Eugene C. Weinbach

Observations on the metabolism of bacteria-free larvae of Trichinella spiralis

Abstract 1. 1. The larvae of Trichinella spiralis consume aerobically and anaerobically about the same amount of glycogen, while lipid consumption occurs only under aerobic conditions. 2. 2. The major endproducts of the carbohydrate fermentation are volatile fatty acids, especially valeric acid, while only traces of lactic acid are produced. Keto substances in small amounts are produced only in the presence of oxygen. Part of the acids is excreted as free acids, while another part may be excreted in the form of ammonium salts. 3. 3. Carbohydrate fermentation is the process which allows the larvae to survive anaerobic processes, but it is insufficient to allow motility. It is probable that a major part of the oxygen consumed is used for the oxidation of lipids and this process may be of importance for the motility of the larvae.

Entamoeba histolytica. I. Aerobic metabolism.

Abstract The respiration of intact trophozoites harvested from axenic cultures of Entamoeba histolytica was studied with the polarographic technique utilizing the Clark oxygen electrode. A typical Qo2 value for the freshly harvested amebae was 1 μatom oxygen/mg protein/hr. It was conclusively demonstrated that this parasite, a putative anaerobe, not only consumes oxygen when provided, but has a high affinity for the gas. Added glucose, galactose, and ethanol increased the respiratory rates, whereas other carbohydrates were without effect on the endogenous respiration. Intermediates of the tricarboxylic acid cycle, amino and fatty acids did not stimulate the respiration of E. histolytica. Inhibitors of the mammalian respiratory chain (cyanide, antimycin) as well as agents that inhibit enzymes catalyzing the tricarboxylic acid cycle (malonate, fluoropyruvate, fluoroacetate, fluorocitrate) had little effect on the endogenous or glucose-supported respiration. Alkylating agents (iodoacetamide, iodoacetate), cinnamate, and N-ethylymaleimide strongly inhibited the oxygen consumption of E. histolytica. The chemotherapeutic agents, Paromomycin, Emetine and Metronidazole, at concentrations that inhibit growth in vitro, did not restrict the respiration. Storage of the trophozoites at 4 C led to progressive deterioraion of the parasites and loss of endogenous and glucose-supported respiration. The deterioration was paralled by loss of SH-materials from the amebae. Likewise, sonication or lysis with detergents abolished both the endogenous respiration and response to glucose. Exogenous NADH or NADPH evoked only marginal increases in oxygen consumption of the freshly harvested amebae, but were effective respiratory substrates with stored or sonicated organisms. Addition of vitamin K3 greatly enhanced the endogenous and glucose-supported respiration of the intact amebae, as well as enhancing the response of stored or sonicated amebae to reduced pyridine nucleotides.

Morphology of mitochondria in the coupled, uncoupled and recoupled states

COII~ELATIVE studies of mitochondrial structure ant1 function often have been predicated on the assumption that major alterations of function are associated with changes in morphology. This assumption is based on the belief that normal mitochondrial morphology is dependent upon a continuing supply of high-energy intermediates generated by oxidativc phosphorylation (for example, see 11, 191). Thus, it might be expectetl that a tlisruption of this bioenergetic process would leatl to market1 disorganization of normal mitochontlrial structure, and that restitution of this function should leacl to a corresponding restitution of structure. \\‘c have shown earlier [%9] that the addition of both albumin ant1 AT1 \vas requiretl to reverse the extensive morphological deterioration of isolated rat liver mitochonclria which occurred when they \vere reacted with pentachlorophenol in a buffered KC1 medium incapable of supporting oxiclative phosphorylation. Our further tlemonstration [27] that bovine serum albumin alone restored oxidative phosphorylation to mitochontlria uncoupled by various substituted phenols provitled a convenient means to test this aspect of the relation between structure and function. In the present communication, the morphology of rat liver mitochondria untlergoing active oxidative phosphorylation is compared with that of mitochondria in which this process is abolished by pentachlorophenol, and \vith that of mitochontiria whose capacity for phosphorylation, ancl hence the generation of high energy intermediates anti endogenous ATP, has been restored by bovine serum albumin. 4 preliminary account of this study has been presented elsewhere [28].

The influence of pentachlorophenol on oxidative and glycolytic phosphorylation in snail tissue

Abstract 1. 1. Oxidative phosphorylation has been demonstrated for the first time in cell-free particulate preparations of the albumen gland of the aquatic snail, Lymnaea stagnalis . 2. 2. Pentachlorophenol (PCP) in low concentrations (2.5 × 10 −5 M ) completely prevented the uptake of inorganic phosphate associated with the oxidation of β-hydroxybutyrate in these preparations. 3. 3. Glycolytic phosphorylation, in the whole homogenate or the soluble fractions, was not interrupted by PCP in concentrations as high as 5 × 10 −4 M . However, 5 × 10 −3 M PCP completely inhibited glycolysis and the associated anaerobic phosphorylations. 4. 4. PCP greatly enhanced the activity of molluscan ATPase (latency effect). 5. 5. Oxidative phosphorylation could not be demonstrated with the hepatopancreas. Possible reasons for this inability are discussed. 6. 6. It is postulated that uncoupling of oxidative phosphorylatiou provides a biochemical mechanism of PCPs molluscicidal action.

Formation, isolation and composition of dense granules from mitochondria

Abstract 1. 1. An investigation was undertaken to determine if the electron-dense granules which accumulate in mitochondria under conditions of massive Ca 2+ -loading could be isolated and identified. 2. 2. Four methods are described for the isolation of dense granules from rat liver mitochondria. Three of these methods involve chemical destruction of the mitochondria. and liberation of the granules; the fourth involves disruption by sonication. 3. 3. The granules are amorphous, and consist of both organic and inorganic constituents. 4. 4. Incineration of the granules destroyed the organic moiety and induced crystallization of the inorganic constituents. 5. 5. Chemical analysis disclosed that Ca 2+ and P i are the major inorganic constituents. Mg 2+ and CO 2− 3 also are present in significant amounts. 6. 6. X-ray diffraction analysis indicated that the patterns of the incinerated granules correspond to either hydroxyapatite, Ca 10 (PO 4 ) 6 (OH) 2 , whitlockite, Ca 3 (PO 4 ) 2 , or to a mixture of both. 7. 7. The isolation of the granules enabled a study of some of the factors which influence this active ion translocation in a more direct way than was hitherto possible.

The effect of pentachlorophenol on the metabolism, of the snail Australorbis glabratus

Abstract 1. 1. Aerobic exposure of living snails to low concentrations (7.5 × 10 −6 M , 2 ppm) of pentachlorophenol (PCP) resulted in the accumulation of acetate, lactate, pyruvate, and inorganic phosphate in their tissues. 2. 2. PCP, in very low concentrations (2 × 10 −6 M ) stimulated the respiration of living snails while higher concentrations (2 × 10 −6 M ) were inhibitory. Similar results were observed with minced snail tissues. 3. 3. These findings are discussed in relation to the hypothesis that the molluscicidal property of PCP is due, at least partially, to its ability to uncouple oxidative phosphorylation.

Biochemistry of enteric parasitic protozoa

Abstract Although the enteric parasitic protozoa are of world-wide public health concern little is known of their physiology and biochemistry. These protozoa were previously considered to be anaerobic, but recent studies show that they have an active respiration. The parasites lack mitochondria and heme proteins; terminal electron transfer to molecular oxygen is accomplished by flavins and non-heme iron-sulfur proteins.

Partial purification and characterization of the isocitric dehydrogenase from trypanosoma cruzi

Abstract A TPN-linked iso citric dehydrogenase was demonstrated in homogenates, cellular fractions, and extracts of acetone powder from Trypanosoma cruzi . The enzyme was purified 45- to 52.5-fold from the combined mitochondrial and soluble fractions of T. cruzi . In the reaction between TPN and iso citrate, stoichiometric relationship was found between TPNH and KG formed. TPNH could be reoxidized upon addition of oxalosuccinate with the use of extracts from acetone powders as source of enzyme, which do not require added Mn ++ for activity. The purified and crude preparations are absolutely dependen on Mn ++ for activity. K m was approximately 5 M , and optimum pH, 7.4.

Purification and biochemical properties of calmodulin in Entamoeba histolica and its distribution during secretion of electron-dense granules

1. Calmodulin (CaM) was detected during secretion of electron-dense granules by Entamoeba histolytica trophozoites with immunofluorescence. 2. It was purified to apparent homogeneity by chromatography with a yield of 2.26 micrograms of calmodulin/mg of protozoan protein. Purity was established by gel electrophoresis. 3. The parasite calmodulin has properties characteristic of calmodulin isolated from other eukaryotes: an apparent molecular weight of 19 or 17 kDa in presence of EGTA or CaCl2, respectively, activation in a calcium dependent manner of bovine heart cyclic nucleotide phosphodiesterase, and its UV spectrum.

Giardia lamblia: detection and characterization of calmodulin

Calmodulin was detected in Giardia lamblia by radioimmunoassay and cyclic AMP phosphodiesterase activation. This protein was purified to apparent homogeneity by fast protein liquid chromatography with a yield of 260 ng of calmodulin/mg of protozoan protein. Purity was established by gel electrophoresis, gel filtration, and ion exchange chromatography. The parasite calmodulin has properties characteristic of calmodulin isolated from other eukaryotes, e.g., an apparent molecular weight of 16.7 kD; activation in calcium dependent manner of bovine heart cyclic nucleotide phosphodiesterase; and sensitivity to known calmodulin antagonists.