of egg production. 0n the other hand, Erasmus (1975) obtained autoradiographic evidence which suggested that the eggshell globules in the vitelline cells of this trematode avidly incorporated tyrosine into proteins. This investi- gator suggested that a tyrosine rich peptide could serve as a sub- strate for S, mansoni phenol oxidase. However, in the absence of detailed biochemical studies, one can conclude very little about the mechanism of eggshell formation in S, mansoni except to say that, based on similarities to the evidence obtained from S, hepatica, it might involve the process of quinone—tanning (Clegg and Smyth, 1968). There is insufficient evidence available from studies on other trema- todes to permit a discussion of the mechanism of eggshell formation in these species (Nollen, 1971). C. Proposed Research Studies by previous investigators (Boros and Warren, 1970; Lichten- berg and Raslavicious, 1967) have established that the presence of an 24 intact eggshell is critical to the development of the egg granuloma which is responsible for most of the pathology associated with schisto- somiasis (Lichtenberg, 1955). Eggshell formation can be inhibited reversibly by a number of drugs which are known to inhibit the enzyme phenol oxidase (Machado 25 gl., 1970; Bennett and Gianutsos, 1978). It is the purpose of this research to l) establish the mechanism by Which the eggshell is formed, 2) determine the role of the enzyme phenol oxidase in tdis process,‘and 3) to evaluate the effects of phenol oxidase inhibitors on the development of schistosomal pathology. MATERIALS AND METHODS A. Schistosomes The schistosomes used in these experiments are derived from a parent stock of Schistosoma mansoni (St. Lucian strain) maintained in outbred laboratory mice at the University of Lowell Research Founda- tion. The mice used in these experiments were outbred (ICR) mice (Spartan Research Animals, Haslett, Mich.) and were infected with 150- 200 cercariae by tail immersion or i.p. injection. Paired mature schistosomes (40-60 days post-infection) were surgically removed from the mesenteric veins of infected mice and placed in a solution containing either Eagles Basal medium.(BME) (Gibco, Long Island, N.Y.), Earle's salts, .05% sodium pentobarbital (Sigma Chemical Co., St. Louis, Mo.) and .02 M Tris buffer, pH 7.4 or phosphate buffered saline, pH 7.4. Sodium pentobarbital (.05%) was included in these media because paired male and female schistosomes cannot be separated without a muscular relaxant being present in the solution. Male and female S, mansoni were separated by gently removing the female with microforceps. B. Activation of Phenol Oxidase in S. mansoni Phenol oxidase was activated by the incubation of whole female schistosomes in the same culture medium as described above with 10% heat-inactivated fetal bovine serum and .05% sodium pentobarbital added. Sodium pentobarbital was included in the incubation medium in order to 25 26 facilitate handling of the female schistosomes. In the absence of pento- barbital the schistosomes tended to cling to the surface of the incuba- tion dish making subsequent removal difficult. Since the presence of pentobarbital also tended to marginally increase the extent of phenol oxidase activation (lo-20%), pentobarbital was 16ft in the incubation medium during the period of enzyme activation. From now on this culture medium without pentobarbital will be referred to as BMEFC. In studies on the effects of protein synthesis inhibitors on induction, the inhibitors and radiolabeled amino acids were added to the BMEFC plus pentobarbital prior to the transfer of the schistosomes. In these experiments, the paired schistosomes were transferred directly from the host into BMEFC before being separated. Males were removed from the induction medium immediately following separation from the female. Some of the drugs and enzymes used in these studies and their sources are: cycloheximide; trypsin, type II; phospholipase A2; protease from Streptomyces griseus, type V; Lubrol wx; Tween 80 (Sigma Chemical Co.); Triton X-100 (Research Products International, Elk Grove, I11.); antimony potassium tartrate (a gift of Dr. Ernest Bueding); 3H—[2,6]-L-tyrosine, 40-60 Ci/mmole (New England Nuclear, Boston, Mass.); and 3H-[4,5]-L-leucine 50-60 Ci/mmole (Amersham/Searle, Arlington, Heights, 111.). C. Phenol Oxidase Assays 1. Standard phenol oxidase assay Phenol oxidase activity was determined in female S, mansoni using a modification of the method described by Mansour (1958) for Fasciola hepatica. Forty female schistosomes were placed in a 27 homogenizing vessel containing 2 m1 of 0.1 M phosphate buffer pH 6.8 and homogenized with a teflon pestle. The homogenate was centrifuged at 3,000 x g for 5 min. The supernatant was discarded and the pellet was resuspended in 2 ml of phosphate buffer pH 6.8. All of the above procedures were performed at 4°C. The resuspended pellet was placed in a Clark-type oxygen electrode (Rank Brothers, Cambridge, England) maintained at 37°C. Three and a half minutes later substrate was added through a small port at the top of the electrode chamber. Oxygen consumption was measured during the first 5 minutes following addition of the substrate. The amount of oxygen consumed was based on the amount of current generated by the reduction of 02 at a constant voltage of 0.6 v. The electrode was calibrated as described by Lessler and Brierley (1969) using air-saturated distilled water as a reference (.410 umoles Ozlml at 37°C). The data are expressed in terms of umoles 02/minemg protein and represent the initial velocity recorded during the first two minutes of reaction. A11 substrates used in this assay were obtained from Sigma Chemical Co. 2. Phenol oxidase assay for protein and peptide substrates Phenol oxidase was activated in eighty mature female schisto- somes as previously described. The schistosomes were homogenized in 4 mls of 5% TX-lOO buffered with 0.1 M sodium phosphate pH 7.0. Two mls of the homogenate were centrifuged at 3,000 x g. The pellet was re- suspended in 2 mls of phosphate buffer and placed in oxygen electrode system maintained at 37°C. L-DOPA was then added to a final concentra- tion of 30 uM. Three minutes following the addition of L-DOPA, 20 ul of a 0.1 M solution of L-tyrosine methylester (TME) was added to the system as substrate. Phenol oxidase activity was measured in terms of 28 the amount of O2 consumed during a 2 minute period immediately following the addition of the substrate. All reagents in this assay were freshly prepared and maintained at room temperature. Homogenization was performed at 4°C. Centrifugation was performed at room temperature. The second portion of the 4 m1 homogenate described above was treated as in the above protocol, with the exception that the pellet was resuspended in phosphate buffer containing a protein or peptide substrate plus 30 uM L-DOPA. In these studies the activity of the protein and peptide substrates‘weredetermined by subtracting the rate of oxidation obtained in the first 3 minutes of assay from the baseline rate of oxidation obtained from the paired sample immediately preceding it. In some studies, TME was added after the 3 min assay period to confirm that the rates of TME oxidation in the two samples were approximately equal. The protein and peptide substrates employed in. these studies and their concentrations were, tri-L-tyrosine methyl ester (Sigma Chemical Co.), 2 mg/ml; a tyrosinezlysine polymer (1:1) (Miles Biochemicals, Elkhart, Ind.), a saturated solution in 0.1 M phosphate buffer; chymotrypsinogen A (Sigma Chemical Co.), 1 mg/ml and endogenous proteins from female S, mansoni, 1 mg/ml. The schistosome proteins were prepared as follows: 100 mature, female schistosomes were homogenized in 2 mls of 1% Triton X-100 buffered with 0.1 M sodium phosphate pH 7.0. The homogenate was dialyzed for 24 hrs against 4 liters of phosphate buffer. The buffer was changed twice during the course of dialysis. The dialyzed proteins were then removed and a 2 ml aliquot assayed as a substrate for phenol oxidase. Residual phenol oxidase activity in the homogenate was assayed as described at the beginning of this section. 29 3. Mushroom phenol oxidase assay In these studies mushroom phenol oxidase (Sigma Chemical Co.) was substituted for schistosome phenol oxidase. The mushroom enzyme, containing 2750 units/mg dry weight was diluted to a concentration of 30 ug/ml. Twenty ul of this solution was then added to phosphate buffer in the oxygen electrode which already contained all substrates and cofactors. This dilution of the enzyme yielded a rate of TME oxidation which approximated that produced by a 3,000 x g pellet ob- tained from a homogenate of 80 female schistosomes, which had been acti- vated by 8 hrs incubation in BMEFC (total activity = 2 units). One uM L-DOPA.was used as a cofactor in these experiments since 10 pM L-DOPA produced a significant rate of oxygen consumption. One unit of phenol oxidase activity is defined as a change in O.D. of .001/min at pH 280 6.5, 25°C in a 3.0 m1 reaction volume using L-tyrosine as a substrate. D. Inhibitor Studies Phenol oxidase inhibitors were obtained from commercial sources as follows: allylthiourea, diethyldithiocarbamate (DDC), disulfiram, l- phenyl-B-(2-thiazolyl)-2-thiourea (PTTU; UK 14624) (Aldrich Chemical Co., Milwaukee, Wis.); penicillamine, bathocupioure sulfonate, L- cysteine, rotenone, EDTA, phenylthiourea, a,a'-dipyridyl, tranylcypro- mains, thiourea, 5-hydroxytryptamine (Sigma Chemical Co.); potassium cyanide (J.T. Baker Chem. Co., Phillipsburg, N.J.). The effects of these inhibitors on phenol oxidase activity was determined by resus- pending the 3,000 x g pellet in 0.1 M phosphate buffer containing the desired concentration of the inhibitor to be used. The inhibitor was then incubated with the resuspended pellet for three and a half-minutes 30 prior to addition of the substrate (2 mM DA). Rate constants of inhibition were determined by the method of Dixon (1951). Additional rate constants of inhibition were determined for DDC and allylthiourea by the method of Lineweaver Burk (1938). E. Histochemistgy l. Catechol method Phenol oxidase was localized histochemically in whole female worms by a modification of the method of Smyth (1954). Whole female schistosomes were fixed in 95% ethanol for a period of 2 hrs. The worms were subsequently incubated for a period of 15 min in an aqueous solution of 0.2% catechol. The worms were then mounted (wet mount) on a slide and examined under a light microscope for the presence of dark~staining material. 2. Fluorescent method Whole female S, mansoni were placed in Hank's balanced salt solution (HBSS) containing 2 mM TME. The worms were allowed to incu- bate for a period of 20 minutes, after which they were mounted on a glass slide in a few drops of HBSS and the relative fluorescence intensity of the vitelline cells was determined using a Leitz micro- spectrofluorimeter. The Optics of the microspectrofluorimeter were essentially the same as described by Jonsson (1969). The light source was a high intensity Leitz xenon lamp. Light emitted from this source was passed through a heat filter, a band pass filter (BC-38) which suppressed all emissions below 300 nm and above 650 and a band-pass filter (UG-S) which suppressed all emissions below 220 nm and above 400 nm. The sample on the slide was visualized with epi-illumination. 31 Light emitted from the sample passed through the objective of the micro- scope and a 500 nm band-pass filter which suppressed U.V. and visible light reflected off the slide from the xenon lamp. This light was then focussed on a photomultiplier tube and readings of fluorescence inten- sity were obtained by monitoring the output from the photomultiplier tube. Readings of fluorescence intensity were made at lOOX magnifica- tion and were obtained from a minimum.of 6 different regions of each worm. The average of these values was treated as a single data point representative of the fluorescence over the entire schistosome. In studies with phenol oxidase inhibitors, the worms were preincubated in HBSS, containing inhibitor, for 10 min prior to being placed in the substrate solution which contained both substrate and inhibitor. F. L-DOPA and DA Determinations L—DOPA and DA levels were determined in fresh, mature (50-60 days post-infection), male and female schistosomes by mass fragmentography. Samples of 10 male schistosomes with a wet weight of approximately 4-5 mg or 20 female schistosomes with a wet weight of 1.5-2 mg were homo- genized in 100 pl of acetone - 0.1N HCl (2:1 v/v) containing 100 pmol of deuterated (d3)-L—D0PA and deuterated (d3)-DA and were centrifuged at 8,000 x g on a Beckman microfuge (Beckman Instruments, Irvine, California) for 6 min. The supernatant was transferred to 1 dram vials which had been coated with dimethyldichlorosilane. The samples were then evaporated to dryness at 55°C under nitrogen. The samples were chemically modified by the addition of 50 pl of a 4:1 mixture of pentafluoropropionic anhydride (PFPA) and pentafluoroprOpanol (PFP) 32 (Regis Chemical Co., Morton Groves, 111.). The vials were sealed and reacted for 30 min. at 75°C. The reagents were subsequently eva- porated under nitrogen at room.temperature. PFPA (30 pl) was added and allowed to react for an additional 10 min at 75°C. The PFPA was again evaporated and the residue dissolved in 20 ul of a 10% solution of PFPA in ethyl acetate. This reaction results in the formation of volatile derivatives of L-DOPA and DA which are suitable for separation on a gas chromatograph and analysis in the mass spectrometer. Amounts of 1-4 ul were injected for analysis. Blank-corrected standard curves for the quantitation of endogenous L—DOPA and dopamine were prepared from a series of standard solutions containing 100 pmol of deuterated L-DOPA and deuterated dopamine and varying amounts of DOPA (0-10 pmole) and dopamine (0-10 pmol). The standards were reacted as described above. Quantitation of L-DOPA and DA in the samples was performed by mass fragmentography using a Finnigan 3200B gas chromatography/mass spectro- metry system. A 1.6 m x 2 mm (i.d.) silanized glass column packed with 3% SP-2250 was used for separation. Temperatures were: column 150°C (isothermal), injector 250°C, separator 250°C. The carrier gas (Helium) was maintained at a flow rate of 15 ml/min. Fragmentation was accom- plished by electron impact at 70 ev and 500 uamp. Ions selected for monitoring from the mass spectra of the derivatives were m/e = 428 and 281 for DA (431 and 284 for deuterated DA) and m/e = 415 and 604 for DOPA (418 and 607 for deuterated L-DOPA) (Figures 4 and 5). The ion at m/e 604 results from cleavage of the bond between the a carbon and nitrogen. The ion at m/e = 415 results from cleavage of the 33 .u0uomuot mnu mcfixfiuum moo“ mo nones: on» cu Hmuofiuuoaoua zauomufiu ma usouuso one .Ofiumu owumaoummma vmuwoatca onu mo muaoewmum “masooaos an commuocmw ucouuso men «0 muwm msu mucmmmuaou Hon comm .fiuo< musumquEmH coauosvcH A¢.m may agave: musuaso m mcfixo Hocmem mo mafia common 0 >ua>fiuo< mmmwfixo Hoomzm mo sowuoamaa mam so esfimmz sowumnaocH wo mama mom muaumnmmeofi mo muommwm may N mAmuw>fiuo< m: ououa mcfimuoum mafimuoua ucmeummua mmmeaxo .me<_ e menmufiaeomuauaom once mmamuaaaompaumq mH< .mfimmnuchm :«muoumamaomOumflaom do mHOufinficcH mammauczw :Hmuoum mo muomuwm mes m mqm<8 64 reduction in protein synthesis was associated with a 17% reduction in phenol oxidase activity. Thus, it appeared that the reduction in phenol oxidase activity was more closely associated with the reduction in total protein content of the schistosome than the reduction in the synthesis of new proteins. In order to control for the possibility that the reduction in synthesis of new proteins was a non-specific artifact due to the large concentrations of cycloheximide, an attempt was made to inhibit protein synthesis with antimony potassium tartrate (SbK). SbK has been shown to inhibit S, mansoni phosphofructokinase, a critical enzyme in the schistosome glycolytic pathway (Mansour and Bueding, 1954). Inasmuch as glycolysis is the sole source of energy for this parasite (Bueding, 1950), inhibition of schistosome phosphofructokinase should result in the inhibition of all energy dependent processes within the schistosome, including protein synthesis. Whole female S, mansoni were incubated in BMEFC containing .05% pentobarbital, 0.2 mM SbK and tritiumslabeled L-leucine or L-tyrosine for a period of 4 hours. ATP concentrations dropped by 70% and this drOp in ATP corresponded with a 90% inhibition of the incorporation of 3H-amino acids into PCA-preci- pitable proteins (Table 3). This decreased incorporation of amino acids into protein could not be attributed to the inhibition of the uptake of amino acids since the uptake of one of the amino acids used in this study (tyrosine) was shown to be a diffusional process (Figure 10). However, deSpite the large decrease in protein synthesis and reduction of ATP concentrations, the activation of phenol oxidase was not significantly altered by SbK. 65 .mcoaumcwaumumm m ummma mm «o .z.m.m H cmme mam mucmmoummu ucHom comm .muamm m.xcm= ca cmumnsocfi mEuoB I A v .mnm quoa wcacamucoo muHmm m.x:m: :H wmumnsocfi meno3 I A v .AHoEE\fio colqu mnemouzu Iglmm mo Ha\Hoa m mam mnemouhulg vmamnmaca mo macaumuuomocoo waamum> mcacfiwuooo muamm m.x:mm :H comm um woumnsoca oum3 memo: .Hcomcma mm mamfimw >3 mcfimouhulq mo mxmums ouuH>.mM .OH muswflm 66 50. CH muswwm ..s. \ mz_mom>.7._ mmnos =\F 3.. an. 8. B. 8. . . . . _ c an QN waom OW-NIW/BNISOUAl-‘I ssnowd 4 67 The results of these studies indicate that the incubation-dependent activation of phenol oxidase is not dependent upon protein synthesis or on any ATP-dependent process. In addition, the mechanism does not appear to involve proteolysis or lipolysis since these treatments do not activate phenol oxidase in female S, mansoni prior to Tp_xi££g_ incubation (Table l). B. Isolation and Histochemical Localization of Phenol Oxidase 1. Isolation of the enzyme 7 0 Previous studies by Mansour (1958) on the trematode T, hepatica have shown that most of the phenol oxidase from this trema- tode is recovered in the 600 x g pellet of whole worm homogenates, thus indicating that most of this enzyme is localized in a crude nuclear and plasma membrane fraction of this trematode (DeDuve ggflaT., 1955; Mahler and Cordes, 1971). This observation is consistent with the results of histochemical studies which have shown that Fasciola phenol oxidase is localized in the membrane globules of vitelline cells (Smyth, 1954). In order to determine if phenol oxidase from female S, mansoni was similarly recovered in the crude nuclear and plasma membrane frac- tions of schistosome homogenates after differential centrifugation, activated female S, mansoni were homogenized in 0.1 M phosphate buffer, pH 6.8 and were centrifuged at 1,000 x g, 3,000 x g and 5,000 x g. The pellets were assayed for both succinate-stimulated oxygen con- sumption and DA—stimulated oxygen consumption since female schisto- somes are known to contain abundant quantities of mitochondria (Erasmus, 1973). Succinate stimulation of oxidative activity in the presence of 68 1 mM malate, glutamate and ADP was used as a marker for the presence of mitochondria (Coles, 1973). In these experiments, l.mM succinate was added 5 minutes prior to the addition of DA in order to determine if mitochondrial respiration had an effect on phenol oxidase activity. The results in Table 4 showed that 96% of the phenol oxidase activity in a 5,000 x g pellet could be recovered in 1,000 x g pellet. How- ever, considerable amounts of succinate stimulated respiration were also noted in all of the pellet fractions. Nonetheless, the presence of this activity did not interfere with the assay of phenol oxidase activity, since the addition of succinate 5 minutes prior to the addition of DA failed to alter the rate of DA stimulated oxygen consumption. 5,000 x g pellets obtained from 3-fold larger quanti- ties of male S, mansoni did not cantain any phenol oxidase activity (data not shown). In further studies on the isolation and purification of phenol oxidase from female S, mansoni, a 3,000 x g pellet was used because it minimized the time of centrifugation (15 min at 1,000 x g vs. 5 min at 3,000 x g) without noticeable loss of phenol oxidase activity. Solubilization of membrane bound phenol oxidase from.mouse melanoma has been noted to occur following treatment with proteolytic enzymes (Miyazaki and Seiji, 1971), phospholipase (Seiji and Yoshida, 1968), detergents (Iwata and Takeuchi, 1977; Burnett §£Hél°: 1967) and combinations of proteolytic enzymes and detergents (Quevedo pg §£-: 1975). In order to determine if phenol oxidase from S, mansoni could be solubilized, female schistosomes which had been incubated in BMEFC 69 TABLE 4 Oxygen Consumption in Pellets of Female Schistosome Homogenates Following Successive Administration of 1 mM Succinate and 2 mM DA Oxygen Consumptiona Substrate 1,000 x g 3,000 x g 5,000 x g pellet ‘ pellet pellet 1 mM succinate, glutamate, malate .062 .018 .054 ADP .014 .031 2 mM DA, 1 me glutamate, malate, .112 .103 .101 ADP, succinate .090 .117 .101 .101 aData expressed as pmoles OZ/min-mg protein. Oxygen consumption stimulated by succinate was subtracted from the total oxygen consumption following the admini— stration of dopamine to give a value which represents the increase in oxygen consumption stimulated by the admini- stration of 2 mM DA. 70 for 4 hrs were either homogenized in detergents, centrifuged and the supernatant and pellet assayed for phenol oxidase, or the phenol oxidase pellet derived from a phosphate buffer homogenate was preincu- bated with proteolytic or lipolytic enzymes for 5 minutes at 37°C prior to recentrifugation and assay. Treatment with proteolytic enzymes or phospholipase decreased the enzyme activity in the pellet without increasing this activity in the supernatant (Table 5). Non- ionic detergents had little or no effect on phenol oxidase activity in the 3,000 x g pellet. However, since nonionic detergents can selectively solubilize a number of membrane-bound proteins (Helenius and Simons, 1975) it is possible that incubation of the phenol oxidase pellet in the presence of a nonionic detergent could aid in the partial purifica- tion of the enzyme by reducing the total protein content of the pellet. Thus, activated female S, mansoni were homogenized and'centrifuged in the presence of 5% Triton X-100 (TX-100) or 0.1 M phOSphate buffer and the 3,000 x g pellets were assayed for both phenol oxidase activity and protein content. Homogenization with 5% TX-lOO resulted in only a slight increase in the purity of the preparation. There was a 23% drop in total protein content of the pellet with no appreciable loss of phenol oxidase activity (Tables 5 and 6). However, treatment with TX-lOO resulted in a significant stabilization of phenol oxidase activity. Incubation of the untreated pellet fraction at 37°C for 30 minutes resulted in a 70% drop in phenol oxidase activity (n=2). Incubation of the TX-lOO treated pellet for 30 min at 37°C resulted in only a 6.8% drOp in phenol oxidase activity (n=3). At this time, it is not known whether this increase in heat stability was due to 71 TABLE 5 The Effects of Enzymes and Detergents on Phenol Oxidase Activitya Treatme t Phenol Oxidase Activityb’c Phenol Oxidase Activity n in Pellet in Supernatant Control 100% (48) not detectable (n.d.) Trypsin 12,500 units/ml 89.6 (2) n.d. Protease .d 8 units/m1 54.8: 4.2% (3) n.d. Phospholipase A . 120 units/ml 2 88.3: 8.1% (3) n.d. Freezing and + .d Thawing 116.3- 3.6% (6) n.d. 2% Triton X-100 107.3:ll.3% (4) n.d. 2% Tween 80 97.5: 4.9% (7) n.d. 2% Lubrol WX 104.8: 4.7% (3) n.d. 0.2% SDS n.d. (2) n.d. aAll phenol oxidase preparations were activated by incubation for 4 hrs in BMEFC containing .05% pentobarbital. Data expressed in terms of percent of control, control being .l62:.011 pmoles Oz/min-mg protein. cNumber of experiments is indicated in parentheses. dSignificantly different from control p<.05. 72 TABLE 6 Protein Levels in Pellet of Female Schistosomes Homogen&zed in Either 5% Triton X-100 or 0.1 M Phosphate Buffer Total proteins in Total proteins in T e of Pellet aqueous fraction of pellet fraction of yp resuspended pellet resuSpended pellet (:13) (us) Triton X-100 248: 13 64.0:l.7b Phosphate buffer 224: 9 83.2:7.2 aData represents mean : S.E.M. of at least 6 determinations. bSignificantly different from phosphate buffer, p<.05. 73 direct interaction of the detergent with phenol oxidase or detergent mediated inhibition of proteolytic enzymes. 2. Histochemical localization The results of the preceding section have indicated that S. mansoni phenol oxidase can be found in a crude membrane and mitochon- drial fraction of female schistosome homogenates as was described previously in E, hepatica (Mansour, 1958). Thus, it is important to determine if S, mansoni phenol oxidase, like T, hepatica phenol oxidase, can be histochemically localized within the eggshell globules of the vitelline cells. Unfortunately, the histochemical technique used by previous investigators to localize phenol oxidase within the eggshell precursor globules of T, hepatica did not permit localization of activity to specific subcellular structures in S, mansoni due to an apparently diffuse reaction to the stain (Figure 11). A fluorescent histochemical assay for phenol oxidase was subsequently develOped, based on the observation that schistosome eggshells fluoresce a characteristic yellow color when viewed under u.v. light using a 500 nm barrier filter to suppress other wavelengths of light emitted by the U.V. source. Incubation of female schistosomes in HBSS in the presence of 2 mM TME resulted in the appearance of yellow fluorescence in the vitelline cells (Figure 12). This fluorescence was localized within small globules located within the vitelline cells. These globules are similar in morphology and distribution to the eggshell precursor glo- bules, previously described by Smyth (1954), Stephenson (1947) and Smyth and Clegg (1959) in S, hepatica and Gonnert (1955) in S, mansoni. 74 Figure 11. Histochemical localization of phenol oxidase activity in female S, mansoni by catechol method. Note that the reaction is diffuse and does not permit identification of specific Cellular structures. Magnification is 35X. 75 Figure 11 76 Figure 12. Histochemical localization of phenol oxidase activity in female S, mansoni by fluorescent method. Fluorescence was induced by incubation in HBSS containing 2 mM TME. Magnification is 140X. 77 Figure 12 78 Incubation of female S, mansoni in HBSS alone did not result in the appearance of a fluorescent color (Table 7). To determine if the tyrosine methyl ester-induced fluorescence of the vitelline cells was being mediated by phenol oxidase, female worms were preincubated for 10 minutes in HBSS containing 0.1 mM DDC prior to the addition of TME. This compound is a well known copper chelator and has classically been described as an inhibitor of phenol oxidase activity (Lerner, 1949, 1953; Pomerantz, 1963). Quantitative analysis of the fluorescence intensity of the vitelline cells by microspectrofluorimetry (see Methods section for details) demonstrated that preincubation of the female worms in DDC significantly reduced (p<.01) the intensity of the fluorescence of the vitelline cells compared to worms incubated in TME without DDC. Other phenolic substrates such as catechol, tyramine, L-DOPA methyl ester and DA were not as effective as TME in inducing fluor- escence. None of these compounds induced fluorescence in the vitelline cells at 2 mM, although L-DOPA methyl ester and catechol did induce fluorescence in these cells at 10 mM. Further evidence was obtained from studies on the fluore- scence characteristics of material extracted from the female worm following incubation in the presence of TME or TME plus DDC as just ’ described. Following incubation in 2 mM TME female worms were homo- genized in 0.1 M phosphate buffer, pH 7.0 and then extracted successively with neutral butanol and butanol acidified with 2 N HCl. The acidified butanol extract demonstrated the presence of fluorescent material with a characteristic excitation maximum of 315 nm and an emission maximum 79 TABLE 7 Effects of Various Treatments on Relative Intensity of Fluorescent Light from Vitelline Glands of Adult Female S, mansoni Treatment Relative Intensity of FTuorescent Light at 537 nm 30 min incubation in.HBSS .071 i .007 30 min incubation in.HBSS + 2.5 mMTME .958 : .090 30 min incubation in HBSS + 2.5 mM TME + 0.1 mM DDC .183 : .024 30 min incubation in HBSS + 0.5 mMTM'E .106 : .009 5 hrs incubation in BMEFC followed by 30 min incubation in HBSS .056 : .002 5 hrs incubation in BMEFC followed by 30 min incubation in HBSS + 0.5 mM tyrosine .186 i .017 aData expressed as mean : S.E.M. of at least 4 determinations. 80 of 420 nm (Figure 13, curve B). The excitation/emission maxima ob- tained in this study were identical to the excitation/emission maxima of the product formed from the reaction of TME with purified mushroom phenol oxidase (Figure 13, dotted curve). Incubation of the female schistosome in the presence of 0.1 mM DDC or in the absence of TME resulted in complete inhibition of this fluorescence (Figure 13, curve A). It has previously been shown (Section I) that incubation of female S, mansoni for extended periods of time in BMEFC at room tempera- ture results in the activation of schistosome phenol oxidase. To determine if the fluorescent assay for phenol oxidase demonstrates similar characteristics, female S, mansoni were preincubated in BMEFC for 4 hours prior to the addition of 2 mM TME. The relative fluore- scence intensity increased in both the butanol extracted schistosomes (Figure 13, curve C) and in the intact schistosome (Table 7). In the latter experiment, the concentration of TME was reduced to 0.5 mM in order to permit visualization of the increased fluorescence intensity. The fluorescence intensity in the intact worm appeared to saturate with 2 mM TME since induction of phenol oxidase activity by incubation in BMEFC failed to increase fluorescence intensity when this concen- tration of substrate was employed. C. Characterization of S. mansoni Phenol Oxidase Phenol oxidase has previously been characterized in a wide variety of vertebrate and invertebrate species (see reviews by Nelson and Dawson, 1944 and Brunet, 1963). In general, phenol oxidase has been reported to be a copper protein (Karlson and Liebau, 1961) and appears to catalyze the conversion of L-tyrosine to L-DOPA and the conversion 81 .cuue> :a may saws mmmwfixo Hocmnm meomoumfinom no Eoousmae mo coauommu msu Eoum vmsuom muoswoum mo momuuxo Hocmusm A IIIII v .ummzm aw cowumcsoca mmmwm.mfl mu: q >n tmum>auom Euoz mo oomuuxo Hocmusn .o m>u=o ”awoa :mmuw mo uomuuxm Hocmusn .m m>u=o napoz commouumua one «o uomuuxm Hoomusn .< m>u=u .cmom coaumufioxm you as owe ou umm cuwcmam>m3 scammHEm .cmom :ofimmaem ecu a: can ou umm :uwcma Im>m3 coaumuaoxm .muomqum Housman mmfimamaom Scum mmcuoomu mums muuommm .mze 25 N cw soaump loose wcfi3oaaow Mwmmmmm 2W mHmEmw Eoum mmuomuuxm muosvoua mo Eauuomam mosmommuoaam .ma muswfim 82 O cozmtoxm MA shaman E: 595.325 0mm com omv 0va 0mm 0 :o_wm_Em v. ngsuazuI annelau 81 .CHuH> :fi mze eufia mmmmfixo Hocmcm esomoumficom no Eoouzmse mo coauommu mzu Eoum wmauom muosmoum mo momuuxm Hocmusm A IIIII V .ommzm :H cowumnsocfi.mmmww.mfi mm: a An vmum>wuom Euoz mo momuuxm Hocmusn .o m>uso mauos :mmum mo momuuxm Hocmusn .m m>uso mauo3 omummuumum one no momuuxm Hocmusn .¢ m>usu .emom :OHumuHoxm mom a: owe Ou mom newsmam>m3 :owwwwem .cmom coammfiem no“ a: can ou umm :uwcma Im>m3 cofiumuaoxm .muomuuxm Hocmusn mofiwflmfiom Eoum couscous mum3 muuommm .mzw 25 N :w mogumn Isoca wefizoaaow fimmmmma 2W onEmw Scum monomuuxm muoswoum mo Esmuommm mosmommuosam .ma muswam 82 O eozsaxm emu HIM. ma muswwm E: 598.652, com com - omv 0 9.2325. ‘2 “4 '" KusuezuI anneleu 5'. 83 of L-DOPA to DOPA-quinone (Lerner 32 21°: 1949). Much of the work on the characterization of phenol oxidase has been performed on exten- sively purified enzymes (Karlson and Liebau, 1961; Aerts and Vercauteren, 1965). However, as was illustrated in Section B, extensive purifi- cation of phenol oxidase from S, mansoni has not been feasible. Therefore, prior to the characterization of this enzyme it was necessary to perform numerous experiments designed to control for the possibility that other oxidative enzymes may contaminate the phenol oxidase prepara- tion in S, mansoni or may in some other way interfere with the assay of phenol oxidase activity (for details of the assay, see the Methods section). 1. Specificity of the assay A number of oxidative enzymes have been identified in S, mansoni which have the potential to interfere with the phenol oxidase assay by virtue of their ability to oxidize dopamine. These enzymes include cytochrome oxidase (Coles, 1972; Coles and Hill, 1972), mono- amine oxidase (Nimmo-Smith and Raison, 1968) and dopamine—B-hydroxylase (Bennett and Gianutsos, 1978). In addition, Byram and Senft (1978) have suggested the possibility that peroxidase might also be present in the schistosome. In order to determine if any one of these enzymes was interfering with the phenol oxidase assay the phenol oxidase pellet was preincubated in the presence of selective inhibitors of the above- mentioned oxidative enzymes prior to the addition of phenol oxidase substrate. The inhibitors used in these studies included catalase (25,000 units), 1 pM rotenone, 10 pM, l-phenyl—Z-thiazolyl-Z-thiourea (PTTU, UK14624), 100 pM a,o'-dipyridyl and 3 pM tranylcypromine (TCP). 84 One pM rotenone was found to inhibit succinate-stimulated oxygen consumption in the schistosome by 50% (n=4). Ten pM PTTU reduced schistosome NE levels from .28:.02 ng/mg worm to non—detectable levels in the catechol-o-methyl transferase radioenzymatic assay (n=3). a,a'-Dipyridyl has previously been characterized as a potent inhibitor of iron containing enzymes such as tyrosine hydroxylase (Taylor 35_ .21°9 1969) and peroxidase (Mason, 1957). Tranylcypromine has been shown to be a potent inhibitor of schistosome monoamine oxidase, Ki - 0.4 pM (Nimmo-Smith and Raison, 1968). None of the inhibitors used in these studies blocked the increase in oxygen consumption pro- duced by the addition of DA to the phenol oxidase pellet (Figure 14). However, when schistosome phenol oxidase was preincubated in the presence of compounds which are classical inhibitors of phenol oxi- dase, e.g., the copper chelating agents DDC, KCN and thiourea (Lerner, 1953), these compounds were found to be potent inhibitors of the oxygen consumption stimulated by the addition of 2 mM DA in the assay (Table 8). Other compounds known to chelate copper, including EDTA, bathocuproine sulfonate and penicillamine, were not found to be inhi- bitors of schistosome phenol oxidase. However, similar results have been obtained by other investigators working with phenol oxidase preparations of considerably greater purity (Pomerantz, 1963). The above results indicated that the assay for schistosome phenol oxidase was not significantly affected by other oxidative enzymes capable of utilizing DA as a substrate. 2. Characteristics of the enzyme Having established the validity of the assay, it was then possible to examine some of the characteristics of schistosome phenol 85 Figure 14. Specificity of the reaction between crude S, mansoni phenol oxidase and DA. Inhibitors of interfering enzymes were added to the phenol oxidase preparation 3.5 min prior to addition of substrate. Each bar represents the mean : S.E.M. of at least 3 determinations. Control is the rate of oxygen consumption by DA in the absence of inhibitors (.l62:.011 pmoles 02/min-mg protein). 96 of Cont I'OI 86 125. 100. +' I 75- 50.. 25.. 0 5 MA 260 um 10 all 1 uM TC P' Catalase UK 14624 Rotenone Figure 14 87 TABLE 8 Effect of Inhibitors on Phenol Oxidase Activity in Female S, mansoni Compoundb Kic Sodium diethyldithio- 0.3 mM carbamate Allylthiourea .02 mM Phenylthiourea .04 mM Potassium cyanide .07 mM L-Cysteine 13 mM Disulfiram 2 mM aFemales were incubated for 4 hr in Earle's BME and assayed as described in Methods. bThe following compounds were found to have no significant effect at the noted concentrations. Penicillamine (0.1 mM), EDTA (0.1 mM), bathocuproine sulfonate (0.1 mM), antimony potassium tartrate (0.2 mM), 5—hydroxytryptamine (0.1 mM), thiourea (0.1 mM). cKi's were determined by Dixon plot. 88 oxidase. The enzyme was active over a fairly broad range of pH with an apparent maximum at pH 7.0 (Figure 15). Substrate specificity studies indicated that the enzyme preferred diphenolic over mono- phenolic substrates (Table 9). On the other hand, kinetic analysis of selected substrates indicated that the enzyme has its greatest affinity for phenolic substrates with an alanine side chain (Table 10). Thus, the enzyme oxidizes DA at a greater maximal rate than L- tyrosine, even though its affinity for DA is lower than its affinity for L-tyrosine. However, the rate of L-tyrosine oxidation can be stimulated by preincubation of the enzyme with 30 pM L-DOPA. This concentration of L-DOPA does not cause any appreciable consumption of oxygen by itself, but results in an 89% increase in the rate of tyrosine oxida- tion (Figure 16). On the other hand, 50 pM DA does not significantly stimulate tyrosine oxidation. Similar results have been obtained in studies on the stimulation of tyrosine hydroxylation by dihydroxy- phenols in.mmmmalian phenol oxidase (Hearing g£_§T,, 1978). Based on its sensitivity to cepper chelating substances, it would appear that the schistosome enzyme is a capper-containing enzyme. Kinetic analysis (Lineweaver-Burk plots) of the mode of inhibition of schistosome phenol oxidase by the capper chelating agents DDC and allylthiourea reveals that both the hydroxylation of tyrosine (Figure 17) and the oxidation of DA (Figure 18) are inhibited by these com- pounds in a non-competitive manner which is consistent with their purported mechanism of action. Although the addition of 20 pM CuSO 4 reversed the inhibition of DA oxidation induced by 10 pM DDC, it was 89 Figure 15. pH optimum of S, mansoni phenol oxidase. Each point represents the mean of 3 determinations. 9O 41 O. .m 9.. £305 mE\:_E\NO «29:: £52 682.5 .825 7.2 6.8 6.4 - 6.0 Figure 15 91 TABLE 9 Effects of Substrates on Phenol Oxidase Agtivity in Homogenates of Female S, mansoni Substrate uMoles O Consumed (2 mM) minute-mg protein L-DOPA methyl ester .283:.021 Dopamine .l62:.018 Catechol . .l38:.015 Tyrosine methyl ester .l44:.020 Epinephrine .072:.008 Norepinephrine .062:.004 Tyramine .053:.004 Phenyl ethyl amine .022:.OO3 aFemale worms were incubated for 4 hours in Eagle's media and then homogenized as described in Methods. Data expressed as mean : S.E.M. for at least 3 determinations. 92 TABLE 10 Michaelis Constants and Maximal Velocities for the Substrages Dopamine, L-DOPA Methyl Ester and L-Tyrosine Methyl Ester Km vmax Compound (moles/liter) (pmoles OZ/min/mg protein) Dopamine 1.8 mM 0.250 L-DOPA methyl 0.45 mM 0.338 ester L-Tyrosine 0.42 mM 0.148 methyl ester aData obtained from double reciprocal plots of substrate con— centration vs. velocity. 93 Figure 16. Stimulation of L-tyrosine oxidation by L-DOPA and DA. L-DOPA and DA were added to the phenol oxidase preparation 3.5 min prior to the addition of IIIMI TME. Asterisk indicates significantly different from control, p<.05. Each bar represents the mean : S.E.M. for 5 determina- tions. Control is .l44:.02 pmole OZ/min-mg protein. PERCENT OF CONTROL 120 ‘ 1OO ‘ 80 " 60 ‘ 4O ‘ 20" 94 ,L 30 uM 100 uM L-DOPA DOPAMINE Figure 16 95 .«m 25 N we mumuumnsm m 96 AH opamam SON mw\F coop com emu OMNH 929:: P.- >\— mm 97 :8 H MH mumHumn—Zm .mdowumcfissmumc q mo .z.m.m H some one muommmuamu ucfiom zoom .mze .mmusofizuahaam we mmmmfixo accuse mo coauanfincfl m>aufiumaaoocoz .we museum or x m\w one. ohm ON on? 98 mg muamwm TON .0” a > dc \F .mo_oE: 99 subsequently found that this phenomenon was not enzyme dependent since 10 pM CuSO4 alone was capable of catalyzing the oxidation of DA. Studies on the phenol oxidase reaction revealed that the product of the reaction between schistosome phenol oxidase and L-DOPA methyl ester fluoresces with a characteristic excitation and emission maximum in neutral buffer of 310 nm and 420 nm, respectively. This same product was formed by the reaction of purified mushroom phenol oxidase with L-DOPA methyl ester and is consistent with previous literature reports on this topic (Haas E£Hél°, 1951; Mason and Peter- son, 1965). D. Identification of the In Vivo Substrate for S. mansoni Phenol Oxidase l. Soluble substrates In:the biochemical characterization of S, mansoni phenol oxidase (Section II), a number of compounds were noted to be potential substrates for this enzyme including L-tyrosine, L-DOPA and DA. These three compounds were the most likely candidates for the Sp.yiyg.sub- strate since two of these compounds, L-tyrosine and DA had previously been demonstrated to be present in the schistosome (Senft §£_§T,, 1969; Gianutsos and Bennett, 1978). L-DOPA was also a likely candi- date by virtue of its being an intermediate in the conversion of L— tyrosine to DA in the classical scheme of catecholamine metabolism (Cooper pg El,, 1978). In order to determine which of these substrates was most likely to serve as the Tp_ygyg substrate for schistosome phenol oxidase, the concentrations of L-tyrosine, L-DOPA and DA were determined in the 100 female schistosome and compared with the levels obtained from the male schistosome. Elevated concentrations of any one of these substrates in the female schistosome would be suggestive of a unique metabolic function for this compound in the female, possibly related to its role in eggshell formation. a. Lnyrosine concentrations: Female levels of tyrosine were found to be relatively constant over all ages tested (Figure 19). The average concentration of tyrosine was 252:7 ng tyrosine/mg wet weight of female schistosome. Tyrosine levels in the male schistosome declined from a concentration of 179:13 ng/mg 30 days post-infection to a plateau level of 92.9:2.l ng/mg 38 days post-infection. In all cases, the concentration of tyrosine in the female was significantly greater than that seen in the male (p<.01). To insure that these differences represented true differences in tyrosine concentration, samples of male and female S, mansoni were assayed in the presence of internal standards or were subjected to thin layer chromatography prior to the assay as described in Methods. These assays failed to demonstrate the presence of any substances which could interfere with the tyrosine determinations. Values obtained for L-tyrosine concen— trations in these studies are approximately 3-fold less than the values obtained by Senft 2E.§$: (1972). The reason for this discre- pancy is apparently due to the use of dry weights by these authors while our studies are based on wet weights. If one assumes a wet/dry weight ratio of 4.48 (Isserhoff ggngT., 1976), one can recalculate the values obtained by Senft EEH§£° (1972) to yield a concentration of 180 ng/mg wet weight. This value is intermediate between the values determined for male and female S, mansoni in our studies. 101 Figure 19. L-Tyrosine concentrations in male and female g. mansoni as a function of age. ( ) Male §, mansoni, (-—---) female g. mansoni. Each point represents the mean i S.E.M. of at least 4 determinations for male §, mansoni and 7 determinations for female g, mansoni. ‘ NG TYROSINE / MG WORM WET WT 102 3°°' ,K 200 100 .. So 54:78 42 is so 54 68 AGE OF WORM (days) Figure 19 103 b. L-DOPA and DA: The concentrations of these substrates in mature male and female §, mansoni (50 days post-infection) were determined by mass fragmentography or COMT radioenzymatic assay (Table 11). There was no significant difference between the DA levels deter- mined by either mass fragmentography or COMT radioenzymatic assay. The concentration of DA in the female as determined by mass fragmento- graphy (.790i.057 ng/mg worm) was approximately 3 times higher than the concentration in the male schistosome (.260i.037 ng/mg worm; p<.Ol). The concentration of L—DOPA in the female schistosome (.954: .129 ng/mg worm) was approximately 5 times higher than in the male schistosome (-l96i.029 ng/mg worm)(P<.Ol). These male-female differ- ences in L-DOPA and DA concentrations are similar to the male-female differences in L-tyrosine concentration in mature schistosomes. Despite this high concentration of L-DOPA in the female schistosome, it was apparent that L-DOPA was not actively accumulated by the female schistosome (Figure 20), which suggests that most of the L-DOPA is derived metabolically from the hydroxylation of L-tyrosine. These results suggest that L-tyrosine may be the i3_ gigg_substrate for schistosome phenol oxidase. However, Erasmus (1975) has presented evidence which suggests that exogenous L-tyrosine is preferentially incorporated into eggshell globule proteins. Thus, it may be that protein-bound tyrosine is the actual i§_zigg substrate for this enzyme, by virtue of its being present in high concentration and in close proximity to schistosome phenol oxidase, which has been histochemically localized in this thesis in the same eggshell globule as the tyrosine-rich proteins. 104 TABLE 11 L-DOPA and DA Concentrations in Male and Female g, mansoni as Determined by Mass Fragmentography (MF) and Catechgl-o~methyl Transferase (COMT) Radioenzymatic Assaya’ Male Female COMT MF COMT MF L—DOPA not determined .l96i.029 not determined .954il.29 DA .234i.022 .260i.037 .736i.986 .790i.057 QMean i S.E.M. for at least 8 determinations. Data expressed in terms of ng/mg schistosome wet weight. 105 .mcofiumcfiaumumm m mo .z.m.m H cmoa onu mucommuamu mowoa comm .AHoEs\fiu ooIOmv wtwmmmucoo muamm m.x:mm ow pmumnsocfi oum3 maho3 .Hcomcma 2W onEmw An oH .om ouswam 106 ON 1. m.. cm ousmam ..z keg-.. 342$ on. muo «e no 107 2. Protein and peptide substrates In order to determine if protein bound tyrosine could serve as a substrate for schistosome phenol oxidase, enzyme activity was determined in the presence of several protein and peptide substrates in 23532, A modified phenol oxidase assay was employed in these studies as described in the Methods section. Only one peptide, tri- L-tyrosine methyl ester (2 mg/ml) was found to produce a significant rate of 02 consumption (Table 12)., However, the rate of oxygen consump- tion produced by this substrate was extremely low (10.6i.7 nmoles 02/min) compared to the rate of oxygen consumption produced by L- tyrosine alone (384:36 nmoles Ozlmin). All other protein and peptide substrates failed to show significant rates of 0 consumption in the 2 presence of a relatively high concentration of schistosome phenol oxidase (l unit/ml). On the other hand, mushroom phenol oxidase, when diluted to levels of activity which were equivalent to those obtained for schistosome phenol oxidase (i.e., 1 unit/ml) was found to oxidize a tyrosine:lysine polymer (1:1) which the schistosome enzyme was incapable of oxidizing. However, the rate of peptide oxidation by mushroom phenol oxidase (12.7i0.l nmoles OZ/min) was only slightly higher than the rate of oxidation of tri-L-tyrosine methyl ester by schistosome phenol oxidase. Neither schistosome phenol oxidase nor mushroom phenol oxidase were capable of oxidizing chymotrypsinogen or homogenates of female schistosomes at protein concentrations of 1 mg/ml. However, chymotrypsinogen has previously been shown to be a substrate for mushroom phenol oxidase, albeit a weak substrate (Sizer, 1946, 1953). Therefore, it might be argued that the dilution of 108 TABLE 12 Proteins and Peptides as Substrates for g, mansoni and Mushroom Phenol Oxidase Enzyme Substrate Concentration Oxygen Consumptiona’b g, mansonic TME 1 mM 384:36 phenol oxidase Tyrosinezlysine Sat'd Solution not detected polymer (1:1) (N.D.) Tri-l—tyrosine 2 mg/ml 10.6t.7 methyl ester - Chymotrypsinogen 1 mg/ml N.D. Female §, mansoni 1 mg/ml N.D. proteins Mushroom TME 1 mM 350:25 phenol oxidase Tyrosine:lysine Sat'd Solution 12.7i.l polymer (1:1) Tri—l-tyrosine 2 mg/ml Not tested methyl ester Chymotrypsinogen 1 mg/ml N.D. Female S, mansoni 1 mg/ml N.D. proteins aData expressed as mean f S.E.M. for at least 3 determinations. Oxygen consumption expressed in terms of nmoles 02/min. cPhenol oxidase pellet produced by incubating 8O worms in BMEFC for 8 hrs. 109 schistosome proteins in this in_yi££g system has effectively prevented them from being oxidized at significant rates. However, it was not feasible to use higher concentrations of female schistosome proteins or phenol oxidase due to the constraints of volume (minimum volume = 1.5 mls) and the numbers of worms required to produce either 1 mg of protein or 1 unit of phenol oxidase (=80 worms). Alternatively, it may be possible to answer the questions concerning the role of protein bound tyrosine in the process of phenol oxidase catalyzed eggshell formation by examining the relative abundance of tyrosine in the male as opposed to the female schistosome. 3. Relative abundance of tyrosine in female proteins In all cases where it appears likely that protein bound tyrosine serves as a substrate for phenol oxidase in the process of protein sclerotization (Hackman, 1953; Brown, 1952) the substrate proteins have been found to be rich in aromatic residues. In order to determine if proteins from female schistosomes possessed a relatively high amount of tyrosine, male and female schistosomes were incubated in;ziggg_in BMEFC in the presence of 3H-L-tyrosine or 3H-L-leucine. 3H-L-leucine was used as a control for L—tyrosine since L-leucine possesses no reactive functional groups other than those involved in the peptide linkage and as such should not be involved in the formation of unusual protein structures such as the hardened eggshell protein. The results of these studies are summarized in Figures 21 and 22. L- tyrosine was incorporated into PCArprecipitated proteins obtained from homogenates of female g, mansoni to a lesser extent than L-leucine overall time points tested (Figure 22). More importantly, there were 110 .m:Ofium:Haumump 0 Mo .z.m.m + cams mam mucmmmudou mafiOQ :omm .mo.va .msemouxu Scum usouowwap haucmoawficwfim mmumofipsfi xmflumum< .mcfimuoua manmufiafiooum I.mH .HN muswam m 111 am ouswfim Adm—3 m2: 20.._.._. I m4<2 -Dma II .II OOOON 88m 880.. . 68: L 880— COCONN Ocean ugezmd 6m /Wd3 112 .mcofiumcwau0umn 0 mo .z.m.m H some mnu mucmmmudou umn comm .xmmau coaumnnonfi 08mm on» Eoum pocHMuao ficomcme .m onEmm mam mama How pmuama mp3 mum: .mcwmuoum manmuwafiomuas.mM .NN muswwm I.“ E U) 0 a: > I'- 113 LEUCINE 1.6 a ~ :3 q o OILVH BTVWSi /31VW INCUBATION TIME (HRS) Figure 22 114 no significant differences between the male/female ratios for the amounts of 3H-L—tyrosine and 3H—L-leucine incorporated into these PCA- precipitated proteins (i.e., the relative abundance of these amino acids in female schistosome proteins is similar to the relative abun- dance of these amino acids in the male schistosome)(Figure 23). However, since female S, mansoni do not produce eggs in_z;££g_in BMEFC, this failure to find evidence of tyrosine rich proteins in female schistosomes might be due to inhibition of the protein synthesis mechanism which is responsible for the generation of eggshell proteins. In order to test this hypothsis, paired S, mansoni were incubated in modified RPMI 1640. In this medium, paired S, mansoni begin producing eggs on the second day of incubation at the rate of 31.4:8.1 eggs per day. In these studies, 3H-L—tyrosine‘was added to the incubation medium for an 18 hour period beginning'on day 2 of incubation. The results are summarized in Tables 13 and 14. These studies showed a significant (p<.05) increase in the amount of 3H—L-tyrosine and 3H-L- leucine incorporated into both male and female schistosome proteins when the incubations were performed in modified RPMI 1640 as opposed to BMEFC (Table 13). However, it was also noted that the relative abundance of both 3H-L-leucine and 3H—L-tyrosine was significantly (p<.01) higher in the proteins obtained from female worms incubated in modified RPMI 1640 as Opposed to BMEFC (Table 14). These results indicated that although both male and female S, mansoni increased the rate of incorporation of exogenous, labeled, amino acids into protein in modified RPMI 1640, there was a greater but non-selective increase in the incorporation of these amino acids into proteins from female schistosomes. 115 .mcoaumcfiaumump c «o .z.m.m H :mmE mnu muammmuamu ucfiom comm .mo.va .mHmE Eoum ucmummwfip xauomofiwficwfim oumofiwsfi mxmwumum< lummam :H .mufifiuflfi .mm muswuu 116 mm muswam Adm—.3 m2: 20:.h clllo H\ \ m._Huomowwficwfim mmumoaucw xmwumum< lummsm 40m Oucfi mpaom ocwemlm mo coaumuomuoosfi ouufi>.mw ecu How mofiumu mHmEmM\mHmz m .+.. Cam:— wanna . mquUMS .sm museum 121 m2.03m._ h .— mz_m0m>._. - «N gunman fiwmIU 92.... 20:23:02. m OllVH ETVWBd / EWVW 122 TABLE 15 Metabolism of 3H-L-Tyrosine in Female S, mansoni During 18 hrs }n_Vitro Incubation in BMEFC or mod. RPMI 1640 Z of Metabolite Recovereda Culture Medium Tyrosine DOPA ’ DA NE 3MT,NM BMEFC 32.0il.8 5.6:1.7 2.2i0.7 1.4i0.2 1.3i0.3 miggoml 38.8:4.3 2.1:o.3 n.d.b n.d. n.d. “Mean : S.E.M. for 4 determinations. bNot detectable. 123 1640. The only significant differences between the metabolism of tyrosine in BMEFC and modified RPMI 1640 were related to the metabo- lism of tyrosine to the catecholamines. Levels of tritiated catechol- amines went from 2.2 and 1.3% for DA and the methoxylated metabolites (3MT and NM), respectively, in BMEFC to nondetectable levels in female schistosomes incubated in RPMI 1640. The remainder of the radioactive material (approximately 60%) was recovered on or near the origin and could not be identified. This material probably represents L-tyrosine present in proteins and peptides or other high molecular weight meta- bolites of L-tyrosine. These results suggest that L-tyrosine in RPMI 1640 may serve as a preferential substrate for another enzyme (e.g. phenol oxidase) which is not part of the classical scheme of tyrosine metabolism to the catecholamines which has been demonstrated in nervous tissues (Cooper g£,§1., 1978). 5. Soluble L:tyrosine and in vitroéprotein crosslink formation It is an essential tenet of this thesis that the reaction of phenol oxidase with its substrate L-tyrosine produces a highly reactive product which is capable of cross-linking proteins from the female schistosome during the process of eggshell formation. In order to determine if the female schistosome contains proteins which are capable of being polymerized by gfquinones, 3H-labeled proteins from female S. mansoni were incubated 12.23252 in the presence or absence of L- tyrosine and phenol oxidase as described in the Methods section. The formation of insoluble prOtein polymers was monitored by measuring the increase in the amount of 3H-labeled protein deposited in a 3,000 x g pellet obtained from homogenates of female schistosomes which had been 124 incubated with phenol oxidase and L-tyrosine. The results of these studies are summarized in Table 16. There was a slight, but signifi- cant (p<.05) increase in the amount of 3H—protein incorporated into the 3,000 x g pellet following incubation in the presence of a half- saturated solution of L-tyrosine and schistosome phenol oxidase. In the absence of L-tyrosine or phenol oxidase, there was no significant increase in the amount of 3H—protein in the 3,000 x g pellet when compared to the amount of 3H—protein in pellets obtained from S. mansoni homogenates incubated in the absence of both L-tyrosine and phenol oxidase. Extraction of the insoluble pellet with 2% SDS and 1% Bdmercaptoethanol did not result in statistically significant increases in the amount of 3H in SDS extracts of the pellets obtained from homogenates of female schistosomes. ‘SDS polyacrylamide gel electropho- resis failed to reveal the presence of any apparent protein polymers when stained with Coomassie blue (Figure 25). However, when these polyacrylamide gels were sliced and counted to determine the extent of 3H present, it became apparent that the SDS extract of the 3,000 x g pellet obtained from homogenates incubated in the presence of both L-tyrosine and phenol oxidase contained a significantly greater (p<.05) amount of tritium label within the first 1-2 mm of these gels when compared to pellets obtained from homogenates incubated in the absence of L-tyrosine and phenol oxidase (Figure 26). However, this increase was not statistically different from the other two controls (L-tyrosine alone or phenol oxidase alone), which in turn were not statistically different from the untreated homogenates. These data I O O 3 provided ev1dence to support the contention that the increase in H 125 .no.vm .maouusoo Scum ucmummwfiv haucmofiMficmeu .moa x 2mm mo maumu ca pommmuaxm momma .msowumcfiaumump m pom .z.m.m H amuse .I . . - . . - . . u . masououa NV +mN HH H ©m+o NOW Q m +® NQN m H...” HQ Hcomama ow. UHGEQW .u . . I . . I . . I . mcaououa “commas am on +oo HH m mm+m mum m mm+o mom a H+¢ mm mamsmm + ommmfixo Hocmsm msaououm “comers aw H0.HH¢.~H o.HHHm.oom «.m Ho.m¢N m.NH~.Hm mamsmH + mcwmouxulq mcfimuoum acmocma am oce.wm~.ca o.mNHH.mcm m.mmww.mam mm.HHm.q¢ mamemm + msfimouzu IA + mmmpfixo Homosm um o s . muo assurances uoHHom unusummua HH m a m H B m mannaomsH Q46 mmmpaxo Hosmnm fisomsme um moan mcamoumalq sues mawmuoum Hoowcma aw mamammlmm moXtoHumnoocH ouua>.mm wcw30HHom mumahaom samuoum mannaomoH mo cowumeuom 0H mAmu=o :owmmfiao mom a o>u=o oofiumuaoxm mo hufimsmucfi m>wumamm .oummmaouchn Hamzmwwm no mam m mo>u=o .mmmpfixo Homosa nuaa uso .9 use m mm>u=o .amom :oHumuaoxm you a: One on now nuwcoam>63 scammfiao use cmom scammaso now as com ou uom nuwcoao>ms soaumuaoxm .0 wow 4 mm>u=o "atom soaumufioxm you a: oaq cu umm zuwsmam>m3 :ofimmfiEo pom snow scammHEQ How 5: can ou umm sumsmao>ms coaumuaoxm .momz z H ca pocwmuno mums muuooam .mHHmSmwwm Mdmmmma aw pmnzaouphn pfiom mo Esuuooam mocoomouonam .mN ousmflm 132 owm 60¢ '4 mm muawwm E: 59.2995 on; own :o_mm...Em II emu—V 0mm own esteem. 0mm Ausuazm aime'ag 133 redissolved as described for the eggshells. The products formed from this reaction had a fluorescence spectrum very similar to the 310/420 peak of the eggshell. However, almost all of the products formed by the reaction of L-DOPA with phenol oxidase in the presence of nucleo- philic amino acids absorb in the U.V. spectrum at 310 nm.except the thiol containing compounds (e.g., cysteine) which absorb at 360 nm. Although the hydrolyzed eggshell has an absorption maximum at 360 nm, both the excitation and emission peaks are very broad, suggesting that the peak seen is composed of multiple fluorescent entities. Therefore, the eggshell hydrolysate was chromatographed on a silica gel thin layer plate and the fluorescence spectrum obtained from slices of this gel were compared with the fluorescence spectrum of the products formed from the reaction of L-DOPA with mushroom phenol oxidase in the presence of various amino acids containing nucleophilic side chains. These latter products were hydrolyzed in acid and were treated the same as the eggshells. The products formed from the reaction of a tyrosine-lysine (1:1) polymer with a quinone derived from the reaction of L-DOPA with phenol oxidase yielded 2 fluorescent products with apparent excitation/ emission maxima which were similar to those found in the eggshell. One product which migrated with a R of between .07 and .13 yielded f excitation/emission maxima of 325/420 which closely corresponded to a compound in the eggshell which appeared to fluoresce at 330/425. The second product migrated between .93 and 1.00 and gave a fluorescent product with excitation/emission maxima at 335/395. These maxima corresponded to an identical peak in the eggshell hydrolysate. No 134 other fluorescent peaks were identified which possessed similar Rf values and fluorescence spectra. The results of these studies suggested that the products formed from the reaction of protein bound lysine with a DOPA or tyrosine derived quinone can be identified in S, mansoni eggshells, thus pro- viding additional evidence to support a concept of schistosome egg- shell formation in which phenol oxidase reacts with tyrosine to form a quinone which cross-reacts with multiple proteins in the eggshell to form a single large and extensively cross-linked protein. In order to determine if there was a good correlation between inhibition of phenol oxidase activity and inhibition of eggshell formation, infected mice were injected with varying quantities of compounds which have been demonstrated to be inhibitors of phenol oxidase in yi££g_(8ection 3, Table 8). Administration of these come pounds had a dramatic effect on egg production. Schistosome eggs in the uterus of female S, mansoni obtained from mice treated with phenol oxidase inhibitors lacked the well-defined eggshell characteristic of S, mansoni eggs (Figure 28). Instead there was a small amount of globular material present in the uterus which fluoresced in a manner similar to the intact eggshell (Figure 29). However, since sulfhydryl groups such as those present in DDC and allylthiourea (C=S) are capable of reacting with quinones (Hirsch, 1955) it is possible that these compounds prevented formation of the intact eggshell by pre- ferentially reacting with the quinones formed from phenol oxidase rather than inhibiting the phenol oxidase catalyzed formation of quinone. In order to control for this possibility, infected mice were 135 Figure 28. Appearance of normal S, mansoni egg in U.V. light. Photo- graphed with dark-field condensor. Color of the egg is yellow. Magnification is 14OX. 136 Figure 28 137 Figure 29. Appearance of S, mansoni egg in U.V. light 1 hr following the administration of 40 mg/kg diethyldithiocarbamate. Photographed with dark-field condensor. Color of material is yellow. Magnification is 140x. 138 Figure 29 139 administered large doses of compounds which were known to be struc- tural analogs of schistosome phenol oxidase inhibitors (e.g., thiambu- tosine, clofazimine, PTTU), but which were not demonstrated to be effective inhibitors of schistosome eggshell formation ig_y飣g, These inactive analogues failed to produce inhibition of eggshell formation at doses which produced 100% inhibition of eggshell formation using known inhibitors of schistosome phenol oxidase (Table 17). Thus, it appeared that inhibition of eggshell formation was closely correlated with inhibition of phenol oxidase activity. However, since DDC and allylthiourea, the two phenol oxidase inhibitors used in these studies, are chelating agents, the possibility existed that the inhi- bition of eggshell formation by these compounds might be related to the inhibition of other metal ion containing enzymes such as peroxidase. In order to control for this possibility, infected mice received a single dose of 100 mg/kg of compounds which either had been demon- strated to inhibit peroxidases involved in eggshell formation (sodium sulfite, phenylhydrazine; Foerder and Shapiro, 1977) or which were known to inhibit iron containing enzymes in general (a,a'-dipyridy1). These drugs failed to inhibit eggshell formation (Table 17). It was noted, however, that these compounds appeared to produce more struc- tural abnormalities (creasing, deformation, elongation of spike) in the eggshell than were apparent in the eggs of untreated schistosomes. It was not certain if these structural changes represented specific .changes due to the action of these drugs on peroxidase or a nonspecific artifact of drug administration. However, it was clear that schisto- some eggshell formation was more susceptible to the actions of phenol oxidase inhibitors than peroxidase inhibitors. 140 TABLE 17 The Effects of Inhibitors of Protein Cross-link gormation on S, mansoni Eggshell Formation lg Vivo ’ Dose # of Percentage % Inhibition of Phenol Compound m /k Ex Abnormal Oxidase In Vitro g g p. Eggs at 10:3M DDC 100 (2) 100 69% 4O (8) 79 20 (7) 20 Allylthiourea 100 (2) 100 74% 4O (3) 83 20 (3) 29 . Phenylthio- 20 (l) Ob 69% urea PTTU 100 (2) 0 5% clofazimine 100 (2) 0 Not Tested thiambutosine 100 (2) 0 3% penicillamine 100 (2) O -15% aAll drugs were administered i.p. in 1% methyl cellulose 1 hr prior to sacrifice. bAnimal died. cThe following drugs did not inhibit eggshell formation at the concentrations indicated and were not found to inhibit phenol oxidase ;n_vitro at 10'4M: a,a-dipyridyl (100 ng/kg), phenhydrazine hydrochloride (100 mg/kg), sodium sulfite (100 mg/kg), dl-u-tocopherol acid succinate (400 m/gkg), ascorbic acid (100 mg/kg). 141 On the other hand, the inhibition of eggshell formation by phenol oxidase inhibitors did not appear to be complete (Figure 28). There was a small amount of residual globular material which fluoresced in a manner similar to the intact eggshell and which persisted following the administration of as much as 200 mg/kg DDC. These globules like eggshells, were brown when visualized with a light microscope and as will be discussed later, were not found to be antigenic. These characteristics of the globules are very similar to the characteristic of purified eggshells (Boros and Warren, 1970) and thus the globules were thought to represent eggshell material. Since doses of DDC well in excess of the dose required to produce 100% inhibition of eggshell formation failed to completely block the formation of eggshell globules, it was thought that some other process such as the autooxidative or peroxidative generation of quinones, or protein cross-linking mediated by free radical formation, might be involved in the generation of this residual eggshell material. In order to determine if one of these mechanisms was responsible for the continued formation of eggshell material in the presence of DDC, a number of antioxidants including ascorbic acid (100 mg/kg), dl-a-tocophenol acid succinate (400 mg/kg) and PTTU (100 mg/kg) were injected into infected mice 1 hr prior to the administration of 200 mg/kg DDC. These antioxidants, in addition to preventing the autooxidative formation of quinones (Hirsch, 1955) also inhibit free radical chain reactions (Tappel, 1972) which may be involved in the formation of protein cross-links (Roubal and Tappel, 1966). None of these compounds caused the complete cessation of eggshell globule formation following DDC administration. Nevertheless, these drugs appear to be capable of inhibiting eggshell formation to 142 a limited extent. When given alone in the doses mentioned above, only ascorbic acid inhibited eggshell formation, and this inhibition occurred in only 1 worm in 20. However, administration of these drugs in doses of 100 mg/kg (dl-a—tocophenol acid succinate) or 50 mglkg (ascorbic acid, PTTU) resulted in an increase in the percentage of abnormal eggs in the uterus following the administration of submaximal doses of DDC (Table 18). It should be noted that these results were based on only 2 experiments, 2 mice/experiment with a minimum of 10 worms/experiment. In addition to the antioxidants, a,a'-dipyridyl was also tested for its ability to inhibit eggshell formation. Like the antioxidants, this compound at 100 mglkg did not inhibit eggshell formation by itself, but did potentiate the DDC—induced inhibition of eggshell formation at a dose of 50 mglkg (Table 18). One hundred mg/kg dipyridyl alone did not inhibit the formation of eggshell globules (Table 17). F. Chemotherapy of Schistosomiasis Using Phenol Oxidase Inhibitors It is apparent from the results of the preceding section that the process of eggshell formation is most susceptible to the inhibitory actions of lipophilic copper chelating agents such as DDC and allyl- thiourea. Thus, in order to evaluate the effectiveness of alleviating the pathology of schistosomiasis by inhibition of eggshell formation, heavily infected mice were fed disulfiram chronically in the diet (0.3%). The effectiveness of disulfiram in alleviating schistosomal pathology was evaluated in terms of its ability to prolong the survival of heavily infected mice and reverse the microscopic pathology asso- ciated with egg deposition. It should be noted that disulfiram per g; 143 TABLE 18 The Effects of Some Inhibitors of Protein Cross-link Formation on the DDC Induced Inhibition of Eggshell Formation lg_Vivo Inhibition of Eggshellb Dose # of Formation Compound m /k Ex 8 g 9’ DDC DDC Control 20 mg/kg 40 mg/kg Control --- (2) —-- 7/29 19/23 dl-a-tocopherol 100 (2) 0/30 12/24 35/38 acid succinate PTTU 50 (2) 0/27 9/31 26/28 a,a'-dipyridyl 50 (2) 0/34 9/27 34/36 ascorbic acid 50 (2) 0/29 9/21 19/21 aVehicle or vehicle plus inhibitor was determined i.p. in 1% methyl cellulose 1 hr prior to the administration of DDC. Animals were sacrificed and worms examined 1 hr following the administration of DDC. bData expressed in terms of the number of abnormal eggs divided by the total number of worms containing egg material in the uterus or uterine canal. 144 is a relatively poor inhibitor of schistosome phenol oxidase (Table 8). However, disulfiram is rapidly metabolized to DDC (Linderholm and Berg, 1951) which, as has previously been mentioned, is a potent inhibitor of phenol oxidase inpyiggg.and eggshell formation ;n_3132, The results of these studies are illustrated in Figure 30. Some early deaths occurred in the drug treated group but these were few in number and ceased by the 48th day after infection. The early deaths in the drug treated and control animals were probably secondary to the severity of the infection. The gross pathology normally asso- ciated with the deposition of schistosome eggs in the liver (distended portal veins, swollen and granular appearance of liver and spleen) did not become apparent until after the 40th day of infection in the untreated mice. After this initial period of increased mortality, no more deaths occurred in the drug—treated animals until the conclusion of the experiment. Deaths in the control group occurred at a more rapid rate, reaching a low of 20% surviving (4/20) by the 96th day after infection. Note that the animals which were withdrawn from the drug diet showed a sudden increase in mortality beginning 12 days following the cessation of drug treatment. Thus, it appears that in the absence of continued drug treatment, the beneficial effects of disulfiram treatment are reversed. Light microscopic examination of the livers, spleens, intestines and lungs from both treated and untreated mice revealed substantial differences in pathology between these groups. Examination of the livers from untreated mice (Figure 31) revealed the presence of characteristic granulomatous lesions surrounding S, mansoni eggs or 145 .umfip wauv may scum :Bmupnufi3 mums ooHE nowz3 um mafia moumoapca Souu< .umfip Emuemanmfip Nm.o Eoum =3mupsuaz mods A.I 1.! v macaw so Emuawasmfiv Nm.o w=fi>wooou mafia A III v “moat Houucoo so woe: A.III V.H=om:ma .m :uHB couommcfl mafia mo huwamuuoa so Emuqmasmfiv mo mucouwm .om muswfim 146 Our 0: on «usage 29.-bum; cub“: m>