P. Ryšavý

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.

The assay of β-galactosidase in soil

The method for the assay of β-galactosidase introduced by Lederberg (1950) was modified and used for studies of the enzyme in the soil.o-Nitrophenyl-β-d-galactoside served as substrate. Trismaleinate buffer was found to be more suitable than phosphate buffer in the assay. The enzyme reaction was stopped by adding sulphuric acid and the incubation mixture was alkalized with sodium carbonate so that the yellow colour ofo-nitrophenol could develop. The method is sensitive and specific and requires small quantities of soil and a short incubation time.

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.

The formation of β-galactosidase in soil

The activity of β-galactosidase during incubation of soil with lactose was studied. Enzyme activity reached its maximum between 6–24 h incubation. The addition of secondary ammonium phosphate stimulated the enzyme formation. Casein hydrolysate alone did not influence significantly enzyme formation even after a 72 h incubation. Enzyme activity in the non-incubated soil was also insignificant. Glucose added to the soil incubated with lactose decreased the formation of the enzyme.

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.

Utilization of glucose and cellobiose by the microflora of soil enriched with cellulose.

The ability of soil microflora to utilize glucose or cellobiose was found to depend on previous incubation of the soil with glucose, cellobiose or cellulose. Glucose was utilized more rapidly than cellobiose in soil preincubated with glucose or cellobiose. The opposite situation was observed in soil preincubated with cellulose. In the presence of a mixture of both sugars the rate of utilization of one of them was decreased by the second and this decrease could be characterized as competitive inhibition. Glucose accumulated in the medium during utilization of cellobiose alone in soil preincubated with cellulose. This phenomenon was not observed during the utilization of cellobiose in soil preincubated with glucose or cellobiose.

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)).

Activity of β -galactosidase in soil continuously supplemented with lactose

The microflora of the chernozem soil mineralized 62 % of the lactose retained on a column consisting of three 10-g layers of the soil at a daily flow of 48 mg of the sugar. Only 45 % of the sugar was mineralized when the daily flow was 136 mg. The highest value of the β-galactosidase activity in the system of heterocontinuous cultivation was two-fold with respect to batch cultivation. At a higher sugar concentration the enzyme activity in steady state was the same in the whole soil column. This value was reached first in the middle layer of the soil. At a lower concentration of the flowing sugar in steady state the highest enzyme activity was detected in the middle layer of the soil. In the upper layer the enzyme activity was one half, in the lower layer it began to decrease after reaching its maximum after 4 d of the incubation.

Mineralization of cellobiose and activity of β-glucosidase in the soil heterocontinuous system

Cellobiose added continuously to the soil was mineralized to CO2 from the beginning of incubation. Daily CO2 production reached a steady state after 4 d (at 0.1 % of supplied cellobiose) or after 8 d (at a 0.25 % concentration). The daily amount of carbon released as CO2 stabilized at 60 –65 % of carbon retained in the soil column in the form of cellobiose after a 3-d incubation, irrespective of the concentration of the supplied cellobiose. The activity of β-glucosidase increased during the entire incubation but it did not reach steady state in any layer of the soil column.