[Calmodulin-dependent regulation of Ca,Mg-ATPase activity in plasma membranes of the swine myometrium]. / Kal'modulinzavisimaia reguliatsiia aktivnosti Ca,Mg-ATPazy plazmaticheskikh membran miometriia svin'i.

Highly purified plasma membrane (PM) preparations of pig myometrium were found to contain 0.91 +/- 0.22 microgram calmodulin per mg of PM protein. Treatment of membranes with 1 mM EGTA in the presence of 0.2 M NaCl causes the diminution of the calmodulin content down to 3% of the original level. The activity of Ca, Mg-ATPase is thereby decreased by 40%. Exogenous calmodulin restores the enzyme activity up to 1.94 +/- +/- 0.30 mumol Pi/mg protein/hour. The maximal activation of Ca, Mg-ATPase is observed with 10(-7) M calmodulin. Calmodulin increases the total ATPase activity of myometrium PM without affecting the Mg-ATPase activity. Trifluoroperazine (20 microM) diminishes the activating effect of exogenous calmodulin on Ca, Mg-ATPase. Calmodulin stimulates Ca, Mg-ATPase at low concentrations of Ca2+(10(-8)-10(-6) M) by decreasing Km for Ca2+ from 0.4.10(-6) M to 2.10(-8) M as well as by increasing Vmax--from 0,8 to 1.42 mumol Pl/mg protein/hour. It is supposed that the activating effect of calmodulin on Ca, Mg-ATPase is based on electrostatic interactions of Ca2+-free calmodulin with the enzyme.

[Ca2+-calmodulin-activated cyclic nucleotide phosphodiesterase from the rabbit myometrium]. / Ca2+-kal'modulinaktiviruemaia fosfodiésteraza tsiklicheskikh nukleotidov miometriia krol'chikh.

Two forms of soluble phosphodiesterase of cyclic nucleotides separating by DEAE-cellulose ion-exchange chromatography and not only differing in physicochemical and catalytic parameters but also differently regulated by calmodulin are found in the doe myometrium. Calmodulin with 10(-7)-10(-5) M concentrations of Ca2+ promotes the two-fold activation of the 3':5'-AMP (but not of 3':5'-GMP) hydrolysis by the first form of phosphodiesterase. Trifluoperazine (10 microM) lowers the activating action of calmodulin. The second form of soluble phosphodiesterase is not sensitive to the action of both calmodulin and Ca2+. 3':5'-GMP (10 microM) inhibits the 3':5'-AMP hydrolysis by the first form of phosphodiesterase; calmodulin exerts no effect on this process. The data obtained testify to the possible participation of Ca2+ and calmodulin in Ca2+-calmodulin-dependent phosphodiesterase regulation of the content of cyclic nucleotides (3':5'-AMP, in particular) in the doe myometrium.

[Cyclic nucleotide content of the rabbit myometrium in various functional states]. / Soderzhanie tsiklicheskikh nukleotidov v miometrii krol'chikh pri razlichnykh funktsional'nykh sostoianiiakh.

Myometrium of female rabbits at the state of functional rest contained 260 pmoles of cAMP per g of tissue and 25 pmoles cGMP. In dynamics of pregnancy content of cAMP was increased up to 400 pmoles/g within the second half of pregnancy; the content of cGMP was decreased down to 10 pmoles/g. During labor content of cAMP and of cGMP became minimal; within the postnatal period concentration of cAMP was increased up to the values observed in the antenatal period and the content of cGMP was unaltered. Under these conditions the content of cAMP, cGMP and the activity of corresponding soluble phosphodiesterases were similarly altered. The regulatory subunit of protein kinase of the type I (RI) was shown to possess two sites for cAMP binding and RII--one site. The data obtained suggest that cyclic nucleotides are important for regulation of the myometrium functional state.

[Properties of cyclic nucleotide phosphodiesterase of the rabbit myometrium in various functional states]. / Svoistva fosfodiésterazy tsiklicheskikh nukleotidov miometriia krol'chikh pri razlichnykh funktsional'nykh sostoianiiakh.

Chromatography on DEAE-cellulose of a soluble sulfate-precipitated fraction of cyclic nucleotide phosphodiesterase from rabbit myometrium revealed two 3':5'-GMP and 3':5'-AMP-hydrolase activities. 3':5'-GMP phosphodiesterase (fraction I) was eluted with 0.15-0.23 M NaCl, while 3':5'-AMP phosphodiesterase (fraction II) with 0.2-0.35 M NaCl. 3':5'-GMP phosphodiesterase hydrolyzed 3':5'-GMP with Km = 14 microM and V = 5.25 nmol . min . mg of protein, while 3':5'-AMP phosphodiesterase hydrolyzed both cyclic nucleotides with Km for 3':5'-GMP equal to 12 microM and V = 1.33 nmol . min . mg of protein; the Km value for 3':5'-AMP was 3.6 and 30.5 microM, respectively; the corresponding values of V were 0.28 and 0.97 nmol . min . mg of protein. In late pregnancy, the level of the 3':5'-AMP hydrolase activity of rabbit myometrium was significantly elevated in parallel with an increase in V, predominantly for the enzyme with a low affinity for 3':5'-AMP. The 3':5'-GMP hydrolase activity and V were largely decreased for both phosphodiesterase fractions; the Km value for fraction I was also diminished. During labour, the rate of 3':5'-AMP hydrolysis by myometrium phosphodiesterase was decreased down to the level typical of functional rest. The rate of 3':5'-GMP hydrolysis during the same period by fraction I remained at a low level, i. e., as in pregnancy, while that of fraction II was increased up to the level typical of functional rest.(ABSTRACT TRUNCATED AT 250 WORDS)

[Characterization of endogenous phosphorylation substrates of sarcoplasmic reticulum fragments from fast skeletal muscles of the rabbit]. / Kharakteristika substratov endogennogo fosforilirovaniia fragmentov sarkoplazmaticheskogo retikuluma bystrykh skeletnykh myshts krolika.

Separation of phosphorylated sarcoplasmic reticulum (SR) fragments by polyacrylamide gel disc electrophoresis in the presence of Na-DS revealed that the radioactivity is distributed in protein zones with molecular weights of 95,000 and 6000-8000. The phosphorylation of the protein with m. w. of 95,000 is Ca2+-dependent. The tryptic hydrolysis of the phosphorylated SR fragments from fast skeletal muscles results in a loss of radioactivity by 60-70%; phospholipase C from Clostridium welchii reduces the labelled phosphate content by 40-50%. The cAMP-dependent protein kinase inhibitor decreases the phosphorylation of both substrates. The substrate of phosphorylation with m. w. of 6000-8000 is not stained with Amidoschwartz 10B or Coumassie brilliant blue. Extraction by an acidified chlorophorm--methanol mixture results in a proteolipid with specific radioactivity exceeding that of the original preparation of phosphorylated SR membranes 3-4-fold. Thin-layer chromatography on Silufol plates and Silicagel KSK showed that the proteolipid is not chromatographically homogeneous after 2-fold precipitation by diethyl ether and is localized in a band with Rf varying from 0.6 to 0.8. The fluorescence spectrum of the proteolipid in a chlorophorm--methanol--HCl solution is represented by an assymmetrical structure-free band with a maximum at 350 nm. A possible role of phosphorylase b and proteolipid in manifestation of the functional activity of the SR fragments is discussed.

[Endogenous phosphorylation of sarcoplasmic reticulum fragments of rabbit fast skeletal muscles]. / Endogennoe fosforilirovanie fragmentov sarkoplazmaticheskogo retikuluma bystrykh skeletnykh myshts krolika.

The purified membrane fragments of sarcoplasmic reticulum (SR) of rabbit fast skeletal muscles were found to incorporate 32P from[gamma-32P]ATP in endogenous membrane substrates and in histone H1. The existence of membrane-bound protein kinase of SR was demonstrated by steady state binding of [3H]-cAMP to the SR membranes. The constant of [3H]cAMP binding to the membranes is 2.5 +/- 0.003 x 10(6) M-1, the number of binding sites is 6.1 +/- 0.8 pmol per 1 mg of protein. The endogenous phosphorylation of SR components was inhibited by cAMP and cGMP at concentrations of 10(-7)-10(-6) and depended on Mg2+ and Ca2+. The thermostable protein inhibitor of cAMP-dependent protein kinase inhibited the endogenous phosphorylation of SR membranes by 30-40%. The protein phosphoproduct of SR membranes revealed the properties of a phosphoester. The membrane-bound protein kinase was active towards the exogenous substrate--histone H1. Phosphorylation in the presence of histones was independent of cyclic nucleotides, Mg2+ and Ca2+. Fractionation of 32P-labelled solubilized membranes in polyacrylamide gel in the presence of Na-SDS showed that the radioactivity is bound to protein zones with molecular weights of 95 000 and 6000.

[Changes in some properties of 3':5'-AMP-dependent protein kinase from rat skeletal muscles during physical exercise]. / Izmenenie nekotorykh svoistv 3':5'-AMP-zavisimykh proteinkinaz skeletnykh myshts krys pri trenirovke fizicheskimi nagruzkami.

During 6-week training of rats the activity of isoenzymes I and II of soluble 3':5'-AMP-dependent protein kinase increases by 22 and 33%, respectively. A long-term physical load does not cause any significant changes in the activity of both isoenzymes. The maximal activity of the isoenzymes from skeletal muscles of the control and experimental rats is observed at the same concentrations of 3':5'-AMP and pH of 6,0-6,5. During training and under physical load the apparent Km values for ATP of both isoenzymes of 3':5'-AMP-dependent protein kinase do not change significantly, whereas that of V shows an increase. The apparent Km and V values for the histone increase for isoenzyme I obtained from skeletal muscles of trained rats both at rest and under physical load. In case of isoenzyme II the Km value for the histone decreases, while that of V remains unchanged. The changes in the properties of isoenzymes I and II of 3':5'-AMP-dependent protein kinase from skeletal muscles suggest the participation of the enzyme in adaptation to systematic muscular activity.

[Effect of training and physical load on 3':5'-AMP content and activity fo the enzymes of its metabolism in rat muscle]. / Vliianie trenirovki i fizicheskoí nagruzki na soderzhanie 3':5'-AMP i aktivnost fermentov ego ovmena v myshtsakh krys.

It is established tha in skeletal muscles of dormant animals under the influence of training the 3':5'-AMP content and the activity of adenylate cyclase increase; that of phosphodiesterase remains unchanged. A long physical load causes no changes in the 3':5"-AMP content in the skeletal muscles of the trained rats as compared to its content in the intact animals but evokes a decrease as compared to its level in the trained rats muscles. The activity of adenylate cyclase in the skeletal muscles of the trained rats under long physical load lowers, that of 3':5'-AMP phosphodiesterase also decreases but to a less extent. The found changes in the 3':5'-AMP metabolism in the trained animals skeletal muscles evidence for a possible participation of the 3':5'-AMP system in development of the organism adaptation to higher physical loads.

[Several properties of 3':5'-AMP-dependent skeletal muscle protein kinases in normal rats and following physical exertion to fatigue]. / Nekotorye svoistva 3':5'-AMP-zavisimykh proteinkinaz skeletnykh myshts krys v norme i pri fizicheskoi nagruzke do utomleniia.

Three fractions of rat adenosine-3',5'-monophosphate-dependent protein kinase were isolated, partially purified in buffer concentration gradient at normal state and after long-term physical loading and studied. It is found that first two fractions of protein kinases at normal state and after intensive muscular work have similar activities with and without cAMP, apparent Km values for ATP and total histone and half-maximal stimulation by cyclic AMP, but they differed from the third fraction. There are differences in some kinetic parameters and in the cyclic AMP stimulated activities between protein kinases after physical loading. The data obtained suggest the existence of at least two kinases in rat skeletal muscle. The isoenzymes differ in their activities during fatigue.