scholarly journals Does pyruvate carboxylase interfere with the radioactive bicarbonate fixation assay of acetyl-CoA carboxylase?

1982 ◽  
Vol 208 (1) ◽  
pp. 247-248 ◽  
Author(s):  
J B Allred ◽  
J Goodson
Keyword(s):  
Pyruvate Carboxylase ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Bicarbonate Fixation

scholarly journals Multiple biotin-containing proteins in 3T3-L1 cells

1986 ◽  
Vol 237 (1) ◽  
pp. 123-130 ◽  
Author(s):  
C S Chandler ◽  
F J Ballard
Keyword(s):  
Pyruvate Carboxylase ◽  
Degradation Products ◽  
Gradient Centrifugation ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Cell Monolayers ◽  
Cell Extracts ◽  
Isoelectric Points ◽  
Molecular Masses ◽  
Partial Degradation

Extracts of 3T3-L1 cells prepared after labelling the monolayer cultures with [3H]biotin contained numerous protein bands that were detected by fluorography of dried SDS/polyacrylamide electrophoresis gels. All labelled proteins in the extracts could be removed by avidin affinity chromatography. The biotin-containing subunits of acetyl-CoA carboxylase, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, with molecular masses of approx. 220, 120, 75 and 72 kDa respectively, were detected together with minor bands at 100, 85 and 37 kDa that did not appear to be partial degradation products. Additional labelled bands increased in amount during incubation of cell extracts or did not occur in extracts prepared with trichloroacetic acid, 9.5 M-urea or proteolytic inhibitors, and were tentatively classified as partial degradation products. The unknown bands were not removed by incubation of cell monolayers for 24 h, a treatment that gave degradation rate constants of 0.47 day-1 for acetyl-CoA carboxylase and 0.28 day-1 for pyruvate carboxylase. Upon two-dimensional electrophoresis, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase had isoelectric points of 6.4, 7.2 and 6.4 respectively. Several additional discrete spots with isoelectric points below 6.2 were also present. All the unknown biotin-containing proteins banded with intact mitochondria during density-gradient centrifugation. We conclude that several unknown biotin-containing proteins are present in the mitochondria of 3T3-L1 cells, whereas others are partial breakdown products of mitochondrial proteolysis.

scholarly journals Distribution and degradation of biotin-containing carboxylases in human cell lines

1985 ◽  
Vol 232 (2) ◽  
pp. 385-393 ◽  
Author(s):  
C S Chandler ◽  
F J Ballard
Keyword(s):  
Cell Lines ◽  
Rate Constants ◽  
Pyruvate Carboxylase ◽  
Degradation Rate ◽  
Human Fibroblasts ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Degradation Rates

Incubation of cultured cells with [3H]biotin leads to the labelling of acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase and methylcrotonyl-CoA carboxylase. The biotin-containing subunits of the last two enzymes from rat cell lines are not separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but adequate separation is achieved with the enzymes from human cells. Since incorporated biotin is only released upon complete protein breakdown, the loss of radioactivity from gel slices coinciding with fluorograph bands was used to quantify degradation rates for each protein. In HE(39)L diploid human fibroblasts, the degradation rate constants are 0.55, 0.40, 0.31 and 0.19 day-1 for acetyl-CoA carboxylase, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase respectively. A similar series of rate constants is found for AG2804 transformed fibroblasts. The degradation rate constants are decreased by 31-67% in the presence of 50 micrograms of leupeptin/ml plus 5 mM-NH4Cl. Although the largest percentage effect was noted with the most stable enzyme, propionyl-CoA carboxylase, the absolute change in rate constant produced by the lysosomotropic inhibitors was similar for the three mitochondrial carboxylases, but greater for the cytosolic enzyme acetyl-CoA carboxylase. The heterogeneity in degradation rate constants for the mitochondrial carboxylases indicates that only part of their catabolism can occur via the autophagy-mediated unit destruction of mitochondria. Calculations showed that the autophagy-linked process had degradation rate constants of 0.084 and 0.102 day-1 respectively in HE(39)L and AG2804 cells. It accounted for two-thirds of the catabolic rate of propionyl-CoA carboxylase and a lesser proportion for the other enzymes.

scholarly journals Fatty Liver and Kidney Syndrome in Chicks II. Biochemical Role of Biotin

1976 ◽  
Vol 29 (6) ◽  
pp. 429 ◽  
Author(s):  
RL Hood ◽  
A RJohnson ◽  
AC Fogerty ◽  
Judith A Pearson
Keyword(s):  
Fatty Liver ◽  
Pyruvate Carboxylase ◽  
Acetyl Coa Carboxylase ◽  
Defence Mechanisms ◽  
Acetyl Coa ◽  
Liver Size ◽  
Glucose Levels ◽  
Blood Glucose Levels ◽  
Liver And Kidney

The role of biotin-dependent enzymes in the fatty liver and kidney syndrome of young chicks was studied. Under conditions of a marginal deficiency of dietary biotin, the level of biotin in the liver has differing effects on the activities of two biotin-dependent enzymes, pyruvate carboxylase and acetyl-CoA carboxylase. The activity of acetyl-CoA carboxylase is increased, but when the dietary deficiency of biotin produces biotin levels which are below o� 8 p,g/g of liver, the activity of pyruvate carboxylase may be insufficient to completely metabolize pyruvate via gluconeogenesis. There is an increase in liver size and in the activities of enzymes involved in alternate pathways for the removal of pyruvate. Blood lactate accumulates and there is increased synthesis of fatty acids, and an accumulation of palmitoleic acid; these steps are accomplished by increased activities of at least the following enzymes: acetyl-CoA carboxylase, malate dehydrogenase (decarboxylating) (NADP+) and the desaturase enzyme. When the biotin level is below 0�35 p,g/g of liver and the chick is subjected to a stress, physiological defence mechanisms of the chick may be inadequate to maintain homeostasis and they finally collapse, resulting in accumulation of triacylglycerol in the liver and blood; the chick is unable to maintain blood glucose levels and death occurs, often only a few hours after the imposition of the stress.

Sign in / Sign up

Export Citation Format

Share Document