A direct spectrophotometric assay for branched-chain 2-oxo acid dehydrogenase activity in intact rat liver mitochondria

1981 ◽  
Vol 9 (1) ◽  
pp. 117-118
Author(s):  
KIM BARTLETT ◽  
H. STANLEY A. SHERRATT
Keyword(s):  
Dehydrogenase Activity ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Spectrophotometric Assay ◽  
Rat Liver Mitochondria ◽  
Acid Dehydrogenase ◽  
Branched Chain

scholarly journals Rat tissue concentrations of branched-chain 2-oxo acid dehydrogenase complex. Re-evaluation by immunoassay and bioassay

1986 ◽  
Vol 235 (2) ◽  
pp. 429-434 ◽  
Author(s):  
P A Patston ◽  
J Espinal ◽  
J M Shaw ◽  
P J Randle
Keyword(s):  
Rat Liver ◽  
Chain Complex ◽  
Liver Mitochondria ◽  
Spectrophotometric Assay ◽  
Normal Diet ◽  
Casein Diet ◽  
Active Complex ◽  
Acid Dehydrogenase ◽  
Branched Chain ◽  
Acid Dehydrogenase Complex

A rabbit polyclonal antibody to purified ox kidney branched-chain oxo acid dehydrogenase complex was shown by a variety of techniques to be an antibody to the E2 (acyltransferase) component. Rocket immunoelectrophoresis showed that the antibody does not discriminate between phosphorylated (inactive) or dephosphorylated (active) complex, and the same technique is used to assay total branched-chain complex (sum of active and inactive forms) in rat liver and heart mitochondrial extracts. The values obtained in normal rats fed on normal diet were comparable with those obtained by spectrophotometric assay of the holocomplex reaction after conversion of inactive complex into active complex. The values obtained in liver mitochondria from rats fed on 0%-casein diet or starved for 48 h were comparable with those in rats fed on normal diet, whereas earlier studies using spectrophotometric assay had shown substantial decreases in rats fed on 0%-casein diet or starved for 48 h. It has been shown that conversion of inactive complex into active complex requires prolonged incubation (120 min) in the presence of ketoleucine (4-methyl-2-oxopentanoate; to inhibit branched-chain oxo acid dehydrogenase kinase) to effect complete conversion in mitochondria from rats fed on 0%-casein diet, or starved for 48 h, or made diabetic with alloxan. By this technique, total activity of the complex in rat liver mitochondria was unaffected by diet or diabetes. The effects of diet and diabetes to decrease the activity of branched-chain complex in rat liver are therefore apparently mediated wholly through inactivation of the complex by phosphorylation.

scholarly journals Factors affecting the translocation of oxaloacetate and l-malate into rat liver mitochondria

1968 ◽  
Vol 109 (5) ◽  
pp. 921-928 ◽  
Author(s):  
J. M. Haslam ◽  
D. E. Griffiths
Keyword(s):  
Rat Liver ◽  
Adenosine Triphosphatase ◽  
Liver Mitochondria ◽  
Spectrophotometric Assay ◽  
Rat Liver Mitochondria ◽  
Anaerobic Conditions ◽  
Factors Affecting ◽  
Adenosine Triphosphatase Activity ◽  
Energy Dependent ◽  
Stimulation Of

1. The rates of translocation of oxaloacetate and l-malate into rat liver mitochondria were measured by a direct spectrophotometric assay. 2. Penetration obeyed Michaelis–Menten kinetics, and apparent Km values were 40μm for oxaloacetate and 0·13mm for l-malate. 3. Arrhenius plots of the temperature-dependence of rates of penetration gave activation energies of +10kcal./mole for oxaloacetate and +8kcal./mole for l-malate. 4. The translocation of both oxaloacetate and l-malate was competitively inhibited by d-malate, succinate, malonate, meso-tartrate, maleate and citraconate. The Ki values of these inhibitors were similar for the penetration of both oxaloacetate and l-malate. 5. Rates of penetration were stimulated by NNN′N′-tetramethyl-p-phenylenediamine dihydrochloride plus ascorbate under aerobic conditions or by ATP under anaerobic conditions. 6. The energy-dependent stimulation of translocation was abolished by uncouplers of oxidative phosphorylation. Oligomycin A, aurovertin, octyl-guanidine and atractyloside prevented the stimulation by ATP, but did not inhibit the stimulation by NNN′N′-tetramethyl-p-phenylenediamine dihydrochloride plus ascorbate. 7. Mitochondria prepared in the presence of ethylene-dioxybis(ethyleneamino)tetra-acetic acid did not exhibit the energy-dependent translocation, but this could be restored by the addition of 50μm-calcium chloride. 8. Valinomycin or gramicidin plus potassium chloride enhanced the energy-dependent translocation of oxaloacetate and l-malate. 9. Addition of oxaloacetate stimulated the adenosine triphosphatase activity of the mitochondria, and the ratio of ‘extra’ oxaloacetate translocation to ‘extra’ adenosine triphosphatase activity was 1·6:1. 10. Possible mechanisms for the energy-dependent entry of oxaloacetate and l-malate into mitochondria are discussed in relation to the above results.

scholarly journals Biochemical heterogeneity of rat liver mitochondria separated by rate zonal centrifugation

1972 ◽  
Vol 129 (1) ◽  
pp. 209-218 ◽  
Author(s):  
M. A. Wilson ◽  
J. Cascarano
Keyword(s):  
Cytochrome C ◽  
Succinate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Cytochrome B ◽  
Rat Liver ◽  
Nadh Dehydrogenase ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Osmotic Gradient ◽  
Glycerophosphate Dehydrogenase

1. Rat liver mitochondria were separated on the basis of their sedimentation coefficients in an iso-osmotic gradient of Ficoll–sucrose by rate zonal centrifugation. The fractions (33, each of 40ml) were collected in order of decreasing density. Fractions were analysed by spectral analysis to determine any differences in the concentrations of the cytochromes and by enzyme analyses to ascertain any differences in the activities of NADH dehydrogenase, succinate dehydrogenase and α-glycerophosphate dehydrogenase. 2. When plotted as% of the highest specific concentration, the contents of cytochrome a+a3 and cytochrome c+c1 were constant in all fractions but cytochrome b was only 65% of its maximal concentration in fraction 7 and increased with subsequent fractions. As a result, the cytochrome b/cytochrome a+a3 ratio almost doubled between fractions 7 and 25 whereas the cytochrome c+c1/cytochrome a+a3 ratio was unchanged. 3. Expression of the dehydrogenase activities as% of highest specific activity showed the following for fractions 6–26: NADH dehydrogenase activity remained fairly constant in all fractions; succinate dehydrogenase activity was 62% in fraction 6 and increased steadily to its maximum in fraction 18 and then decreased; the activity of α-glycerophosphate dehydrogenase was only 53% in fraction 6 and increased slowly to its peak in fractions 22 and 24. 4. These differences did not result from damaged or fragmented mitochondria or from microsomal contamination. 5. These results demonstrate that isolated liver mitochondria are biochemically heterogeneous. The importance of using a system for separating biochemically different mitochondria in studies of mitochondrial biogenesis is discussed.

scholarly journals Characterization of the glucose 6-phosphate dehydrogenase activity in rat liver mitochondria

1976 ◽  
Vol 159 (3) ◽  
pp. 683-687 ◽  
Author(s):  
M Grunwald ◽  
H Z Hill
Keyword(s):  
Dehydrogenase Activity ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
The Other ◽  
Rat Liver Mitochondria ◽  
Kinetic Behaviour ◽  
Two Peaks ◽  
Glucose 6 Phosphate Dehydrogenase

Glucose 6-phosphate dehydrogenase activity in rat liver mitochondria can be released by detergent. The released activity is separated by chromatography into two peaks. One peak has the kinetic behaviour and mobility similar to the soluble sex-linked enzyme, whereas the other peak is similar to the microsomal hexose 6-phosphate dehydrogenase. There is no evidence for the existence of a new glucose 6-phosphate dehydrogenase activity in rat liver mitochondria.

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