J. Náprstek

Protein phosphorylation in Streptomyces albus.

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.

Characterization of adenylate cyclase fromEscherichia coli

Adenylate cyclase activity was detected and characterized in cell-free preparations of different strains ofEscherichia 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.

Cyclic 3′,5′-adenosine monophosphate and catabolite repression in Escherichia coli

Abstract Several strains of E. coli were grown on different sources of carbon and β-galactosidase activity as well as intracellular and extracellular concentrations of c-AMP were determined. There was a good (inverse) correlation between extracellular concentrations of c-AMP and the intensity of catabolite repression, whereas the relationship between intracellular c-AMP levels and catabolite repression was not clear-cut.

Purification of DNA-dependent RNA polymerase fromStreptomyces granaticolor

RNA nucleotidyltransferase (EC 2.7.7.6) ofStreptomyces granaticolor was purified by precipitation with polymin P and ammonium sulphate, affinity chromatography on DNA-cellulose and gell filtration on Biogel A 1.5 m. SDS-polyacrylamide gel electrophoresis revealed 8 protein bands of molar mass ranging from 37 to 130 kg/mol. Proteins of molar mass of 130 and 120 kg/mol were identified to be β and β subunits, respectively. The role of other subunits of the enzyme is discussed.

Induction of β-D-Glucosidase inStreptomyces granaticolor

Abstractβ-d-Glucosidase inStreptomyces granaticolor is an inducible enzyme. Methyl-β-d-glucoside or cellobiose, added to a glycerol-containing medium, are most suitable inducers. The activity of β-d-glucosidase in a culture fully induced by cellobiose is 50 times higher than the basal level of the enzyme. β-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.

Some Properties of DNA-Dependent RNA Polymerase from Streptomyces granaticolor

RNA nueleotidyltransferase fromStreptomyces granaticolor was purified and some of its properties were investigated. The temperature optimum of the enzyme is 35 °C, pH optimum 7.8—8.4. Antibodies against the β subunit of the enzyme fromBacillus subtilis immunologieally cross-react with the β subunit of the enzyme fromS. granaticolor. Antibodies against the β′ subunit of the enzyme fromB. subtilis immunologically cross-react with both β and β′subunits of the enzyme fromS. granaticolor.

DNA-Dependent RNA Polymerase from Streptomyces Granaticolor

DNA-Dependent RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6) plays an important role in the transcription of genetic information. This process is assumed to be regulated by the level and specificity of RNA polymerase. Although RNA polymerases of E.coli and B.subtilis have been studied in most detail, a. number of papers concerned with the isolation and characterization of this enzyme from other microorganisms have already been published (for a review see refs.l and 2). Data concerning the isolation of the enzyme from streptomycetes are quite scant and include primarily the results obtained with S.mediterranei (3), S.antibioticus (4,5,6), S.aureofaciens (7), S.hygroscopicus (8) and S.coelicolor (Westfehling and Losick, cited after (9)).

Catabolite repression during single and multiple induction inEscherichia coli

Intracellular concentration of cAMP regulates the synthesis of enzymes sensitive to catabolite repression. The relationship between the single and multiple induction of β-galactosidase (EC 3.2.1.23), L-tryptophanase (EC 4.1.99.1), D-serine deaminase (EC 4.2.1.14), L-asparaginase (EC 3.5.1.1) and L-malate dehydrogenase (EC 1.1.1.37) was studied and the effect of cAMP level on the induction inEscherichia coli Crookes (ATCC 8739) was investigated. A varying degree of catabolite repression was observed during induction of individual enzymes induced separately on different energy sources. The synthesis of L-tryptophanase was most sensitive, whereas L-asparaginase was not influenced at all.Exogenous cAMP was found to overcome partially the catabolite repression of β-galactosidase and d-serine deaminase, both during single induction. The synthesis of L-malate dehydrogenase was negatively influenced by the multiple induction even in the presence of cAMP; on the other hand, the synthesis of L-tryptophanase was stimulated, independently of the level of the exogenous cAMP. Similarly, the activity of L-asparaginase slightly but significantly increased during the multiple induction of all five enzymes; here too the activity increase did not depend on exogenous cAMP.

The control of adenylate cyclase activity in Escherichia coli.

Cell-free extract ofE. coli possessed an inhíbíted adenylate cyclase activíty 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.

The effect of glucose on cellobiose uptake and β-D-glucosidase activity inStreptomyces granaticolor

Glucose inhibits the inducible synthesis of β-D-glucosidase inStreptomyces 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 β-D-glucosidase inS.granaticolor.