Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Accelerated calcium transport into the sarcoplasmic reticulum (SR) of the heart may mediate the inotropic actions of agents that act to increase adenosine 3',5'-monophosphate (cyclic AMP) within the cell. Studies in our laboratory have shown that ATP-dependent Ca uptake by cardiac microsomes rich in SR is enhanced by pretreatment with bovine cardiac cyclic AMP-dependent protein kinase (cyclic AMP-PK). Ca2+-activated ATPase is increased concomitantly with Ca uptake, stoichiometric coupling of 2 moles of Ca2+ taken up per mole of ATP hydrolyzed remaining constant. The steady state level of Ca binding is not increased by cyclic AMP-PK pretreatment, suggesting that the turnover rate of the transport system rather than the number of transport sites is increased. Phosphorylation of the SR by protein kinase is half-maximal at approximately 10(-7) M cyclic AMP, a value similar to that which gives half-maximal stimulation of both Ca uptake and Ca2+-activated ATPase. Over 80 percent of the 32P associated with membrane protein is identifiable as phosphoserine and phosphothreonine. The 32P is incorporated into a 22,000-dalton protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein, which we have tentatively named phospholamban (lambda alpha mu beta alpha psi usilon epsilon omega = to receive) appears to particiapte in the regulation of calcium transport by the heart's SR and may play a role in the inotropic actions of drugs, such as epinephrine, which act upon the cyclic AMP-PK system.
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PMID:Phospholamban: a regulatory protein of the cardiac sarcoplasmic reticulum. 12 51

Six electrophoretically distinct species of oligomeric phospholamban were identified immunologically following phosphorylation of sarcoplasmic reticulum vesicles by cAMP-dependent protein kinase. The phosphate content of each was determined, confirming that the discrete sequential retardation of phospholamban oligomers was the result of ascending mole ratios of phosphate (P0-P5) per oligomer. These data afford support to the pentameric arrangement of oligomeric phospholamban and offer a means of determining phosphorylation stoichiometry independent of the absolute phospholamban concentration. Detection of the relative concentration of individual species during phosphorylation facilitated the description of a random mechanism of phosphorylation by cAMP-dependent protein kinase. By contrast, dephosphorylation of cAMP-dependent protein kinase phosphorylated phospholamban was shown to exhibit strong positive cooperativity in its reaction mechanism.
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PMID:Immunological detection of phospholamban phosphorylation states facilitates the description of the mechanism of phosphorylation and dephosphorylation. 237 37

A Ca2+-calmodulin-dependent protein kinase was purified to apparent homogeneity from the cytosolic fraction of canine myocardium, with phospholamban as substrate. Purification involved sequential chromatography on DEAE-cellulose, calmodulin-agarose, DEAE-Bio-Gel A, and phosphocellulose. This procedure resulted in a 987-fold purification with a 5.4% yield. The purified enzyme migrated as a single band on native polyacrylamide gels, and it exhibited an apparent molecular weight of 550,000 upon gel filtration. Gel electrophoresis under denaturing conditions revealed a single protein band with Mr 55,000. The purified kinase could be autophosphorylated in a Ca2+-calmodulin-dependent manner, and under optimal conditions, 6 mol of Pi was incorporated per mole of 55,000-dalton subunit. The activity of the enzyme was dependent on Ca2+, calmodulin, and ATP.Mg2+. Other ions which could partially substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations were Sr2+ greater than Mn2+ greater than Zn2+ greater than Fe2+. The substrate specificity of the purified Ca2+-calmodulin-dependent protein kinase for cardiac proteins was determined by using phospholamban, troponin I, sarcoplasmic reticulum membranes, myofibrils, highly enriched sarcolemma, and mitochondria. The protein kinase could only phosphorylate phospholamban and troponin I either in their purified forms or in sarcoplasmic reticulum membranes and myofibrils, respectively. Exogenous proteins which could also be phosphorylated by the purified protein kinase were skeletal muscle glycogen synthase greater than gizzard myosin light chain greater than brain myelin basic protein greater than casein. However, phospholamban appeared to be phosphorylated with a higher rate as well as affinity than glycogen synthase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and characterization of a calcium-calmodulin-dependent phospholamban kinase from canine myocardium. 277 41

Abnormalities in calcium flux have been linked to abnormal contractile behavior of the heart in patients with congestive heart failure as well as in animal models. Decreased activity or levels of the calcium adenosine triphosphatase of the sarco(endo)plasmic reticulum (SERCA2) particularly have been known to cause a delay in calcium transients. The SERCA2 protein pumps 2 moles of calcium per mole of adenosine triphosphate (ATP) split from the cytoplasm into the sarcoplasmic reticulum, thus lowering the free cytoplasmic calcium concentration. It therefore is of interest to identify mechanisms by which SERCA activity could be increased in the heart. To determine influences of increased expression of the SERCA2 gene on calcium transient and contractile behavior, we constructed transgenic mice and rats expressing a SERCA2 transgene in their heart. In these animals, a 20% increase in SERCA levels occurs due to additional expression of the SERCA transgene. This leads to a corresponding increase in contractile activity as determined by the increase in left ventricular pressure measured as dP/dt(max) and decrease in diastolic ventricular pressure determined as dP/dt(min). Similarly, isolated cardiac myocytes obtained from the heart of transgenic mice showed an accelerated calcium transient and increased speed of shortening and relengthening as determined by edge detection. To determine if SERCA2 transgene expression could have a compensatory effect on the contractile behavior of the heart in transgenic mice expressing SERCA2, these mice were made hypothyroid, and papillary muscle function was determined. Contractile behavior of the papillary muscle of wild-type hypothyroid mice showed a significant increase in muscle relaxation time (RT50). In contrast, SERCA2 transgenic hypothyroid mice showed normal contractile behavior of papillary muscle. A compensatory effect of SERCA transgene expression was therefore demonstrated. In addition, we constructed transgenic rats expressing a SERCA2 transgene in which constriction of the ascending aorta induced cardiac hypertrophy and a delayed contraction of papillary muscle. In preliminary results, we found that SERCA2 transgenic rats submitted to ascending aortic constriction did not show the delayed relaxation of papillary muscle as was found in wild-type rats submitted to aortic constriction. In addition, adenoviral vectors expressing transgenes for calcium-handling proteins can be used to improve cardiac myocyte contraction. Adenoviruses expressing a SERCA transgene or a mutant phospholamban transgene exhibiting dominant negative action were used to infect isolated myocytes treated with a phorbol ester (phorbol 12-myristate 13-acetate), which delays the calcium transients. The calcium transients and contractile behavior of the isolated myocytes indicated that increased SERCA expression or increased expression of mutant phospholamban transgene led to increased SERCA2 activity, resulting in an increased contractile phenotype. Recent findings by other investigators also indicate that decreased SERCA2 activity can be increased under in vivo conditions using adenoviral vector-based SERCA2 expression. A gene therapy type of approach delivering increased amounts of SERCA or phospholamban mutants leading to increased SERCA activity should therefore be considered in the future.
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PMID:Calcium regulatory proteins and their alteration by transgenic approaches. 1075 May 95

We have developed a quantitative immunoblot method to measure the mole fraction of phospholamban (PLB) phosphorylated at Ser16 (X(p)) in biological samples. In cardiomyocytes, PLB phosphorylation activates the sarcoplasmic reticulum calcium ATPase (SERCA), which reduces cytoplasmic Ca(2+) to relax the heart during diastole. Unphosphorylated PLB (uPLB) inhibits SERCA at low [Ca(2+)] but phosphorylated PLB (pPLB) is less inhibitory, so myocardial physiology and pathology depend critically on X(p). Current methods of X(p) determination by immunoblot provide moderate precision but poor accuracy. We have solved this problem using purified uPLB and pPLB standards produced by solid-phase peptide synthesis. In each assay, a pair of blots is performed with identical standards and unknowns using antibodies partially selective for uPLB and pPLB, respectively. When performed on mixtures of uPLB and pPLB, the assay measures both total PLB (tPLB) and X(p) with accuracy of 96% or better. We assayed pig cardiac sarcoplasmic reticulum (SR) and found that X(p) varied widely among four animals, from 0.08 to 0.38, but there was remarkably little variation in the ratios of X(p)/tPLB and uPLB/SERCA, suggesting that PLB phosphorylation is tuned to maintain homeostasis in SERCA regulation.
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PMID:Accurate quantitation of phospholamban phosphorylation by immunoblot. 2236 95

We have used site-directed spin labeling and electron paramagnetic resonance (EPR) to map interactions between the transmembrane (TM) domains of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) and phospholamban (PLB) as affected by PLB phosphorylation. In the cardiac sarcoplasmic reticulum, PLB binding to SERCA results in Ca-dependent enzyme inhibition, which is reversed by PLB phosphorylation at Ser16. Previous spectroscopic studies on SERCA-PLB have largely focused on the cytoplasmic domain of PLB, showing that phosphorylation induces a structural shift in this domain relative to SERCA. However, SERCA inhibition is due entirely to TM domain interactions. Therefore, we focus here on PLB's TM domain, attaching Cys-reactive spin labels at five different positions. In each case, continuous-wave EPR indicated moderate spin-label mobility, with the addition of SERCA revealing two populations, one indistinguishable from PLB alone and another with more restricted rotational mobility, presumably due to SERCA-binding. Phosphorylation had no effect on the rotational mobility of either component but significantly decreased the mole fraction of the restricted component. Solvent-accessibility experiments using power-saturation EPR and saturation-recovery EPR confirmed that these two spectral components were SERCA-bound and unbound PLB and showed that phosphorylation increased the overall lipid accessibility of the TM domain by increasing the fraction of unbound PLB. However-based on these results-at physiological levels of SERCA and PLB, most SERCA would have bound PLB even after phosphorylation. Additionally, no structural shift in the TM domain of SERCA-bound PLB was detected, as there were no significant changes in membrane insertion depth or its accessibility. Therefore, we conclude that under physiological conditions, the phosphorylation of PLB induces little or no change in the interaction of the TM domain with SERCA, so relief of inhibition is predominantly due to the previously observed structural shift in the cytoplasmic domain.
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PMID:Effect of Phosphorylation on Interactions between Transmembrane Domains of SERCA and Phospholamban. 2987 8