Ca2+ (calmodulin)-dependent phosphorylation of plasma membranes of pig myometrium

The high-purified vesicles of pig myometrium sarcolemma closed, mainly, so that the cytoplasmatic side is outside possess the Ca2+ (calmodulin)-dependent protein kinase activity. The initial rate of the endogenic phosphorylation without exogenic calmodulin is 6.3 and with its presence--10.7 pmol of 32Pi 1 min per 1 mg of protein. Km for ATP is equal to 164 microM, and Vmax--0.27 nmol of 32Pi 1 min per 1 mg of protein. Exogenic calmodulin increases the affinity to ATP (50 microM), Vmax being unchanged. Under optimal concentrations of calmodulin (10(-7)-10(-6) M) and 10(-4) M Ca2+ the protein kinase activity is 0.132 nmol of 32Pi min per 1 mg of protein. Electrophoresis in DS-PAAG has shown that membrane proteins with molecular weight of 105, 58, 25, 12 and 2 kDa are basic substrates of Ca2+ (calmodulin)-dependent phosphorylation. Trifluoperazine++ in the concentration of 40 microM inhibits phosphorylation of all five proteins. Ca2+ (calmodulin)-dependent phosphorylation is supposed to be a regulator of Ca2+-transport processes of sarcolemma.     

Ca2+-calmodulin-dependent phosphorylation and passive transport of Ca2+ in the myocardial sarcolemma

Highly purified vesicles of rabbit myocardium sarcolemma with predominant inside-out orientation possess the Ca2+-calmodulin-dependent protein kinase activity. At optimal concentrations of calmodulin (0.5 microM) and Ca2+ (0.1 mM), the activity of protein kinase is 0.21 nmol 32P X min X mg of protein. The Km(app) value for ATP is 3.0 X 10(-6) M, V = 0.27 nmol 32P X mg of protein X min. Endogenous Ca2+-calmodulin-dependent protein kinase phosphorylates four protein substrates in sarcolemmal vesicles (Mr = 145, 22, 11.5, and 6-8 KD). Studies with passive efflux of Ca2+ from the SL vesicles showed that the Ca2+-calmodulin-dependent phosphorylation of protein components of sarcolemma inhibits this reaction.     

The effect of Ca2+-phospholipid dependent phosphorylation on passive calcium transport in the myocardial sarcolemma

Protein kinase C in vesicular preparations of the myocardium sarcolemma is shown to phosphorylate proteins with the molecular weight of 250, 140, 67, 58, 24 and 11 kD. The exogenic protein kinase C catalyzed phosphorylation of the sarcolemma preparations lowers the initial rate of the passive calcium transport from 0.56 down to 0.18 mmol per 1 mg second. Activation of endogenic protein kinase C by 4 beta-phorbol-12 beta-myristate-13 alpha-acetate is also accompanied by phosphorylation of vesicular preparations of sarcolemma and by inhibition of the passive calcium transport. Polymyxin B, being an inhibitor of protein kinase C, suppresses the phosphorylation and thus prevents the inhibitory action of phosphorylation on the passive calcium transport.     

Cyclic nucleotide-dependent endogenous phosphorylation of rabbit myometrium membranes

The membrane-bound protein kinase activity in plasma membranes (PM) and sarcoplasmic reticulum (SR) of rabbit myometrium was revealed, which catalyzes the synthesis of protein phosphoester products. cAMP had no effect on the phosphorylation of membrane substrates by soluble protein kinases I and II as well as by the membrane-bound enzyme of SR. At the same time, cAMP (10(-8) stimulated by 200% the phosphorylation of sarcolemmal components at functional rest (FR). In preparations obtained from pregnant animals, cAMP (10(-8) and 10(-5) M) stimulated the phosphorylation of PM 7- and 3-fold, respectively. cGMP had no effect on the phosphorylation of PM and SR proteins at FR. At 10(-5) and 10(-8) M, cGMP stimulated endogenous phosphorylation of PM and SR 7- and 4-fold, respectively. In pregnancy, the degree of endogenous phosphorylation of PM and SR increased by 70% and 260% as compared to that at FR; the activity of soluble protein kinases decreased two times under these conditions. At FR, the sarcolemmal proteins with Mr 35 000, 57 000, 89 000 and 174 000 underwent phosphorylation. The phosphorylation of the proteins with Mr 35 000 and 57 000 was cAMP-dependent. In pregnant animals sarcolemma, the phosphorylation affected the proteins with Mr 47 000, 57 000 and 174 000 and was cAMP-dependent for the former two proteins and cGMP-dependent for the latter protein. At FR, two SR proteins with Mr 47 000 and 168 000, while in pregnant animals the proteins with Mr 47 000, 132 000 and 168 000 were phosphorylatable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of purified bovine cardiac sarcolemma and potassium-stimulated calcium uptake

Sarcolemmal vesicles were prepared from bovine cardiac muscle by differential and discontinuous sucrose density gradient centrifugation. Na+/K+-ATPase was purified 33-fold to a specific activity of 53 +/- 0.5 (12) mumol Pi X mg-1 X h-1, binding sites for strophantin 20-fold to a density of 56.3 +/- 5.3 (14) pmol/mg and that for the calcium antagonist nitrendipine 5.5-fold to a density of 0.72 +/- 0.07 (6) pmol/mg. The specific activity of the Na+/Ca2+ exchanger was 61.1 +/- 3.7 (6) nmol/mg. The vesicles had an intravesicular volume of 20 +/- 4 (4) microliter/mg and 56.9 +/- 6 (4)% of the vesicles were right-side-out oriented. Several peptides of the purified membranes were phosphorylated in the presence of Mg . ATP and EGTA. Most of the radioactive phosphate was incorporated into a peptide with an apparent molecular mass of 22 kDa. Denaturation of the membranes at 100 degrees C changed the mobility of this peptide to 15 kDa and 11 kDa. This peptide could not be distinguished from a sarcoplasmic reticulum peptide of similar molecular mass. The phosphorylation of the sarcolemmal peptide was stimulated by Ca2+/calmodulin, cAMP and the catalytic subunit of cAMP-dependent protein kinase. A comparison of the phosphorylation of sarcolemmal membranes with that of sarcoplasmic reticulum showed that Ca2+/calmodulin stimulated in each membrane, the phosphorylation of the 22-kDa peptide and a 44-kDa peptide, and in the sarcoplasmic reticulum the phosphorylation of an additional peptide of 55-kDa. Ca2+/calmodulin-dependent phosphorylation of a 55-kDa peptide could not be demonstrated in sarcolemma, regardless if sarcolemmal membranes were incubated together with sarcoplasmic reticulum or if the phosphorylation was carried out in the presence of purified cardiac myosin light chain kinase or phosphorylase kinase. 'Depolarization' induced Ca2+ uptake which was measured according to Bartschat, D.K., Cyr, D.L. and Lindenmayer, G.E. [(1980) J. Biol. Chem. 255, 10044-10047] was 5 nmol/mg protein. This uptake was not enhanced after preincubation of the vesicles with Mg . ATP or Mg . ATP and cAMP-dependent protein kinase. The value of 5 nmol/mg protein is in agreement with the theoretical amount of Ca2+ which can be accumulated by the bovine cardiac sarcolemma in the absence of a driving force other than the Ca2+ gradient. The potassium-stimulated Ca2+ uptake was not blocked by the organic Ca2+ channel blockers. Prolonged incubation of Mg . ATP with sarcolemmal vesicles in the presence of various ATPase inhibitors led to the hydrolysis of ATP. The liberated phosphate precipitated with Ca2+ in the presence of LaCl3. These precipitates amounted to an apparent Ca2+ uptake ranging from 50 to over 1000 nmol/mg. The results suggest that potassium-stimulated Ca2+ uptake of bovine cardiac sarcolemmal vesicles is not enhanced in the presence of ATP or by phosphorylation of a 22-kDa peptide.     

Phosphorylation of cardiac sarcolemma proteins in isadrine myocarditis

The incorporation of 33P into 54, 43 and 23 kDalton proteins of rabbit cardiac sarcolemma is demonstrated both in intact muscle and under isadrine myocarditis. The phosphorylation by cAMP-dependent protein kinase is shown only for the 23.5 kDalton protein. The level of this protein phosphorylation decreases under isadrine myocarditis in the presence of 10(-6) M cAMP and absence of "exogenic" protein kinase. In the preparations of the sarcolemma of the cardiac muscle under myocarditis the phosphorylation of the 23.5 kDalton protein is not stimulated by beta-agonists in the medium without "exogenic" protein kinase and cAMP. The latter being present, the phosphorylation reaches the control values.     

Membrane localization of myocardial type II cyclic AMP-dependent protein kinase activity

Crude cardiac membrane vesicles were separated into subfractions of sarcolemma and sarcoplasmic reticulum. The subfractions were used to determine the origin and type of cyclic AMP-dependent protein kinase activity present in myocardial membranes. A cyclic AMP-binding protein of molecular weight 55,000 was covalently labeled with the photoaffinity probe 8-azido adenosine 3',5'-mono[32P]phosphate, and found to copurify with the (Na+ + K+)-ATPase activity of sarcolemma, and away from the (Ca2+ + K+)-ATPase activity of sarcoplasmic reticulum. Endogenous cyclic AMP-dependent protein kinase activity also copurified with sarcolemma. Protein substrates phosphorylated by cyclic AMP-dependent protein kinase activity had apparent molecular weights of 21,000 and 8000 and were present in both sarcolemma and sarcoplasmic reticulum. However, while addition of cyclic AMP alone resulted in phosphorylation of sarcolemma proteins, both cyclic AMP and exogenous, soluble cyclic AMP-dependent kinase were required for phosphorylation of sarcoplasmic reticulum proteins. Addition of the calcium-binding protein, calmodulin, to either sarcolemma or sarcoplasmic reticulum resulted in phosphorylation of the 21,000 and 8000-dalton proteins, as well. The results suggest that cardiac sarcolemma contains an intrinsic type II cyclic AMP-dependent protein kinase activity that is not present in sarcoplasmic reticulum. On the other hand, Ca2+- and calmodulin-dependent protein kinase activity is present in both sarcolemma and sarcoplasmic reticulum.     

Characterization of the membrane-bound protein kinase C and its substrate proteins in canine cardiac sarcolemma

Cardiac sarcolemma was purified from canine ventricles. Enrichment of the sarcolemmal membranes was demonstrated by the high (Na+ + K+)-ATPase activity of 28.0 +/- 1.5 mumol Pi/mg protein per h and the high concentration of muscarinic receptors with the Bmax of 8.2 +/- 2.5 pmol/mg protein as determined by [3H]QNB binding. The purified sarcolemma also contains significant levels of a membrane-bound Ca2+ and phospholipid-dependent protein kinase (protein kinase C). To elucidate the protein kinase C activity in sarcolemma, a prior incubation of the membranes with EGTA and Triton X-100 was necessary. The specific activity of protein kinase C was found to be 131.4 pmol Pi/mg per min, in the presence of 6.25 micrograms phosphatidylserine and 0.5 mM CaCl2. Treatment of sarcolemma with 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBu2) resulted in a concentration-dependent activation of protein kinase C activity. The effect of TPA and PBu2 on protein kinase C in sarcolemma was independent of exogenous Ca2+ and phosphatidylserine. Polymyxin B inhibited phorbol-ester-induced activation of protein kinase C activity. The distribution of protein kinase C in the cytosolic fraction was also examined. The specific activity of the kinase in the cytosolic fraction was 59.7 pmol Pi/mg per min. However, the total protein kinase C activity in the cytosol was 213500 pmol Pi/min, compared to that of 1025 pmol Pi/min in the sarcolemma isolated from approx. 100 g of canine ventricular muscle. Several endogenous proteins in cardiac sarcolemma were phosphorylated in the presence of Ca2+ and phosphatidylserine. The major substrates for protein kinase C were proteins of Mr 94 000, 87 000, 78 000, 51 000, 46 000, 11 500 and 10 000. Most of these substrate proteins have not been identified before. Other proteins of Mr 38 000, 31 000 and 15 000 were markedly phosphorylated in the presence of Ca2+ only. Phosphorylation of phospholamban (Mr 27 000 and 11 000) was also stimulated in the presence of Ca2+ and phosphatidylserine, but the low Mr form of phospholamban was distinct from two other low Mr substrate proteins for protein kinase C. Polymyxin B was more selective in inhibiting the protein kinase C dependent phosphorylation. On the other hand, trifluoperazine selectively inhibited the phosphorylation of phospholamban and Mr 15 000 protein. Although the exact function of this kinase is unknown, based on these observations, we believe that protein kinase C in the cardiac sarcolemma may play an important role in the cell-surface-signal regulated cardiac function.     

Cyclic GMP- and AMP-modified calcium binding by the myocardial sarcolemma in circulatory hypoxia

10(-6) M cAMP were shown to induce a 61% and 21% increase in 45Ca binding to sarcolemma proteins in intact and injured (circulatory hypoxia) hearts, respectively. The addition of exogenous protein kinase equalized 45Ca-binding levels in normal and impaired sarcolemma. The decrease in 45Ca-binding capacity by 16 and 36% was detected in the presence of 10(-7) and 10(-6) M of cGMP, respectively. In impaired hearts, cAMP and Ca-ATPase activity levels remain constant, while cGMP content increases, as compared to normal myocardial level.     

Adenosine 3′,5′-monophosphate-dependent phosphorylation of a 6000 and A 22000 dalton protein from cardiac sarcoplasmic reticulum

In canine cardiac sarcoplasmic reticulum, adenosine 3′,5′-monophosphate (cyclic AMP)-dependent protein kinase specifically phosphorylates two proteins, as seen by sodium dodecyl sulfate-slab gel electrophoresis and autoradiography. One protein has a molecular weight ranging between 22 000 and 24 000 daltons and has previously been identified and named phospholamban (Tada, M., Kirchberger, M.A. and Katz, A.M. (1975) J. Biol. Chem. 250, 2640–2647). The other protein that the ³²P label incorporates into has a molecular weight of approximately 6000. Like the 22 000 dalton protein, the 6000 dalton protein has characteristic of phosphoester bonding. The time-dependent course of phosphorylation shows that initially the ³²P label is incorporated more rapidly into the 22 000 dalton protein than the 6000 dalton protein, with both proteins reaching a steady-state level of phosphorylation after 10 min of incubation. When both protein kinase and cyclic AMP are eliminated from the incubation medium, both the 22 000 and the 6000 dalton protein are still phosphorylated but only to about a quarter of the activity found when cyclic AMP and protein kinase are included in the incubation mixture. The addition of phosphodiesterase completely eliminates the phosphorylation of both proteins. Treating the microsomes with trypsin prevents subsequent phosphorylation of either protein. Phosphorylating the microsomes first, then treating with trypsin, renders both the 22 000 and the 6000 dalton proteins resistant to even prolonged trypsin attack. Unphosphorylated, both proteins are solubilized by a very low concentration of deoxycholate. After phosphorylation the proteins cannot be solubilized by deoxycholate. Phosphorylation appears to alter greatly the physical properties of these proteins.Control experiments exclude the possibility that a lipid is being phosphorylated. After phosphorylation, the phosphorylated 22 000 dalton protein is separated from the 6000 dalton protein by proteolipid extraction. After first treating the microsomes with methanol, the 22 000 dalton protein is then soluble in acidified chloroform/methanol, while the 6000 dalton protein remains insoluble. The finding that both proteins have much different biochemical properties when phosphorylated than when not, may be relevant in how they regulate calcium transport in the sarcoplasmic reticulum.     

Ca2+-phospholipid-dependent phosphorylation of vesicular preparations of myocardial sarcolemma

A Ca2+-phospholipid-dependent protein kinase C was isolated from the soluble fraction of bovine brain, using hydrophobic chromatography on phenyl-Sepharose CL-4B and high performance liquid chromatography on a Mono Q column. The enzyme had a specific activity of 822 nmol 32P/mg protein/min with histone H1 as a substrate. Phosphorylation of pig myocardium sarcolemma protein substrates was stimulated by Ca2+ and phosphatidylserine; the optimal concentrations of these compounds were 10(-4) M and 200 micrograms/ml, respectively. The value of Km(app) for Ca2+ was 3.10(-6) M. An addition of exogenous dioleine increased the enzyme affinity for Ca2+ which led to a decrease of Ca2+ concentration necessary for the maximal activation to occur. The optimal concentration of ATP needed for sarcolemmal preparation phosphorylation was 0.3-0.4 mM, which seems to be due to the high activity of sarcolemmal ATPases. The proteins phosphorylated in sarcolemmal preparations were identified, using SDS polyacrylamide gel electrophoresis with subsequent autoradiography. The 250, 140, 67, 58, 25 and 11 kD proteins appeared to be phosphorylated in the greatest degree. Since in myocardial sarcolemma protein kinase C predominantly phosphorylates the same proteins as does the cAMP-dependent protein kinase, it was assumed that protein kinase C can also play a role in the regulation of Ca2+-transporting systems of sarcolemma.     

cAMP-dependent phosphorylation inhibits potential-dependent passive transport of Ca2+ in myometrial sarcolemma vesicles

It is established that Ca2+ transport from the predominantly inverted vesicles of pig myometrium sarcolemma depends on the value of the membrane potential which is created on vesicles by the K+-valinomycin system. It is shown that variations in the membrane potential from -60 to +30 mV cause acceleration of the calcium transport from the vesicles, the maximal transport being observed at delta psi from 0 up to +30 mV. The endogenic and exogenic cAMP-dependent phosphorylation of plasma membrane proteins inhibits the passive transport of calcium at all the membrane potential values studied. A degree of potential-dependent Ca2+ transport inhibition correlates with the value of cAMP-dependent phosphorylation of sarcolemma proteins.     

Partial characterization of protein kinase-catalyzed phosphorylation of low molecular weight proteins in purified preparations of pigeon heart sarcolemma and sarcoplasmic reticulum.

Pigeon heart microsomes contain three minor size protein kinase substrates of minimal molecular weights of 22 000, 15 000, and 11500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the microsomes were partially loaded with calcium oxalate and subjected to rate zonal and isopycnic centrifugations in sucrose density gradient columns, the 22 000 and the 15 000 dalton proteins settled in the heaviest fraction, which was composed mainly of vesicles of sarcoplasmic reticular membranes; the 11 500 dalton protein was concentrated in the lightest fractions, which consisted chiefly of vesicles of sarcolemmal origin. During incubation of the membrane fractions with Mg [gamma-32P]ATP significant amounts of 32P were incorporated into all these proteins. Incorporation of 32P into the 15 000 dalton protein was moderately and 32P incorporation into the 22 000 dalton protein was markedly enhanced in the presence of exogenous soluble cyclic AMP-dependent protein kinase and cyclic AMP. The phosphorylation of the three proteins was virtually unaffected by Ca2+ concentrations up to 0.1 mM and by ethyleneglycol-bis-(beta-aminoethyl-ether)-N,N'-tetraacetic acid in the absence of added Ca2+. Phosphorylation of the 22 000 and the 11 500 dalton proteins occurred mainly at serine residues. In the 15 000 dalton protein threonine residues were the main site of endogenous phosphorylation. Nearly equal amounts of [32P]-phosphate were incorporated into threonine and serine residues of this protein, when phosphorylation was supported by exogenous cyclic AMP-dependent protein kinase and cyclic AMP. The 15 000 dalton protein could be removed from its membrane attachment by extraction with an acidic chloroform/methanol mixture. This step opens the way for the purification of this membrane-bound protein kinase substrate.     

Free radical production from the aerobic oxidation of reduced pyridine nucleotides catalysed by phenazine derivatives

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a (Ca2+ + Mg2+)-ATPase activity of about 10 nmol (min . mg)-1 and an ATP-dependent Ca2+ uptake of about 10 nmol (min . mg)-1. When incubated in the presence of Mg[gamma-32P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [32P]phosphate-labeled Ca2+ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca2+ or Ca2+ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of 125I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol (Mr = 94 000, 87 000, 60 000 and 43 000). Some minor calmodulin-binding proteins were enriched in the membrane fractions (Mr = 69 000, 57 000, 39 000 and 37 000). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca2+ uptake is essentially unaltered, while the Ca2+ capacity is diminished as a consequence of a doubling in the rate of Ca2+ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca2+ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 microM calmodulin result in increased levels of vesicle phosphorylation.     

A sarcolemma-associated inhibitor is capable of modulating calcium-dependent proteinase activity

Purified bovine myocardial sarcolemma vesicles were shown to contain calcium-dependent proteinase inhibitor protein by direct assay and by immunoblot analysis following gel electrophoresis (Western blotting). Calcium-dependent proteinase (calpain, EC 3.4.22.17) was not detected in the sarcolemma vesicles. The inhibitor protein was not solubilized when the vesicles were ruptured by repetitive freezing and thawing. However, a large amount of latent inhibitor activity was exposed after freezing and thawing the sarcolemma, and the inhibitor was much more susceptible to removal by 1.0 M NaCl or proteolysis following this treatment. Since the vesicles were predominantly right-side-out, the latter observations suggested that the inhibitor was associated with the cytoplasmic face of the sarcolemma. The endogenous inhibitor was capable of protecting sarcolemmal protein kinase C from proteolytic conversion to soluble protein kinase M by type I or type II calcium-dependent proteinase. Thus, the inhibitor is probably important in controlling calcium-dependent proteolysis of sarcolemmal proteins.     

Partial characterization of protein kinase-catalyzed phosphorylation of low molecular weight proteins in purified preparations of pigeon heart sarcolemma and sarcoplasmic reticulum

Pigeon heart microsomes contain three minor size protein kinase substrates of minimal molecular weights of 22 000, 15 000, and 11 500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the microsomes were partially loaded with calcium oxalate and subjected to rate zonal and isopynic centrifugations in sucrose density gradient columns, the 22 000 and the 15 000 dalton proteins settled in the heaviest fraction, which was composed mainly of vesicles of sarcoplasmic reticular membranes; the 11 500 dalton protein was concentrated in the lightest fractions, which consisted chiefly of vesicles of sarcolemmal origin. During incubation of the membrane fractions with Mg[γ-³²P]ATP significant amounts of ³²P were incorporated into all these proteins. Incorporation of ³²P into the 15 000 dalton protein was moderately and ³²P incorporation into the 22 000 dalton protein was markedly enhanced in the presence of exogenous soluble cyclic AMP-dependent protein kinase and cyclic AMP. The phosphorylation of the three proteins was virtually unaffected by CA²⁺ concentrations up to 0.1 mM and by ethyleneglycol-bis(β-aminoethylether)-N,N′-tetraacetic acid in the absence of added Ca²⁺.Phosphorylation of the 22 000 and the 11 500 dalton proteins occurred mainly at serine residues. In the 15 000 dalton protein threonine residues were the main site of endogenous phosphorylation. Nearly equal amounts of [³²P]-phosphate were incorporated into threonine and serine residues of this protein when phosphorylation was supported by exogenous cyclic AMP-dependent protein kinase and cyclic AMP.The 15 000 dalton protein could be removed from its membrane attachment by extraction with an acidic chloroform/methanol mixture. This step opens the way for the purification of this membrane-bound protein kinase substrate.     

Nucleoside-dependent protein kinase from Trypanosoma gambiense.

A nucleoside-dependent protein kinase (EC 2.7.1.37) was partially purified from Trypanosoma gambiense, the pathogenic agent of sleeping sickness. This enzyme catalyzes the phosphorylation of histone and protamine. Various nucleosides at the concentration of 10(-4) M stimulated the histone kinase activity about two-fold, whereas cyclic AMP and cyclic GMP were without effect. The pH-optimum for histone phosphorylation was at about pH 7.0. The enzyme activity absolutely depends on Mg2+, Mn2+ or Co2+. The apparent Km-value for histone was 0.3 mg/ml and those for ATP were 2 - 10(-4) M and 6 - 10(-5) M in the absence or presence of 10(-4) M adenosine respectively. IDP and ADP complete with ATP. The inhibition constants were calculated to be 2 - 10(-4) M and 2.5 - 10(-4) M, respectively. The molecular weight of the histone kinase was found to be 95 000 by gel filtration and 88 000 by sedimentation in a sucrose gradient.     

Physiological Ca2+ level and Ca2+-induced Permeability Transition Pore control protein phosphorylation in rat brain mitochondria

Phosphorylation of several low molecular mass proteins (3.5, 17, 23 and 29kDa) was observed in rat brain mitochondria (RBM) at ATP concentration close to that in the mitochondrial matrix. Furthermore, regulatory effects of Ca2+ on phosphorylation of these proteins were investigated. Protein phosphorylation was found to be modulated by Ca2+ in the physiological concentration range (10(-8) to 10(-6)M free Ca2+). Incorporation of 32P from [gamma-32P]ATP into the 17kDa protein was dramatically increased within the 10(-7) to 10(-6)M free Ca2+ range, whereas an opposite effect was observed for the 3.5kDa polypeptide. Strong de-phosphorylation of the 3.5kDa polypeptide and enhanced 32P-incorporation into the 17 and 23kDa proteins were found with supra-threshold Ca2+ loads and these effects were eliminated or reduced in the presence of cyclosporin A, an inhibitor of Permeability Transition Pore (PTP) opening. In the presence of calmidazolium (Cmz), a calmodulin antagonist, enhanced levels of phosphorylation of the 17 and 3.5kDa polypeptides were observed and the 17kDa protein phosphorylation was suppressed by H-8, a protein kinase A inhibitor. It is concluded that Ca2+ in physiological concentrations, as a second messenger, can control phosphorylation of the low molecular mass phospoproteins in RBM, in addition to well known regulation of some Krebs cycle dehydrogenases by Ca2+. The protein phosphorylation was strongly dependent on the Ca2+-induced PTP opening.     

Demonstration of cGMP-dependent protein kinase and cGMP-dependent phosphorylation in cell-free extracts of platelets

Homogenates, membranes and cytosol of rat and human platelets were found to contain cGMP-dependent protein kinase immunoreactivity. Specific cGMP-dependent protein kinase immunoreactivity was about 1.7 pmol protein kinase/mg protein for homogenates of human platelets and 0.7 pmol/mg for homogenates of rat platelets; the majority appeared to be associated with the membrane fraction. In membranes of platelets low concentrations of cAMP (0.5-2 microM) stimulated the phosphorylation of five major proteins with apparent relative molecular masses, Mr, of 240 000, 130 000, 50 000, 42 000 and 22 000 while low concentrations of cGMP (0.5-2 microM) stimulated the phosphorylation of three major proteins with apparent Mr of 130 000, 50 000 and 46 000. An affinity-purified antibody against the cGMP-dependent protein kinase was prepared which specifically inhibited the activity of cGMP-dependent protein kinase. In membranes of human platelets this affinity-purified antibody inhibited the cGMP-stimulated phosphorylation of the three proteins with Mr of 130 000, 50 000 and 46 000 while it had no effect on the cAMP-dependent and cyclic-nucleotide-independent protein phosphorylation. The results demonstrate that platelets contain a cGMP-dependent protein kinase and at least three specific substrates for this enzyme. Two of these substrates, the proteins with apparent molecular Mr of 130 000 and 50 000, are substrates for both cAMP- and cGMP-dependent protein kinase. The protein with apparent Mr of 130 000 appears to be closely related to an intrinsic plasma membrane protein of vascular smooth muscle cells which is a substrate for a membrane-associated cGMP-dependent protein kinase. Therefore, cGMP-dependent protein kinase and cGMP-regulated phosphoproteins may mediate in platelets the intracellular effects of those hormones, vasodilators and drugs which elevate the level of cGMP and inhibit platelet aggregation.     

The Ca2+-pumping ATPase and the major substrates of the cGMP-dependent protein kinase in smooth muscle sarcolemma are distinct entities

It has been proposed that the plasma membrane Ca2+ pump of smooth muscle tissues may be regulated by cGMP-dependent phosphorylation [Popescu, L. M., Panoiu, C., Hinescu, M. & Nutu, O. (1985) Eur. J. Pharmacol. 107, 393-394; Furukawa, K. & Nakamura, H. (1987) J. Biochem. (Tokyo) 101, 287-290]. This hypothesis has been tested on a smooth muscle sarcolemma preparation from pig thoracic aorta. The actomyosin-extracted membranes showed ATP-dependent Ca2+ uptake as well as cGMP-dependent protein kinase (G-kinase) activity. The molecular masses of the major protein substrates of the G-kinase (G1) and that of the Ca2+ pump were compared. Electrophoretic analysis of the phosphorylated intermediate of the sarcolemmal Ca2+-ATPase and the G1 phosphoprotein showed that these two proteins are not identical. The results were confirmed by using a 125I-calmodulin overlay technique and an antibody against human erythrocyte Ca2+-ATPase. Ca2+-uptake experiments with prephosphorylated membrane vesicles were carried out to elucidate possible effects of cGMP-dependent phosphorylation of membrane proteins on the activity of the Ca2+ pump. The cGMP-dependent phosphorylation was found to be extremely sensitive to temperature leading to very low steady-state phosphorylation levels at 37 degrees C. The difficulty was overcome by ATP[gamma S], which produced full and stable thiophosphorylation of G1 during the Ca2+-uptake experiments at 37 degrees C. However, the cGMP-dependent thiophosphorylation failed to influence the Ca2+-uptake properties of sarcolemmal vesicles. The results show that the Ca2+ pump of smooth muscle plasma membrane is not a direct target of the cGMP-dependent protein kinase and is not regulated by the cGMP-dependent phosphorylation of membrane proteins.