Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity.

To define catalytically essential residues of bacteriophage T7 RNA polymerase, we have generated five mutants of the polymerase, D537N, K631M, Y639F, H811Q and D812N, by site-directed mutagenesis and purified them to homogeneity. The choice of specific amino acids for mutagenesis was based upon photoaffinity-labeling studies with 8-azido-ATP and homology comparisons with the Klenow fragment and other DNA/RNA polymerases. Secondary structural analysis by circular dichroism indicates that the protein folding is intact in these mutants. The mutants D537N and D812N are totally inactive. The mutant K631M has 1% activity, confined to short oligonucleotide synthesis. The mutant H811Q has 25% activity for synthesis of both short and long oligonucleotides. The mutant Y639F retains full enzymatic activity although individual kinetic parameters are somewhat different. Kinetic parameters, (kcat)app and (Km)app for the nucleotides, reveal that the mutation of Lys to Met has a much more drastic effect on (kcat)app than on (Km)app, indicating the involvement of K631 primarily in phosphodiester bond formation. The mutation of His to Gln has effects on both (kcat)app and (Km)app; namely, three- to fivefold reduction in (kcat)app and two- to threefold increase in (Km)app, implying that His811 may be involved in both nucleotide binding and phosphodiester bond formation. The ability of the mutant T7 RNA polymerases to bind template has not been greatly impaired. We have shown that amino acids D537 and D812 are essential, that amino acids K631 and H811 play significant roles in catalysis, and that the active site of T7 RNA polymerase is composed of different regions of the polypeptide chain. Possible roles for these catalytically significant residues in the polymerase mechanism are discussed.     

Additives and Enzyme Stability

The polypeptide chain of an enzyme is folded so that the necessary functional groups are brought together in the active site. Conformational changes may disrupt this arrangement and cause loss of enzymic activity. The effect of soluble additives on the unfolding process is discussed. Additives may be classified as substrates and similar ligands, small uncharged organic molecules, specific and non-specific ionic species, and polymers.     

Characterization of phosphotyrosyl-protein phosphatase activity associated with calcineurin

Calcineurin purified from bovine brain is shown to possess phosphotyrosyl -protein phosphatase activity towards proteins phosphorylated by the epidermal growth factor receptor/kinase. The phosphatase activity is augmented by Ca2+/calmodulin or divalent cation (Ni2+ greater than Mn2+ greater than Mg2+ greater than Co2+). In the simultaneous presence of all three effectors, the enzymatic activity is synergistically increased. Ca2+/calmodulin activates the Mg2+-supported activity by decreasing the Km value for phosphotyrosyl -casein from 2.2 to 0.6 microM, and increasing the Vmax from 0.4 to 4.6 nmol/min/mg. These results represent the first demonstration that calcineurin can dephosphorylate phosphotyrosyl -proteins and suggest a novel mechanism of activation of this enzyme.     

Degradation of sugar cane bagasse by several white-rot fungi

Forty-two white-rot fungi isolated in South America were incubated with long fibre sugar cane bagasse (LFB). The residual composition of LFB was determined after white-rot decay at 30 and 60 days. The ratio of residual lignin to residual lignin to residual cellulose (RL/RC) of untreated material (LFB) was 0.48. After white-rot-decay, the residual material with lower RL/RC ratios indicated that mainly lignin was degraded. In only 30 days, Phlebia sp. MVHC 5535, Athelia sp. MVHC 5509 and Spongipellis pachyodon MVHC 5019 caused a decrease in the RL/RC ratio to 0.36, 0.37 and 0.38, respectively, while it took 60 days for Ganoderma applanatum MVHC 5347, Hyphodontia sp. MVHC 5544, Panus tigrinus MVHC 5400, Stereum sp. MVHC 5113, Phellinus punctatus MVHC 5346 and MVHC 6388 to reach a ratio lower than 0.40. No correlation was found between the amount of some ligninolytic enzymes secreted and the residual composition of bagasse after white-rot fungi fermentation. Most of the fungal strains caused an increase in the relative amount of residual cellulose, indicating that hemicellulose was the preferred energy source.     

Gastrointestinal pH measurement in rats: influence of the microbial flora, diet and fasting

The pH of the rat intestinal tract was decreased by the presence of a microbial flora, but its influence in the forestomach is less clear. Stomach pH values varied according to the amount of food present at the time of measurement. Fasting increased the pH of the gastrointestinal tract in conventional rats but had little effect in germfree rats. In the conventional rat, feeding a purified diet compared with a commercial diet resulted in a lower pH in the forestomach and a higher pH in the caecal contents. Magnesium trisilicate promoted gastric emptying in conventional rats and its antacid effect was observed only in the caecum and colon.