Biomedical subjects
Publications and source records attributed to Z Dobrová.
The existence of phosphoprotein phosphatase (PPP) in aerial mycelium of S. granaticolor was demonstrated. Using inhibitors of serine and/or threonine PPP and specifically labeled substrate it was found that the PPP is of the serine and/or threonine type.
Transition from vegetative cells to aerial mycelium and spores of Streptomyces collinus is accompanied by changes in the pattern of proteins phosphorylated. Preparation from spores exhibits lower phosphorylation activity than those of vegetative cells and aerial mycelium. Phosphorylation of proteins from aerial mycelium was markedly stimulated by the presence of Mn2+. Our data indicate that phosphorylation of proteins on Ser/Thr residues is involved in transition of vegetative cells to aerial mycelium.
Protein kinase from Streptomyces lincolnensis was purified nearly to homogeneity using a high performance liquid chromatography (HPLC) and a Pharmacia FPLC system. The procedure used employed column chromatography on DE-53, followed by FPLC affinity chromatography with serine- or threonine-Sepharose (prepared as described in this paper) and gel filtration using a Superose 12 or TSK G3000SW column. Starting with 3.5 g of mycelial proteins, approximately 1 mg of pure enzyme was obtained. The procedure is simple and highly reproducible. The protein kinase thus obtained was nearly pure by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein kinase phosphorylated substrate proteins at the seryl residues.
RNA polymerase was isolated from Streptomyces granaticolor and protein kinase was partially purified from Streptomyces albus. When RNA polymerase was treated with protein kinase in vitro the activity of RNA polymerase was markedly enhanced. Furthermore, a protein of M = 65 kDa was isolated which, after being phosphorylated, stimulated RNA polymerase activity in vitro. Because neither the beta-subunits nor the alpha-subunits of RNA polymerase were phosphorylated it is assumed that phosphorylation of the 65 kDa protein may regulate the activity of RNA polymerase in streptomycetes.
The phosphorylated proteins of Streptomyces albus, radioactively labeled with [32P]orthophosphate have been analyzed by gel electrophoresis and autoradiography. More than 10 protein species were found to be phosphorylated. With [32P]ATP as substrate cell free extracts phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. From cell extract which exhibited active phosphorylated in vitro, a protein kinase has been partially purified. The kinase activity was identified in fractions corresponding to a 90 kDa protein.
The covalent modification of proteins by phosphorylation constitutes a major regulatory mechanism. It was first recognized in mammalian tissues. A conclusive evidence for the occurrence of protein phosphorylation and protein kinases in coliform bacteria was obtained in 1978. Several phosphate labeled proteins were found when Salmonella typhimurium was pulse-labeled with 32p(i) and solubilized bacterial contents were analyzed by SDS-polyacrylamide gel electrophoresis. In streptomycetes protein phosphorylation has not yet been demonstrated. We found that Streptomyces albus possesses a protein kinase activity. This in vitro protein phosphorylation is cAMP-independent.
Cell-free extract of E. coli possessed an inhibited adenylate cyclase activity after a previous anaerobic incubation of cells with glucose which is transported and metabolized. The degree of the inhibition depends on incubation conditions. Glucose analogues that are only transported but not metabolized, are not inhibitory. To restore the adenylate cyclase activity, the cells have to be cultivated aerobically prior to disintegration for a defined period of time without glucose.
Glucose inhibits the inducible synthesis of beta-D-glucosidase in Streptomyces granaticolor. Neither cAMP nor cGMP influence the inhibitory effect of glucose. Glucose also inhibits the inducible synthesis of the cellobiose uptake system but has no effect on its activity. This may be the mechanism underlying glucose inhibition of induction of beta-D-glucosidase in S. granaticolor.
beta-D-glucosidase in Streptomyces granaticolor is an inducible enzyme. Methyl-beta-D-glucoside or cellobiose, added to a glycerol-containing medium, are most suitable inducers. The activity of beta-D-glucosidase in a culture fully induced by cellobiose is 50 times higher than the basal level of the enzyme. beta-D-glucosidase is an intracellular enzyme, whose inducibility differ with culture age and reaches its maximum in a 10-h-old mycelium. The enzyme synthesis begins 2 h after the addition of the induced and reaches its maximum after a 10-h-induction.
Simultaneous induction of two enzymes sensitive to catabolite repression does not lead to an additive decrease of the specific activity of the two. Exogenously added cAMP increases the specific activity of catabolically repressed enzymes, irrespective of whether the enzyme is induced separately or simultaneously with another enzyme. In the presence of 12 different substrates metabolized by inducible enzymes glucose does not bring about catabolite repression. Synthesis of cAMP is identical with that occurring under conditions when glucose brings about catabolite repression.
Adenylate cyclase activity was detected and characterized in cell-free preparations of different strains of Escherichia coli; it was localized not only in the membrane fraction but also in the cytoplasm, the localization differing from strain to strain. The adenylate cyclase activity is highly dependent on the method used for disintegration of cells. The best results were obtained when using vortexing of the cell suspension with ballotini beads. The pH optimum of adenylate cyclase in cell-free preparations was found to be 9.0--9.5. The enzyme has an absolute requirement for Mg2+ and is inhibited by sodium fluoride and inorganic diphosphate. Release of adenylate cyclase from the membrane leads to an immediate loss of the activity; it was found that adenylate cyclase is quite labile and hence it could not yet been purified. The method used to determine adenylate cyclase activity and cyclic AMP is described.
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