scholarly journals Purification and properties of the soluble carnitine palmitoyltransferase from bovine liver mitochondria

1987 ◽  
Vol 244 (2) ◽  
pp. 271-278 ◽  
Author(s):  
R R Ramsay ◽  
J P Derrick ◽  
A S Friend ◽  
P K Tubbs
Keyword(s):  
Glutamate Dehydrogenase ◽  
Gel Filtration ◽  
Random Order ◽  
Bovine Liver ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Purification And Properties ◽  
Rapid Equilibrium ◽  
Variable Substrate ◽  
Myristoyl Coa

A new carnitine palmitoyltransferase (CPT) was purified to homogeneity from bovine liver mitochondria which were 96% free of peroxisomal contamination, as judged by catalase and glutamate dehydrogenase activities. The enzyme is easily removed from mitochondria, without the use of detergent. It is monomeric (Mr 63,500), unlike other preparations of CPT from mitochondria, and is most active with myristoyl-CoA and palmitoyl-CoA. The Km values are between 0.8 and 4 microM for a range of substrates from hexanoyl-CoA to stearoyl-CoA; these are much lower than values reported for other purified CPT preparations. The Km for L-carnitine is 185 microM measured with palmitoyl-CoA, and does not vary greatly with the chain length. This is also lower than the values reported for other CPT preparations, but higher than those cited for the medium-chain transferases. Kinetic and inhibitor studies were consistent with a rapid-equilibrium random-order mechanism. 2-Bromopalmitoyl-CoA, which is an inhibitor of the outer CPT, inhibited the enzyme competitively with palmitoyl-CoA as the variable substrate, when added without preincubation. If the enzyme was preincubated with 2-bromopalmitoyl-CoA and carnitine, the activity did not reappear after gel filtration of the protein. The inhibitor was bound in a 1:1 stoichiometry per subunit of enzyme.

scholarly journals A product-inhibition study of bovine liver glutamate dehydrogenase

1975 ◽  
Vol 151 (2) ◽  
pp. 305-318 ◽  
Author(s):  
P C Engel ◽  
S S Chen
Keyword(s):  
Glutamate Dehydrogenase ◽  
Competitive Inhibition ◽  
Dissociation Constants ◽  
Random Order ◽  
Bovine Liver ◽  
Product Inhibition ◽  
Rate Equations ◽  
Sodium Phosphate Buffer ◽  
Rapid Equilibrium ◽  
Variable Substrate

1. Initial rates of oxidative deamination of L-glutamate with NAD+ as coenzyme, and of reductive aminiation of 2-oxoglutarate with NADH as coenzyme, catalysed by bovine liver glutamate dehydrogenase were measured in 0.111 M-sodium phosphate buffer, pH 7, at 25 degrees C, in the absence and presence of product inhibitors. All 12 possible combinations of variable substrate and product inhibitor were used. 2. Strict competition was observed between NAD+ and NADH, and between glutamate and 2-oxoglutarate. All other inhibition patterns were clearly non-competitive, except for inhibition by NH4+ with NAD+ as variable substrate. Here the extrapolation did not permit a clear distinction between competitive and non-competitive inhibition. 3. Mutually non-competitive behaviour between glutamate and NH4+ indicates that these substrates can be bound at the active site simultaneously. 4. Primary Lineweaver-Burk plots and derived secondary plots of slopes and intercepts against inhibitor concentration were linear, with one exception: with 2-oxoglutarate as variable substrate, the replot of primary intercepts against inhibitory NAD+ concentration was curved. 5. Separate Ki values were evaluated for the effect of each product inhibitor on the individual terms in the reciprocal initial-rate equations. With this information it is possible to calculate rates for any combination of substrate concentrations within the experimental range with any concentration of a single product inhibitor. 6. The inhibition patterns are consistent with neither a simple compulsory-order mechanism nor a rapid-equilibrium random-order mechanism without modification. They can, however, be reconciled with either type of mechanism by postulating appropirate abortive complexes. Of the two compulsory sequences that have been proposed, one, that in which the order of binding is NADH, NH4+, 2-oxoglutarate, requires an implausible pattern of abortive complex-formation to account for the results. 7. On the basis of a rapid-equilibrium random-order mechanism, dissociation constants can be calculated from the Ki values. Where these can be compared with independent estimates from the kinetics of the uninhibited reaction or from direct measurements of substrate binding, the agreement is reasonable good. On balance, therefore, the results provide further support for the rapid-equilibrium random-order mechanism under these conditions.

Purification and properties of different isoforms of bovine cathepsin B

1990 ◽  
Vol 68 (4) ◽  
pp. 822-826 ◽  
Author(s):  
Christiane Deval ◽  
Daniel Bechet ◽  
Alain Obled ◽  
Marc Ferrara
Keyword(s):  
Cathepsin B ◽  
Cathepsin L ◽  
Gel Filtration ◽  
Specific Inhibitor ◽  
Bovine Liver ◽  
Relative Mass ◽  
Low Concentrations ◽  
Purification And Properties ◽  
Different Populations ◽  
Rapid Purification

A rapid purification procedure is described for cathepsin B from bovine liver. After preparation of crude lysosomal extracts, the method only involves DEAE Zeta-Prep-Disk chromatography, gel filtration, and fast protein liquid chromatography on Mono-S column. Two active peaks (P1 and P2) of cathepsin B were distinguished. Both presented uncleaved (relative mass (Mr) 30 000) and cleaved (Mr 25 000 + Mr 5000) chains, but different isoforms as revealed by isoelectrofocusing. These two different populations of cathepsin B isoforms nevertheless exhibited similar enzymatic properties. Km and kcat were 114 μM and 52 s−1, and 125 μM and 75 s−1, for hydrolysis of Z-Arg-Arg-NMec by P1 and P2, respectively. Both were rapidly inhibited by low concentrations of E-64 or leupeptin, but were unaffected by cathepsin-L-specific inhibitor Z-Phe-Phe-CHN2.Key words: protein/enzyme purification, cathepsin B, isoforms, lysosomes.

scholarly journals A steady-state random-order mechanism for the oxidative deamination of norvaline by glutamate dehydrogenase

1983 ◽  
Vol 211 (1) ◽  
pp. 99-107 ◽  
Author(s):  
C LiMuti ◽  
J E Bell
Keyword(s):  
Steady State ◽  
Glutamate Dehydrogenase ◽  
Dissociation Constants ◽  
Random Order ◽  
Kinetic Mechanism ◽  
Order Model ◽  
Oxidative Deamination ◽  
Steady State Kinetics ◽  
Rapid Equilibrium ◽  
Good Agreement

The kinetic mechanism of glutamate dehydrogenase with the monocarboxylic substrate norvaline was examined by using initial-rate steady-state kinetics and inhibition kinetics. To a first approximation the reaction mechanism can be described as a rapid-equilibrium random-order one. Binding synergism between the monocarboxylic substrate and coenzyme is not observed. Dissociation constants for NAD+ and 2-oxoglutarate calculated from the kinetic data assuming a rapid-equilibrium random-order model are in good agreement with independently obtained estimates. Lineweaver-Burk plots with varied norvaline concentration are not strictly linear, and it is concluded that a steady-state random-order model more accurately reflects the observed kinetics with norvaline as substrate.

The Effect of Guanidinium Chloride on the Self-Association of Bovine Liver Glutamate Dehydrogenase: A Gel Filtration Study

1987 ◽  
Vol 42 (3) ◽  
pp. 217-220
Author(s):  
Alberto Mazzini ◽  
Roberto Favilla
Keyword(s):  
Sodium Chloride ◽  
Glutamate Dehydrogenase ◽  
Gel Filtration ◽  
Bovine Liver ◽  
The Self ◽  
Gel Chromatography ◽  
Elution Volume ◽  
Guanidinium Chloride ◽  
Trimer Formation ◽  
Self Association

The associative behaviour of bovine liver glutamate dehydrogenase has been studied by gel chromatography at neutral pH in 1 ᴍ guanidinium chloride and 1 ᴍ sodium chloride. In guanidinium chloride both the elution volume and the elution profile of the enzyme are independ­ent of protein concentration, whereas in sodium chloride they are strongly dependent on it. In NaCl the enzyme behaves as expected according to the well established random association model, whereas in guanidinium chloride it appears to have completely lost the self-associative property. Furthermore, since the elution volume of the enzyme in guanidinium chloride corre­sponds to that of an hexamer, trimer formation reported to occur in these conditions is not confirmed by this technique.

scholarly journals Lysine and tyrosine in the NADH inhibitory site of bovine liver glutamate dehydrogenase.

1981 ◽  
Vol 256 (22) ◽  
pp. 11866-11872
Author(s):  
K.V. Saradambal ◽  
R.A. Bednar ◽  
R.F. Colman
Keyword(s):  
Glutamate Dehydrogenase ◽  
Bovine Liver ◽  
Inhibitory Site

scholarly journals Sequence of Bovine Liver Glutamate Dehydrogenase

1971 ◽  
Vol 246 (8) ◽  
pp. 2374-2399 ◽  
Author(s):  
Michael Landon ◽  
Dennis Piszkiewicz ◽  
Emil L. Smith
Keyword(s):  
Glutamate Dehydrogenase ◽  
Bovine Liver
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