Studies II. lsoenzyme
Encarnita Conde-del Pino, Alma M. Annexy-Martfnez, Marin& P&ez-Vilar, and A. A. Cintrbn-Rivera General
Center, School of Medicine, San Juan, Puerto Rico for
CONDE-DEL Pmo, ENCARNITA, ANNEXY-MARTINEZ, ALMA M., P&z-VU, MARIN~S, AND CINTR~N-RIVERA, A. A. 1968. Studies in Schistosoma mansoni. II. Isoenzyme patterns for alkaline phosphatase, isocitric dehydrogenase, glutamic oxalacetic transaminase, and glucose B-phosphate dehydrogenase of adult worms and cercariae. Experimental Parasitology 22, 288-294. The results of a study of isoenzymes of alkaline phosphatase, isocitric dehydrogenase, glutamic oxalacetic transaminase, and glucose 6-phosphate dehydrogenase in Schistosoma mansoni adults and cercariae have been presented. TWO bands of alkaline phosphatase activity were found in adults and cercariae. No activity could be elicited from snail tissue extracts. Mouse liver showed two closely migrating bands. Isocitric dehydrogenase activity could not be demonstrated in adult S. mansoni. Mouse liver showed two bands, and snail and cercariae exhibited only one. Differences in the mobility of the fractions were observed between these extracts. Cercariae and adult extracts yielded three bands of glutamic oxalacetic transaminase activity. Two were anodic and one cathodic. Mouse liver and snail homogenates evidenced two bands of activity, one anodic and one cathodic. Comparison of adult S. mansoni homogenates with mouse liver and snail extracts showed that the cathodic bands had similar mobilities, but none of the anodic bands corresponded to one another in electrophoretic migration. One band of glucose 6-phosphate dehydrogenase activity was obtained from cercariae, adult, and mouse liver extracts. The cercarial band had a shorter migratory path. The band of activity from extracts of adult S. mansoni obtained from the peritoneal cavity of the mouse and by perfusion had the same mobility as the mouse liver band. Snail tissue homogenates showed no activity.
Various enzymes have been shown to operate in Schistosoma mansoni (Mansour and Bueding, 1954; Bueding and Ma&in‘These studies were supported by grant AI07557-01 from the National Institute of Allergy and Infectious Diseases and grants FR-63-04 and FR-63-05 from the Division of Research Facilities and Resources, National Institutes of Health, U.S. Public Health Service.
non, 1955a; Timms and Bueding, 1959; Bueding, 1962), and several from the adult parasite have been shown to have kinetic and immunological differences when compared with their counterparts from mammalian tissues (Mansour and Bueding, 1953; Bueding and MacKinnon, 19.5517; Henion et al., 1955; Bueding, 1962). More recently, isoenzymes of lactic and malic
STUDIES IN %histo~OTTUI
dehydrogenases have been demonstrated in S. mansoni (Conde-de1 Pino et al., 1966). This study is part of a series of investigations aimed at elucidating the molecular similarities or diflerences in size and charge among selected enzymes of two stages of S. mansoni, the adult male and female and the cercaria. At the same time a comparison is made between the same enzymes from tissues of the mouse and those from snail hosts. MATERIAL
Electrophoresis was used to separate the different proteins, and these were then characterized by their enzymatic activities and relative mobilities. The preparation of mouse tissue and S. mansoni extracts and their fractionation by ceIIuIose acetate and disc electrophoresis was accomplished as previously described (Conde-de1 Pino et al., 1966). Total protein of the homogenates was determined by the method of GornaII et al. (1949) by using a Beckman model 150 uItramicro analytical system. Al1 extracts were diluted with saline to a concentration of 30 mg protein/ml. Snail tissue was obtained after removing the shells from uninfected 8 weeks old Biomphularia glabrata which had fasted 24 hours prior to use. For experiments with glucose 6-phosphate dehydrogenase, S. mansoni adults grown intraperitoneally were required. The infection was obtained by intraperitoneal injection of mice with 350 cercsriae. The mice were killed 8 weeks after infection. The peritoneal cavity was flushed with tissue culture solution medium 199, pH 7.4. The schistosomes were collected, washed ten times with the same solution, and frozen immediately in liquid nitrogen. (Ornstein and Disc electrophoiesis Davis, 1962) was used to fractionate alkaline phosphatase, since this enzyme gave very poor resoIution by celluIose acetate electrophoresis.
Staining of Isoenzymes Alkaline phosphatase was stained by the method of Lawrence et al. (1960), in which sodium alpha-naphthyl phosphate served as substrate. The pH of the medium was maintained at 9.2. Isocitric dehydrogenase activity was localized by the technique of Bell and Baron (1962). Determinations were made using dl-trisodium isocitrate or threo-n, ( + ) -isocitrate as substrate. Nicotinamide adenine dinucleotide phosphate (NADP) was the coenzyme used. Glutamic oxalacetic transaminase and glucose 6-phosphate dehydrogenases were visualized by the methods of Rome1 and La Mancusa (1965) and Haut et al. ( 1964)) respectively. Controls were incubated in a medium from which the substrate was omitted. Eight determinations were performed on each enzyme studied. Patterns were constant and reproducible. Preliminary experiments using livers from normal and infected mice gave no evidence of differences in the number, electrophoretic mobility, or staining intensity of the isoenzymes tested. Subsequently perfused livers from normal animals were used. The mice were 3 months old and weighed approximately 20 gm. RESULTS
1. Alkaline phosphatase (Orthophosphoric monoester phosphohydrolase, EC. 184.108.40.206.).” Adult S. mansoni and cercariae contained high activity as evidenced by the intensity of the reaction (Fig. 1). Discrete bands were not obtained by cell&se acetate electrophoresis even after dilution of the extracts. By disc electrophoresis the parasites’ extracts resolved into two fractions. The slowest moving contained the greatest activity. Mouse liver contained two bands which migrated very close together. There were no apparent differences in ’ Enzyme Commission Report (1964).
CERCARIA ADULT S. MANSONI
ADULT S. MANSONI
staining intensity between the two mouse liver bands (Fig. 1 ), No alkaline phosphatase activity could be elicited fi;om snail tissue extracts. Comparative electrophoretic mobilities could not be determined accurately by the disc electrophoresis technique. 2. Isocitric dehydrogenase ( threo-n,-isocitrate: NADP oxidoreductase, E.C. 1.1. 1.42). One band of activity could be demonstrated in cercarial extracts but not in male oi female S. munsoni adults. Mouse liver showed two bands and snail tissue only one. The snail isocitric dehydrogenase had a longer migration path as compared with mouse liver and cercariae. No cathodic bands could be elicited from cercarial or snail homogenates (Fig. 2). Identical results were obtained when either dl-trisodium isocitrate or threo-n, ( + ) -isocitr’ate were used as substrate. 3. Glutamic oxalacetic transaminase (LAspartate: 2-oxoglutarate aminotransferase, E.C. 220.127.116.11. ) . Cercarial and adult S. mansoni extracts yielded three bands of activity with identical mobilities. Two were anodic and one was cathodic (Fig. 3a). Mouse liver and snail homogenates evidenced two bands of activity, one anodic and one cathodic (Fig. 3b). When these extracts were
ELECTROPHORESIS S. mans&
compared with adult male S. mansoni homogenates, it was observed that the cathodic bands had slight variations in ability. None of the anodic bands corresponded to one another in electrophoretic migration (Fig. 3~). 4. Glucose B-phosphate dehydrogenase (D-Glucose 6-phosphate: NADP oxidoreductase, E.C. 18.104.22.168). Cercaria and adult S. mansoni evidenced one anodic band of
+ MOUSE LIVER
Isocitric dehydrogenase cercariae, and snail
ADULT S. MANSONI
IN Schistosoma mansoni
a FIG. 3. Glutamic snail tissue.
ADULT 6 S.MANSONI
activity. Male and female bands had identical mobilities while the cercarial band had a shorter path of migration (Fig. 4). When adult S. mansoni was compared with mouse liver, the electrophoretic mobility of the fractions proved to be closely similar. Extracts made from adult schistosomes obtained from the peritoneal cavity of the mouse had one band of activity with the same mobility as that of mouse liver and of
c in adult
S. mansoni obtained by conventional perfusion (Fig. 4). Snail tissue homogenates showed no activity. DISCUSSIOX
There have been several studies of phosphatase activity in S. mansoni. Enzyme activity has been reported in whole worm homogenates and localized histochemically in the cuticle, gut, and reproductive strucI
ADULT S. MANSONI
3 d INTRAPERlTONEAL cercaria,
tures of adults (Dusanic, 1959; Robinson, 1961; Nimmo-Smith and Standen, 1963; Halton, 1967) and in the body of cercariae (Dusanic, 1959; Fripp, 1966). These findings have often been interpreted as indicating that phosphatases of the adult may be involved in phosphorylated transfer mechanisms and carbohydr’ate metabolism (Halton, 1967). A difficulty encountered in the attempt to characterize different phosphatases by histochemical reaction resides in the fact that biochemically different phosphatases may have the same histological localization. Electrophoresis allows for more detailed characterization of different types of alkaline phosphatases. This study discloses the muhiple nature of a naphthyl phosphatase from adults and cercariae. The size, intensity, and position of the zones of activity of the parasite differ from the pattern obtained with mouse liver. The use of different substrates and inhibitors may help to distinguish different enzymes. Isocitric
In mammalian tissue, isocitric dehydrogenase exists as two components, one intraand the other extramitochondrial in origin. fractionation has shown Electrophoretic that the extramitochondrial band migrates towards the anode while the mitochondrial band moves towards the cathode (Lowenstein and Smith, 1962). These two bands appear in the mouse liver extracts. Cercariae and snail tissue exhibit only an anodic moving band. Restriction of a particular member of an isoenzymic set to a specific cellular region is known to occur (Allen, 1961). Thus it is possible that many of the multiple forms of enzymes as separated by electrophoresis may represent enzymes with specific cellular locahzations. If we interpret our data accordinalv. it avnears that cercarial and
snail tissue evidence the extramitochondrial isocitric dehydrogenase. The failure to elicit any activity from S. mansoni adult extracts may only reflect a deficiency in the experimental conditions used as these are designed to demonstrate mainly isoenzymes in mammalian tissues. Von Brand (1966) has stressed that the mere demonstration of enzymes in an organism does not necessarily prove the presence of a functional cycle, nor does the failure to demonstrate certain enzymes constitute certain proof of the absence of the cycle. However, since all other extracts showed isocitric dehydrogenase activity, it can be concluded that even if isocitric dehydrogenase is present in adult S. mansoni, it differs from mouse, snail, and cercarial isocitric dehydrogenase. GlzGtamic Oxalucetic
The metabolism of proteins plays an important part in the growth and reproduclink the tion of parasites. Transaminases metabolism o’f carbohydrates, fats, and proteins. Garson and Williams (1957) have shown that transamination occurs in S. mansoni. Huang et al. (1962) have demonstrated the presence of glutamic pyruvic transaminase and glutamic oxalacetic transaminase in S. japonicum. In mouse and snail tissues two bands of activity appear in the extracts. The cathodic bands have similar mobilities. Schistosoma mansoni, maIe, female and cercariae, evidence, in addition, a third band with anodal mobility. The significance of this extra band of activity, presumably of cytoplasmic origin, is not known at present. Experiments are in progress to determine whether this is characteristic of other S. mansoni transaminases. Glucose
De Ley and Vercruysse (1955) have shown that a number of worms, free-living and narasites. contain glucose 6-nhosnhate
dehydrogenase and 6-phosphogluconate dehydrogenase. Waitz (1964) has reported that extracts of adult S. mansoni contain both enzymes. Histochemical studies by Dodin et aE. (1966) indicate that glucose B-phosphate dehydrogenase is localized in the subcuticular tegumental papillae of adult male and the vitellaria of female S. munsoni. In our studies, all extracts with the exception of snail tissue homogenates gave one band of activity. The ceicarial band differs in mobility from that of S. mansoni adults. Mouse liver homogenates revealed an anodal band that corresponds in mobility to that of S. mansoni adults obtained by perfusion. Because of the possibility that host enzymes, if they remain undigested in the worm’s gut, could contaminate worm several experiments were perextracts, formed with extracts from S. mansoni adults obtained from the peritoneal cavity of mice 8 weeks after intraperitoneal infection. It was presumed that these worms would have less contact with host tissue and blood enzymes. Results, however, have been the same as those obtained with perfused S. mansoni. This similarity in mobility of the host and the parasite’s isoenzymes may be merely a coincidence, identity of mobility not necessarily indicating an identity of the proteins. The origin of an enzyme is relevant to any description of the enzymatic composition of a parasitic organism. Experiments are in progress to determine whether this enzyme differs in other properties from the host enzyme and is, therefore, endogenous to the parasite. These studies show that molecular variations among similar enzymes from the different species analyzed are usually the rule. Even among the different stages of a parasitic worm, metabolic differences may prevail. Characterization of the biochemical differences of parasitic worms as compared with their hosts offers opportunities for
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