Dehalogenation of 4-chlorobenzoate by 4-chlorobenzoate dehalogenase from pseudomonas sp. CBS3: an ATP/coenzyme A dependent reaction

Pseudomonas sp. CBS3 was grown with 4-chlorobenzoate as sole source of carbon and energy. Freshly prepared cell-free extracts converted 4-chlorobenzoate to 4-hydroxybenzoate. After storage for 16 hours at 25 degrees C only about 50% of the initial activity was left. Treatment at 55 degrees C for 10 minutes, dialysis or desalting of the extracts by gel filtration caused a total loss of the activity of the 4-chlorobenzoate dehalogenase. The activity could be restored by the addition of ATP, coenzyme A and Mg2+. If one of these cofactors was missing, no dehalogenating activity was detectable. The amount of 4-hydroxybenzoate formed was proportional to the amount of ATP available in the test system whereas CoA served as a real coenzyme. A novel ATP/coenzyme A dependent reaction mechanism for the dehalogenation of 4-chlorobenzoate by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 is proposed.     

Antioxidant effect of diethyldithiocarbamate on microsomal lipid peroxidation assessed by low-level chemiluminescence and alkane production

Different thiol-containing compounds, such as diethyldithiocarbamate (DDC), glutathione, penicillamine, and dithioerythritol have been chosen to study their effect on ascorbate/Fe-ADP-induced lipid peroxidation, detected by low-level chemiluminescence and alkane production. In the concentration range used, these thiols exerted a temporary protection against lipid peroxidation by lengthening the induction period; after overcoming this induction period, no substantial inhibition of either chemiluminescence or alkane production was observed. DDC was effective in protecting against lipid peroxidation in the nanomolar range, whereas the group of other thiol-containing molecules operated in the millimolar range.     

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cell. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ‘Crabtree effect’, was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate hydrogenase was subject to glucose inactivation.     

Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45

By chemoenzymatic synthesis the gene for a (Leu27) analogue of human growth hormone releasing hormone-Gly45 [(Leu27)GHRH-Gly45] was constructed, cloned and expressed in Escherichia coli as a fusion protein with beta-galactosidase under the control of the lac promoter and operator. Upon induction with isopropyl-D-thio-beta-galactopyranoside the fusion protein accumulated to a yield of 15-20% of the total cellular protein. After cyanogen bromide cleavage of the fusion protein the precursor peptide (Leu27)hGHRH-Gly45 was separated by extraction and purified by ion exchange and h.p.l.c.-RP18 chromatography. The purified peptide was analysed by sequencing, isoelectric focusing, amino acid analysis and amino acid analysis after V8 protease digestion. The carboxy-terminal glycine was subsequently amidated by PAM (peptidylglycine-alpha-amidating-monooxygenase), an enzyme which was isolated and characterized from fresh bovine pituitaries. Correct amidation of the penultimate amino acid, leucine, was verified by peptide sequencing with an authentic leucine amide reference.     

Direct selection for curing and deletion of Rhizobium plasmids using transposons carrying the Bacillus subtilis sacB gene

We have constructed derivatives of the transposon Tn5 carrying the mob site (oriT) of plasmid RP4, and an nptI-sacB-sacR cassette [Ried and Collmer, Gene 57 (1987) 239-246]. The mob site, in conjunction with the antibiotic-resistance markers carried on the transposons, allows identification of transposon inserts in cryptic plasmids by mobilisation to other strains. The sacB-sacR genes allow direct selection for the loss or curing of plasmids, because only strains which no longer contain an active sacB gene are able to grow on media containing sucrose. We have tested these transposons in four strains of Rhizobium leguminosarum and two strains of Rhizobium meliloti, and have been able to demonstrate curing of several large cryptic plasmids, and generation of large deletions in many other plasmids. This method has enabled us to show that the R. leguminosarum plasmids pRL12JI and pR1eVF39f carry auxotrophic markers, and that the plasmid pR1eVF39c carries genes which affect colony morphology.