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 Localization and characterization of two structurally different forms of acetyl-CoA carboxylase in young pea leaves, of which one is sensitive to aryloxyphenoxypropionate herbicides

1994 ◽  
Vol 300 (2) ◽  
pp. 557-565 ◽  
Author(s):  
C Alban ◽  
P Baldet ◽  
R Douce
Keyword(s):  
Molecular Mass ◽  
Subunit Structure ◽  
Acetyl Coa Carboxylase ◽  
Leaf Extracts ◽  
Acetyl Coa ◽  
Native Molecular Mass ◽  
Molecular Masses ◽  
Minor Form ◽  
A Minor

Young pea leaves contain two structurally different forms of acetyl-CoA carboxylase (EC 6.4.1.2; ACCase). A minor form, which accounted for about 20% of the total ACCase activity in the whole leaf, was detected in the epidermal tissue. This enzyme was soluble and was purified to homogeneity from young pea leaf extracts. It consisted of a dimer of two identical biotinyl subunits of molecular mass 220 kDa. In this respect, this multifunctional enzyme was comparable with that described in other plants and in other eukaryotes. A predominant form was present in both the epidermal and mesophyll tissues. In mesophyll protoplasts, ACCase was detected exclusively in the soluble phase of chloroplasts. This enzyme was partially purified from pea chloroplasts and consisted of a freely dissociating complex, the activity of which may be restored by combination of its separated constituents. The partially purified enzyme was composed of several subunits of molecular masses ranging from 32 to 79 kDa, for a native molecular mass > 600 kDa. One of these subunits, of molecular mass 38 kDa, was biotinylated. This complex subunit structure was comparable with that of microorganisms and was referred to as a ‘prokaryotic’ form of ACCase. Biochemical parameters were determined for both ACCase forms. Finally, both pea leaf ACCases exhibited different sensitivities towards the grass ACCase herbicide, diclofop. This compound had no effect on the ‘prokaryotic’ form of ACCase, while the ‘eukaryotic’ form was strongly inhibited.

scholarly journals Aminoacyl-tRNA synthetase complex in Saccharomyces cerevisiae

1995 ◽  
Vol 309 (1) ◽  
pp. 321-324 ◽  
Author(s):  
C L Harris ◽  
C J Kolanko
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Aminoacyl Trna Synthetase ◽  
Sucrose Density Gradient Centrifugation ◽  
Cell Extracts ◽  
Molecular Masses

The size distribution of aminoacyl-tRNA synthetase activity was investigated in cell extracts prepared from Saccharomyces cerevisiae. Bio-Gel A-5M chromatography of 105,000 g supernatants separated isoleucyl-tRNA synthetase activity into three peaks, with apparent molecular masses (Da) of about 100,000, 350,000 and 10(6) or greater. Similar results were obtained with synthetases specific for glutamic acid, serine and tyrosine. Sucrose-density-gradient centrifugation of yeast supernatants also provided evidence for the existence of synthetase complexes. These data provide the first evidence for the existence of a high-molecular-mass aminoacyl-tRNA synthetase complex in yeast, perhaps similar to those reported in higher eukaryotes.

scholarly journals Studies on the assay, activity and sedimentation behaviour of acetyl-CoA carboxylase from isolated hepatocytes incubated with insulin or glucagon

1984 ◽  
Vol 221 (3) ◽  
pp. 869-874 ◽  
Author(s):  
K F Buechler ◽  
A C Beynen ◽  
M J H Geelen
Keyword(s):  
Enzyme Activity ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Acid Synthesis ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Cell Extracts ◽  
Malonyl Coa ◽  
Triton X ◽  
Acid Stable

The activity of acetyl-CoA carboxylase, measured in various ways, was studied in 15000g extracts of rat liver hepatocytes and compared with the rate of fatty acid synthesis in intact hepatocytes incubated with insulin or glucagon. Hepatocyte extracts were prepared by disruption of cells with a Dounce homogenizer or by solubilization with 1.5% (v/v) Triton X-100. Sucrose-density-gradient centrifugation demonstrated that the sedimentation coefficient of acetyl-CoA carboxylase from cell extracts was 30-35S, regardless of the conditions of incubation or disruption of hepatocytes. Solubilization of cells with 1.5% Triton X-100 yielded twice as much enzyme activity (measured by [14C]bicarbonate fixation) in the sucrose-gradient fractions as did cell disruption by the Dounce homogenizer. Analysis by high-performance liquid chromatography of acetyl-CoA carboxylase reaction mixtures showed that [14C]malonyl-CoA accounted for 10-60% of the total acid-stable radioactivity, depending on the method for disrupting hepatocytes and on the preincubation of the 15000g extract, with or without citrate, before assay. Under conditions in which incubation of cells with insulin or glucagon caused an activation or inhibition, respectively, of acetyl-CoA carboxylase, only 25% of the acid-stable radioactivity was [14C]malonyl-CoA and enzyme activity was only 13% (control), 16% (insulin), and 57% (glucagon) of the rate of fatty acid synthesis. Under conditions when up to 60% of the acid-stable radioactivity was [14C]malonyl-CoA and acetyl-CoA carboxylase activity was comparable with the rate of fatty acid synthesis, there was no effect of insulin or glucagon on enzyme activity.

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.

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