Principal component analysis of mass spectra of peptides generated from the tryptic digestion of protein mixtures.

Principal component analysis (PCA) has been used to analyse mass spectral peptide profiles obtained from the enzymatic digestion of standard protein mixtures. Scores and loadings plots clearly revealed peptide fragments that differentiated one protein mixture from another. Peptide map search results identified with a high degree of certainty any additional proteins in these mixtures. As a proof-of-concept this methodology was applied to hepatic protein mixtures obtained from rats treated with two hepatotoxic compounds: methapyriline and SB-219994. Liver proteins were extracted, pre-separated by one-dimensional polyacrylamide gel electrophoresis, subjected to tryptic digestion and analysed by mass spectrometry. Two up-regulated proteins, glutathione S-transferase with methapyrilene and peroxisomal bifunctional enzyme with SB-219994, were identified in this manner.

The binding of propionyl-CoA and carboxymethyl-CoA to Escherichia coli citrate synthase.

The interaction of propionyl-CoA and acetyl-CoA with E. coli citrate synthase has been studied in order to gain insight into the structural requirements for substrate binding by this enzyme. In contrast to the enzyme from pig heart, the E. coli enzyme was unable to catalyse significant exchange of the methylene protons of propionyl-CoA while overall activity was very low with this enzyme. Carboxymethyl-CoA is a presumptive transition state analogue of acetyl-CoA using pig heart citrate synthase. The effect of carboxymethyl-CoA on both the native enzyme from E. coli and a catalytically active aspartate mutant (D362E) was investigated. Whereas the native enzyme was inhibited by carboxymethyl-CoA, the mutant enzyme (D362E) shows either no inhibition or minimal inhibition depending on the assay conditions. The binding of acetyl-CoA is not inhibited as a result of the mutation. The results with propionyl-CoA and carboxymethyl-CoA suggest that the active site of the E. coli enzyme is more restricted as compared with the enzyme from pig heart and, in the case of propionyl-CoA, this restriction prevents the formation of a catalytically productive enzyme-substrate complex.

Influence of unsaturated fatty acids on the production of tumour necrosis factor and interleukin-6 by rat peritoneal macrophages.

The effect of individual unsaturated fatty acids on the release of tumour necrosis factor (TNF) and interleukin 6 (IL6) was investigated in thioglycollate-induced rat peritoneal macrophages. The intracellular mechanisms associated with the changes of cytokine production in response to fatty acids were also studied. Incubation of macrophages with 100 microM docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) increased TNF (21% and 15% respectively) and IL6 (69% and 40% respectively) production. Linoleic acid (LA) diminished TNF production by 16%. At 100 microM oleic acid (OA), LA and EPA concentration an increase in macrophage adenylate cyclase activity (110%, 72% and 39% respectively) and a decrease (14%) in the presence of DHA was observed. PGE2 production in the presence of 100 microM DHA was reduced by 36%, whereas in the presence of 100 microM LA an increase (75%) was observed. Phospholipase A2 (PLA2) activity was also found to be modified in the presence of EPA and DHA at 50 microM (20% and 60% respectively) and 100 microM (34% and 62% respectively) concentrations. The activities of both protein kinase A (PKA) and protein kinase C (PKC) were effected by the different fatty acids. At 50 microM all fatty acids suppressed PKA activity except OA which enhanced PKA activity by 14%. At 100 microM fatty acid concentration, EPA suppressed PKA activity by 40%. PKC activity was enhanced by LA and OA, by 18% and 21% respectively. However, at 100 microM EPA and DHA, PKC activity was suppressed by 37% and 17% respectively, whereas PKC activity was enhanced by 146% in the presence of 100 microM LA. These results show for the first time that unsaturated fatty acids have an effect on macrophage PLA2 activity and that PGE2 may be a potent modulator of IL6 production. From these studies it is tempting to speculate that macrophage TNF and IL6 release may, in part, occur via a PKC and PKA independent pathway and that PLA2 activity and PGE2 concentration are inversely related to production of TNF and IL6.

The effect of replacing the conserved active-site residues His-264, Asp-312 and Arg-314 on the binding and catalytic properties of Escherichia coli citrate synthase.

The first step in the overall catalytic mechanism of citrate synthase is the binding and polarization of oxaloacetate. Active-site residues Arg-314, Asp-312 and His-264 in Escherichia coli citrate synthase, which are involved in oxaloacetate binding, were converted by site-directed mutagenesis to Gln-314, Asn-312 and Asn-264 respectively. The R314Q and D312N mutants expressed negligible overall catalytic activity at pH 8.0, the normal assay pH, but substantial activities for the partial reactions that reflect the cleavage and hydrolysis of the substrate intermediate citryl-CoA. However, when the pH was lowered to 7.0, the overall reaction of the mutants became significant, in contrast to the wild-type enzyme, whereas the two mutants exhibited reduced activities for the partial reactions. This result is consistent with the existence of a rate-limiting step between the two partial reactions for these mutants that is pH-dependent. The Km for oxaloacetate for the two mutants was increased 10-fold and was paralleled by an increase in the Km for citryl-CoA, whereas the Km for acetyl-CoA was increased only 2-fold. Overall, there was a striking parallel between the results obtained for these two mutants, which suggests that they are functionally linked in the E. coli enzyme. The equivalent of these two residues form a salt bridge in the pig heart citrate synthase crystal structure. The H264N mutant, in which the amide nitrogen of asparagine should mimic the delta-nitrogen of histidine, showed negligible activity in terms of both overall and partial catalysis, which may result from a hindrance of conformational change upon oxaloacetate binding. The affinity of this mutant for oxaloacetate appeared to be greatly reduced when investigated using indirect fluorescence and chemical modification techniques.

Conversion of citrate synthase into citryl-CoA lyase as a result of mutation of the active-site aspartic acid residue to glutamic acid.

The active-site aspartic acid residue, Asp-362, of Escherichia coli citrate synthase was changed by site-directed mutagenesis to Glu-362, Asn-362 or Gly-362. Only very low catalytic activity could be detected with the Asp----Asn and Asp----Gly mutations. The Asp----Glu mutation produced an enzyme that expressed about 0.8% of the overall catalytic rate, and the hydrolysis step in the reaction, monitored as citryl-CoA hydrolysis, was inhibited to a similar extent. However, the condensation reaction, measured in the reverse direction as citryl-CoA cleavage to oxaloacetate and acetyl-CoA, was not affected by the mutation, and this citryl-CoA lyase activity was the major catalytic activity of the mutant enzyme. This high condensation activity in an enzyme in which the subsequent hydrolysis step was about 98% inhibited permitted considerable exchange of the methyl protons of acetyl-CoA during catalysis by the mutant enzyme. The Km for oxaloacetate was not significantly altered in the D362E mutant enzyme, whereas the Km for acetyl-CoA was about 5 times lower. A mechanism is proposed in which Asp-362 is involved in the hydrolysis reaction of this enzyme, and not as a base in the deprotonation of acetyl-CoA as recently suggested by others. [Karpusas, Branchaud & Remington (1990) Biochemistry 29, 2213-2219; Alter, Casazza, Zhi, Nemeth, Srere & Evans, (1990) Biochemistry 29, 7557-7563].