Rat Liver Pyruvate Carboxylase

McClure, William R.; Lardy, Henry A.; Wagner, Marion J.; Cleland, W. W.

doi:10.1016/s0021-9258(18)62168-4

Cited by 89 publications

(4 citation statements)

References 12 publications

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“…However, pyruvate carboxylase did show some inhibition at high concentrations of a-cyanocinnamate and acyano-4-hydroxycinnamate. This inhibition was investigated further by using a partially purified preparation of the rat liver enzyme which showed similar kinetic properties to those reported by other workers (Seufert et al, 1971;McLure, 1969;McLure et al, 1971 (Halestrap, 1975). Initially it appeared that a-cyano-4-hydroxycinnamate inhibited pyruvate dehydrogenase, but further investigation showed that this apparent inhibition was due to inhibition of arylamine acetyltransferase, the enzyme used to follow the reaction.…”

Section: Resultssupporting

confidence: 60%

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by α-Cyano-4-hydroxycinnamate and related compounds

Halestrap

Denton

1975

Biochemical Journal

196

123

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1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.

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Section: Resultssupporting

confidence: 60%

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by α-Cyano-4-hydroxycinnamate and related compounds

Halestrap

Denton

1975

Biochemical Journal

196

123

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“…The parallel pattern results from the release of glutamate before the NH3 reacts •U the other subunit. This situation is similar to the biotin enzymes which have a two site ping-pong mechanism (Northrop, 1 969;McClure et al, 1971). In this case ammonia is being transferred instead of carboxy biotin.…”

Section: Discussionmentioning

confidence: 61%

Kinetic mechanism of Escherichia coli carbamoyl-phosphate synthetase

Raushel¹,

Anderson²,

Villafranca³

1978

Biochemistry

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The kinetic mechanism of Escherichia coli carbamoyl-phosphate synthetase has been determined at pH 7.5, 25 degrees C. With ammonia as the nitrogen source, the initial velocity and product inhibition patterns are consistent with the ordered addition of MgATP, HCO3-, and NH3. Phosphate is then released and the second MgATP adds to the enzyme, which is followed by the ordered release of MgADP, carbamoyl phosphate, and MgADP. With glutamine as the ammonia donor, the patterns are consistent with a two-site mechanism in which glutamine binds randomly to the small molecular weight subunit producing glutamate and ammonia. Glutamate is released and the ammonia is transferred to the larger subunit. Carbamoyl-phosphate synthetase has also been shown to require a free divalent cation for full activity.

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“…We show that the P450R steady-state kinetic mechanism is a two-site ping-pong mechanism in which the "carrier" between the substrate binding sites is the FAD/ FMN prosthetic group pair. A number of other enzymes possess multi-site ping-pong mechanisms (Barden et al, 1972; Katiyar et al, 1975;McClure et al, 1971;Northrop, 1969;Tsai et al, 1973), and the theory describing the kinetic behavior of such enzymes is well established (Northrop, 1969;Cleland, 1973Cleland, , 1977. Although most multi-site ping-pong enzymes possess a mobile carrier (biotin, lipoic acid, or 4/-phosphopantetheine) to transfer a chemical group between substrate binding sites, P450R is unique in that the carrier is stationary since it is electrons that are being transferred between sites, which can be accomplished via tunneling (McClendon, 1988).…”

mentioning

confidence: 99%

Kinetic Mechanism for the Model Reaction of NADPH-Cytochrome P450 Oxidoreductase with Cytochrome c

Sem¹,

Kasper²

1994

Biochemistry

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The kinetic mechanism of NADPH-cytochrome P450 oxidoreductase (P450R) has been determined for the model reaction with cytochrome c3+. Although initial velocity studies show parallel patterns, consistent with a classical (one-site) ping-pong mechanism that precludes the formation of a ternary NADPH-P450R-cytochrome c3+ complex, product and dead-end inhibition results suggest a nonclassical (two-site) ping-pong mechanism [Northrop, D. B. (1969) J. Biol. Chem. 244, 5808-5819]. This mechanism is a hybrid of the random sequential (ternary complex) and ping-pong mechanisms, since ternary complexes can form as well as intermediate, modified forms of the enzyme that can be present in the absence of any bound substrate. The complete rate equation is derived for this mechanism, and values for Vmax, (V/K)NADPH, (V/K)cytc, and the corresponding Michaelis constants are presented in terms of microscopic rate constants along with the expected product inhibition patterns (Appendix). Inhibition by NADP+ is competitive versus NADPH and uncompetitive versus cytochrome c3+, while inhibition by cytochrome c2+ is competitive versus cytochrome c3+ and noncompetitive versus NADPH. These inhibition patterns are consistent with the proposed two-site mechanism. This mechanism would give the same initial velocity patterns as the classical one-site ping-pong mechanism, but it allows for the formation of a ternary complex, with NADPH and cytochrome c3+ reacting independently at two separate sites on P450R. The D(V/K)NADPH isotope effect is not affected by cytochrome c3+ concentration, consistent with our assumption (in deriving the rate equation) that binding at the two sites is independent. At the high ionic strength used in this study (850 mM), the mechanism is two-site ping-pong, with the electron acceptor site itself reacting with cytochrome c3+ in a tetra uni ping-pong manner.

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Rat Liver Pyruvate Carboxylase

Cited by 89 publications

References 12 publications

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by α-Cyano-4-hydroxycinnamate and related compounds

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by α-Cyano-4-hydroxycinnamate and related compounds

Kinetic mechanism of Escherichia coli carbamoyl-phosphate synthetase

Kinetic Mechanism for the Model Reaction of NADPH-Cytochrome P450 Oxidoreductase with Cytochrome c

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