J. Janeček

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.

Synthesis of β-galactosidase inEscherichia coli in the presence of phenylalanine analogues

The effect of phenylalanine analogues (p-F-phenylalanine, phenylserine and furylalanine) is described on the synthesis of inducible β-galactosidase inEscherichia coli ML-30 and phenylalanine requiring mutant ML-48. The incorporation of these analogues into the enzyme molecule results in the formation of a protein sensitive to a different extent to heat, urea and trypsin. The influence of the analogues on the ability to concentrate inducer inside the cells is also described. The different effect of the analogues on the synthesis and stability of the enzyme is discussed.

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.

Effect of ethionine on the synthesis of β-galactosidase inEscherichia coli

Ethionine at concentrations of 10−3M, 5×10−3M and 10−2M inhibits growth, both of β-galactosidase inducible ML-30 and constitutive ML-308Escherichia coli strains. The protein synthesis (measured by the incorporation of l-leucine-14C and l-aspartic-14C acid into proteins) of these strains is inhibited to the same extent as their growth. The synthesis of inducible and constitutive β-galactosidase produced by the strains ML-30 and ML-308, respectively, is considerably inhibited by ethionine.

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.

Regulation of protein synthesis inEscherichia coli by amino acid analogues

SummaryThe effect of phenylserine, an analogue of phenylalanine, on proteosynthesis and on the formation of induced β-galactosi-dase was studied in different strains ofEscherichia coli deficient in amino acids. It was found that inEscherichia coli ser- gly- phenylserine inhibited the incorporation of alanine and β-galactosidase formation in the presence of these essential amino acids. In deficient medium it stimulated both incorporation and β-ga-lactosidase synthesis.The incorporation of amino acids and formation of the enzyme were not affected inEscherichia coli phe- in complete medium. In deficient medium phenylserine increased incorporation, but had no effect on β-galactosidase synthesis. It was also found that phenylserine-1-14C was incorporated into the proteins ofEscherichia coli phe-. The stimulant effect of phenylserine inEscherichia coli ser-, gly- was investigated and it was found that the analogue was broken down into benzaldehyde and glycine.The mechanism of the action of this analogue on different species of deficient mutants ofEscherichia coli is discussed.AbstractЭффект phenylserine, аналоговый от Фенилаланин, и по proteosynthesis на создание искусственных β-galactosidase был изучен в разных штаммов Escherichia коли-либо аминокисл от. Было установлено, что в Escherichia титр сер (вс-gly) вс-phenylserine препятствует включе нию от аланин и β-galactosidase формирование в присутствии этих важнейших аминокислот. В среднесрочной его недостатками стимулируется как включение и β-galactosidase синтез.Включение аминокисл от и формирование энзима не были затронуты в Escherichia титр phe-в полной средой. В недостаткам и среднего phenylserine увеличился регистра ции, но не влияет на β-galactosidase синте за. Было также установле но, что phenylserine-1-14C была включена в белк и из Escherichia коли-phe. Стимуляторы эффект в phenylserine Escherichia титр услуг, gly-было расследовано и было установлено, чт о аналог было разбито и benzaldehyde глицин.Механизм действия эт ого Аналог на разных в идов недостатками мутантов из Escherichia коли обсуждается.

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.

The synthesis of β-galactosidase inEscherichia coli in the presence of 7-azatryptophan

The presence of 7-azatryptophan an analogue of tryptophan in the growth medium ofEscherichia coli resulted in a considerable inhibition of the synthesis of active β-galactosidase. No synthesis of an immunologically cross-reacting protein was detected. In addition, the replacement of tryptophan by the analogue rendered the enzyme more susceptible to heat, urea and trypsin as compared with the normal enzyme. The inhibition of growth and enzyme synthesis by 7-azatryptophan was reversed by tryptophan. The analogue did not exhibit any effect on the synthesis and activity of β-galactoside permease.

Further observations on the effect of ethionine on the synthesis of β-galactosidase inEscherichia coli: Formation of an immunologically cross-reacting protein

It was found that ethionine partially inhibits the transport of the inducer (TMG) of β-galactosidase into the cells ofEscherichia coli ML-30. The synthesis of β-galactosidase-specific messenger RNA is not inhibited. Ethionine appears to be incorporated into proteins synthesized by the strains used. The incorporation of ethionine into the molecule of β-galactosidase results in the synthesis of an enzymically inactive, immunologically cross-reacting protein.