Increased adipose protein carbonylation in human obesity

Insulin resistance is associated with obesity but mechanisms controlling this relationship in humans are not fully understood. Studies in animal models suggest a linkage between adipose reactive oxygen species (ROS) and insulin resistance. ROS oxidize cellular lipids to produce a variety of lipid hydroperoxides that in turn generate reactive lipid aldehydes that covalently modify cellular proteins in a process termed carbonylation. Mammalian cells defend against reactive lipid aldehydes and protein carbonylation by glutathionylation using glutathione-S-transferase A4 (GSTA4) or carbonyl reduction/oxidation via reductases and/or dehydrogenases. Insulin resistance in mice is linked to ROS production and increased level of protein carbonylation, mitochondrial dysfunction, decreased insulin-stimulated glucose transport, and altered adipokine secretion. To assess protein carbonylation and insulin resistance in humans, eight healthy participants underwent subcutaneous fat biopsy from the periumbilical region for protein analysis and frequently sampled intravenous glucose tolerance testing to measure insulin sensitivity. Soluble proteins from adipose tissue were analyzed using two-dimensional gel electrophoresis and the major carbonylated proteins identified as the adipocyte and epithelial fatty acid-binding proteins. The level of protein carbonylation was directly correlated with adiposity and serum free fatty acids (FFAs). These results suggest that in human obesity oxidative stress is linked to protein carbonylation and such events may contribute to the development of insulin resistance.     

Defects in the nutrient-dependent methylation of a membrane-associated protein in spo mutants of Bacillus subtilis

Summary Methylation of a membrane-associated protein with an apparent molecular mass of 40000 daltons has been observed in Bacillus subtilis. The methylation was nutrient dependent and occurred with a doubling time of 4 ± 1 min. In wild-type strains, the half-life of turnover of the methyl group(s) was 17 ± 6 min. Several isogenic strains of B. subtilis containing spo0 mutations (spo0A and spo0H) were found to be normal in glutamate-dependent methylation of the protein and turnover of the methyl group(s). In strains containing spo0B and spo0E mutations, the methyl group(s) were incorporated in response to glutamate addition but turnover was not at a normal rate. The half-life of methyl group turnover was extended to 45 ± 3 min in these strains. In a spo0K mutant and in spoILI and spoIIF mutants, the protein was not significantly methylated. The methylation of a 40000 dalton protein was also found to be dependent on phosphate. This methylation was observed in wild-type and spo0A and spo0H strains with a doubling time of 4 ± 1 min and a half-life of turnover of the methyl group(s) of 11 + 3 min. In strains containing spo0B, spo0E, and spo0F mutations, the phosphate-dependent incorporation of the methyl group(s) was normal (5 ± 1 min) but the turnover half-life was extended to 46 ± 8 min. It is not known whether the nitrogen-dependent and phosphate-dependent systems methylated the same protein. The spo0 mutants are defective in the initial stages of sporulation, and it has been proposed that the spo0 gene products may play a role in nutrient sensing. The discovery of defects in the methylation of the 40 kDa protein in some of these spo0 mutants supports the proposal that the protein methylation may be part of a nutrient sensing system for the control of growth and sporulation in Bacillus species.     

Regulation of the activity of the Bacillus licheniformis A5 glutamine synthetase

The regulation of glutamine synthetase activity by positive and negative effectors of enzyme activity singularly and in combinations was studied by using a homogeneous enzyme preparation from Bacillus licheniformis A5. Phosphorylribosyl pyrophosphate at concentrations greater than 2mM stimulated glutamine synthetase activity by approximately 70%. The concentration of phosphorylribosyl pyrophosphate required for half-maximal stimulation of enzyme activity was 0.4 mM. Results obtained from studies of fractional inhibition of glutamine synthetase activity were consistent with the presence of one allosteric site for glutamine binding (apparent I0.5, 2.2mM) per active enzyme unit at a glutamate concentration of 50 mM. At a glutamate concentration of 30 mM or less, the data were consistent with the enzyme containing two binding sites for glutamine (one of which was an allosteric site with an apparent I0.5 of 0.4 mM). Bases on an analysis of the response of glutamine synthetase activity to positive and negative effectors in vitro and to the intracellular concentration of these effectors in vivo, the primary modulators of glutamine synthetase activity in B. licheniformis A5 appear to be glutamine and alanine (apparent I0.5, 5.2mM).     

Regulation of nitrogen catabolic enzymes in Bacillus spp.

The levels of the inducible nitrogen catabolic enzymes arginase (L-arginine amidinohydrolase, EC 3.5.3.1) and alanine dehydrogenase (L-alanine:NAD+ oxidoreductase [deaminating], EC 1.4.1.1) from Bacillus licheniformis and histidase (L-histidine ammonia-lyase, EC 4.3.1.3) from Bacillus subtilis and the ammonia assimilatory enzymes from B. licheniformis were determined in cultures grown in the presence of different nitrogen sources. Although the levels of these enzymes were dependent upon the nitrogen source present, induction of the catabolic enzymes in response to the addition of inducer occurred even in the presence of preferred nitrogen sources. Intracellular pool sizes of ammonia, glutamate, glutamine, and alpha-ketoglutarate were measured in continuous cultures of b. licheniformis growing in the presence of different nitrogen sources. A comparison of the pool sizes of these metabolites with the ammonia assimilatory enzyme levels showed that the pools of the metabolites did not change in a manner consistent with their use as regulators of the synthesis of any of these enzymes.     

Is cyclic guanosine 3'',5''-monophosphate a cell cycle regulator?

Transient increases in the intracellular level of cyclic guanosine 3',5'-monophosphate have been observed at a periodicity of one generation time in two spoT strains of Escherichia coli and in Bacillus licheniformis.     

Analysis of the ACP1 gene product: classification as an FMN phosphatase.

The relationship between the ACP1 gene product, an 18kDa acid phosphatase (E.C. 3.1.3.2) postulated to function as a protein tyrosyl phosphatase, and the cellular flavin mononucleotide (FMN) phosphatase has been examined in vitro and by using cultured Chinese hamster ovary (CHO) cells. Kinetic analysis indicated that at pH 6 the acid phosphatase utilized a variety of phosphate monoesters as substrates. While small molecules such as FMN were effectively utilized as substrates (kcat/Km = 7.3 x 10(3) s-1M-1), the tyrosyl phosphorylated form of the adipocyte lipid binding protein was a relatively poor substrate (kcat/Km = 1.7 x 10(-1) s-1M-1) suggesting a role for the phosphatase in flavin metabolism. Fractionation of CHO cell extracts revealed that 90% of the FMN phosphatase activity was soluble and that all of the soluble activity eluted from a Sephadex G-75 column with the acid phosphatase. All of the soluble FMN phosphatase activity was inhibited by immunospecific antibodies directed against the bovine heart ACP1 gene product. These results suggest that the ACP1 gene product functions cellularly not as a protein tyrosyl phosphatase but as a soluble FMN phosphatase.     

Characterization and Regulation of Pyruvate Carboxylase of Bacillus licheniformis

Cell-free extracts of Bacillus licheniformis were found to contain pyruvate carboxylase which catalyzes the reaction between pyruvate and bicarbonate to yield oxalacetate in the presence of adenosine triphosphate (ATP), acetylcoenzyme A (CoA), and manganese. The plot between the reaction velocity of the carboxylation by the partially purified pyruvate carboxylase (25-fold) and the concentration of pyruvate, bicarbonate, manganese, and ATP did not indicate a pronounced deviation from the Michaelis-Menten hyperbola. The enzyme was inhibited by avidin and aspartate. Biotin partially protected the enzyme from avidin inhibition, whereas the amount of inhibition by aspartate was dependent on the concentration of acetyl-CoA present. The intracellular concentration of acetyl-CoA did not vary significantly enough to allow control of the enzyme by this method. Extracts of 4-hr postexponential-phase cells of B. licheniformis were also found to contain phosphoenolpyruvate carboxykinase, which appears to be under catabolite repression control. It is suggested that the endogenous induction of this enzyme is the determining factor allowing the shift to gluconeogenesis from glycolysis during sporulation of glucose-grown cells.