Inhibitory effect of vanadate on protein phosphatase, glycogen phosphorylase and protein kinase in extracts from several rat tissues.

The purpose of this study was to determine the in vitro effect of increasing vanadate concentrations (1 nm-I mm) on protein phosphatase (PP) and glycogen phosphorylase (GP) of extracts from several rat tissues. PP activity was inhibited in various brain structures by moderate concentrations of vanadate; however, in the spinal cord, comparatively low concentrations (10 nm) of vanadate caused a steep increase in enzyme activity. Only at a high concentration (1 mm) did vanadate produce moderate PP inhibition. Reciprocal correlation was obtained between vanadate effects on PP and GP in various parts of the nervous system. In the last series of experiments the effect of vanadate upon protein kinase (PK) activity in various regions of the CNS was determined; low and intermediate vanadate concentrations produced a moderate activation; higher vanadate concentrations suppressed the enzyme activity. Vanadate at a concentration of 0.1 mm produced activation of PK in the medulla oblongata, whereas at 1 mm vanadate there was significant inhibition of PK. The spinal cord enzyme was more sensitive to vanadate, being inhibited at concentrations of 10 nm-1 mm.

Changes in the activity of enzymes, participating in glycogen metabolism of alloxan diabetic rats.

Alloxan diabetes induced in white rats by intraperitoneal injection of alloxan-monohydrate (15 mg/100 g body weight) was used to study changes in the glycogen phosphorylase a and b, phosphoprotein phosphatases and hexokinase activities under insulin deficiency conditions. Among the enzymes studied, an increase in muscle phosphorylase a activity as well as the a/b ratio have been obtained. In diabetic muscle phosphoprotein phosphatases and hexokinase activities were diminished. AMP increased the liver glycogen phosphorylase activity twice in diabetic rats whereas in normal animals the enzyme was less sensitive to this effector. The changes in liver hexokinase activity at diabetes were not connected and correlated with the altered phosphorylase and protein phosphatase activities. The logical chain of probable molecular events taking place in muscle glycogen metabolism under the conditions of insulin deficiency is offered.

[Phosphoprotein phosphatase of the myocardium. Isolation of individual molecular forms and their regulation]. / Fosfoproteinfosfataza serdechnoi myshtsy. Vydelenie otdel'nykh molekuliarnykh form i ikh reguliatsiia.

Individual molecular forms of phosphoprotein phosphatase (PPase) and their inhibitors of the protein nature were isolated from rat heart muscle by means of column chromatography on DEAE Sephadex A-50. All the three fractions of PPase were capable to dephosphorylate casein but did not affect pyrophosphate. PPase I dephosphorylated trimethaphosphate or casein. The enzyme molecular forms were different from each other by the value of specific activity and sensitivity to heat treatment. Two protein inhibitors eluted by buffers of different ionic strength - 0.2 M and 1.6 M - were identified. One of them was inactivated during heat treatment (95 degrees 5 min), while the second inhibitor maintained completely its activity in these conditions.

Multiple forms of phosphoprotein phosphatase and its protein inhibitors from rat brain.

By means of Sephadex A-50 ion exchange chromatography 5 peaks of phosphoprotein phosphatase (PPase) activity have been detected in rat brain. These molecular forms of the enzyme differ from each other by their specific activity as well as by a number of other properties--pH optima, heat and storage stability, and substrate specificity. It was shown that cyclic AMP (10(-5)-10(-6) M) decreased PPase activity of the majority of the peaks (I-III, V) and increased that of the peak IV. Ion exchange chromatography of brain tissue extracts revealed 4 peaks of PPase protein inhibitors. The results obtained on dialysis and tryptic digestion point to the protein nature of the inhibitors that differed from each other by their effect on the enzyme activity and heat stability (95 degrees C, 5 min). The significance of the specificity of separate molecular forms of PPases is discussed.

[Separation of fractions of phosphoprotein phosphatase and its protein inhibitors by isoelectrofocusing]. / Razdelenie fraktsii fosfoproteinfosfatazy i eë belkovykh ingibitorov metodom izoelektrofokusirovaniia.

Individual molecular forms of phosphoprotein phosphatase from albino rat cardiac muscle were separated by isoelectrofocusing, resulting in a few fractions differing in pI (5.1, 5.4, 5.9-6.1 (double peak) and 7.1, respectively). Isoelectrofocusing of a purified enzyme preparation also allowed to isolate and characterize two protein inhibitors of the enzyme. The first one with pH 6.5-6.7 is similar to the thermostable phosphoprotein phosphatase inhibitor known from literature. This protein inhibits the enzyme activity by 90%; its effect is not decreased after 5-min heating at 95 degrees. The other inhibitor protein with pH 5.6-5.8 is thermolabile. When the enzyme activity was decreased 2.5-fold prior to thermal treatment, the latter protein lost this ability after heating at 95 degrees and inhibited the enzyme only by 9%. It is assumed that inhibitory proteins beside low molecular weight effectors can be involved in the mechanisms of the post-synthetical operative modification of phosphoprotein phosphatase function.

[Molecular mechanisms of hypothalamic hexapeptide regulation of the links in glycogen metabolism]. / Molekuliarnye mekhanizmy reguliatsii gipotalamicheskim geksapeptidom nekotorykh zven'ev obmena glikogena.

Alterations in content of glycogen as well as in activity of phosphorylase and phosphoprotein phosphatase were studied in various rat tissues after intravenous administration of a new hypothalamic hexapeptide Tyr-Gly-Leu-Pro-Gly-NH2. An increase in phosphoprotein phosphatase activity was observed in heart muscle, which correlated with transformation of phosphorylase A to the B form and with accumulation of glycogen. The opposite effect was found in liver tissue and sceletal muscle.

[Participation of some nucleotides in regulation of phosphoprotein phosphates activity in rat and chicken myocardium]. / Uchastie nekotor'kh nukleotidov v reguliatsii fosfoproteinfosfataznoi aktivnosti v serdechnoi myshtse krys i kur.

Adenine- and uridine di- and triphosphates (in a 3 mM concentration) increase considerably phosphoprotein phosphatase (PPPase) (EC 3.1.3.16) activity of rat and chicken myocardium homogenates. AMP and Pi are effective inhibitors of the enzyme. The ATP activating effect is also shown in partially purified preparations of rat myocardium PPPase. ATP is able of protecting significantly the enzyme during its thermodenaturation.