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L A Fahien

Publications and source records attributed to L A Fahien.

16 recordsLinked to original sources
PubMed1992

Glutamate-malate metabolism in liver mitochondria. A model constructed on the basis of mitochondrial levels of enzymes, specificity, dissociation constants, and stoichiometry of hetero-enzyme complexes.

The level of aspartate aminotransferase in liver mitochondria was found to be approximately 140 microM, or 2-3 orders of magnitude higher than its dissociation constant in complexes with the inner mitochondrial membrane and the high molecular weight enzymes (M(r) = 1.6 x 10(5) to 2.7 x 10(6)) carbamyl-phosphate synthase I, glutamate dehydrogenase, and the alpha-ketoglutarate dehydrogenase complex. The total concentration of aminotransferase-binding sites on these structures in liver mitochondria was more than sufficient to accommodate all of the aminotransferase. Therefore, in liver mitochondria, the aminotransferase could be associated with the inner mitochondrial membrane and/or these high molecular weight enzymes. The aminotransferase in these hetero-enzyme complexes could be supplied with oxalacetate because binding of aminotransferase to the high molecular weight enzymes can enhance binding of malate dehydrogenase, and binding of both malate dehydrogenase and the aminotransferase facilitated binding of fumarase. The level of malate dehydrogenase was found to be so high (140 microM) in liver mitochondria, compared with that of citrate synthase (25 microM) and the pyruvate dehydrogenase complex (0.3 microM), that there would also be a sufficient supply of oxalacetate to citrate synthase-pyruvate dehydrogenase.

Animals↗
PubMed1992

Kinetics and regulation of hepatoma mitochondrial NAD(P) malic enzyme.

Kinetic studies of Morris 7777 hepatoma mitochondrial NAD(P) malic enzyme were consistent with an ordered mechanism where NAD adds to the enzyme before malate and dissociation of NADH from the enzyme is rate-limiting. In addition to its active site, malate apparently also associates with a lower affinity with an activator site. The activator fumarate competes with malate at the activator site and facilitates dissociation of NADH from the enzyme. The ratio of NAD(P) malic enzyme to malate dehydrogenase activity in the hepatoma mitochondrial extract was found to be too low, even in the presence of known inhibitors of malate dehydrogenase, to account for the known ability of NAD(P) malic enzyme to intercept exogenous malate from malate dehydrogenase in intact tumor mitochondria (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221). However, NAD(P) malic enzyme may be able to intercept exogenous malate because according to the present results, it can associate with the pyruvate dehydrogenase complex, which could localize NAD(P) malic enzyme in the vicinity of the inner mitochondrial membrane. The activity levels of some key metabolic enzymes were found to be different in Morris 7777 mitochondria than in liver or mitochondria of other rapidly dividing tumors. These results are discussed in terms of differences among tumors in their ability to utilize malate, glutamate, and citrate as respiratory fuels.

Animals↗
PubMed1992

Functional and morphological studies of mitochondria exposed to undecagold clusters: biologic surfaces labeling with gold clusters.

This study reports morphological and functional alterations observed in respiring isolated mitochondria when they are exposed to nonpenetrating, positive electrostatically charged synthetic undecagold clusters. Modification of the undecagold clusters positive charges change or prevent the functional effects and the binding to the outside surface of the mitochondria. The mitochondrial functional alterations are dependent on the oxidative phosphorylation capacity of the isolated organelles. The results of these experiments indicate that artificial undecagold may be useful to explore the molecular mechanisms of biological energy transducers which require electric charges separation, ionic fluxes, and electric surface properties.

Animals↗
PubMed1991

Glucose regulates leucine-induced insulin release and the expression of the branched chain ketoacid dehydrogenase E1 alpha subunit gene in pancreatic islets.

Much evidence has accumulated to support the idea that leucine can stimulate insulin release by allosterically activating glutamate dehydrogenase thus enhancing glutamate metabolism. It is less clear how the metabolism of leucine itself contributes to the signal for insulin release. We recently found that culturing pancreatic islets for 1 day at low glucose (1 mM) suppressed glucose-induced insulin release, but preserved leucine-induced insulin release. When islets were cultured at high glucose (20 mM), glucose-induced insulin release was preserved, but leucine-induced insulin release was suppressed (MacDonald, M. J., Fahien, L. A., McKenzie, D. I., and Moran, S. M. (1990) Am. J. Physiol., 259, E548-E554). The suppression of leucine-induced insulin release can be explained by glucose's suppression of the synthesis of the enzyme that catalyzes the first committed step of leucine metabolism, branched chain ketoacid dehydrogenase complex (BCKDH). High glucose suppressed the enzyme activity of the E1 component of the BCKDH complex, as well as the total activity of the BCKDH complex, to usually negligible levels in islets and decreased by an average of 90% the mRNA which encodes E1 alpha, the catalytic subunit of the E1 component of BCKDH, in islets and rat insulinoma cells. Time course studies showed that about 24 h in culture was required to maximally induce or suppress the expression of BCKDH E1 alpha. Culture at high glutamine with or without leucine mimicked to a lesser and more variable degree the effects of high glucose on leucine-induced insulin release and BCKDH E1 alpha mRNA. Leucine-plus-glutamine-induced insulin release was present after culture of islets with glucose and with or without any other secretagogue. Also, glutamate dehydrogenase transcripts and enzyme activity were not significantly altered by varying the concentration of glucose in the culture medium. Thus, leucine's insulinotropism via activation of glutamate dehydrogenase is constitutive. Preproinsulin mRNA levels were markedly increased at high glucose and glyceraldehyde phosphate dehydrogenase transcripts were either unaffected or slightly increased by glucose. Glutamine did not significantly effect the expression of genes other than BCKDH E1 alpha, and leucine had little or no effect on the expression of any of the four genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Interactions among mitochondrial aspartate aminotransferase, malate dehydrogenase, and the inner mitochondrial membrane from heart, hepatoma, and liver.

The inner mitochondrial membranes from bovine heart, rat liver, and Morris hepatoma 7777 all bound the mitochondrial isozymes of aspartate aminotransferase and malate dehydrogenase with comparable affinities and binding ratios (mg of enzyme bound per mg of membrane protein). A low molecular weight fraction separated from a detergent extract of the heart membrane by chromatography on Sephacryl S-300 contained most of the binding activity of the extract for the aminotransferase and had a dissociation constant for the aminotransferase of 0.2 microM. The protein component of the membrane binding sites for the aminotransferase was apparently present in this fraction because binding activity was largely eliminated by proteolysis with trypsin. When this fraction was chromatographed on an aminotransferase affinity column, only the portion that was bound and eluted by 0.25 M KCl associated with added aminotransferase. Unlike the membrane, which was markedly inhibited by the non-ionic detergent Genapol but was inhibited only 20% by trypsin, the binding activity of this subfraction was completely inhibited by trypsin but not by Genapol. This suggests, on the membrane, that the aminotransferase binds to the binding protein and is then transferred to lipids specifically associated with the binding protein. These putative lipids are presumably removed on the affinity column. Although the yield of the binding protein was low, there is probably ample binding protein in mitochondria to accommodate the aminotransferase. In every case, binding of the aminotransferase to the membrane inactivated the malate dehydrogenase binding site whereas malate dehydrogenase had little effect on the binding of the aminotransferase and only associated with the higher molecular weight fractions from the Sephacryl column that contained Complex I activity. Inactivation of the malate dehydrogenase site by the aminotransferase, but not vice versa, could result from aminotransferase associating with the binding protein and malate dehydrogenase with Complex I followed by association of the enzymes with lipids located in the same region of the membrane. However, since aminotransferase is more cationic, it is not displaced readily from the lipids by malate dehydrogenase. The relevance of these interactions to the organization of the enzymes is discussed.

Animals↗
PubMed1990

Insulin release in pancreatic islets by a glycolytic and a Krebs cycle intermediate: contrasting patterns of glyceraldehyde phosphate and succinate.

Glyceraldehyde phosphate, a glycolytic intermediate, and succinic acid (as its methyl ester to make it permeable to the cell), a citric acid cycle intermediate, were the only glucose metabolites of many recently tested that stimulated insulin release. The effects of these two "new" insulin secretagogues on several pancreatic islet parameters were compared. Glyceraldehyde phosphate stimulated all of the insulin it released during the first 5 min after islets were exposed to it, and its maximum effect on calcium uptake was observed at 5 min. Monomethyl succinate stimulated insulin release mostly during the last 30 min of a 1-h incubation and its maximum effect on calcium uptake was at 60 min after it was applied to islets. Monomethyl succinate-induced insulin release, but not glyceraldehyde phosphate-induced insulin release, was inhibited by metabolic inhibitors (antimycin A, rotenone, cyanide, FCCP, fluoride, and iodoacetamide). This is consistent with the idea that monomethyl succinate is hydrolyzed to succinate which is metabolized intramitochondrially. The effects of glyceraldehyde suggest that glucose signals the first phase of insulin release by an agonist-like mechanism that originates in the cytosol and requires minimal energy. The effects of monomethyl succinate suggest that the signal for the second phase of glucose-induced insulin release originates in the mitochondrion and requires a large amount of energy.

Animals↗
PubMed1990

Purification of tumor mitochondrial malic enzyme by specific ligand affinity chromatography.

A two-step chromatographic procedure, based on a specific ligand-binding approach, for the purification of tumor NAD(P)(+)-dependent malic enzyme is described. The enzyme was purified to near homogeneity by extraction from mitochondria, negative cellulose phosphate chromatography, ammonium sulfate precipitation, and application of specific elution from a malate-agarose column. The rationale for the use of the affinity column is also described.

Ammonium Sulfate↗
PubMed1990

Novel effects of insulin secretagogues on capacitation of insulin release and survival of cultured pancreatic islets.

Agents that stimulate insulin release from fresh pancreatic islets were tested for their ability to capacitate pancreatic islets to secrete insulin and to support beta-cell survival in tissue culture. Capacitation was defined as the ability to release insulin after 24 h in culture in the presence of an insulinotropic concentration of a secretagogue. Viable islets that lose glucose-induced insulin release gradually regain it during culture for 24 h in 20 mM glucose. Survival was defined as the ability to regain glucose-induced insulin release. To measure insulin release after culture, islets were incubated with various secretagogues in Krebs-Ringer buffer for 1 h. Examples of the diverse patterns of responses included the following. Glucose was the only secretagogue that capacitated glucose-induced release. Leucine-, leucine plus glutamine-, and glyceraldehyde-induced release remained capacitated after culture with no secretagogue. Culture at high glucose completely inhibited leucine-induced release. Culture at low glucose (1 mM) or at both high leucine and glutamine abolished glucose-induced release. Only leucine and glutamine capacitated monomethyl succinate-induced release. All agents including subinsulinotropic glucose (1 mM), except D-glyceraldehyde, permitted islet survival. Thus the metabolic pathways for initiation, capacitation, and survival are not identical between and within secretagogues. There is a reciprocal relationship between leucine and glucose with respect to capacitation. Capacitation follows a time course, which suggests that it is regulated by enzyme induction.

Animals↗
PubMed1990

Regulation of glutamate dehydrogenase by Mg2+ and magnification of leucine activation by Mg2+.

In the presence of Mg2+, pure glutamate dehydrogenase is more reactive with NADPH than with NADH and is markedly activated by elevations in the ADP/ATP ratio or the addition of leucine. Because these are properties of glutamate dehydrogenase in mitochondria but not properties of the pure enzyme studied in the absence of Mg2+, Mg2+ could be a ligand that confers upon glutamate dehydrogenase the regulatory properties of this enzyme found in situ. In the absence of the allosteric activators ADP, leucine, or succinyl-CoA, Mg2+ is an inhibitor and increases product inhibition by alpha-ketoglutarate in the forward reaction and substrate inhibition by alpha-ketoglutarate in the reverse reaction. However, the allosteric activators convert Mg2+ from an inhibitor into an activator of the forward reaction. In the reverse reaction, ADP also converts Mg2+ from an inhibitor into an activator and leucine eliminates inhibition by Mg2+. Because Mg2+ is an inhibitor in the absence of activator that also increases inhibition by alpha-ketoglutarate, whereas in the presence of activator Mg2+ has no effect or is itself an activator, Mg2+ magnifies the effect of the activator, and magnification increases with increases in the concentration of alpha-ketoglutarate. Leucine and its analog 2-aminobicyclo (2.2.1) heptane 2-carboxylic acid (BCH) have almost identical effects on both human and bovine glutamate dehydrogenase in both the presence and absence of Mg2+. However, advantages of BCH over leucine as a potential pharmacological activator of glutamate dehydrogenase are that BCH is not metabolized and, unlike leucine, BCH does not inhibit ornithine transcarbamylase. Isoleucine and valine alone have little effect on human glutamate dehydrogenase, but isoleucine slightly inhibits the enzyme in the presence of leucine.

Allosteric Regulation↗
PubMed1977

Effect of aspartate on complexes between glutamate dehydrogenase and various aminotransferases.

In previous studies it was found that: (a) aspartate aminotransferase increases the aspartate dehydrogenase activity of glutamate dehydrogenase; (b) the pyridoxamine-P form of this aminotransferase can form an enzyme-enzyme complex with glutamate dehydrogenase; and (c) the pyridoxamine-P form can be dehydrogenated to the pyridoxal-P form by glutamate dehydrogenase. It was therefore concluded (Fahien, L.A., and Smith, S.E. (1974) J. Biol. Chem 249, 2696-2703) that in the aspartate dehydrogenase reaction, aspartate converts the aminotransferase into the pyridoxamine-P form which is then dehydrogenated by glutamate dehydrogenase. The present results support this mechanism and essentially exclude the possibility that aspartate actually reacts with glutamate dehydrogenase and the aminotransferase is an allosteric activator. Indeed, it was found that aspartate is actually an activator of the reaction between glutamate dehydrogenase and the pyridoxamine-P form of the aminotransferase. Aspartate also markedly activated the alanine dehydrogenase reaction catalyzed by glutamate dehydrogenase plus alanine aminotransferase and the ornithine dehydrogenase reaction catalyzed by ornithine aminotransferase plus glutamate dehydrogenase. In these latter two reactions, there is no significant conversion of aspartate to oxalecetate and other compounds tested (including oxalacetate) would not substitute for aspartate. Thus aspartate is apparently bound to glutamate dehydrogenase and this increases the reactivity of this enzyme with the pyridoxamine-P form of aminotransferases. This could be of physiological importance because aspartate enables the aspartate and ornithine dehydrogenase reactions to be catalyzed almost as rapidly by complexes between glutamate dehydrogenase and the appropriate mitochondrial aminotransferase in the absence of alpha-ketoglutarate as they are in the presence of this substrate. Furthermore, in the presence of aspartate, alpha-ketoglutarate can have little or no affect on these reactions. Consequently, in the mitochondria of some organs these reactions could be catalyzed exclusively by enzyme-enzyme complexes even in the presence of alpha-ketoglutarate. Rat liver glutamate dehydrogenase is essentially as active as thebovine liver enzyme with aminotransferases. Since the rat liver enzyme does not polymerize, this unambiguously demonstrates that monomeric forms of glutamate dehydrogenase can react with aminotransferases.

Alanine Transaminase↗