Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A rat heart sarcolemmal preparation could be obtained in which both 5'-nucleotidase and adenylate cyclase were enriched approx. 9-fold by subjecting a homogenate to a discontinuous sucrose gradient, without the use of a high salt extraction. After incubation of this fraction with Mg[gamma-32P]ATP, the majority of 32P incorporated was present in 24 000- and 9000-dalton protein components. Only when a heart cytosol fraction or a purified cyclic AMP-dependent protein kinase was added, was enhancement of 32P-incorporaton found by addition of cyclic AMP. The 9000- and 24 000-dalton proteins appeared to be interconvertible. The degree of conversion could be affected by changing the temperature during solubilizaion of the membranes in SDS prior to electrophoresis. This suggested that the 24 000-dalton protein does not correspond to phospholamban, first identified by others in canine heart sarcoplasmic reticulum. Moreover, it could be excluded that the 24 000-dalton protein was derived from contaminating myofibrillar troponin I. When the sarcolemmal fraction was preincubated with Ca2+, Mg2+, ATP and oxalate, contaminating sarcoplasmic reticulum vesicles, loaded with calcium oxalate, settled to a greater density in the sucrose gradient. Membrane constituents other than those with enzymatic activity were monitored to confirm the separation between sarcolemmal and sarcoplasmic reticulum membranes: Coomassie blue staining material, sialic acid, cholesterol and phospholipid. The 24 000- and 9000-dalton proteins were equally distributed among the sarolemmal and sarcoplasmic reticulum fractions present in the sucrose gradient. However, the rate of 32P-incorporation in the presence of heart cytosol fraction was much slowr in the sarcoplasmic reticulum than in the sarcolemmal fraction.
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PMID:Phosphorylation of low molecular weight proteins in purified preparations of rat heart sarcolemma and sarcoplasmic reticulum. 625

1 A microsomal fraction was prepared from human umbilical arteries by differential centrifugation. The preparation was capable of an oxalate-stimulated Ca2+ uptake at a mean rate of 0.74 nmol Ca2+ mg-1 protein min-1 which could be inhibited by a Ca2+ ionophore, A 23 187, and by Tween 80. 2 The rate of Ca2+ uptake in the fraction obtained by density gradient fractionation paralleled 5'-nucleotidase activity suggesting that vesicles of predominantly sarcolemmal origin were responsible for the microsomal Ca2+ uptake. 3 Cyclic adenosine 3',5'-monophosphate-dependent protein kinase enhanced membrane phosphorylation but did not affect Ca2+ uptake. Preincubation with alkaline phosphatase reduced membrane phosphorylation to a greater extent than Ca2+ uptake. These data are not in favour of a close correlation between Ca2+ uptake and phosphorylation. 4 None of 15 vasodilator drugs (bencyclane, carbocromen, diazoxide, dilazep, hydralazine, indapamide, isosorbide dinitrate, methyl-isobutyl-xanthine, minoxidil, naftidrofuryl, nitroglycerine, prenylamine, sodium nitroprusside, tetracaine, and verapamil) had any effect on Ca2+ uptake at 10(-5) M. This suggests that vasodilator drugs do not act by a direct influence on the Ca2+ pumps of vascular smooth muscle cells.
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PMID:Effects of vasodilator drugs, alkaline phosphatase, and cyclic AMP-dependent protein kinase on the 45calcium uptake of sarcolemmal microsomes from human umbilical arteries. 625 79

In previous efforts to characterize sarcoplasmic reticulum function in human muscles, it has not been possible to distinguish the relative contributions of fast-twitch and slow-twitch fibers. In this study, we have used light scattering and 45Ca to monitor Ca accumulation by the sarcoplasmic reticulum of isolated, chemically skinned human muscle fibers in the presence and absence of oxalate. Oxalate (5 mM) increased the capacity for Ca accumulation by a factor of 35 and made it possible to assess both rate of Ca uptake and relative sarcoplasmic reticulum volume in individual fibers. At a fixed ionized Ca concentration, the rate and maximal capacity (an index of sarcoplasmic reticulum volume) both varied over a wide range, but fibers fell into two distinct groups (fast and slow). Between the two groups, there was a 2- to 2.5-fold difference in oxalate-supported Ca uptake rates, but no difference in average sarcoplasmic reticulum volumes. Intrinsic differences in sarcoplasmic reticulum function (Vmax, K0.5, and n) were sought to account for the distinction between fast and slow groups. In both groups, rate of Ca accumulation increased sigmoidally as [Ca++] was increased from 0.1 to 1 microM. Apparent affinities for Ca++ (K0.5) were similar in the two groups, but slow fibers had a lower Vmax and larger n values. Slow fibers also differed from fast fibers in responding with enhanced Ca uptake upon addition of cyclic AMP (10(-6) M, alone or with protein kinase). Acceleration by cyclic AMP was adequate to account for adrenaline-induced increases in relaxation rates previously observed in human muscles containing mixtures in fast-twitch and slow-twitch fibers.
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PMID:Calcium accumulation by the sarcoplasmic reticulum in two populations of chemically skinned human muscle fibers. Effects of calcium and cyclic AMP. 627 58

Highly purified sarcolemmal membranes were prepared from pig heart homogenates by differential and density gradient centrifugations. The membrane fragments exhibit ATP-dependent Ca2+-transport and Na+/Ca2+-exchange activities. ATP-dependent Ca2+-transport (K0.5Ca2+ = 0.3 microM; Vmax = 4.6 nmol Ca2+.mg protein-1.min-1) is not stimulated by oxalate. Ca2+-uptake is also not supported by p-nitro-phenylphosphate. Preincubation of sarcolemma with MgATP, calmodulin and catalytic subunit of cyclic AMP-dependent protein kinase stimulates active Ca2+-transport 1.8-fold. The effects of calmodulin and catalytic subunit are potentiating rather than additive. A large portion of the Ca2+ additionally accumulated after prephosphorylation of membranes is exchangeable for Na+ via the Na+/Ca2+-exchange system.
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PMID:Potentiating effect of calmodulin and catalytic subunit of cyclic AMP-dependent protein kinase on ATP-dependent Ca2+-transport by cardiac sarcolemma. 629 9

Two substrate proteins for cAMP-dependent protein kinase detected in a rat heart sarcolemma preparation displayed molecular weights of 24,000 and 9000 in sodium dodecyl sulfate gels and were shown to be interconvertible. The 9000-dalton protein could readily be separated from other low molecular weight phosphoproteins (mol. wt. 14,000 and 7000) by the use of 15% polyacrylamide gels. In addition to an endogenous cAMP-dependent protein kinase the membrane preparation also contained a protein-phosphorylation system that required Ca2+ and calmodulin. It appeared that both 24,000- and 55,000-dalton proteins were substrates for the endogenous Ca2+- and calmodulin-dependent protein kinase. Contaminating sarcoplasmic reticulum vesicles, first loaded with calcium oxalate, could be separated from the enriched sarcolemma preparation by sucrose gradient centrifugation. The separation was confirmed by comparative analysis of 5'-nucleotidase, Na+ -Ca2+ antiporter, and (Ca2+ + Mg2+)-dependent ATPase activities and by determination of gel electrophoretic (phospho)protein composition, sialic acid, cholesterol, and phospholipid contents. The 24,000-dalton phosphoprotein complex was equally distributed between sarcolemmal and sarcoplasmic reticulum fractions, whereas the 55,000- and 7000-dalton proteins were predominantly found in the sarcolemmal fraction. The 24,000-dalton protein was most likely phospholamban, because no other phosphoprotein was found in the 20,000 molecular weight range.
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PMID:Phosphorylation of low-molecular-weight proteins in preparations of rat heart sarcolemma and sarcoplasmic reticulum. 630 73

A procedure has been developed for isolating canine cardiac sarcoplasmic reticulum with considerably improved Ca2+ transport properties and stability. Contamination by mitochondria and sarcolemma is low, and the preparation is at least 85% pure sarcoplasmic reticulum. The preparation exhibits efficient, high activity ATP-dependent, oxalate-facilitated Ca2+ accumulation. At 13-16 microM ionized Ca2+, loading at 37 degrees C is 2.55 +/- 0.08 mumol of Ca2+/mg of protein in 1 min and reaches 9.08 +/- 0.64 mumol of Ca2+/mg of protein. Approximately 1 mol of Ca2+ is transported per mol of ATP hydrolyzed. Ca2+-insensitive ATPase is low. The Ca2+ loading rate and the Ca2+/ATP efficiency are increased by addition of ryanodine. At 1.2 microM ionized Ca2+, where the control rate is significantly higher than values previously reported, the Ca2+ transport rate is further increased 64% by calmodulin, 2.3-fold by cAMP plus cAMP-dependent protein kinase, and 2.5-fold by the combination of these components. The preparation is stable for 24 h at room temperature and for 48 hr at 0 degrees C and can be stored at -70 degrees C with retention of function for more than 1 month. The preparation is further characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by phospholipid analysis, and by thin section, freeze-fracture, and negative staining electron microscopy. The analyses indicate that the orientation and turnover number of the cardiac Ca2+ pump protein are similar to those of the skeletal muscle enzyme, and that the major factor in the lower Ca2+ transport rate of cardiac sarcoplasmic reticulum is a lower density of Ca2+ pump polypeptides in the membrane.
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PMID:Isolation and characterization of canine cardiac sarcoplasmic reticulum with improved Ca2+ transport properties. 630 48

The rate of calcium transport by sarcoplasmic reticulum vesicles from dog heart assayed at 25 degrees C, pH 7.0, in the presence of oxalate and a low free Ca2+ concentration (approx. 0.5 microM) was increased from 0.091 to 0.162 mumol . mg-1 . min-1 with 100 nM calmodulin, when the calcium-, calmodulin-dependent phosphorylation was carried out prior to the determination of calcium uptake in the presence of a higher concentration of free Ca2+ (preincubation with magnesium, ATP and 100 microM CaCl2; approx. 75 microM free Ca2+). Half-maximal activation of calcium uptake occurs under these conditions at 10-20 nM calmodulin. The rate of calcium-activated ATP hydrolysis by the Ca2+-, Mg2+-dependent transport ATPase of sarcoplasmic reticulum was increased by 100 nM calmodulin in parallel with the increase in calcium transport; calcium-independent ATP splitting was unaffected. The calcium-, calmodulin-dependent phosphorylation of sarcoplasmic reticulum, preincubated with approx. 75 microM Ca2+ and assayed at approx. 10 microM Ca2+ approaches maximally 3 nmol/mg protein, with a half-maximal activation at about 8 nM calmodulin; it is abolished by 0.5 mM trifluperazine. More than 90% of the incorporated [32P]phosphate is confined to a 9-11 kDa protein, which is also phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and most probably represents a subunit of phospholamban. The stimulatory effect of 100 nM calmodulin on the rate of calcium uptake assayed at 0.5 microM Ca2+ was smaller following preincubation of sarcoplasmic reticulum vesicles with calmodulin in the presence of approx. 75 microM Ca2+, but in the absence of ATP, and was associated with a significant degree of calmodulin-dependent phosphorylation. However, the stimulatory effect on calcium uptake and that on calmodulin-dependent phosphorylation were both absent after preincubation with calmodulin, without calcium and ATP, suggestive of a causal relationship between these processes.
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PMID:Calmodulin-dependent elevation of calcium transport associated with calmodulin-dependent phosphorylation in cardiac sarcoplasmic reticulum. 630 68

A microsomal fraction was isolated from guinea pig taenia caecum by differential centrifugation. Activities of ouabain-sensitive (Na+, K+)-ATPase, 5'-nucleotidase and NADPH-cytochrome c reductase were enriched in the microsomal fraction. On the other hand, less cytochrome c oxidase and monoamine oxidase were contained in this fraction. These results suggest that the microsomal fraction used in this study was derived from both sarcolemma and sarcoplasmic reticulum. Ca2+ uptake by this fraction was strictly dependent on the presence of ATP and was facilitated by oxalate. An ATP-regenerating system was required for the determination of Ca2+ uptake, when a lower concentration of ATP (e.g. 0.25 mM) was used. Phosphorylation of the microsomal fraction was doubled when these membranes were incubated in the presence of cyclic AMP plus cyclic AMP-dependent protein kinase (protein kinase). When the microsomal fraction was pretreated with cyclic AMP plus protein kinase, Ca2+ uptake was stimulated. The increases in microsomal phosphorylation and Ca2+ uptake were significantly correlated (P less than 0.01). This stimulation of Ca2+ uptake by microsomal phosphorylation was observed only in the presence of protein kinase, oxalate, and low Ca2+ and Mg2+ concentrations. The results suggest that stimulation of Ca2+ uptake may be the mechanism by which cyclic AMP is involved in beta-adrenergic relaxation of smooth muscle.
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PMID:Effects of cyclic AMP and protein kinase on calcium uptake in a microsomal fraction from guinea pig taenia caecum. 631 21

Calcium efflux and EGTA-induced calcium release from an internal platelet membrane fraction have been studied after the oxalate-supported calcium uptake had reached steady state. Increasing external calcium concentrations stimulate the calcium efflux velocity, with an apparent half-maximal stimulation at about 5 microM outside calcium concentration and a maximal velocity of calcium efflux of 4.66 +/- 2.32 nmol X min-1 X mg-1. Moreover, the ratio of the liberated calcium on the loaded calcium seems to be independent of the increasing external calcium concentration. Increasing the calculated internal calcium concentration by varying the oxalate potassium concentration from 10 mM to 1 mM results in an increase of the liberated calcium from the membrane vesicles from 7.4% to 63%, respectively, without changing the calcium efflux velocity. Similar conclusions can be drawn from the observation of results from the calcium efflux and EGTA-induced calcium release methods. Moreover, calcium pump reversal does not seem to be responsible for the calcium efflux or calcium release. All these different points added to the previously described regulation of calcium efflux by the catalytic subunit of cAMP protein kinase suggest us that the mechanism of calcium liberation by the platelet membranes is different from the calcium uptake.
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PMID:Characterization of calcium liberation from a human platelet membrane fraction. 642 15

The oxalate transport system along with protein phosphorylation appears to be deranged in stone formers. This study was undertaken to characterize in LLC-PK1 cells in culture the effect of altering specific intracellular second messenger systems on oxalate uptake. Cellular uptake experiments were performed at 37 degrees C in buffer [265 mM mannitol, 5 mM NaOH, 5 mM KOH, 10 mM Ca-EGTA, 25 mM HEPES/TRIS, pH = 7.4 or in Hank's balanced salt solution (HBSS)] containing 200 microM labeled oxalate (1-14C, 0.3 microCi). Cells were preincubated with DAG (final concentration of 100 microM), phorbol myristate acetate (10 microM), forskolin (50 microM), 8-bromo-cyclic AMP (50 microM), trifluoroperazine (20 microM) and low molecular weight heparin (1 mg/ml) for 10 min in the presence and absence of the anion transport inhibitor DIDS (100 microM) and the effect(s) on oxalate uptake at 10, 25 and 45 min incubation were determined. Chemicals (DAG, forskolin, TPA and 8-bromo-cAMP) which stimulate protein kinase A or C activity resulted in an increased uptake of oxalate while inhibitors of these systems (trifluoroperazine and low molecular weight heparin) resulted in decreased oxalate uptake. The results demonstrate that oxalate uptake in renal tubular cells is modulated by protein kinase C and A dependent mechanisms.
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PMID:Effect of second messenger systems on oxalate uptake in renal epithelial cells. 767 38


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