Moises Agosin

STUDIES ON THE METABOLISM OF ECHINOCOCCUS GRANULOSUS. VII. REACTIONS OF THE TRICARBOXYLIC ACID CYCLE IN E. GRANULOSUS SCOLICES.

Abstract 1. 1. E. granulosus intact scolices as well as cell-free preparations oxidize several Krebs acid cycle intermediates. Cell-free preparations contain condensing enzyme, a TPN- and DPN-dependent isocitric dehydrogenase, succinic, malic and α-ketoglutaric dehydrogenases, as well as fumarase, aconitase, pyruvic oxidase and α-carboxylase. 2. 2. Phosphoenolpyruvic, carboxydismutase and probably “malic” enzyme are involved in CO 2 -fixation reactions in E. granulosus . 3. 3. Acetate is activated by aceto-CoA-kinase. No evidence was found for acetokinase and transacetylase, although the extracts contained an active acetyl-phosphatase. The glyoxylate cycle seems to be ruled out since neither isocitriatase nor malate synthetase was present in homogenates or acetone powder extracts. 4. 4. C 14 -labeled carbon dioxide is utilized by intact scolices for the synthesis of Krebs acid cycle intermediates, protein, lipids and phospholipids, nucleic acids and polysaccharides. Most of the C 14 is fixed into succinic acid, which is excreted into the medium. Acetate-2-C 14 is also utilized for the synthesis of various cellular materials, including polysaccharides. 5. 5. The above findings, together with the labeling patterns from C 14 O 2 -fixation of the amino acids obtained from protein hydrolysates as well as of the Krebs acid cycle intermediates, are considered as an indication of the presence of the Krebs acid cycle in E. granulosus . 6. 6. A C 3 -C 1 -type of C 14 O 2 -fixation with the formation of oxalacetate which is turn reduced into succinate via malate-fumarate is advanced as a possible mechanism for the production of succinate.

Studies on the metabolism of Echinococcus granulosus—VIII. The pathway to succinate in E. Granulosus scolices☆

Abstract 1. 1. The mechanism of succinate production has been investigated in E. granulosus scolices. 2. 2. The evidence obtained is in agreement with the formation of pyruvate by glycolysis, followed by CO2-fixation into pyruvate via “malic enzyme” and the reduction of the carboxylation product into succinate. 3. 3. The pentose phosphate pathway contributes, although to a minor extent, to succinate formation.

Studies on the metabolism of Echinococcus granulosus. III. Glycolysis, with special reference to hexokinases and related glycolytic enzymes.

Abstract 1. 1. Phosphorylative glycolysis has been demonstrated in cell-free extracts of hydatid cyst scolices. An appropriate phosphate pool appears to be necessary for optimal in vitro activity. 2. 2. Hydatid cyst scolices contain four hexokinases catalyzing specifically the phosphorylation of glucose, fructose, mannose and glucosamine. 2-deoxyglucose is not phosphorylated by crude or purified preparations. The four hexoses are phosphorylated in position 6. Gluco-, fructo-, and mannokinase activities are inhibited by glucose-6-phosphate, while mannose-6-phosphate inhibits only gluco- and manno-kinases. ADP inhibits competitively fructose phosphorylation by fructokinase. Glucokinase is sensitive to PCMB, the inhibition being partially reversed by cysteine. 3. 3. Cell-free preparations of hydatid cyst scolices contain in addition to various phosphatases, myokinase, phosphoglucomutase, phosphoglucose, and phosphomannose isomerases, phosphofructokinase, aldolase, glycerophosphate dehydrogenase, and lactic dehydrogenase. The extracts could also catalyze mutation of fructose-1-phosphate to fructose-6-phosphate and isomerization of glucosamine-6-phosphate to glucose-6-phosphate.

Studies on the metabolism of Echinococcus granulosus. II. Some observations on the carbohydrate metabolism of hydatid cyst scolices.

Abstract 1. 1. Intracellular glycogen disappears upon incubation under aerobic and anaerobic conditions, 11.97 μmoles and 13.50 μmoles/gram fresh tissue/three hours being consumed respectively. 2. 2. Under aerobic conditions, glycogen is converted to a mixture of lactic, acetic, succinic, and pyruvic acids, and ethyl alcohol. From a quantitative standpoint, lactic acid was the most important endproduct. 3. 3. Under anaerobic conditions, glycogen is fermented to a mixture of lactic, succinic, and acetic acids, as well as ethyl alcohol. No α-ketoacids are produced; lactic and succinic acids are quantitatively the most important endproducts.

Some properties of the microsomal system metabolizing DDT in Triatoma infestans

Abstract 1. 1. Microsomes prepared from Triatoma infestans fifth-instar larvae metabolize DDT to a compound similar to Kelthane (metabolite No. 3), and to a derivative of apparently a phenolic nature (metabolite No. 2). 2. 2. The production of both metabolites requires either NADPH or NADH, but only the formation of metabolite No. 3 is nicotinamide—and magnesium— dependent. 3. 3. Optimum pH for the formation of metabolite No. 3 is 8·5, while it is 9·0 for metabolite No. 2. The K m values for each activity were also slightly different. 4. 4. The activity for each metabolite was increased by pretreatment with phenobarbital, but the induction kinetics was different in each case. 5. 5. These observations suggest that two rather than one microsomal enzyme are involved in DDT metabolism in T. infestans .

The effect of DDT on the incorporation of glucose and glycine into various intermediates in DDT-resistant strains of Musca domestica L.

Abstract 1. 1. Housefly strains showing various patterns of cross-resistance to insecticides have remarkable metabolic differences as to levels of NADP, relative participation of glycolysis and the pentose phosphate pathway in glucose utilization, rates of protein synthesis and glutathione turnover. 2. 2. DDT treatment stimulates the pentose phosphate pathway, increasing the availability of NADPH, and enhances the rates of protein synthesis and glutathione turnover in a pyrethrin-resistant strain, which is also cross-resistant to DDT. On the other hand, a susceptible strain did not evidence any of the above DDT effects. 3. 3. It is suggested that the metabolic changes observed are closely linked to the activity of detoxifying enzymes and constitute an expression of insecticide resistance.

Intermediary carbohydrate metabolism of Triatoma infestans (insecta; hemiptera)—I. Glycolytic and pentose phosphate pathway enzymes and the effect of DDT☆

Abstract 1. (1) Cell-free preparations obtained from thoraces and legs of T. infestans glycolyse glucose, as well as HDP, with the production of pyruvate and α-glycerophosphate in equimolar amounts in the proportion of 1 mole of pyruvate and 1 mole of α-glycerophosphate per mole of glucose utilized. Added HDP is necessary for optimal activity, but the effect of its omission can be partially reversed by increasing the concentration of added ATP. Fluoride is not required for glucose phosphorylation. 2. (2) Cell-free extracts contain enzymes for glycolysis. GAP dehydrogenase is markedly sensitive to iodoacetate and shows appreciable activity in the absence of added l -cysteine. Lactic dehydrogenase activity is negligible, which explains the non-accumulation of lactate as an end-product of glycolysis. 3. (3) The presence of the pentose phosphate pathway in T. infestans is indicated by the presence of several reactions of this pathway in cell-free extracts; 6-PG dehydrogenase and probably G-6-P dehydrogenase appear to be rate-limiting enzymes for carbohydrate degradation through the pentose pathway. 4. (4) The enzyme content of cell-free extracts is related to the developmental stage of the insect and also to the sex. 5. (5) In vivo topically applied DDT (300 μg per specimen) inhibits the glycolytic pyruvate production by cell-free preparations of male and nymph specimens after 22 hr of exposure. Pyruvate production is restored to normal after 48 hr of exposure, but probably some degree of inhibition of α-glycerophosphate production persist in male specimens. This effect is shared by DDE, a non-toxic DDT-analog. 6. (6) Hexokinase, G-6-P dehydrogenase, 6-PG dehydrogenase, aldolase, GAP dehydrogenase, Gly-P dehydrogenase, phosphopyruvic kinase and ribolytic activity are inhibited by DDT in male specimens after 48 hr of exposure to DDT. Phosphopentose isomerase apparently is not inhibited. On the other hand, only G-6-P dehydrogenase and aldolase are inhibited in nymph specimens by DDT.

Anaerobic glycolysis in homogenates of Trichinella spiralis larvae.

Abstract 1. 1. Phosphorylative glycolysis has been demonstrated in T. spiralis larvae; fructose-1,6-diphosphate, diphosphopyridine nucleotide, magnesium, and pyruvate, were required for maximum activities. 2. 2. Lactic acid was an end product of glycolysis, although apparently not a major one. Acetic, valeric, and caproic acids were also found and the possibility is discussed that pyruvate may be their precursor. 3. 3. Hexokinase, ATP-ase, phosphohexose isomerase, phosphohexose kinase, aldolase, glycerophosphate dehydrogenase, triosephosphate dehydrygenase, lactic dehydrogenase, and glucose-6-phosphate dehydrogenase have been demonstrated in T. spiralis homogenates or acetone powder extracts. Alcohol dehydrogenase was not detected. 4. 4. The existence of a sequence similar to the Warburg-Dickens-Lipmann cycle is suggested by the presence of the enzymes involved in the two initial steps, i.e. hexokinase, and glucose-6-phosphate dehydrogenase.

Protein synthesis in cell-free preparations from Musca domestica L.

Abstract 1. 1. Protein synthesis in a cell-free system from Musca domestica L. has been shown to occur by the pathway involving amino acyl-adenylates and amino acyl-soluble ribonucleic acid as intermediates. 2. 2. The system requires an ATP-generating system, a divalent cation and a monovalent cation, guanosine-5′-triphosphate and microsomes or ribosomes and pH 5 enzymes for optimal activity. 3. 3. The requirement of guanosine-5′-triphosphate is not evidence in larval specimens. 4. 4. An insecticide-susceptible strain did not show a dependence on the pH 5 fraction, suggesting that the corresponding enzymes may be tightly bound to ribosomes. 5. 5. Phenylthiourea is required for optimal activity in pupal preparations, but its effect is not significant in other developmental stages.

ENHANCED PROTEIN SYNTHESIS IN TRIATOMA INFESTANS TREATED WITH DDT.

Abstract The effect of DDT and DDE, a DDT-non toxic analog, on protein biosynthesis in Triatoma infestans nymphs and adult specimens has been studied. DDT increases the in vivo rate of incorporation of dl -leucine-1-C14 into total proteins, while a slight inhibitory effect is observed in adult males. DDE does not appreciably change the rate of protein biosynthesis in nymphs, although it appears to be inhibitory in males. Nymphal microsomes and sarcosomes show the highest specific activity after dl -leucine-1-C14 injection. This activity is markedly increased by DDT, while DDE again is inhibitory. A cell-free system obtained from nymphal specimens incorporates dl -leucine-1-C14 into proteins in the presence of ATP, an ATP-generating system, GTP, and magnesium ions. In vivo pretreatment with DDT at low concentrations increases the rate of dl -leucine-1-C14 incorporation into protein by this system. In vitro added DDT also is stimulatory at very low concentrations, while higher ones are inhibitory. The results obtained indicate that protein biosynthesis is stimulated by DDT, but not DDE; an effect that is only evident in nymphal T. infestans. This appears to be consistent with the inductive effect of DDT previously shown with NAD-kinase in this organism (Ilevicky et al., 1964).