Pharmacological characterization of protein phosphatase activities in preparations from failing human hearts.
(1/155)
beta-Adrenoceptor stimulation acts in the heart in part by increasing the phosphorylation state of phospholamban and phospholemman. There is evidence that the beta-adrenoceptor-mediated increase in phospholamban phosphorylation is in part due to inhibition of type 1 phosphatases. The aim of the present study was to elucidate which phosphatases dephosphorylate phospholamban and phospholemman in the human heart. In the past, cardiac serine/threonine phosphatases have been studied using phosphorylase a as substrate. Here, type 1 and type 2A phosphatase activities were studied in preparations from failing human hearts using phosphorylated phospholamban and phospholemman as substrates. Phospholamban and phospholemman phosphatase activity was detectable in human cardiac homogenates. Moreover, using a heparin-Sepharose column, the catalytic subunits of type 1 and type 2A phosphatases could be separated from human ventricles. Okadaic acid and cantharidin inhibited phosphatase activities dephosphorylating phospholamban, phospholemman, and phosphorylase a in homogenates in a concentration-dependent manner. However, okadaic acid was more potent. Cantharidin inhibited type 2A and type 1 activities against all substrates studied with IC50 values <15 nM and >290 nM, respectively. Okadaic acid inhibited type 1 and type 2A phosphatase activities as effectively but 10-30 times more potently than cantharidin. This work provides evidence that in the human heart, type 1 and 2A phosphatases are involved in the dephosphorylation of phospholamban and phospholemman and could play a role in the effects of beta-adrenergic stimulation in the heart. (+info)
Kinetic study on the dimer-tetramer interconversion of glycogen phosphorylase a.
(2/155)
Kinetic theory of dissociating enzyme systems has been applied to a study of the dimer-tetramer interconversion of glycogen phosphorylase a. All kinetic constants for the dissociating-associating reaction of phosphorylase a have been determined. The results indicate that (a) the presence of glucose-1-phosphate has no influence on either the rate of dissociation or the rate of association, and hence does not shift the dimer-tetramer equilibrium of phosphorylase a; (b) the binding og glycogen to the enzyme decreases the association rate of the dimer to form the tetramer, but has no effect on the dissociation rate of the tetramer; (c) both the dimeric and tetrameric form of phosphorylase a can bind glycogen, but the tetrameric form has a lower affinity for glycogen and is catalytically inactive. (+info)
Effects of microcystins on phosphorylase-a binding to phosphatase-2A: kinetic analysis by surface plasmon resonance biosensor.
(3/155)
Cyclic heptapeptide microcystins are a group of hepatoxicants which exert the cytotoxic effects by inhibiting the catalytic activities of phosphatase-2A (PP-2A) and phosphatase-1 (PP-1) and thus disrupt the normal signal transduction pathways. Microcystins interact with PP-2A and PP-1 by a two-step mechanism involving rapid binding and inactivation of protein phosphatase catalytic subunit, followed by a slower covalent interaction. It was proposed that inactivation of PP-2A/PP-1 catalytic activity by microcystins precedes covalent adduct formation. In this study, we used a biosensor based on surface plasmon resonance (SPR) to examine the effects of three microcystins, MCLR, MCRR and MCYR, on the binding between PP-2A and its substrate, phosphorylase-a (PL-a), during the first step of the interaction. The SPR biosensor provides real-time information on the association and dissociation kinetics of PL-a with immobilized PP-2A in the absence and presence of microcystins. It was found that the affinity of PL-a to microcystin-bound PP-2A was four times smaller compared to unbound PP-2A, due to 50% decreases in the association rates and two-fold increases in dissociation rates of PL-a binding to PP-2A. The results suggest that the rapid binding of microcystins to the PP-2A catalytic site leads to the formation of a noncovalent microcystin/PP-2A adduct. While the adduct formation fully inhibits the catalytic activity of PP-2A, it only results in partial inhibition of the substrate binding. The similar effects of the three microcystins on PP-2A suggest that the toxins bind to PP-2A at the same site and cause similar conformational changes. The present work also demonstrates the potential application of biosensor technology in environmental toxicological research. (+info)
beta2-adrenergic cAMP signaling is uncoupled from phosphorylation of cytoplasmic proteins in canine heart.
(4/155)
BACKGROUND: Recent studies of beta-adrenergic receptor (beta-AR) subtype signaling in in vitro preparations have raised doubts as to whether the cAMP/protein kinase A (PKA) signaling is activated in the same manner in response to beta2-AR versus beta1-AR stimulation. METHODS AND RESULTS: The present study compared, in the intact dog, the magnitude and characteristics of chronotropic, inotropic, and lusitropic effects of cAMP accumulation, PKA activation, and PKA-dependent phosphorylation of key effector proteins in response to beta-AR subtype stimulation. In addition, many of these parameters and L-type Ca2+ current (ICa) were also measured in single canine ventricular myocytes. The results indicate that although the cAMP/PKA-dependent phosphorylation cascade activated by beta1-AR stimulation could explain the resultant modulation of cardiac function, substantial beta2-AR-mediated chronotropic, inotropic, and lusitropic responses occurred in the absence of PKA activation and phosphorylation of nonsarcolemmal proteins, including phospholamban, troponin I, C protein, and glycogen phosphorylase kinase. However, in single canine myocytes, we found that beta2-AR-stimulated increases in both ICa and contraction were abolished by PKA inhibition. Thus, the beta2-AR-directed cAMP/PKA signaling modulates sarcolemmal L-type Ca2+ channels but does not regulate PKA-dependent phosphorylation of cytoplasmic proteins. CONCLUSIONS: These results indicate that the dissociation of beta2-AR signaling from cAMP regulatory systems is only apparent and that beta2-AR-stimulated cAMP/PKA signaling is uncoupled from phosphorylation of nonsarcolemmal regulatory proteins involved in excitation-contraction coupling. (+info)
The carboxyl-terminal region of the retinoblastoma protein binds non-competitively to protein phosphatase type 1alpha and inhibits catalytic activity.
(5/155)
pRB, a negative-growth regulatory protein, is a demonstrated substrate for type 1 serine/threonine protein phosphatases (PP1). In a recent report from this laboratory, we demonstrated that select forms of phosphorylated as well as hypophosphorylated pRB can be found complexed with the alpha-isotype of PP1 (PP1alpha). This complex can also be observed when PP1 is rendered catalytically dead by toxin inhibition. These data suggested to us that pRB may bind to PP1 at one or more sites other than the catalytically active one on the enzyme and that such binding may play a role other than bringing the substrate into contact with the enzyme to facilitate catalysis. To address this possibility we utilized a series of pRB deletion mutants and coprecipitation studies to map the pRB domain involved in binding to PP1. Together with competition assays using in vivo expression of SV40 T-antigen, we show here that the carboxyl-terminal region of pRB is both necessary and sufficient for physical interaction with PP1. Subsequent biochemical analyses demonstrated inhibition of PP1 catalytic activity toward the standard substrate phosphorylase a when this enzyme is bound to pRB containing this region. K(m) and V(max) calculations revealed that pRB binds to PP1 in a non-competitive manner. These data support the notion that pRB, in addition to being a substrate for PP1, also functions as a PP1 inhibitor. The significance of this finding with respect to the functional importance of this interaction is discussed. (+info)
Molecular mode of inhibition of glycogenolysis in rat liver by the dihydropyridine derivative, BAY R3401: inhibition and inactivation of glycogen phosphorylase by an activated metabolite.
(6/155)
The racemic prodrug BAY R3401 suppresses hepatic glycogenolysis. BAY W1807, the active metabolite of BAY R3401, inhibits muscle glycogen phosphorylase a and b. We investigated whether BAY R3401 reduces hepatic glycogenolysis by allosteric inhibition or by phosphatase-catalyzed inactivation of phosphorylase. In gel-filtered liver extracts, racemic BAY U6751 (containing active BAY W1807) was tested for inhibition of phosphorylase in the glycogenolytic (in which only phosphorylase a is active) and glycogen-synthetic (for the evaluation of a:b ratios) directions. Phosphorylase inactivation by endogenous phosphatase was also studied. In liver extracts, BAY U6751 (0.9-36 micromol/l) inhibited glycogen synthesis by phosphorylase b (notwithstanding the inclusion of AMP), but not by phosphorylase a. Inhibition of phosphorylase-a-catalyzed glycogenolysis was partially relieved by AMP (500 micromol/l). BAY U6751 facilitated phosphorylase-a dephosphorylation. Isolated hepatocytes and perfused livers were tested for BAY R3401-induced changes in phosphorylase-a:b ratios and glycogenolytic output. Though ineffective in extracts, BAY R3401 (0.25 micromol/l-0.5 mmol/l) promoted phosphorylase-a dephosphorylation in hepatocytes. In perfused livers exposed to dibutyryl cAMP (100 micromol/l) for maximal activation of phosphorylase, BAY R3401 (125 micromol/l) inactivated phosphorylase by 63% but glucose output dropped by 83%. Inhibition of glycogenolysis suppressed glucose-6-phosphate (G6P) levels. Activation of glycogen synthase after phosphorylase inactivation depended on the maintenance of G6P levels by supplementing glucose (50 mmol/l). We conclude that the metabolites of BAY R3401 suppress hepatic glycogenolysis by allosteric inhibition and by the dephosphorylation of phosphorylase a. (+info)
Uric acid inhibits liver phosphorylase a activity under simulated in vivo conditions.
(7/155)
We have reported that glycogen synthesis and degradation can occur in vivo without a significant change in the amount of phosphorylase a present. These data suggest the presence of a regulatable mechanism for inhibiting phosphorylase a activity in vivo. Several effectors have been described. AMP stimulates, whereas ADP, ATP, and glucose inhibit activity. Of these effectors, only the glucose concentration changes under normal conditions; thus it could regulate phosphorylase a activity in vivo. We previously have reported that, when all of these effectors were present at physiological concentrations, the net effect was no change in phosphorylase a activity. Addition of caffeine, an independent inhibitor of activity, to the above effectors not only resulted in inhibition but also restored a glucose concentration-dependent inhibition. Because uric acid is an endogenous xanthine derivative, we decided to determine whether it had an effect on phosphorylase a activity. Independently, uric acid did not affect activity; however, when added at a presumed physiological concentration in combination with AMP, ADP, ATP, and glucose, it inhibited activity. A modest but not statistically significant glucose concentration-dependent inhibition was also present. Thus uric acid may play an important role in regulating phosphorylase a activity in vivo. (+info)
Skeletal muscle glycogen phosphorylase a kinetics: effects of adenine nucleotides and caffeine.
(8/155)
This study aimed to determine physiologically relevant kinetic and allosteric effects of P(i), AMP, ADP, and caffeine on isolated skeletal muscle glycogen phosphorylase a (Phos a). In the absence of effectors, Phos a had Vmax = 221 +/- 2 U/mg and Km = 5.6 +/- 0.3 mM P(i) at 30 degrees C. AMP and ADP each increased Phos a Vmax and decreased Km in a dose-dependent manner. AMP was more effective than ADP (e.g., 1 microM AMP vs. ADP: Vmax = 354 +/- 2 vs. 209 +/- 8 U/mg, and Km = 2.3 +/- 0.1 vs. 4.1 +/- 0.3 mM). Both nucleotides were relatively more effective at lower P(i) levels. Experiments simulating a range of contraction (exercise) conditions in which P(i), AMP, and ADP were used at appropriate physiological concentrations demonstrated that each agent singly and in combination influences Phos a activity. Caffeine (50-100 microM) inhibited Phos a (Km approximately 8-14 mM, approximately 40-50% reduction in activity at 2-10 mM P(i)). The present in vitro data support a possible contribution of substrate (P(i)) and allosteric effects to Phos a regulation in many physiological states, independent of covalent modulation of the percentage of total Phos in the Phos a form and suggest that caffeine inhibition of Phos a activity may contribute to the glycogen-sparing effect of caffeine. (+info)