ATPase Reaction Sequence Research Articles

The role of protein kinases and protein phosphatases in the regulation of cardiac sarcoplasmic reticulum function.

Canine cardiac sarcoplasmic reticulum is phosphorylated by adenosine 3',5'-monophosphate (cAMP)-dependent and by calcium.calmodulin-dependent protein kinases on a 27,000 proteolipid, called phospholamban. Both types of phosphorylation are associated with an increase in the initial rates of Ca2+ transport by SR vesicles which reflects an increased turnover of elementary steps of the calcium ATPase reaction sequence. The stimulatory effects of the protein kinases on the calcium pump may be reversed by an endogenous protein phosphatase, which can dephosphorylate both the cAMP-dependent and the calcium.calmodulin-dependent sites on phospholamban. Thus, the calcium pump in cardiac sarcoplasmic reticulum appears to be under reversible regulation mediated by protein kinases and protein phosphatases.

Reaction sequences for (Na + + K +)-dependent ATPase hydrolytic activities: new quantitative kinetic models

To delineate better the reaction sequence of the (Na + + K +)-ATPase and illuminate properties of the active site, kinetic data were fitted to specific quantitative models. For the (Na + + K +)-ATPase reaction, double-reciprocal plots of velocity against ATP (in the millimolar range), with a series of fixed KCl concentrations, are nearly parallel, in accord with the ping pong kinetics of ATP binding at the low-affinity sites only after P i release. However, contrary to requirements of usual formulations, P i is not a competitor toward ATP. A new steady-state kinetic model accommodates these data quantitatively, requiring that under usual assay conditions most of the enzyme activity follows a sequence in which ATP adds after P i release, but also requiring a minor alternative pathway with ATP adding after K + binds but before P i release. The fit to the data also reveals that P i binds nearly as rapidly to E 2 · K · ATP as to E 2 · K, whereas ATP binds quite slowly to E 2 · P · K: the site resembles a cul-de-sac with distal ATP and proximal P i sites. For the K +-nitrophenyl phosphatase reaction also catalyzed by this enzyme, the apparent affinities for both substrate and P i (as inhibitor) decrease with higher KCl concentrations, and both P i and TNP-ATP appear to be competitive inhibitors toward substrate with 10 mM KCl but noncompetitive inhibitors with 1 mM KCl. These data are accommodated quantitatively by a steady-state model allowing cyclic hydrolytic activity without obligatory release of K +, and with exclusive binding of substrate vs. either P i or TNP-ATP. The greater sensitivity of the phosphatase reaction to both P i and arsenate is attributable to the weaker binding by the occuluded-K + enzyme form occurring in the (Na + + K +)-ATPase reaction sequence. The steady-state models are consistent with cyclical interconversion of high- and low-affinity substrate sites accompanying E 1/E 2 transitions, with distortion to low-affinity sites altering not only affinity and route of access but also separating the adenine- and phosphate-binding regions, the latter serving in the E 2 conformation as the active site for the phosphatase reaction.

Divalent cations and the phosphatase activity of the (Na + K)-dependent ATPase.

Phosphatase activity of a kidney (Na + K)-ATPase preparation was optimally active with Mg2+ plus K+. Mn2+ was less effective and Ca2+ could not substitute for Mg2+. However, adding Ca2+ with Mg2+ or substituting Mn2+ for Mg2+ activated it appreciably in the absence of added K+, and all three divalent cations decreased apparent affinity for K+. Inhibition by Na+ decreased with higher Mg2+ concentrations, when Ca2+ was added, and when Mn2+ was substituted for Mg2+. Dimethyl sulfoxide, which favors E2 conformations of the enzyme, increased apparent affinity for K+, whereas oligomycin, which favors E1 conformations, decreased it. These observations are interpretable in terms of activation through two cases of cation sites. (i) At divalent cation sites, Mg2+ and Mn2+, favoring (under these conditions) E2 conformations, are effective, whereas Ca2+, favoring E1, is not, and monovalent cations complete. (ii) At monovalent cation sites divalent cations compete with K+, while Na+ at these sites favors E1 conformations. K+ increases the Km for substrate, but both Ca2+ and Mn2+ decrease it, perhaps by competing with K+. On the other hand, phosphatase activity in the presence of Na+ plus K+ is stimulated by dimethyl sulfoxide, by higher concentrations of Mg2+ and Mn2+, but not by adding Ca2+; this is consistent with stimulation occurring through facilitation of an E1 to E2 transition, perhaps an E1-P to E2-P step like that in the (Na + K)-ATPase reaction sequence. However, oligomycin stimulates phosphatase activity with Mg2+ plus Na+ alone or Mg2+ plus low K+: this effect of oligomycin may reflect acceleration, in the absence of adequate K+, of an alternative E2-P to E1 pathway bypassing the monovalent cation-activated steps in the hydrolytic sequence.

Mechanism of the stimulation of Ca 2+-dependent ATPase of skeletal muscle sarcoplasmic reticulum by protein kinase

Sarcoplasmic reticulum isolated from moderately fast rabbit skeletal muscle contains intrinsic adenosine 3′,5′-monophosphate (cAMP)-independent protein kinase activity and a substrate of 100 000 M r . Phosphorylation of skeletal sarcoplasmic reticulum by either endogenous membrane bound or exogenous cAMP-dependent protein kinase results in stimulation of the initial rates of Ca 2+ transport and Ca 2+-ATPase activity. To determine the molecular mechanism by which protein kinase-dependent phosphorylation regulates the calcium pump in skeletal sarcoplasmic reticulum, we examined the effects of protein kinase on the individual steps of the Ca 2+-ATPase reaction sequence. Skeletal sarcoplasmic reticulum vesicles were preincubated with cAMP and cAMP-dependent protein kinase in the presence (phosphorylated sarcoplasmic reticulum) and absence (control sarcoplasmic reticulum) of adenosine 5′-triphosphate (ATP). Control and phosphorylated sarcoplasmic reticulum were subsequently assayed for formation (5–100 ms) and decomposition (0–73 ms) of the acid-stable phosphorylated enzyme ( E ∼ P ) of Ca 2+-ATPase. Protein kinase mediated phosphorylation of skeletal sarcoplasmic reticulum resulted in pronounced stimulation of initial rates and levels of E ∼ P in sarcoplasmic reticulum preincubated with either ethylene glycol bis(β- aminoethyl ether)-N,N′- tetraacetic acid (EGTA) prior to assay (Ca 2+-free sarcoplasmic reticulum), or with calcium/EGTA buffer (Ca 2+-bound sarcoplasmic reticulum). These effects were evident within a wide range of ionized Ca 2+. Phosphorylation of skeletal sarcoplasmic reticulum by protein kinase also increased the initial rate of E ∼ P decomposition. These findings suggest that protein kinase-dependent phosphorylation of skeletal sarcoplasmic reticulum regulates several steps in the Ca 2+-ATPase reaction sequence which result in an overall stimulation of the active calcium transport observed at steady state.

Differences between CTP and ATP as substrates for the (Na + K)-ATPase

CTP was a poorer substrate than ATP when substituted in the (Na + K)-ATPase reaction assay, not only in terms of K m but also of V. CDP was a poorer inhibitor than ADP, so product inhibition cannot account for CTP being a poorer substrate. In the Na-ATPase reaction, which the enzyme also catalyzes, substituting CTP for ATP resulted in greater activity, arguing against CTP being less effective than ATP in forming the enzyme-phosphate intermediate common to both reactions. Ligands that favor the E 2 conformational state of the enzyme, K +, Mg 2+, and Mn 2 +, inhibited the (Na + K)-CTPase reaction more than the (Na + K)-ATPase. Conversely, Triton X-100, which favors the E 1 conformational state of the enzyme, K +, Mg 2+, and Mn 2+, inhibited the (Na + K)-CTPase ATPase reaction but stimulated the (Na + K)-CTPase. Although the (Na + K)-ATPase reaction sequence probably involves cyclical interconversion between E 1 and E 2 conformational states (and is thus inhibitable by ligands favoring either state), the K-phosphatase reaction catalyzed by the enzyme apparently functions entirely in the E 2 state. This reaction is better stimulated by CTP plus Na + than by ATP plus Na +; moreover, CTP lessens inhibition by Triton X-100, and ATP lessens inhibition by inorganic phosphate (which reacts with the E 2 state). These observations indicate that CTP is a poorer substrate than ATP because it is less effective in promoting conversion of E 2 to E 1, essential for the (Na + K)-dependent reaction mechanism. However, contrary to this rationale, dimethyl sulfoxide stimulated the (Na + K)-CTPase reaction although by other criteria, including inhibition of the (Na + K)-ATPase, the reagent appears to favor the E 2 over the E 1 conformational state.

Mechanism of the stimulation of calcium ion dependent adenosine triphosphatase of cardiac sarcoplasmic reticulum by adenosine 3',5'-monophosphate dependent protein kinase.

Canine cardiac sarcoplasmic reticulum (SR) is known to be phosphorylated by adenosine 3',5'-monophosphate (cAMP) dependent protein kinase on a 22 000-dalton protein. Phosphorylation enhances the initial rate of Ca2+ uptake and Ca2+-ATPase activity. To determine the molecular mechanism by which phosphorylation regulates the calcium pump in SR, we examined the effect of cAMP-dependent protein kinase on the individual steps of the Ca2+-ATPase reaction sequence. Cardiac sarcoplasmic reticulum was preincubated with cAMP and cAMP-dependent protein kinse in the presence (phosphorylated SR) and absence (control) of adenosine 5'-triphosphate (ATP). Control and phosphorylated SR were subsequently assayed for formation (4-200 ms) and decomposition (0-73 ms) of the acid-stable phosphorylated enzyme (E approximately P) of Ca2+-ATPase in media containing 100 microM [ATP] and various free [Ca2+]. cAMP-dependent phosphorylation of SR resulted in pronounced stimulation of initial rates and levels of E approximately P formed at low free [Ca2+] (less than or equal to 7 microM), but the effect was less at high free Ca2+ (greater than or equal to 10 microM). This stimulation was associated with a decrease in the dissociation constant for Ca2+ binding and a possible increase in Ca2+ sites. The observed rate constant for E approximately P formation of calcium-preincubated SR was not significantly altered by phosphorylation. Phosphorylation also increased the initial rate of E approximately P decomposition. These findings indicate that phosphorylation of cardiac SR by cAMP-dependent protein kinase regulates several steps in the Ca2+-ATPase reaction sequence which result in an overall stimulation of the calcium pump observed at steady state.

In vitro studies of skeletal muscle membranes characterization of a phosphorylated intermediate of sarcolemmal ( [formula omitted

The sarcolemmal membranes isolated from rat skeletal muscle are capable of incorporating 32P from [γ− 32P]ATP. The membrane protein phosphorylation requires Mg 2+. Cyclic AMP, cyclic GMP and their dibutyrul derivatives showed no marked effect on sarcolemmal phosphorylation. The Mg 2+-dependent 32P labeling was significantly enhanced by Na +. The rate of Na + -stimulated 32P incorporation was quite rapid reaching steady state levels within 5 s at 0 °C. K + reduced the Na + -stimulated 32P-incorporation but enhanced the 32P i release. This inhibitory effect of K + on Na + -stimulated 32P incorporation was prevented by the cardiac glycoside, ouabain. The Na + -dependent 32P labeling showed substrate dependency and the Na + site was saturable. The apparent K m for ATP was 2 · 10−5 M. The optimum pH for 32P labeling was between 7 and 8. Na + -dependent membrane phosphorylation showed a direct relationship with the (Na + + K +ATPase activity. The high turnover rate of 32P intermediate (12 000 min −1) suggested its functional significance in the overall transport ATPase reaction sequence. The predominate portion (> 90%) of the phosphorylated membrane complex was sensitive to acidified hydroxylamine and to alkaline pH suggesting an acylphosphate nature of the phosphoprotein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that 32P incorporation occurred predominately into a 108 000 dalton subunit which is a major protein component of sarcolemmal membranes. A very low level of 32P incorporation was also observed into a 25 000 dalton subunit and Ca 2+ slightly enhanced the phosphorylation of this component. The size ( M r 108 000 ) and some properties of the sarcolemmal phosphoprotein are closely similar to other (Na + + K +ATPase preparations reported so far.

Further studies on the activation of microsomal (Na + + K +-ATPase by a leukocytic product

Previous studies showed that microsomal (Na + + K +)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) is activated by a proteinaeous material released by polymorphonuclear leukocytes. Investigations on the mode of action of the activator have been conducted by the isolation of 32P-labeled phosphoenzyme intermediates formed in the reaction of ATP and (Na + + K +)-ATPase, which has been postulated to occur through the formation and hydrolysis of acyl phosphate intermediates. The activator caused a concentration-dependent decrease in the recovery of phosphoenzyme intermediates that was not quantitatively altered by the Na + or K + concentration of the reaction mixture or by the presence of 1 mM ouabain. A decline in phosphoenzyme intermediate recovery was promoted by the addition of the activator to performed phosphoenzyme intermediates but not by activator that had been pretreated with protease or phenol. In addition, the activator caused a concentration-dependent stimulation of the p-nitrophenyl phosphatase and acetyl phosphatase activities of microsomal (Na + + K +)-ATPase. It was proposed that the activator stimulates the dephosphorylation step of the (Na + + K +)-ATPase reaction sequence.

Stimulation of the (Na + + K +)-dependent adenosine triphosphatase by amino acids and phosphatidylserine: Chelation of trace metal inhibitors

Amino acids stimulated the (Na + + K +)-dependent ATPase activity of a rabbit kidney preparation without affecting the Mg 2+-ATPase activity; the most effective was histidine, producing a 2-fold increase in activity. Similar stimulation was produced by the well-known chelators EDTA, EGTA, and 8-hydroxyquinoline, and by the chelating phospholipid phosphatidylserine. In the presence of maximally effective concentrations of one agent, the other agents were unable to produce additional stimulation. It is suggested that the amino acids, phosphatidylserine, and the conventional chelators all stimulate the ATPase by a common mechanism: the removal of inhibitory trace metal (s). From measurements of the metal content of the enzyme preparation and experiments with extracted reagents it was concluded that the chelatable inhibitor was in the reagents used in the incubation medium rather than being endogenous to the enzyme; attempts to identify the inhibitor (s) were unsuccessful. The chelators also stimulated the K +-dependent phosphatase activity in the preparation but had no major effect on Na +-dependent incorporation of 32P from [ 32P]ATP. On monovalent cation activation the chelators appeared to relieve an uncompetitive inhibition of Na 32 activation and a noncompetitive inhibition of K 32 activation, also suggesting an action of the chelatable inhibitor on the later stages of the ATPase reaction sequence.

Interaction of [formula omitted] and Ca 2+ with human erythrocyte membrane ATPase

Human erythrocyte membranes were treated with N- ethylmaleimide and then assayed for cation-sensitive components of ATPase activity. Inhibition of Na +-activated ATPase and (Na +-K +)-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3), observed following N- ethylmaleimide treatment, was abolished when ethylene glycol bis-(β-aminoethyl)-N,N- tetraacetate (EGTA) was included in the assay system. N- Ethylmaleimide pretreatment reduced Mg 2+-activated ATPase activity both in the absence and presence of EGTA. The Mg 2+-dependent components of [ 14C]ADP-ATP exchange and membrane phosphorylation using [ γ- 32 P] ATP were reduced following N- ethylmaleimide pretreatment whereas the Na +-stimulated components of these activities were unaffected. At very low ATP concentration, chelation of endogenous Ca 2+ increases the sensitivity of the ATPase system to stimulation by low concentrations of Na +. In contrast a Ca 2+-requirement at a Na +-dependent step of (Na +-K +) activated ATPase reaction sequence was observed at higher ATP concentration. These results suggest both inhibitory and activating effects of Ca 2+ on alkali cation-sensitive ATPase.

SODIUM-POTASSIUM-ACTIVATED ATPASE AND POTASSIUM-ACTIVATED P-NITROPHENYLPHOSPHATASE: A COMPARISON OF THEIR SUBCELLULAR LOCALIZATIONS IN RAT BRAIN.

The association of a Na+-K+-activated ATPase (ATP phosphohydrolase, EC 3.6.1.4.) with the active transport of these cations is supported by substantial evidence.l-4 An effect of sodiuml ions on a brain ATPase was first reported by Utter.5 Further work has led to the concept that the Na+-K+-ATPase activity may be a resultant of transphosphorylations within the cell membrane which could couple phosphate bond energy directly to cation transport.6-s Consequently, the enzyme system would be an integral component of specialized membranes. Several workers have suggested that the final step in the ATPase reaction sequence may involve a K+-activated phosphatase6 9 and in particular, a phosphoprotein phosphatase.6 Since phenyl phosphate serves as substrate for some phosphoprotein phosphatasesl? and a K+-activated p-nitrophenylphosphatase occurs in brain,ll the structural association of a K+-activated phosphatase with the Na+-K+-ATPase in well-defined subcellular units would support their possible functional relationship. However, the operationally defined subcellular fractions of brain as prepared in various laboratories have produced discordant accounts of ATPase localizations: Deul and McIlwainl2 found Na+-activated ATPase mainly in cerebral microsomes; Schwartz et al.13 found 75 per cent of the Na+-activated ATPase associated with microsomal material other than ribosomes; JirnefeltL4 studied brain homogenates in 0.25 Mf sucrose in which the major ATPase activity was found in a heavy microsomal fraction recovered after centrifuging at 25,000 g X 45 min. In a more recent paper by Hayashi et al.,15 a large percentage of activity was found in a nuclear fraction containing cell debris and intact cells and also in the mitochondrial and microsomlal fractions.