M. Jirešová

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.

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.

Inhibition of β-galactosidase synthesis inEscherichia coli after infection with different DNA and RNA phages

Infection ofEscherichia coli with T1, T2r+, T3 and T4 phages leads to an immediate inhibition of β-galactosidase synthesis. Similar results were obtained with the virulent mutant of phage lambda. The degree of inhibition of β-galactosidase synthesis depends on the time delay between the addition of the inducer and the phage particles, and on the amount of phage DNA, which has penetrated into the host cell. RNA phage MS2 exhibited no inhibitory effect on enzyme synthesis.

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.

Catabolite repression of different inducible enzymes inEscherichia coli and the effect of cAMP

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.

The specific inhibition of the expression of the lactose operon by D, L-threo-phenylserine inEscherichia coli

Phenylserine, one of the phenylalanine analogues, is incorporated into proteins ofEscherichia coli and replaces the natural amino acid. The incorporation results in the inhibition of the synthesis of both inducible and constitutive β-galactosidase. The rate of the synthesis of β-galactosidase specific m-RNA is only slightly influenced by phenylserine, the steady-state level being decreased by about 40%. The m-RNA formed in the present of the analogue functions normally and its translation after the removal of the inhibitor results in the formation of normal β-galactosidase. The character of the inhibition of the enzyme synthesis by phenylserine is similar to that caused by chloramphenicol. However, phenylserine specifically inhibits only the synthesis of β-galactosidase, whereas other cell proteins are synthesized. No protein immunologically cross-reacting with the antiserum against normal β-galactosidase is formed by inducible ánd constitutiveEscherichia coli strains. The active transport is completely inhibited as the cells induced in the presence of phenylserine do not accumulate14C-TMG. It follows from the results that phenylserine inhibits both the formation of TMG-specific permease and the synthesis of the active molecule of β-galactosidase inEscherichia coli.

Regulation of cAMP Synthesis in Streptomyces Granaticolor

Adenosine-3′,5′-monophosphate (cAMP) and guanosine-3′,5′-monophosphate (cGMP) are found in a variety of microbial cells (1,2). The important role of cAMP as a regulator of transcription of some catabolite-sensitive operons has been described in several Gram-negative bacteria (3). The mechanism involved and some of the physiological consequences of changes in the levels of cAMP and cGMP are not certain, even in Escherichia coli. In most other microorganisms, all that is known is that the nucleotides can be isolated and identified, and, when added exogenously, that they can influence some physiological activities (2).

The activity ofEscherichia coli DNA-dependent RNA polymerase on DNA templates of different origin. The effect of cAMP

DNA isolated from different T phages served as a better template for the synthetic activity of unmodifiedEscherichia coli RNA polymerase in thein vitro system than did the host DNA. cAMP significantly stimulated the activity of such a preparation of RNA polymerase. The stimulation was more pronounced with the host DNA template than with phage DNA. However, the synthetic activity ofEscherichia coli RNA polymerase was greater in the presence of cAMP than without it when phage DNA served as the template.