Changes in the properties of adenylate cyclase from guinea pig lungs in tuberculosis

Changes in the properties of adenylate cyclase from the lungs of tuberculotic guinea pigs were revealed. The number of beta-adrenergic receptors in the lungs was found to be reduced by 30% at the second and by 70% at the third stage of the disease. The degree and the value of Ka for adenylate cyclase activation by isoproterenol remained thereby unchanged. The basal activity of adenylate cyclase was increased by 20% against the control level at the second stage and decreased by 20% at the third stage of the disease. At these periods, the stimulating effects of guanylyl imidodiphosphate, NaF and forskolin on lung adenylate cyclase were diminished. The experimental results point to the significant role of the enzymes of cAMP metabolism and reflect the course of the tuberculosis process in experimental animals.     

Effects of some protein kinases, cyclic nucleotides and specific inhibitors of phosphorylation on the mitotic cycle of Physarum polycephalum

The true slime mould Physarum polycephalum was treated with various agents by spraying them upon the cell surface 4 hrs before the second synchronous mitosis. The onset of mitosis was considerably approximated after the plasmodium treatment with protein kinases from rat hepatoma or Ph. polycephalum at the late G2 phase. The catalytic and regulatory subunits of cAMP-dependent pig brain protein kinase caused retardation of mitosis, while the holoenzyme, casein kinase and alkaline phosphatase did not affect the timing of mitosis. The cyclic nucleotides and inhibitors of their metabolic enzymes were used to investigate the role of phosphorylation processes in the mitotic cycle.     

Use of monoclonal antibodies for purifying the regulatory subunit of cAMP-dependent protein kinase

Monoclonal antibodies against regulatory subunit of cAMP-dependent protein kinase, type II, were obtained from pig brain (R II). The immune-affinity sorbent has been synthesized on the basis of monoclonal antibodies against R II. The method was proposed for the purification of homogeneous R II with high cAMP-binding activity using immune-affinity sorbent.     

Analysis of the H+/sugar symport in yeast under conditions of depolarized plasma membrane

H⁺/sugar symport in the obligatory aerobic yeastRhodotorula glutinis was analyzed under conditions where the plasma membrane was selectively depolarized by the lipophilic cation tetraphenylphosphonium (TPP⁺). Control experiments showed that this treatment did not impair the transmembrane pH, the cell energy charge, and the function of plasma membrane H⁺-ATPase. The kinetic data were fitted to elementary functions derived from a model constructed on the basis of some simplifying premises for ordered (either C + H⁺ + S or C + S + H⁺) and random reaction mechanisms. In addition, the comparison of the kinetic parameters in fully energized and depolarized cells provided information about the free carrier charge. It was concluded that the binding sequence of formation of the ternary carrier/H⁺/substrate complex follows a random mechanism and that the carrier bears a negative charge.     

Antioxidative properties of histidine-containing dipeptides from skeletal muscles of vertebrates

1. Ascorbate-dependent peroxidation of lipid components of biological membranes is inhibited by the natural histidine-containing dipeptides, carnosine and anserine, used at physiological concentrations. 2. Carnosine and anserine exhibit an equal antioxidative activity, whereas the preventing effect of homocarnosine is manifested only at low concentrations of oxidized lipid material. 3. The inhibiting effect of the dipeptides is enhanced either by the rise in the dipeptide concentration or by the decrease in the level of membrane components. 4. Addition of the dipeptides results in a marked decrease in the level of primary molecular products of lipid peroxidation. 5. In this case the optical spectrum of primary molecular products of polyunsaturated fatty acids changes significantly.     

Dynamic distribution of the regulatory subunit of cAMP-dependent protein kinase type II in NIH 3T3 cells

Experiments on the introduction of the regulatory subunit of cAMP-dependent protein kinase type II (RII) into NIH 3T3 cells clearly demonstrated its translocation into the nucleus. The labelled protein was incorporated into erythrocyte ghosts and their fusion with the cells was carried out. The dynamics of distribution of the labelled RII in NIH 3T3 cells was studied by the method of historadiography. It was found that during the next few hours after its penetration into the cytoplasm, the protein translocates into the nucleus and concentrates in the immediate proximity to the nucleoli.     

Calibration of a flow cytometric assay of glucose-6-phosphate dehydrogenase activity.

The reduction of tetrazolium salts to colored formazans is a reaction which has been exploited both in histo- and cytochemistry. Tetrazolium salts forming fluorescent formazans prove suitable for measuring defined cellular dehydrogenase activities in automated processes. This study considers an important aspect of formazan measurement in flow cytometry, namely, calibration. Calibration is performed by correlating the number (and fluorescence intensity) of formazan-bearing cells measured by flow cytometry with simultaneously performed biochemical analyses of the same material. The method is demonstrated by an example of glucose-6-phosphate dehydrogenase. Using the data of a typical experiment, the enzyme activity is expressed in femtomol of hydrogen transferred per cell during incubation time. Furthermore, through spatially resolved double excitation of formazan and nuclear DAPI fluorescence, an independent analysis of cell cycle and cellular enzymatic activity is established.     

Enzymes of a bacteriolytic lysoamidase preparation. Various properties of bacteriolytic protease L2]

Bacteriolytic proteinase L2 is able to cleave fluorogenic synthetic tripeptide anthranoyl-alanyl-alanyl-phenylalanyl-nitroanilide (Abz-Ala-Ala-Phe-pNA) at the bond between phenylalanine and p-nitroaniline. Optimal conditions of the tripeptide cleavage have been determined: pH 6.7 + 0.1; mu = 2 (by NaCl); t = 40 degrees C; KM = 2.6 x 10(-5) M. Metal cations reduced the enzyme activity. The enzyme was inhibited by EDTA, p-CMB, DIF. The synthetic tripeptide can be used to determine the activity of the L2 enzyme.