Subcellular Distribution of Aspartate Transaminase,Alanine Amino Transferase,Glutamate Dehydrogenases,and Lactate Dehydrogenase in Tetrahymena*

SYNOPSIS. Mitochondria and peroxisomes were isolated from homogenates of Tetrahymena pyriformis by sedimentation through a sucrose gradient. Succinate dehydrogenase was used as a mitochondrial marker; catalase and isocitrate lyase were used to mark the peroxisomal fraction. Lactate dehydrogenase, glutamate dehydrogenase, and alanine aminotransferase were found only in the mitochondrial fraction. Aspartate transaminase was found in both mitochondrial and peroxisomal fractions.     

Subcellular localization of long-chain alcohol dehydrogenase and aldehyde dehydrogenase in n-alkane-grown Candida tropicalis

Long-chain alcohol dehydrogenase and longchain aldehyde dehydrogenase were induced in the cells of Candida tropicalis grown on n-alkanes. Subcellular localization of these dehydrogenases, together with that of acyl-CoA synthetase and glycerol-3-phosphate acyltransferase, was studied in terms of the metabolism of fatty acids derived from n-alkane substrates. Both longchain alcohol and aldehyde dehydrogenases distributed in the fractions of microsomes, mitochondria and peroxisomes obtained from the alkane-grown cells of C. tropicalis. Acyl-CoA synthetase was also located in these three fractions. Glycerol-3-phosphate acyltransferase was found in microsomes and mitochondria, in contrast to fatty acid -oxidation system localized exclusively in peroxisomes. Similar results of the enzyme localization were also obtained with C. lipolytica grown on n-alkanes. These results suggest strongly that microsomal and mitochondrial dehydrogenases provide long-chain fatty acids to be utilized for lipid synthesis, whereas those in peroxisomes supply fatty acids to be degraded via -oxidation to yield energy and cell constituents.     

Tricarboxylic Acid Cycle Enzymes in Tetrahymena pyriformis after Infection of the Cockroach Periplaneta americana1

SYNOPSIS. Activities of enzymes of the tricarboxylic acid cycle in extracts of Tetrahymena pyriformis S, axenically recovered after living in the hemocoel of female cockroaches Periplaneta americana for 48 hr, were compared with activities in ciliates not exposed to the cockroach. Malic dehydrogenase activity was depressed after recovery from the cockroach; isocitric and succinic dehydrogenases and α-ketoglutaric oxidase activities were unchanged. Citrate synthetase activity was increased, and pyruvi oxidase activity decreased, after ciliates had been in the cockroach. These alterations persisted for several hundred generations after recovery from the insect.     

Growth of Bacillus fastidiosus on glycerol and the enzymes of ammonia assimilation

Bacillus fastidiosus was able to grow on glycerol as a carbon source when allantoin or urate was used as nitrogen source. The primary assimilatory enzyme for glycerol was glycerol kinase; glycerol dehydrogenase could not be detected. The glycerol kinase activity was increased 30-fold in allantoin/glycerol-grown cells as compared to alantoin-grown cells. Under both growth conditions high levels of glutamate dehydrogenase were found. Glutamine synthetase and glutamate synthase activities could not be demonstrated, while low levels of alanine dehydrogenase were present. It is concluded that B. fastidiosus assimilates ammonia by the NADP-dependent glutamate dehydrogenase.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase     

Lysosomes in Tetrahymena pyriformis. I. Some properties and lysosomal localization of acid hydrolases

In homogenates of Tetrahymena pyriformis, five hydrolases — phosphatase, ribonuclease, deoxyribonuclease, proteinase, amylase — with acid pH optima were found. Over 75% of their activity is sedimentable with a centrifugal force of 250,000 g. min. Only 17% of the acid phosphatase and ribonuclease is active when assayed in the presence of 0.25 M sucrose at 0°. Exposure to a lowered osmotic pressure, freezing and thawing, and incubation at temperatures over 0° result in activation of the latent phosphatase and ribonuclease. After isopycnic centrifugation in a sucrose density gradient the hydrolases show a broad distribution which differs greatly from those of enzymes associated with mitochondria (succinate dehydrogenase) or with peroxisomes (catalase). The results are interpreted as evidence that the five acid hydrolases studied are localized in lysosomes which represent a distinct population of subcellular particles in Tetrahymena.     

Thymidylate Synthetase as a Chemotherapeutic Target in the Treatment of Avian Coccidiosis*

SYNOPSIS. Thymidylate synthetase (E.C.2.1.1.45) has been demonstrated in unsporulated oocysts of Eimeria tenella. The properties of this enzyme have also been investigated in Tetrahymena pyriformis, as a protozoan model, and 7-day-old chick embryo, as a host model. The enzymes from E. tenella and chick embryo were inhibited by all concentrations of MnCl₂ and MgCl₂ tested. Tetrahymena pyriformis thymidylate synthetase was stimulated by low concentrations of both these cations but was inhibited by high concentrations. Subsequent data refer to chick embryo, E. tenella and T. pyriformis respectively: the apparent K_m was 5.89 μM, 5.94 μM, and 0.53 M for the substrate dUMP: and 5.13 μM, 1.10 μM and 4.65 μM, respectively for the cofactor N⁵N¹⁰-methylenetetrahydrofolate. The pH optimum for the enzyme from both chick embryo and T. pyriformis was 8.0, with Tris-HCl buffer; activity of E. tenella thymidylate synthetase was still increasing at pH 8.2. The E. tenella enzyme was found to have a molecular weight of 4.6–4.9 × 10⁵ daltons. The effects of nucleotides, inhibitors, and the omission of assay components on each enzyme are presented. Thymidylate synthetase from E. tenella is not greatly different from that of chick embryo, but does not resemble the enzyme from T. pyriformis. A case for using thymidylate synthetase as a chemotherapeutic target in the treatment of Eimeria infections remains. Indeed Eimeria may be considered as a model for infections caused by other protozoan parasites, such as Toxoplasma and Plasmodium, provided that suitable inhibitors can be found that are not toxic to the host.     

Peroxisomes in the Alga Vaucheria are Neither of the Leaf Peroxisomal Nor of the Glyoxysomal Type*,1

Microbodies were isolated from the freshwater alga Vaucheria sessilis as well as from a marine Vaucheria. The organelles equilibrated on sucrose gradients at densities 1.23 g . cm^?3 and 1.24g . cm^?3, respectively. On electron micrographs they showed an ovoid or spheroid shape with a diameter of 0.5 to 0.8 μm. Besides catalase, the peroxisomes of both algae possess glycolate oxidase and glutamate-glyoxylate aminotransferase, but no other leaf-peroxisomal enzymes. Instead, the enzymes malate synthase and isocitrate lyase, which are markers of glyoxysomes in higher plants, are constituents of the peroxisomes in the marine as well as in the freshwater alga. Citrate synthase, aconitase, malate dehydrogenase and enzymes of the fatty acid β-oxidation pathway are located exclusively in the mitochondria. Therefore, the peroxisomes from Vaucheria do not belong to either the type of leaf peroxisomes or to the type of glyoxysomes.     

Enzymes of beta-Oxidation in Different Types of Algal Microbodies

The algae Mougeotia and Eremosphaera were used for isolation of microbodies with the characteristics of leaf peroxisomes and unspecialized peroxisomes, respectively. In both types of organelles, the following enzymes of the β-oxidation pathway were determined: acyl-CoA oxido-reductase, enoyl-CoA hydratase, and 3-hydroxyacyl-CoA dehydrogenase. There are indications that the peroxisomal oxidoreductase of both algae is a H₂O₂-forming oxidase rather than a dehydrogenase.

The enzymes enoyl-CoA hydratase and acyl-CoA oxidoreductase are located also in the mitochondria from Eremosphaera but not from Mougeotia. The mitochondrial acyl-CoA oxidizing enzyme was found to be a dehydrogenase. The specific activities of acyl-CoA oxidase and enoyl-CoA hydratase are lower than in spinach leaf peroxisomes. However, the activity of 3-hydroxyacyl-CoA dehydrogenase in the peroxisomes of both algae is almost 2-fold higher. The capability for degradation of fatty acids is a common feature of all different types of peroxisomes from algae.

     

Enzymes of ammonia assimilation and their regulation inAzospirillum lipoferum strain D-2

Azospirillum lipoferum strain D-2 possesses the following enzymes for the assimilation of N₂ and NH ₄ ⁺ : nitrogenase, glutamine synthetase, NADPH-dependent glutamate synthase, NADH-/NADPH-dependent glutamate dehydrogenase, and NADH-dependent alanine dehydrogenase. Nitrogenase and glutamine synthetase are repressed, whereas glutamate dehydrogenase and alanine dehydrogenase are induced by NH ₄ ⁺ . Glutamine synthetase activity is modulated by both repression and depression and also by adenylylation.     

Isocitrate dehydrogenase of Tetrahymena pyriformis

Summary We have studied the isocitrate dehydrogenase ofTetrahymena pyriformis. This enzyme is able to utilize both NAD and NADP, but kinetic studies suggest that the enzymatic activity with NAD is not of physiological significance.Some of the factors that might regulate the NADP-dependent isocitrate dehydrogenase were also studied. This enzyme has an absolute requirement for divalent cations; Mg²⁺ and Mn²⁺ will serve as cofactors but the latter is more effective than the former.It is known that this enzyme is subject to a concerted inhibition by oxaloacetate and glyoxylate. Either glyoxylate or oxaloacetate alone also are capable of inhibiting the enzyme although higher concentrations are required. We have found concerted inhibition also for the NAD-dependent isocitrate dehydrogenase from rat liver and yeast. The activity of theTetrahymena pyriformis enzyme is inhibited by NADPH. This inhibition is competitive with NADP. The Ki and Km values are, respectively, 23µ m and 18µ m.     

Nitrogen assimilation in facultative methylotrophic bacteria

A study was done of the pathways of nitrogen assimilation in the facultative methylotrophsPseudomonas MA andPseudomonas AM1, with ammonia or methylamine as nitrogen sources and with methylamine or succinate as carbon sources. When methylamine was the sole carbon and/or nitrogen source, both organisms possessed enzymes of the glutamine synthetase/glutamate synthase pathway, but when ammonia was the nitrogen sourcePseudomonas AM1 also synthesized glutamate dehydrogenase with a pH optimum of 9.0, andPseudomonas MA elaborated both glutamate dehydrogenase (pH optimum 7.5) and alanine dehydrogenase (pH optimum 9.0). Glutamate dehydrogenase and glutamate synthase from both organisms were solely NADPH-dependent; alanine dehydrogenase was NADH-dependent. No evidence was obtained for regulation of glutamine synthetase by adenylylation in either organism, nor did glutamine synthetase appear to regulate glutamate dehydrogenase synthesis.     

Induction and subcellular localization of enzymes participating in propionate metabolism in Candida tropicalis

Candida tropicalis, a representative alkane- and higher fatty acid-utilizing yeast, can grow on propionate used as sole carbon and energy source. Initial pH of the medium markedly affected the growth of the yeast on propionate. In propionate-grown cells, several enzymes associated with peroxisomes and/or participating in propionate metabolism were induced in connection with the appearance of the characteristic peroxisomes. Acetate-grown cells of this yeast had only few peroxisomes, while alkane-grown cells contained conspicuous numbers of the organelles. As compared with alkane-grown cells, some specific features were observed in peroxisomes and enzymes associated with the organelles of propionate-grown cells: The shape of peroxisomes was large but the number was small; unlike localization of catalase in peroxisomes of alkane-grown cells, the enzyme of propionate-grown cells was mainly localized in cytoplasm; as for carnitine acetyltransferase localized almost equally in peroxisomes and mitochondria in alkane-grown cells, propionate-grown cells contained mainly the mitochondrial type enzyme. A propionate-activating enzyme, which was different from acetyl-CoA synthetase, was also induced in cytoplasm of propionate-grown cells. The role of carnitine acetyltransferase and the propionate-activating enzyme in propionate metabolism is discussed in comparison with the role of carnitine acetyltransferase and acetyl-CoA synthetase in acetate metabolism.     

Beta-Oxidation in Glyoxysomes from Euglena*

SYNOPSIS. We demonstrated previously the presence of glyoxysomes containing the glyoxylate cycle enzymes in Euglena gracilis grown in the dark on ethanol. We have now established that the glyoxysomes of Euglena grown on hexanoate also contain the following enzymes of the pathway for β-oxidation of fatty acids: hexanoyl-CoA synthetase, 3-β-hydroxyacyl-CoA dehydrogenase and thiolase. Estimations of specific activities indicate that these enzymes are over 20 times as active in glyoxysomes as they are in mitochondria, suggesting that the β-oxidation of fatty acids occurs almost entirely in Euglena glyoxysomes under these conditions. Thus, the entire portion of the gluconeogenic pathway from fatty acid to succinate is localized in the glyoxysome of Euglena.     

N2 fixation and NH 4 + assimilation in the thermophilic anaerobes Clostridium thermosaccharolyticum and Clostridium thermoautotrophicum

Inorganic nitrogen metabolism in the obligate anaerobic thermophiles Chlostridium thermosaccharolyticum and Clostridium thermoautotrophicum differs in several respects. C. thermosaccharolyticum contains a nitrogenase as inferred from NH ₄ ⁺ repressible C₂H₂ reduction, a glutamine synthetase which is partially repressed by ammonium, very labile glutamate synthase activities with both NADH and NADPH, NADPH-dependent glutamate dehydrogenase, and NH ₄ ⁺ -dependent asparagine synthetase. C. thermoautotrophicum contains no nitrogenase, but glutamine synthetase, no glutamate synthase, no glutamate dehydrogenase, but a NADH-dependent alanine dehydrogenase and a NH ₄ ⁺ -dependent asparagine synthetase.Abbreviation GOGAT glutamine-oxoglutarate amidotransferase amidotransferase (glutamate synthase)     

Ligninolytic activity and levels of ammonia assimilating enzymes in Sporotrichum pulverulentum

Levels of ammonia-assimilating enzymes (glutamate dehydrogenase, glutamine synthetase, glutamate synthase) were determined in extracts of Sporotrichum pulverulentum grown under different conditions with respect to both nitrogen source and concentration. Evolution of ¹⁴CO₂ from ¹⁴C-synthetic lignin by fungal cultures grown under parallel conditions was also determined as a measure of lignin decomposition and the suppressive effect of nitrogen on ligninolysis confirmed. Under low nitrogen conditions, fungal extracts exhibited relatively high levels of NADP-dependent glutamate dehydrogenase and glutamine synthetase dehydrogenase. Conversely, in high nitrogen extracts, lower levels of NADP-dependent glutamate dehydrogenase and glutamine synthetase activity, and higher levels of NAD-dependent glutamate dehydrogenase, were recorded. Possible effects of enzyme activities on intracellular pool concentrations of glutamate/glutamine, and the implications for the regulation of lignin metabolism, are discussed.A preliminary report was presented at The Ekman Days 1981, International Symposium on Wood and Pulping Chemistry, Stockholm, Sweden, June 9–12, 1981.     

Nitrogen metabolism inRhodopseudomonas globiformis

Rhodopseudomonas globiformis strain 7950 grew with a variety of amino acids, urea, or N₂ as sole nitrogen sources. Cultures grown on N₂ reduced acetylene to ethylene; this activity was absent from cells grown on nonlimiting NH ₄ ⁺ . Glutamate dehydrogenase could not be detected in extracts of cells of strain 7950, although low levels of an alanine dehydrogenase were present. Growth ofR. globiformis on NH ₄ ⁺ was severely inhibited by the glutamate analogue and glutamine synthetase inhibitor, methionine sulfoximine. High levels of glutamine synthetase (as measured in the -glutamyl transferase assay) were observed in cell extracts of strain 7950 regardless of the nitrogen source, although N₂ and amino acid grown cells contained somewhat higher glutamine synthetase contents than cells grown on excess NH ₄ ⁺ . Levels of glutamate synthase inR. globiformis were consistent with that reported from other phototrophic bacteria. Both glutamate synthase and alanine dehydrogenase were linked to NADH as coenzyme. We conclude thatR. globiformis is capable of fixing N₂, and assimilates NH ₄ ⁺ primarily via the glutamine synthetase/glutamate synthase pathway.Abbreviations GS glutamine synthetase - GOGAT Glutamineoxoglutarate aminotransferase - GDH Glutamate dehydrogenase - ADH Alanine dehydrogenase - MSO Methionine sulfoximine     

Methanol metabolism in yeasts: Regulation of the synthesis of catabolic enzymes

The regulation of the synthesis of four dissimilatory enzymes involved in methanol metabolism, namely alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase and catalase was investigated in the yeasts Hansenula polymorpha and Kloeckera sp. 2201. Enzyme profiles in cell-free extracts of the two organisms grown under glucose limitation at various dilution rates, suggested that the synthesis of these enzymes is controlled by derepression — represion rather than by induction — repression. Except for alcohol oxidase, the extent to which catabolite repression of the catabolic enzymes was relieved at low dilution rates was similar in both organisms. In Hansenula polymorpha the level of alcohol oxidase in the cells gradually increased with decreasing dilution rate, whilst in Kloeckera sp. 2201 derepression of alcohol oxidase synthesis was only observed at dilution rates below 0.10 h^–1 and occurred to a much smaller extent than in Hansenula polymorpha.Derepression of alcohol oxidase and catalase in cells of Hansenula polymorpha was accompanied by synthesis of peroxisomes. Moreover, peroxisomes were degraded with a concurrent loss of alcohol oxidase and catalase activities when excess glucose was introduced into the culture. This process of catabolite inactivation of peroxisomal enzymes did not affect cytoplasmic formaldehyde dehydrogenase.     

Localization of glycolate dehydrogenase in two species of Dunaliella

The occurrence of glycolate oxidase in addition to glycolate dehydrogenase in Dunaliella salina and D. primolecta, as reported in the literature, could not be confirmed. Both species were demonstrated to possess only glycolate dehydrogenase. After separation of organelles by gradient centrifugation, glycolate dehydrogenase along with hydroxypyruvate reductase was found exclusively in the mitochondria. Thus the peroxisomes from Dunaliella are not of the leaf-type: because of their content of catalase, uricase and hydroxyacyl-CoA dehydrogenase they appear to be of the same type as in Eremosphaera and other chlorophycean algae. No activity of glycolate dehydrogenase was found in the chloroplast fraction when the 2,6-dichlorophenol-indophenol test was used.This work was supported by the Deutsche Forschungsgemeinschaft.     

Isolation and characterization of peroxisomes from diatoms

Peroxisomes were isolated by gradient centrifugation from two different diatoms: Nitzschia laevis (subgroup of Pennales) and Thalassiosira fluviatilis (subgroup of Centrales). In neither of these organelles could catalase or any H₂O₂-forming oxidase be demonstrated. The glycolate-oxidizing enzyme present in the peroxisomes is a dehydrogenase capable of oxidizing l-lactate as well. The peroxisomes also contain the glyoxysomal markers isocitrate lyase and malate synthase. However, enzymes of the fatty-acid -oxidation pathway are located exclusively in the mitochondria. The mitochondria additionally possess glutamate-glyoxylate aminotransferase and a glycolate dehydrogenase which differs from the peroxisomal glycolate dehydrogenase since it preferably utilizes d-lactate as an alternative substrate. Hydroxypyruvate reductase and glyoxylate carboligase were not found in the cells of either diatom. By culturing Nitzschia laevis it could be demonstrated that decreasing the CO₂ concentration in the aeration mixture from 2% to 0.03% and increasing the irradiance from 40 to 250 mol quanta · m^–2 · s^–1 resulted in an increase of all peroxisomal enzyme activities. In addition, enzyme activities of the -oxidation pathway were increased. However, mitochondrial glycolate dehydrogenase and aminotransferase did not alter their activities under these conditions. Summarizing all results, it is postulated that there are two different pathways for the metabolism of glycolate in the diatoms.This work was supported by the Deutsche Forschungsgemeinschaft.