Biomedical subjects
Publications and source records attributed to I S Pashkovskaia.
The data concerning proteolytic enzymes in human and animal malignant tumours are reviewed. The activity of these proteinases, its changes in the course of the disease, intracellular localization and secretion into the extracellular space and blood are discussed. The role of proteolytic enzymes including the role of plasminogen activator in tumour progress and during the metastasis development is considered.
Insulin-degrading neutral proteinase with molecular weight of 70 kDa was partly purified from the rat liver and erythrocyte plasma membranes. Incubation of membranes with [gamma-32P]ATP resulted in the enzyme phosphorylation. Intensity of this process greatly increased in the presence of insulin (100 microU/ml), and correlated with the elevation of the insulin-degrading activity in proteinase. Ca2+, Mn2+, dithiothreitol, cysteine were shown to have a stimulatory effect on insulin degradation; p-chloromercuribenzoate significantly repressed this process. Phenylmethylsulphonyl fluoride and soybean trypsin inhibitor did not affect the activity of the proteinase. It was concluded that the investigated enzyme was a calpain and may participate in the mechanism of insulin action.
Insulin-degrading, Ca2+-activated, neutral proteinases of molecular weight about 150 kDa and 70 kDa were purified from plasma membranes of the loach liver and embryo cells. It was shown that dithiothreitol and cysteine enhanced the enzyme activity, whereas p-chloromercuribenzoate and iodoacetic acid inhibited its level. Incubation of isolated plasma membranes with 5'[gamma 32P]ATP resulted in phosphorylation of these proteinases. The intensity of the process increased under the influence of insulin (100 microU/ml), that correlated with a decrease in the activity of proteinase with molecular weight of 150 kDa and an increase in 70 kDa enzyme activity. The data suggest the existence of common regulatory pathways of insulin degradation in plasma membranes of the loach liver and embryo cells.
It is established that insulin enhances the ability of the loach liver plasma membranes to phosphorylate lactate dehydrogenase. In the case of insulin-treated plasma membranes the amount of incorporated 32P is more than 4 times higher than that of the basal level. It is concluded that insulin-stimulated plasma membrane-dependent phosphorylation of the enzyme is one of the possible molecular mechanisms of hormone action on intracellular metabolism.
When lactate dehydrogenase obtained from Misgurnus muscles and purified to the homogeneous state is incubated for 16 hat 38 degrees C, its activity lowers down to 10% of the initial value. Extracts of egg cells, embryos or skeletal muscles of the mentioned fish species added to the enzyme solution decrease considerably its inactivation. Proteins stabilizing the activity of lactate dehydrogenase are revealed in the supernatant liquid obtained after salting out of the above extracts with 60% sulphate ammonium saturation. These proteins are in fractions with the molecular weight below 45 kDa. Among proteins with the molecular weight 10 kDa there are polypeptides which exert an activation effect on lactate dehydrogenase. This effect is intensified with the presence of insulin.
The action and some properties of cathepsin D, partly purified from unfertilized loach eggs, embryos and skeletal muscles were determined. The enzyme from embryo cells displays the activity maximum at pH 3.0 and pH 4.8 while enzyme from skeletal muscles--only at pH 3.0. Cathepsin D purified from all three sources splits actively hemoglobin, albumin, alpha-glycerophosphate dehydrogenase, pyruvate kinase and practically does not influence casein, hexokinase, glucose-6-phosphate dehydrogenase. The enzyme is comparatively thermolabile and its activity decreases in the presence of thiol compounds. The main part of cathepsin D in skeletal muscle cells and in embryo cells is precipitated after differential centrifugation of homogenates (25000 g; 60 min).
Explore the source record for details and available documents.
Studies of the pig muscle lactate dehydrogenase (LDH) activity were permormed after incubating the enzyme solution with extracts of loach (Misgurnus fossilis) eggs and embryos, which were subjected to the influence of insulin hydrocortisone as well as to insulin combination with actinomycin D, cycloheximide or puromycin. The insulin alone is established to decrease the inactivating ability of the investigated extracts on the lactate dehydrogenase activity, when antibiotics removed to a considerable extent the influence of hormone. In the eggs and embryos there are proteins activating and stabilizing the LDH molecule. The level of LDH activation under the influence of the eggs and embryos extracts subjected to the insulin action was decreased. The addition of hydrocortisone to the medium for the incubation of eggs and embryos does not affect significantly the LDH-inactivating ability of their extracts.
Loach eggs are stated to possess some quantities of insulin or insulin-like substances with a positive reaction to antiinsulin antiserum. The level of these components is five times as high 30 minutes after fertilization, and does not change for the next 20 hours of the embryo development. No 17-oxycorticosteroids were detected in loach eggs and embryos during first four hours of the development; they appeared only at the blastula stage and at the state of gastrula their level increases considerably. It is supposed that insulin or insulin-like substances participate in metabolism regulation and cell differentiation of fish embryos during early stages of embryogenesis.
The paper deals with studies in proteolytic activity of loach eggs and embryos incubated in water and in insulin solutions. It is established that after fertilization the above-mentioned activity level increased considerably, but even at the cleavage stage of embryo development it is 7-10 times as low as in the liver and 3-5 times as low as in the skeletal muscle tissue of the adults. After incubation of unfertilized eggs in insulin solutions at relatively low concentrations (0.1 and 1.0 micro/ml) proteolytic activity of their extracts at the acid and alkaline pH values increased. At higher hormone concentrations (2.0 and 4.0 micro/ml) the level of this activity, on the contrary, decreased. Analogous changes in proteolytic activity were observed in embryos which were incubated in insulin solution up to the cleavage stage of development. However in this case the increase in the activity under the effect of lower hormone concentrations was observed only at the acid pH zone. Higher insulin concentrations at the blastula stage of embryo development exerted their inhibitory influence on proteolytic activity at the acid and neutral pH values.
It is established that the incubation of glucose-6-phosphate dehydrogenase solution (33 micrograms of enzymic protein per 1 ml) with extracts of unfertilized loach eggs and embryos results in a marked stabilization of the enzyme with respect to its inactivation. Extracts from the embryos preliminary affected by insulin possessed a lower stabilizing ability, the hormonal effect being removed to a considerable extent by actinomycin D and cycloheximide.
Explore the source record for details and available documents.