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PubMed1985

End products and enzyme levels of aerobic glucose fermentation in trypanosomatids.

Trypanosoma cruzi (epimastigotes), Crithidia fasciculata and Leishmania mexicana (promastigotes) were grown in a brain-heart-tryptose medium supplemented with heat-inactivated fetal calf serum. T. cruzi and C. fasciculata utilized glucose completely during the log phase of growth, whereas L. mexicana used significant amounts of the carbohydrate only at the end of the log phase and at the beginning of the stationary phase. In all cases glucose consumption resulted in excretion of succinate, and much smaller amounts of acetate. C. fasciculata and L. mexicana produced very small amounts of pyruvate. C. fasciculata produced ethanol, which was taken up again and metabolysed after glucose was exhausted. Lactate and malate were not produced. The cells were disrupted by sonic disintegration, and the activities of some key enzymes of carbohydrate and amino acid catabolism were assayed in the whole homogenates. Phosphoenolpyruvate carboxykinase was present in the three organisms; L. mexicana presented the highest specific activity. The activity of this enzyme was maximal during glucose consumption, and slightly decreased after glucose was exhausted. This suggests that the role played by the enzyme is glycolytic and not gluconeogenic; the latter is the case in most higher organisms. Hexokinase and pyruvate kinase presented their highest levels in C. fasciculata and T. cruzi during glucose consumption. L. mexicana, which was in active glycolysis during the whole experimental period, presented the highest specific activities of both enzymes. Citrate synthase, on the other hand, increased in C. fasciculata and, to a lesser extent, in T. cruzi, after glucose was exhausted; the enzyme could not be detected in L. mexicana. The NAD-linked glutamate dehydrogenase increased considerably in C. fasciculata and T. cruzi after glucose was exhausted, suggesting a catabolic role for the enzyme. This increase coincided with an increase in NH3 production by both organisms after glucose consumption. The NADP-linked glutamate dehydrogenase, on the other hand, presented a maximum about the time when glucose was exhausted, and then decreased again, which suggests a catabolic role for the enzyme. Both glutamate dehydrogenases had low activities in L. mexicana; this fits in well with the low NH3 production throughout the culture of this organism. The results are in good agreement with current ideas on the mechanism of aerobic glucose fermentation by trypanosomatids, and suggest that, under the experimental conditions used, both T. cruzi and C. fasciculata used glucose perferentially over amino acids for growth.

Animals↗
PubMed2003

Apicoplast fatty acid biosynthesis as a target for medical intervention in apicomplexan parasites.

New chemotherapies for human and animal apicomplexan infections are needed as a component of future strategies to deal with these diseases. An extensive search for new treatments exploring the unique developmental physiology, metabolism and molecular structures of Apicomplexa is under way. The description of the full complement of about 5,300 Plasmodium falciparum genes and fast growing sequence databases for other Apicomplexa allow reconstruction of metabolic pathways of these parasites and thus accelerate identification and biochemical analysis of potential targets. The apicoplast de novo fatty acid biosynthetic pathway shows great potential as a target for small-molecule inhibitors in a stand-alone or combination chemotherapy. Three enzymatic activities, acetyl-CoA carboxylase, beta-ketoacyl-ACP synthase and enoyl-ACP reductase, respond to inhibitors previously identified for bacteria and plants, and deserve to be explored in depth. In this connection, screening systems have been established to seek more potent and specific antiparasitic compounds that are harmless to the host. To this end the interconnections of fatty acid biosynthesis in Apicomplexa with other metabolic and cellular processes must be investigated.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed1987

Purification and characterization of dihydrodipicolinate synthase from wheat suspension cultures.

Dihydrodipicolinate synthase, the first enzyme unique to lysine biosynthesis in higher plants, was purified about 5100-fold from suspension-cultured cells of wheat (Triticum aestivum var Chinese Spring). The synthase has an average molecular weight of 123,000 as determined by gel filtration and exhibited maximum activity at pH 8.0. The kinetics of the condensation reaction are compatible with a "Ping Pong" mechanism in which pyruvate reacts first with the enzyme to form a Schiff base. Pyruvate and l-aspartic-beta-semialdehyde (ASA) have respective K(m) values of 11.76 and 0.80 millimolar. Allosteric inhibition was observed with increasing concentrations of l-lysine and its structural analogs, including threo-4-hydroxy-l-lysine and S-(2-aminoethyl)-l-cysteine, with respective I(0.5) values of 51, 141, and 288 micromolar. These amino acids were competitive inhibitors with respect to ASA and noncompetitive inhibitors with respect to pyruvate. We propose that the binding site for lysine overlaps with the ASA binding site, possibly by an attachment of the common alanyl moiety. The wheat enzyme was inhibited by Zn(2+), Cd(2+), and Hg(2+) and also by sulfhydryl inhibitors, p-(hydroxymercuri)benzoic acid and p-chloromercuribenzenesulfonic acid.

Journal Article↗
PubMed1995

Effect of BDM, verapamil, and cardiac work on mitochondrial membrane potential in perfused rat hearts.

The biochemical link providing effective coordination between the mitochondrial ATP synthetic machinery and the contractile apparatus following transitions in cardiac work remains enigmatic. Studies were designed to determine whether activation of the actomyosin adenosinetriphosphatase (ATPase) is a necessary part of the signaling mechanism to the mitochondrial ATP synthase or whether a rise in cytosolic free Ca2+ is sufficient to activate the synthase. With the use of Langendorff-perfused rat hearts, cardiac work was varied via changes in perfusion pressure and by the inclusion of a beta-adrenergic agent. Furthermore, 2,3-butanedione monoxime and verapamil were used to vary independently either the activity of the actomyosin ATPase or the level of cytosolic free Ca2+. Determinations of the in vivo mitochondrial membrane potential [delta psi m; see Wan et al. Am. J. Physiol. 265 (Heart Circ. Physiol. 34): H445-H452, 1993] and its vectorial displacement during work transitions provide valuable information concerning direct activation of the ATP synthase and proton movement through the membrane domain of the synthase. Increased cardiac work in the presence of the beta-adrenergic agent resulted in a decrease in delta psi m. Addition of 2,3-butanedione monoxime decreased cardiac work but did not change delta psi m. The inclusion of verapamil resulted in similar decreases in cardiac work. However, delta psi m reversed back to a value observed under control, low-work conditions. These results in conjunction with data regarding levels of high-energy phosphates, free Mg2+, and adenosine 3',5'-cyclic monophosphate suggest a Ca(2+)-mediated increase in the activity of the ATP synthase.

Animals↗
PubMed2000

Insulin increases NO-stimulated guanylate cyclase activity in cultured VSMC while raising redox potential.

Insulin acutely stimulates cyclic guanosine monophosphate (cGMP) production in primary confluent cultured vascular smooth muscle cells (VSMC) from canine femoral artery, but the mechanism is not known. These cells contain the inducible isoform of nitric oxide (NO) synthase (iNOS), and insulin-stimulated cGMP production in confluent cultured cells is blocked by the NOS inhibitor, N(G)-monomethyl-L-arginine (L-NMMA). In the present study, it is shown that iNOS is also present in freshly dispersed VSMC from this artery, indicating that iNOS expression in cultured VSMC is not an artifact of the culture process. Insulin did not stimulate NOS activity in primary confluent cultured cells because it did not affect citrulline or combined NO(-)(3)/NO(-)(2) production. To see whether insulin required the permissive presence of NO to stimulate cGMP production, iNOS and basal cGMP production were inhibited with L-NMMA, and the cells were incubated with or without 1 nM insulin and/or the NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at a concentration (0.1 microM) that restored cGMP production to the basal value. In the presence of L-NMMA, insulin no longer affected cGMP production but when insulin was added to L-NMMA plus SNAP, cGMP production was increased by 69% (P < 0.05 vs. L-NMMA plus SNAP). Insulin, which increases glucose uptake by these cells, increased the cell lactate content and the lactate-to-pyruvate ratio (LPR) by 81 and 97%, respectively (both P < 0.05), indicating that the hormone increased aerobic glycolysis and the redox potential. The effects of insulin on LPR and cGMP production were blocked by removing glucose or by adding 2-deoxyglucose to the incubation media and were duplicated by the reducing substrate, beta-hydroxybutyrate. We conclude that insulin does not acutely affect iNOS activity in these VSMC but it does augment cGMP production induced by the NO already present in the cell while increasing aerobic glycolysis and the cell redox potential.

Aerobiosis↗
PubMed2003

Changes in mitochondrial electron partitioning in response to herbicides inhibiting branched-chain amino acid biosynthesis in soybean.

The adaptation of the respiratory metabolism in roots of soybean (Glycine max L. Merr. cv Ransom) treated with herbicides that inhibit the enzyme acetolactate synthase (ALS) was analyzed. A new gas phase dual-inlet mass spectrometry system for simultaneous measurement of 34O2 to 32O2 and O2 to N2 ratios has been developed. This system is more accurate than previously described systems, allows measurements of much smaller oxygen gradients, and, as a consequence, works with tissues that have lower respiration rates. ALS inhibition caused an increase of the alternative oxidase (AOX) protein and an accumulation of pyruvate. The combination of these two effects is likely to induce the activation of the alternative pathway and its participation in the total respiration. Moreover, the start of the alternative pathway activation and the increase of AOX protein were before the decline in the activity of cytochrome pathway. The possible role of AOX under ALS inhibition is discussed.

Acetolactate Synthase↗
PubMed2006

Overexpressing GLT1 in gpd1Delta mutant to improve the production of ethanol of Saccharomyces cerevisiae.

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant, KAM-4, the GPD1 gene, which encodes a glycerol 3-phosphate dehydrogenase of S. cerevisiae to synthesize glycerol, was deleted. The mutant KAM-12 had the GLT1 gene (encodes glutamate synthase) placed under the PGK1 promoter while harboring the GPD1 deletion. Notably, overexpression of GLT1 by the PGK1 promoter along with GPD1 deletion resulted in a 10.8% higher ethanol production and a 25.0% lower glycerol formation compared to the wild type in anaerobic fermentations. The growth rate of KAM-4 was slightly lower than that of the wild type under the exponential phase whereas KAM-12 and the wild type were indistinguishable in the biomass concentration at the end of growth period. Meanwhile, dramatic reduction of formation of acetate and pyruvic acid was observed in all the mutants compared to the wild type.

Biomass↗
PubMed2001

Functional properties of the active core of human cystathionine beta-synthase crystals.

Human cystathionine beta-synthase is a pyridoxal 5'-phosphate enzyme containing a heme binding domain and an S-adenosyl-l-methionine regulatory site. We have investigated by single crystal microspectrophotometry the functional properties of a mutant lacking the S-adenosylmethionine binding domain. Polarized absorption spectra indicate that oxidized and reduced hemes are reversibly formed. Exposure of the reduced form of enzyme crystals to carbon monoxide led to the complete release of the heme moiety. This process, which takes place reversibly and without apparent crystal damage, facilitates the preparation of a heme-free human enzyme. The heme-free enzyme crystals exhibited polarized absorption spectra typical of a pyridoxal 5'-phosphate-dependent protein. The exposure of these crystals to increasing concentrations of the natural substrate l-serine readily led to the formation of the key catalytic intermediate alpha-aminoacrylate. The dissociation constant of l-serine was found to be 6 mm, close to that determined in solution. The amount of the alpha-aminoacrylate Schiff base formed in the presence of l-serine was pH independent between 6 and 9. However, the rate of the disappearance of the alpha-aminoacrylate, likely forming pyruvate and ammonia, was found to increase at pH values higher than 8. Finally, in the presence of homocysteine the alpha-aminoacrylate-enzyme absorption band readily disappears with the concomitant formation of the absorption band of the internal aldimine, indicating that cystathionine beta-synthase crystals catalyze both beta-elimination and beta-replacement reactions. Taken together, these findings demonstrate that the heme moiety is not directly involved in the condensation reaction catalyzed by cystathionine beta-synthase.

Binding Sites↗
PubMed1996

Hepatic gluconeogenesis and the activity of PDH in individual tissues of GTG-obese mice following adrenalectomy.

Adrenalectomy (ADX) lowers circulating glucose levels in animal models of non-insulin dependent diabetes (NIDDM) and obesity. To investigate the role of hepatic glucose production (HGP) and tissue glucose oxidation in the improvement in glucose tolerance, hepatocyte gluconeogenesis and the activity of pyruvate dehydrogenase (PDH) were examined in different tissues of gold thioglucose (GTG) obese mice 2 weeks after ADX or sham ADX. GTG-obese mice which had undergone ADX weighed significantly less than their adrenal intact counterparts (GTG ADX: 37.5 +/- 0.7 g; GTG: 44.1 +/- 0.4; p < 0.05), and demonstrated lower serum glucose (GTG ADX: 22.5 +/- 1.6 mmol/L; GTG: 29.4 +/- 1.9 mmol/L; p < 0.05) and serum insulin levels (GTG ADX: 76 +/- 10 microU/mL; GTG: 470 +/- 63 microU/mL; p < 0.05). Lactate conversion to glucose by hepatocytes isolated from ADX GTG mice was significantly reduced compared with that of hepatocytes from GTG mice (GTG ADX: 125 +/- 10 nmol glucose/10(6) cells; GTG: 403 +/- 65 nmol glucose/10(6) cells; p < 0.05). ADX also significantly reduced both the glycogen (GTG ADX: 165 +/- 27 mumol/liver; GTG: 614 +/- 60 mumol/liver; p < 0.05) and fatty acid content (GTG ADX: 101 +/- 9 mg fatty acid/g liver; GTG: 404 +/- 40 mg fatty acid/g liver; p < 0.05) of the liver of GTG-obese mice. ADX of GTG-obese mice reduced PDH activity by varying degrees in all tissues, except quadriceps muscle. These observations are consistent with an ADX induced decrease in hepatic lipid stores removing fatty acid-induced increases in gluconeogenesis and increased peripheral availability of fatty acids inhibiting PDH activity via the glucose/fatty acid cycle. It is also evident that the improvement in glucose tolerance which accompanies ADX of GTG-obese mice is not due to increased PDH activity resulting in enhanced peripheral glucose oxidation. Instead, it is more likely that reduced blood glucose levels after ADX of GTG-obese mice are the result of decreased gluconeogenesis in the liver.

Adrenalectomy↗
PubMed1996

Evolution of the self-reproducing system to the biosynthesis of the membrane: an approach from the amino acid sequence similarity in proteins.

In order to study the problem of how the biomembrane synthesis started in the evolutionary process of the self-reproducing system, we carry out an extensive similarity search of the sequence data stored in databases, using the acetyl-CoA carboxylase, fatty acid synthase and the enzyme proteins leading to the combination of sn-glycerol 3-phosphate and fatty acid as the query sequences. With the use of the FASTA program (Pearson & Lipman, 1988), the proteins that carry an amino acid sequence showing similarity to any of the query sequences are picked up under the criterion of statistical significance of more than 6.0 for the homology, then classified according to the functional blocks where they operate. Finally they are filtered to the enzyme proteins in the metabolic pathways and to the DNA- or RNA-interacting proteins in the translation, transcription and replication apparatuses by eliminating proteins such as membrane proteins, lipase etc. which seem to have been generated after the appearance of the biomembrane. The distribution of the proteins thus selected shows a clear pattern that the amino acid sequences showing considerable similarity to the biomembrane synthetic proteins are concentrically found in the enzyme proteins in and around the section of glycolytic pathway from glyceraldehyde 3-phosphate to pyruvate while the DNA- or RNA-interacting proteins similar to the query sequences are distributed sparsely over the translation, transcription and replication systems. The assignment of similarity regions ascertains that considerable regions of most biomembrane synthetic proteins are covered by the enzyme proteins in and around the glycolytic pathway. Although acetyl-CoA carboxylase and fatty acid synthase are full of variety in the constitution of active domains depending on species, the above-mentioned pattern is also obtained by using either the monofunctional or the multifunctional type of proteins as the query sequences. Thus, the evolution towards biomembrane synthesis may be positioned as an event following the establishment of a section of glycolytic pathway from glyceraldehyde 3-phosphate to pyruvate. The causality of this evolution from the glycolytic pathway to the biomembrane synthesis is also discussed in connection with the absorption of protons released in the glycolytic process.

Amino Acid Sequence↗
PubMed1975

Some aspects of fatty acid oxidation in isolated fat-cell mitochondria from rat.

Mitochondrial were prepared from fat-cells isolated from rat epididymal adipose tissues of fed and 48 h-starved rats to study some aspects of fatty acid oxidation in this tissue. The data were compared with values obtained in parallel experiments with liver mitochondria that were prepared and incubated under identical conditions. 2. In the presence of malonate, fluorocitrate and arsenite, malate, but not pyruvate-bicarbonate, facilitated palmitoyl-group oxidation in both types of mitochondria. In the presence of malate, fat-cell mitochondria exhibited slightly higher rates of palmitoylcarnitine oxidation than liver. Rates of octanoylcarnitine oxidation were similar in liver and fat-cell mitochondria. Uncoupling stimulated acylcarnitine oxidation in liver, but not in fat-cell mitochondria. Oxidation of palmitoyl- and octanoyl-carnitine was partially additive in fat-cell but not in liver mitochondria. Starvation for 48 h significantly decreased both palmitoylcarnitine oxidation and latent carnitine palmitoyltransferase activity in fat-cell mitochondria. Starvation increased latent carnitine palmitoyltransferase activity in liver mitochondria but did not alter palmitoylcarnitine oxidation. These results suggested that palmitoylcarnitine oxidation in fat-cell but not in liver mitochondria may be limited by carnitine palmitoyltransferase 2 activity. 3. Fat-cell mitochondria also differed from liver mitochondria in exhibiting considerably lower rates of carnitine-dependent oxidation of palmitoyl-CoA or palmitate, suggesting that carnitine palmitoyltransferase 1 activity may severely rate-limit palmitoyl-CoA oxidation in adipose tissue.

Adenosine Triphosphate↗