Phosphorylation of the inhibitory subunit of troponin in perfused hearts of mice deficient in phosphorylase kinase. Evidence for the phosphorylation of troponin by adenosine 3':5'-phosphate-dependent protein kinase in vivo.
Ontology highlight
ABSTRACT:
SUBMITTER: England PJ
PROVIDER: S-EPMC1183765 | biostudies-other | 1977 Nov
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Cardiac phospholipid-sensitive Ca2+-dependent protein kinase phosphorylated cardiac troponin inhibitory subunit (troponin I) and tropomyosin-binding subunit (troponin T), present either as the free form or as the troponin-tropomyosin complex. Exhaustive phosphorylation of troponin I and of troponin T revealed that 1.7 and 2 mol of phosphate was incorporated/mol of the subunits respectively. Cyclic AMP-dependent protein kinase, though incorporating 0.8 mol of phosphate/mol of troponin I, was unable to phosphorylate troponin T. Phosphorylation of troponin I (apparent Km = 3.4 microM; Vmax. = 2.6 mumol/min per mg of enzyme) or troponin T (apparent Km = 0.3 microM; Vmax. = 0.5 mumol/min per mg of enzyme) by the Ca2+-dependent enzyme was inhibited by various agents, such as adriamycin, palmitoylcarnitine, trifluoperazine, melittin and N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide (compound W-7). Ca2+ antagonists (such as verapamil), forskolin and ouabain were ineffective. These findings indicate that troponin I and troponin T were effective substrates for this species of Ca2+-dependent protein kinase, suggesting its potential regulatory role in the contractile activity of myofibrils modulated by troponin.
| S-EPMC1154071 | biostudies-other
Project description:Rabbit skeletal-muscle troponin T was phosphorylated by a standard preparation of phosphorylase kinase [Cohen (1973) Eur. J. Biochem. 34, 1--14] and by fractions obtained after chromatography of phosphorylase kinase on phosphocellulose. The original preparation of phosphorylase kinase phosphorylated at least two sites, one of which was serine-1. The second and probably the third sites were presumably located in the peptide flanked by amino-acid residues 147 and 161 of troponin T. Fractions of phosphorylase kinase was adsorbed on phosphocellulose phosphorylated only the second site. Tightly adsorbed fractions possessed high troponin T kinase and phosvitin kinase activities and phosphorylated only serine-1 of troponin T. The results suggest that standard preparations of phosphorylase kinase are contaminated by troponin T kinase, which can phosphorylate serine-1 of troponin T.
| S-EPMC1162285 | biostudies-other
Project description:1. Rat hearts were perfused with 32Pi, and contractile force was increased by positive inotropic agents (agents that increase contractility). The inhibitory subunit of troponin (troponin I) was then isolated by affinity chromatography in 8M-urea, and its 32P content measured. Incorporation of phosphate into the subunit was calculated on the basis of the [gamma-32P]ATP specific radioactivity in the hearts. 2. When hearts were perfused with 30 nM-DL-isoprenaline (N-isopropylnoradrenaline), there was an increase in contractile force over 30s which was paralleled by an increase in troponin I phosphorylation. When hearts were perfused for 25s with increasing concentrations of isoprenaline from 1 NM to 0.6 muM, there was again a parallel increase in contractile force and troponin I phosphorylation. The maximum phosphorylation observed was 1.5 mol of phosphate/mol of troponin I, which was reached after 25s with 0.1 muM-isoprenaline. 3. Hearts were stimulated with a 15s pulse perfusion of 30nM-DL-isoprenaline. There was an increase in contractile force which was followed by a return to the control value within 50s. Troponin I phosphorylation increased to a plateau value which was reached within 30s, and remained constant for 60s after the isoprenaline pulse. Phosphorylase a and 3':5'-cyclic AMP concentration showed changes similar to that of the contractile force. There was no change in 3':5'-cyclic GMP concentration. 4. When hearts stimulated with a 15S pulse of isoprenaline were subsequently perfused with 0.6 muM-acetylcholine, the changes in contractile force, phosphorylase a and 3':5'-cyclic AMP were very similar to those seen with the 15s pulse of isoprenaline alone. Troponin I phosphorylation increased to a maximum 30s after the end of the isoprenaline pulse, but then rapidly decreased during the subsequent 30s. This decrease was preceded by a 60% increase in the concentration of 3':5'-cyclic GMP. 5. Hearts were perfused with 0.2 muM-glucagon for periods up to 60s. Contractile force showed little change for the first 30s, but then increased rapidly. This was paralleled by changes in 3':5'-cyclic AMP concentration. Troponin I phosphorylation increased slowly, but the increase in contractile force had reached a maximum before significant phosphorylation had occurred. 6. It is concluded that under certain conditions, e.g. immediately after beta-adrenergic stimulation, there is a good correlation between contractile force and troponin I phosphorylation. However, under other conditions, e.g. when contractile force is decreasing after removal of beta-adrenergic stimulation or in the presence of glucagon, contractile force and troponin I phosphorylation are not well correlated. These results suggest that mechanisms for modifying cardiac contractility, other than troponin I phosphorylation, must be present in rat heart.
| S-EPMC1164236 | biostudies-other
Project description:1. Incubation of rabbit cardiac-muscle troponin I with phosphorylase b kinase leads to the incorporation of .07-1.2 mol of Pi/mol. 2. The major site of phosphorylation is a serine residue at position 72. 3. Lesser amounts of phosphate are incorporated into threonine-138, threonine-162 and serine 20. 4. Serine-20 is the only site that contains a significant amount of phosphate before incubation with phosphorylase b kinase. 5. Unlike the situation with serine-20, the extent of phosphorylation of serine-72 and threonine-138 in the perfused rabbit heart does not change when the heart is exposed to adrenaline (4 microM).
| S-EPMC1162246 | biostudies-other
Project description:1. The phosphorylation of troponin T from rabbit white sketetal muscle is catalysed by phosphorylase kinase, but not at a significant rate by bovine 3':5'-cyclic AMP-dependent protein kinase. 2. The amino acid sequences adjacent to the three major phosphorylation sites of troponin T were determined. 3. The serine in the N-terminal peptide (Asx,SerP, Glx)Glu-Val-Glu, is that phosphorylated (SerP, phosphoserine) when the troponin complex is isolated. 4. The other two sites of phosphorylation are located in the sequence Ala-Leu-(Ser, SerP)-Met-Gly-Ala-Asn-Tyr(Ser,SerP)Tyr. 5. When troponin T is phosphorylated in the presence of troponin C, the extent of phosphorylation at each site is considerably decreased. 6. CNBr fragments of troponin T are also phosphorylated by phosphorylase kinase, but the rate of phosphorylation at each site in the CNBr fragments is considerably slower than in the native protein. 7. From these studies it is suggested that troponin C interacts with troponin T in the region containing the two closely situated phosphorylation sites.
| S-EPMC1164514 | biostudies-other
Project description:Phosphorylation of myofibrillar and sacroplasmic-reticulum (SR) proteins was studied in Langendorff-perfused rabbit hearts subjected to various inotropic interventions. Stimulation of hearts with isoprenaline resulted in the phosphorylation of both troponin I (TnI) and C-protein in myofibrils and phospholamban in SR. Phosphorylation of phospholamban could be reversed by a 15 min perfusion with drug-free buffer, after a 1 minute pulse perfusion with isoprenaline, at which time the mechanical effects of isoprenaline stimulation had also been reversed. However, both TnI and C-protein remained phosphorylated at this time. Moreover, the inhibition of Ca2+ activation of the Mg2+-dependent ATPase (Mg-ATPase) activity associated with myofibrillar phosphorylation persisted in myofibrils prepared from hearts frozen after 15 min of washout of isoprenaline. To assess the contribution of C-protein phosphorylation in the decrease of Ca2+ activation of the myofibrillar Mg-ATPase activity, we reconstituted a regulated actomyosin system in which only C-protein was phosphorylated. In this system, C-protein phosphorylation did not contribute to the decrease in Ca2+ activation of Mg-ATPase activity, indicating that TnI phosphorylation is responsible for the diminished sensitivity of the myofibrils to Ca2+. These observations support the hypothesis that phospholamban phosphorylation plays a more dominant role than TnI or C-protein phosphorylation in the mechanical response of the mammalian heart to beta-adrenergic stimulation.
| S-EPMC1148764 | biostudies-other
Project description:Schistosoma mansoni do not have de novo purine pathways and rely on purine salvage for their purine supply. It has been demonstrated that, unlike humans, the S. mansoni is able to produce adenine directly from adenosine, although the enzyme responsible for this activity was unknown. In the present work we show that S. mansoni 5´-deoxy-5´-methylthioadenosine phosphorylase (MTAP, E.C. 2.4.2.28) is capable of use adenosine as a substrate to the production of adenine. Through kinetics assays, we show that the Schistosoma mansoni MTAP (SmMTAP), unlike the mammalian MTAP, uses adenosine substrate with the same efficiency as MTA phosphorolysis, which suggests that this enzyme is part of the purine pathway salvage in S. mansoni and could be a promising target for anti-schistosoma therapies. Here, we present 13 SmMTAP structures from the wild type (WT), including three single and one double mutant, and generate a solid structural framework for structure description. These crystal structures of SmMTAP reveal that the active site contains three substitutions within and near the active site when compared to it mammalian counterpart, thus opening up the possibility of developing specific inhibitors to the parasite MTAP. The structural and kinetic data for 5 substrates reveal the structural basis for this interaction, providing substract for inteligent design of new compounds for block this enzyme activity.
| S-EPMC5147791 | biostudies-literature
Project description:Skeletal muscle phosphorylase kinase (PhK) is an (alphabetagammadelta) 4 hetero-oligomeric enzyme complex that phosphorylates and activates glycogen phosphorylase b (GP b) in a Ca (2+)-dependent reaction that couples muscle contraction with glycogen breakdown. GP b is PhK's only known in vivo substrate; however, given the great size and multiple subunits of the PhK complex, we screened muscle extracts for other potential targets. Extracts of P/J (control) and I/lnJ (PhK deficient) mice were incubated with [gamma- (32)P]ATP with or without Ca (2+) and compared to identify potential substrates. Candidate targets were resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified by matrix-assisted laser desorption ionization mass spectroscopy. In vitro studies showed GAPDH to be a Ca (2+)-dependent substrate of PhK, although the rate of phosphorylation is very slow. GAPDH does, however, bind tightly to PhK, inhibiting at low concentrations (IC 50 approximately 0.45 microM) PhK's conversion of GP b. When a short synthetic peptide substrate was substituted for GP b, the inhibition was negligible, suggesting that GAPDH may inhibit predominantly by binding to the PhK complex at a locus distinct from its active site on the gamma subunit. To test this notion, the PhK-GAPDH complex was incubated with a chemical cross-linker, and a dimer between the regulatory beta subunit of PhK and GAPDH was formed. This interaction was confirmed by the fact that a subcomplex of PhK missing the beta subunit, specifically an alphagammadelta subcomplex, was unable to phosphorylate GAPDH, even though it is catalytically active toward GP b. Moreover, GAPDH had no effect on the conversion of GP b by the alphagammadelta subcomplex. The interactions described herein between the beta subunit of PhK and GAPDH provide a possible mechanism for the direct linkage of glycogenolysis and glycolysis in skeletal muscle.
| S-EPMC3674776 | biostudies-literature
Project description:Phosphorylation of phospholamban (PLB), a membrane-bound 15 kDa protein and troponin I (TNI) was studied in isolated perfused rat hearts by using the back-phosphorylation technique with [32P]ATP catalysed by an excess of exogenous catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase, followed by protein separation. This standardized method allows the quantitative detection of protein phosphorylation specifically stimulated by cAMP. In control hearts the extent of specific phosphorylation was equivalent to 3.3 nmol of PLB and 11.0 mumol of TNI per g of cardiac tissue. In hearts freeze-clamped 30 s after exposure to isoprenaline (10 pM-10 microM), there was a dose-dependent decrease in phosphate incorporation in vitro, indicating a phosphorylation of the respective proteins in vivo. A differential sensitivity of TNI and PLB phosphorylation towards the beta-adrenergic agonist and the subsequent increase in tissue cAMP was found, favouring TNI phosphorylation. K0.5 values for isoprenaline were 2.94 +/- 0.04 nM and 4.46 +/- 0.24 nM for PLB and the 15 kDa protein, but 0.13 +/- 0.01 nM for TNI phosphorylation in the intact tissue. At an isoprenaline-induced increase in cAMP less than 3 pmol/mg of protein there was no or only a small increase in PLB phosphorylation, whereas TNI phosphorylation was nearly maximal. By plotting phosphorylation data against changes in contractile parameters a strong correlation was obtained for TNI (r = 0.95), assuming a linear relationship. For PLB a complex relationship is likely to exist. Our data (i) indicate a functional compartmentalization of the cAMP signal cascade and (ii) confirm that phosphorylation of TNI rather than of PLB is related to changes in mechanical myocardial responses.
| S-EPMC1131103 | biostudies-other
Project description:Phosphorylase kinase (PhK) coordinates hormonal and neuronal signals to initiate the breakdown of glycogen. The enzyme catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a. We present a 9.9 angstroms resolution structure of PhK heterotetramer (alphabetagammadelta)4 determined by cryo-electron microscopy single-particle reconstruction. The enzyme has a butterfly-like shape comprising two lobes with 222 symmetry. This three-dimensional structure has allowed us to dock the catalytic gamma subunit to the PhK holoenzyme at a location that is toward the ends of the lobes. We have also determined the structure of PhK decorated with GPb at 18 angstroms resolution, which shows the location of the substrate near the kinase subunit. The PhK preparation contained a number of smaller particles whose structure at 9.8 angstroms resolution was consistent with a proteolysed activated form of PhK that had lost the alpha subunits and possibly the gamma subunits.
| S-EPMC2639635 | biostudies-literature