Role of divalent cation bound to phosphoenzyme intermediate of sarcoplasmic reticulum ATPase

Effect of divalent cations bound to the phosphoenzyme intermediate of the ATPase of sarcoplasmic reticulum was investigated at 0 degree C and pH 7.0 using the purified ATPase preparations. Our previous study (Shigekawa, M., Wakabayashi, S., and Nakamura, H. (1983) J. Biol. Chem. 258, 14157-14161) indicated that 1 mol of the ADP-sensitive phosphoenzyme (E1P) formed from CaATP has 3 mol of high affinity binding sites for Ca2+, of which two are transport sites for calcium while the remainder is the acceptor site for calcium derived from the substrate, CaATP ("substrate site"). When incubated with a chelator of divalent cation, E1P formed from CaATP released all of its bound calcium to form a divalent cation-free phosphoenzyme. Evidence was presented that calcium dissociation from the substrate site was faster than that from the transport sites and primarily responsible for the ADP sensitivity loss of E1P induced by the chelator. Divalent cation-free phosphoenzyme was kinetically stable but when treated with divalent cations, it behaved similarly to the ADP-insensitive phosphoenzyme (E2P) which is the normal reaction intermediate of ATP hydrolysis. 45Ca bound at the substrate site on E1P formed from 45CaATP exchanged readily with nonradioactive ionized Ca2+ in the reaction medium whereas 45Ca at the transport sites on E1P was displaced only at a very slow rate which was almost the same as that for the phosphoenzyme hydrolysis. It was suggested that calcium at the transport sites on E1P formed from CaATP is released only after the rate-limiting conformational transition of the phosphoenzyme from E1P to E2P and that removal of calcium by a chelator from the substrate site facilitates this conformational transition, thereby allowing calcium bound at the transport sites to be released readily from the phosphoenzyme.     

Effect of divalent cation bound to the ATPase of sarcoplasmic reticulum. Activation of phosphoenzyme hydrolysis by Mg2+

In order to study the mechanism for activation of ATP hydrolysis by Mg2+, the stoichiometry of the high affinity calcium-binding sites with respect to each form of reaction intermediate of sarcoplasmic reticulum ATPase was determined at 0 degrees C and pH 7.0 in the presence and absence of added Mg2+ using the purified ATPase preparation. High affinity calcium binding to the enzyme-ATP complex and to ADP-sensitive (E1P) and ADP-insensitive (E2P) phosphoenzymes occurred with stoichiometric ratios of 2, 2, and 0, and 3, 3, and 1 in the presence and absence of added Mg2+, respectively. The results were interpreted to indicate that in addition to 2 mol of calcium bound to the transport sites of the ATPase, 1 mol of divalent cation, which is derived from the metal component of the substrate, the metal-ATP complex, remains bound to each mole of the enzyme at least until E2P is hydrolyzed. As activation of phosphoenzyme hydrolysis by Mg2+ was blocked by the low concentrations of Ca2+ used in the calcium binding experiments, it was concluded that it is the magnesium derived from MgATP that is responsible for rapid hydrolysis of the phosphoenzyme intermediate.     

Distinction between the intermediates in Na+-ATPase and Na+,K+-ATPase reactions. II. Exchange and hydrolysis kinetics at micromolar nucleotide concentrations

The ATP hydrolysis rate and the ADP-ATP exchange rate of (Na+ + K+)-ATPase from ox brain were measured at 10 microM Mg2+free and at micromolar concentrations of free ATP and ADP. (1) In the absence of K+, substrate inhibition of the hydrolysis rate was observed. It disappeared at low Na+ and diminished at increasing concentrations of ADP. This was interpreted in terms of free ATP binding to E1P. In support of this interpretation, free ATP was found to competitively inhibit ADP-ATP exchange. (2) In the presence of K+, substrate activation of the hydrolysis rate was observed. Increasing (microM) concentrations of ADP did not give rise to competitive inhibition in contrast to the situation in the absence of K+ (cf. 1, above). This was interpreted to show that at micromolar substrate, some low-affinity, high-turnover Na+ + K+ activity is possible, provided the Mg2+ concentration is low. (3) While small concentrations of K+ increased the hydrolysis rate (cf. 2) they decreased the rate of ADP-ATP exchange. To elucidate this phenomenon, parallel measurements of exchange and hydrolysis rates were performed over a wide range of ATP and ADP concentrations, with and without K+. If, in the presence and absence of K+, ADP (and ATP competing) are binding to the same phosphorylated intermediate for the backward reaction, it places quantitative restrictions on the ratio of rate constants with and without K+. The results did not conform to these restrictions, and the discrepancy is taken as evidence for the necessity for a bicyclic scheme for the action of the (Na+ + K+)-ATPase. (4) An earlier statement concerning the nature of the phosphoenzyme obtained in the presence of Na+ and K+ is amended.     

The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations

In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stability and partial reactions of soluble and membrane-bound sarcoplasmic reticulum ATPase

The Ca-ATPase of sarcoplasmic reticulum was solubilized at pH 6.5 and 30 degrees C using different nonionic detergents, Triton X-100, C12E8, Lubrol PX, or Tween 20. After full solubilization by any of these detergents, the enzyme was unstable (t1/2 = 2-3 min) in the absence of Ca2+. The soluble enzyme was stable in the presence of calcium, half-maximal protection being attained in the presence of 0.2 mM Ca2+. In the absence of Ca2+, stability was restored by addition of co-solvents dimethyl sulfoxide or glycerol. In the presence of 4 mM Ca2+, the progressive addition of nonionic detergents to a medium containing leaky vesicles promoted an increase, up to 3-fold, in the rate of ATP hydrolysis. This was not observed when ITP was used as substrate. The small amount of ADP accumulated in the medium during ATP hydrolysis was sufficient to inhibit the ATPase activity of the membrane-bound enzyme but had no effect on the soluble enzyme. Increasing concentrations of detergent promoted a progressive inhibition of the ATP----Pi exchange reaction. The ATP hydrolysis/synthesis ratio of soluble enzyme was 10 times higher than that of membranous enzyme. Addition of co-solvent restored this ratio to values similar to those obtained with membrane-bound Ca-ATPase. Soluble enzyme prepared from native sarcoplasmic reticulum vesicles was able to catalyze the net synthesis of ATP when phosphorylated by Pi in the presence of dimethyl sulfoxide and then diluted in a medium containing 10 mM CaCl2 and 2 mM ADP. This was not observed when the soluble enzyme was prepared from purified Ca-ATPase. The results suggest that some of the partial reactions of the catalytic cycle of Ca-ATPase are dependent on the hydrophobic environment found in the native membrane. This environment can be mimicked by co-solvents.     

The effect of monovalent and divalent cations on the ATP-dependent Ca2+-binding and phosphorylation during the reaction cycle of the sarcoplasmic reticulum Ca2+-transport ATPase

The coupling of Ca2+ movements and phosphate fluxes as well as the time-dependent occurrence of sequential reaction intermediates in the forward mode of the Ca,Mg-dependent ATPase reaction have been investigated using leaky vesicles (A23187) in the presence of varying Ca2+, Mg2+, and K+ concentrations. The employed ATP concentration of 2 microM does not allow more than one reaction cycle to occur. The respective fractions of ADP-sensitive and ADP-insensitive phosphoenzyme have been determined. The chosen experimental conditions (0-1 degree C, pH 6.0, absence of solubilizers) allow a prolonged time of observation and exclude interfering alterations of coupling and binding parameters, respectively. It is shown that under the experimental conditions K+ interacts with at least four different reaction steps (phosphoenzyme formation, E1P----E2P transition, E2P hydrolysis, and E2----E1 transformation). Mg2+ represents the sole ionic co-factor for the formation of the substrate MgATP if it is present in high concentrations (5 mM). Additional Ca2+ is bound to the substrate as well as to unspecific sites otherwise occupied by Mg2+ if Mg2+ is reduced to 0.1 mM. In this case the E1P----E2P transition rate (including Ca2+ translocation and Ca2+ release from low-affinity sites) is little diminished. If, in the absence of K+, both Mg2+ and Ca2+ are deficient E2P hydrolysis is vastly retarded. We find Ca2+ release to occur time-coincidently with E1P formation and not concomitantly with the comparably slow appearance of E2P; the molar amount of Ca2+ released, however, rather agreed with that of E2P formed. This suggests that under the prevailing conditions of a high proton concentration, phosphoenzyme states containing occluded Ca2+ or Ca2+ bound to low-affinity sites are transitional and not detectable. Preliminary findings on this subject have been published by us and colleagues from this laboratory [Hasselbach, W., Agostini, B., Medda, P., Migala, A. & Waas, W. (1985) in The sarcoplasmic reticulum calcium pump: Early and recent developments critically overviewed (Fleischer, S. & Tonomura, Y., eds) pp. 19-49, Academic Press, Orlando].     

Modulation of phosphatidylinositol-4,5-bisphosphate hydrolysis in rat aorta by guanine nucleotides, calcium and magnesium

Rat aortic smooth muscle homogenates and membrane preparations contain a phospholipase C which hydrolyzes phosphatidylinositol 4,5-biphosphate (PIP2). We discovered that guanyl-5'yl-imidodiphosphate (Gpp(NH)p) activated the hydrolysis of exogenous PIP2 but not of phosphatidylinositol (PI) in rat aortic membranes. Further, maximal Gpp(NH)p-dependent hydrolysis was dependent on physiological levels of calcium. Also, magnesium inhibited PIP2 hydrolysis and catalyzed the dephosphorylation of PIP2 to phosphatidylinositol-4-phosphate (PIP). The results imply that PIP2 is the primary substrate of the nucleotide-regulated phospholipase C in rat aorta and that calcium and magnesium are physiological regulators of this activity.     

Monolayers of long chain lecithins at the air/water interface and their hydrolysis by phospholipase A2

The behavior of phosphatidylcholine monolayers at the air/water interface was studied by measuring their surface isotherm, surface potential, surface viscosity, and rate of hydrolysis by the dimeric phospholipase A2 from the venom of Crotalus atrox. The monolayers showed typical liquid-expanded behavior. In this phase, the surface potential was linearly dependent on surface concentration and extrapolated at zero concentration to a value characteristic of a liquid hydrocarbon/water interface. The rate of the reaction was measured by monitoring changes in area at constant surface pressure for 1,2-dioctanoyl- and 1,2-didecanoyl-3-sn-phosphatidylcholines, and by monitoring changes in surface potential for 1,2-dimyristoyl-, 1,2-dipalmitoyl-, 1-palmitoyl-2-oleoyl-, and 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholines. The enzymatic hydrolysis is first order with respect to the enzyme-calcium complex which forms with a Kd = 1.5 mM. A mechanism is proposed to account for the dependency of the reaction rates on the surface concentration of the substrate. We postulate that the rate-limiting step is the decomposition of a quaternary complex formed from two phospholipid molecules, one calcium ion and one dimeric enzyme. The rate is independent of the surface pressure per se; addition of inert lipids to a monolayer at constant area, and hence constant surface concentration of the substrate, increases the surface pressure without changing the surface density of the substrate yielding maximal enzymatic rate. The enzyme is specific for loosely packed substrate molecules in the liquid-expanded state: transition into the liquid-condensed state or compression of the liquid-expanded layer beyond 80 A2/phospholipid strongly inhibits the enzymatic reaction. Our results show that surface recognition is a direct consequence of a bifunctional active site since it is only at a phospholipid surface that the distance between two substrate molecules is optimal for forming a catalytically competent enzyme-Ca2+-(substrate)2 complex.     

Activation of phospholipase A2 by adriamycin in vitro. Role of drug-lipid interactions

To probe adriamycin-phospholipid interactions, the effects of this cytotoxin on the hydrolysis of a pyrene-labeled acidic alkyl-acyl phospholipid analog 1-octa-cosanyl-2-(6-pyren-1-yl)hexanoyl-sn-glycero-3-phos p hatidylmethanol (C28-O-PHPM) by porcine pancreatic phospholipase A2 (PLA2) were studied. In the absence of added Ca2+ adriamycin caused a 3-4-fold activation of hydrolysis of this pyrenelipid whereas an inhibition of action of PLA2 on the corresponding phosphatidylcholine derivative C28-O-PHPC was observed. Under similar conditions adriamycin also enhanced the rate of hydrolysis of the pyrene-labeled diacyl lipid 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphatidylgly cer ol and inhibited the hydrolysis of PLA2 on the phosphatidylcholine derivative. Increasing calcium concentrations abolished the activating and most of the inhibitory effects of adriamycin with the above phospholipid substrates. Quenching of pyrene excimer fluorescence by adriamycin revealed efficient binding of the drug to acidic lipids. Addition of 1 mM calcium reduced fluorescence quenching by adriamycin maximally by approximately 90%. In comparison, quenching by adriamycin of pyrene-labeled phosphatidylcholine was much weaker and calcium had only an insignificant effect. Monolayer experiments at an air/water interface showed a rapid and surface pressure-dependent penetration of the drug into a film of C28-O-PHPM. Increase in surface pressure was reversed by 80% by the inclusion of 1 mM Ca2+ into the subphase. Penetration of adriamycin into a monolayer of C28-O-PHPC was much weaker. In agreement with earlier studies two types of binding of adriamycin to C28-O-PHPM are proposed.     

Interactions of metal ions with mu-monothiopyrophosphate.

mu-Monothiopyrophosphate (MTP) binds monovalent and divalent metal ions with dissociation constants (Kd) similar to those for pyrophosphate (PPi). The values of Kd for metal-MTP complexes are the following, as measured kinetically in the hydrolysis of MTP (microM): Mg2+, 32 +/- 4; Mn2+, 5.4 +/- 1.4; and Co2+, 27 +/- 15. The thermodynamically measured (EPR) values for Mg2+ and Co2+ are 28 +/- 13 microns and 11 +/- 4 microM, respectively; and the Kd for the complex MnPPi is 3.4 +/- 0.5 microM. The metal-MTP complexes undergo hydrolysis at rates modestly faster or slower than the rate at which MTP itself reacts. The complexes MgMTP2-, CoMTP2-, and MnMTP2- undergo hydrolytic cleavage with release of thiophosphate with observed first-order rate constants of 1.6 x 10(-2) min-1, 2.3 x 10(-2) min-1, and 0.6 x 10(-2) min-1, respectively, at 35 degrees C, compared with 1.1 x 10(-2) min-1 for MTP4- under the same conditions. Alkali metal cations also stimulate or retard the hydrolysis of MTP. At 25 degrees C and pH 12.2, the observed rate constant for tetramethylammonium MTP4- is 2.1 x 10(-3) min-1, and the estimated rate constants (min-1) for saturating alkali metals under the same conditions are as follows: Li+, 0.25 x 10(-3); Na+, 3.9 x 10(-3), K+, 6.7 x 10(-3); and Cs+, 6.7 x 10(-3). Divalent metal ions markedly retard the hydrolysis of MTP at pH 7 and 8 because complexation shifts the pH rate profile more than 2 pH units toward the acid side.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and properties of an extracellular beta-glucosidase from the polycentric rumen fungus Orpinomyces sp. strain PC-2.

An extracellular beta-glucosidase (EC 3.2.1.21) was purified from culture filtrate of the anaerobic rumen fungus Orpinomyces sp. strain PC-2 grown on 0.3% (wt vol-1) Avicel by using Q Sepharose anion-exchange chromatography, ammonium sulfate precipitation, chromatofocusing ion-exchange chromatography, and Superose 12 gel filtration. The enzyme is monomeric with a M(r) of 85,400, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, has a pI of 3.95, and contains about 8.5% (wt vol-1) carbohydrate. The N terminus appears to be blocked. The enzyme catalyzes the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (PNPG). The Km and Vmax values with cellobiose as the substrate at pH 6.0 and 40 degrees C are 0.25 mM and 27.1 mumol.min-1 x mg-1, respectively; with PNPG as the substrate, the corresponding values are of 0.35 mM and 27.7 mumol.min-1 x mg-1. Glucose (Ki = 8.75 mM, with PNPG as the substrate) and gluconolactone (Ki = 1.68 x 10(-2) and 2.57 mM, with PNPG and cellobiose as the substrates, respectively) are competitive inhibitors. Optimal activity with PNPG and cellobiose as the substrates is at pH 6.2 and 50 degrees C. The enzyme has high activity against sophorose (beta-1,2-glucobiose) and laminaribiose (beta-1,3-glucobiose) but has no activity against gentiobiose (beta-1,6-glucobiose). The activity of the beta-glucosidase is stimulated by Mg2+, Mn2+, Co2+, and Ni2+ and inhibited by Ag+, Fe2+, Cu2+, Hg2+, SDS, and p-chloromercuribenzoate.     

Activation of calcium transport in skeletal muscle sarcoplasmic reticulum by monovalent cations.

The rates of calcium transport and Ca2+-dependent ATP hydrolysis by rabbit skeletal muscle sarcoplasmic reticulum were stimulated by monovalent cations. The rate of decomposition of phosphoprotein intermediate of the Ca2+-dependent ATPase of sarcoplasmic reticulum was also increased by these ions to an extent that is sufficient to account for the stimulation of calcium transport and Ca2+-dependent ATPase activity. The order of effectiveness of monovalent cations tested at saturating concentrations in increasing rate of phosphoprotein decomposition is: K+, Na+ greater than Rb+, NH4+ greater than Cs+ greater than Li+, choline+, Tris+.     

Manganese-phospholipid-stimulated protein kinase activity of human leukemic cells

A protein kinase system with unusual characteristics was noted in extracts of HL-60 cells using endogenous proteins as substrates. This system exhibited a cation preference for manganese at an optimal concentration of 0.5 mM. Moderate activity was detectable with magnesium at an optimal concentration of 5.0 mM, but calcium was inactive. Activity was markedly stimulated by phospholipid, with phosphatidylglycerol and phosphatidylinositol exhibiting greater activity than phosphatidylserine. In isolated plasma membranes, the major substrate of this system was a 73-kDa protein, while cytoplasmic extracts exhibited larger amounts of a 42-kDa substrate. 73-kDa phosphorylating activity of membranes was comparably active at 0 and 31 degrees C, although in cytosol activity was greater at 31 degrees C. No 73-kDa protein phosphorylation was observed in the presence of Ca2+, Mg2+, and phosphatidylserine. Phosphoamino acid analysis of the 73-kD band revealed phosphothreonine and phosphoserine. The 42-kDa substrate was distinguishable from actin by two-dimensional gel electrophoresis, which disclosed that both major substrates were highly basic in the isoelectric focusing dimension. Protamine and histones (H2B greater than H1 greater than H3) exhibited phospholipid-stimulated phosphorylation in the presence of Mn2+, but phosvitin, casein, and vinculin were not substrates. High levels of phosphorylative activity involving the 73-kDa substrate were noted in nuclear extracts. Complex patterns of increase of this activity were noted in both cytosol and nuclear extracts following induction of differentiation with dimethyl sulfoxide, retinoic acid, or phorbol 12-myristate 13-acetate. This study thus demonstrated the presence of a previously undescribed type of protein kinase activity which exhibited alterations during leukemic cellular differentiation.     

The temporal sequence of events in the activation of phospholipase A2 by lipid vesicles. Studies with the monomeric enzyme from Agkistrodon piscivorus piscivorus

The substrate dependence of the time courses of hydrolysis of both small and large unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) by Agkistrodon piscivorus piscivorus monomeric phospholipase A2 is consistent with an activation process involving enzyme aggregation on the vesicle surface. The time course of hydrolysis of large unilamellar vesicles is particularly complex; a slow initial rate of hydrolysis is followed by an extremely abrupt increase in enzyme activity. The length of this slow phase is a minimum at the phase transition temperature of the vesicles. The intrinsic fluorescence intensity of the phospholipase A2 also abruptly increases (50-60%) after a latency period revealing a strong temporal correlation between enzyme activity and the increase in fluorescence intensity. The length of the latency period before the sudden increase in fluorescence intensity is directly proportional to substrate concentration at DPPC concentrations above 20-100 microM. At lower concentrations, the length of the latency period is inversely proportional to the DPPC concentration. Such biphasic substrate dependence is predicted by a previously proposed enzyme activation model involving dimerization on the surface vesicle. Simultaneous monitoring of the protein fluorescence and hydrolysis demonstrates that the magnitude of the fluorescence change and the rate of hydrolysis are in exact temporal correlation. Furthermore, simultaneous monitoring of the fluorescence of the protein and that of a lipid probe, trimethylammonium diphenylhexatriene, indicates a change in lipid vesicle structure prior to, or coincident with, the abrupt change in protein activation. These results are consistent with the hypothesis that the monomeric phospholipase A2 from A. piscivorus piscivorus initially possesses a low level of intrinsic activity toward large unilamellar DPPC vesicles and that the enzyme slowly becomes further activated on the vesicle surface via dimerization. Eventually, the vesicles undergo an abrupt transition in internal structure leading to sudden rapid activation of the enzyme.     

Increased substrate selectivity during transition from Ca2+-activated to K+,EDTA-activated nucleoside triphosphatase activity of heavy meromyosin

A comparison of kinetic parameters (Km(app) and V) of hydrolysis by heavy meromyosin of natural (ATP and ITP) and modified nucleoside triphosphates showed that in the K+, EDTA-ATPase conformation the enzyme exhibited a higher selectivity towards the structure of the substrate nucleoside moiety than in the case of the Ca2+-stimulated nucleoside triphosphatase activity. In the presence of Ca2+, all the N1- and N6-substituted analogs of ATP as well as ITP, etheno-ATP and the dialdehyde derivative of ATP were hydrolyzed at a high rate irrespective of their markedly decreased affinity for heavy meromyosin. In the presence of K+, EDTA the ATPase activity showed a tendency for a total decrease of the analog affinity for nucleoside triphosphates, i.e., the impossibility of tight binding of the substrate phosphate residues to the protein in the absence of bivalent cations, which was concomitant with an increase in the hydrolysis rate. However, it was found that only in N1-substituted analogs any appreciable changes in the substrate properties were absent. All the other nucleoside triphosphates tested (N6-carboxy-methoxy-ATP, N6-(N'-acetylaminoethoxy)-ATP, etheno-ATP, ITP and the dialdehyde derivative of ATP having a rupture in the ribose ring) lost their ability to be hydrolyzed by heavy meromyosin. The experimental results as well as the literature data are suggestive of differences in the spatial structure of the active center in two different myosin conformations associated with a high catalytic activity, i.e., K+, EDTA-ATPase and Ca2+-ATPase.     

Phosphorylation of the plasma membrane calcium pump at high ATP concentration. On the mechanism of ATP hydrolysis

The plasma membrane calcium ATPase (PMCA) reacts with ATP to form acid-stable phosphorylated intermediates (EP) that can be measured using (gamma-32P)ATP. However, the steady-state level of EP at [ATP] higher than 100 microM has not yet been studied due to methodological problems. Using a microscale method and a purified preparation of PMCA from human red blood cells, we measured the steady-state concentration of EP as a function of [ATP] up to 2 mM at different concentrations of Mg2+, both at 4 and 25 degrees C. We have measured the Ca2+-ATPase activity (v) under the same conditions as those used for phosphorylation experiments. While the curves of ATPase activity vs [ATP] were well described by the Michaelis-Menten equation, the corresponding curves of EP required more complex fitting equations, exhibiting at least a high- and a low-affinity component. Mg2+ increases the apparent affinity for ATP of this latter component, but it shows no significant effect on its high-affinity one or on the Ca2+-ATPase activity. We calculated the turnover of EP (k(pEP)) as the ratio v/EP. At 1 mM Mg2+, k(pEP) increases hyperbolically with [ATP], while at 8 microM Mg2+, it exhibits a behavior that cannot be explained by the currently accepted mechanism for ATP hydrolysis. These results, together with measurements of the rate of dephosphorylation at 4 degrees C, suggest that ATP is acting in additional steps involving the interconversion of phosphorylated intermediates during the hydrolysis of the nucleotide.     

Reaction mechanism of (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum. The role of Mg2+ that activates hydrolysis of the phosphoenzyme

The role of Mg2+ in the activation of phosphoenzyme hydrolysis has been investigated with the (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles. The enzyme of the native and solubilized vesicles was phosphorylated with ATP at 0 degrees C, pH 7.0, in the presence of Ca2+ and Mg2+. When Ca2+ and Mg2+ in the medium were chelated, phosphoenzyme hydrolysis continued for about 15 s and then ceased. The extent of this hydrolysis increased with increasing concentrations of Mg2+ added before the start of phosphorylation. This shows that the hydrolysis was activated by the Mg2+ added. The Mg2+ which activated phosphoenzyme hydrolysis was distinct from Mg2+ derived from MgATP bound to the substrate site. The Mg2+ site at which Mg2+ combined to activate phosphoenzyme hydrolysis was located on the outer surface of the vesicular membranes. During the catalytic cycle, Mg2+ combined with the Mg2+ site before Ca2+ dissociated from the Ca2+ transport site of the ADP-sensitive phosphoenzyme with bound Ca2+. This Mg2+ did not activate hydrolysis of the ADP-sensitive phosphoenzyme with bound Ca2+, but markedly activated hydrolysis of the ADP-insensitive phosphoenzyme without bound Ca2+. It is concluded that during the catalytic cycle, Mg2+ activates phosphoenzyme hydrolysis only after Ca2+ has dissociated from the Ca2+ transport site of phosphoenzyme.     

Purification and characterization of xylanases from<Emphasis Type="Italic">Aspergillus giganteus</Emphasis>

A strain of Aspergillus giganteus cultivated in a medium with xylan produced two xylanases (xylanase I and II) which were purified to homogeneity. Their molar mass, estimated by SDS-PAGE, were 21 and 24 kDa, respectively. Both enzymes are glycoproteins with 50 degrees C temperature optimum; optimum pH was 6.0-6.5 for xylanase I and 6.0 for xylanase II. At 50 degrees C xylanase I exhibited higher thermostability than xylanase II. Hg2+, Cu2+ and SDS were strong inhibitors, 1,4-dithiothreitol stimulated the reaction of both enzymes. Both xylanases are xylan-specific; kinetic parameters indicated higher efficiency in the hydrolysis of oat spelts xylan. In hydrolysis of this substrate, xylotriose, xylotetraose and larger xylooligosaccharides were released and hence the enzymes were classified as endoxylanases.     

Lanthanum inhibits steady-state turnover of the sarcoplasmic reticulum calcium ATPase by replacing magnesium as the catalytic ion

LaATP is shown to be an effective inhibitor of the calcium ATPase of sarcoplasmic reticulum because the binding of LaATP to cE.Ca2 results in the formation of lanthanum phosphoenzyme, which decays slowly. Steady-state activity of the calcium ATPase in leaky sarcoplasmic reticulum vesicles is inhibited 50% by 0.16 microM LaCl3 (15 nM free La3+, 21 nM LaATP) in the presence of 25 microM Ca2+ and 49 microM MgATP (5 mM MgSO4, 100 mM KCl, 40 mM 4-morpholinepropanesulfonic acid, pH 7.0, 25 degrees C). However, 50% inhibition of the uptake of 45Ca and phosphorylation by [gamma-32P]ATP in a single turnover experiment requires 100 microM LaCl3 (28 microM free La3+) in the presence of 25 microM Ca2+; this inhibition is reversed by calcium but inhibition of steady-state turnover is not. Therefore, binding of La3+ to the cytoplasmic calcium transport site is not responsible for the inhibition of steady-state ATPase activity. The addition of 6.7 microM LaCl3 (1.1 microM free La3+) has no effect on the rate of dephosphorylation of phosphoenzyme formed from MgATP and enzyme in leaky vesicles, while 6.7 mM CaCl2 slows the rate of phosphoenzyme hydrolysis as expected; 6.7 microM LaCl3 and 6.7 mM CaCl2 cause 95 and 98% inhibition of steady-state ATPase activity, respectively. This shows that inhibition of ATPase activity in the steady state is not caused by binding of La3+ to the intravesicular calcium transport site of the phosphoenzyme. Inhibition of ATPase activity by 2 microM LaCl3 (0.16 microM free La3+, 0.31 microM LaATP) requires greater than 5 s, which corresponds to approximately 50 turnovers, to reach a steady-state level of greater than or equal to 80% inhibition. Inhibition by La3+ is fully reversed by the addition of 0.55 mM CaCl2 and 0.50 mM EGTA; this reactivation is slow with t1/2 approximately 9 s. Two forms of phosphoenzyme are present in reactions that are partially inhibited by La3+: phosphoenzyme with Mg2+ at the catalytic site and phosphoenzyme with La3+ at the catalytic site, which undergo hydrolysis with observed rate constants of greater than 4 and 0.05 s-1, respectively. We conclude, therefore, that La3+ inhibits steady-state ATPase activity under these conditions by replacing Mg2+ as the catalytic ion for phosphoryl transfer. The slow development of inhibition corresponds to the accumulation of lanthanum phosphoenzyme. Initially, most of the enzyme catalyzes MgATP hydrolysis, but the fraction of enzyme with La3+ bound to the catalytic site gradually increases because lanthanum phosphoenzyme undergoes hydrolysis much more slowly than does magnesium phosphoenzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

β-Galactosidase from a cold-adapted bacterium: purification, characterization and application for lactose hydrolysis

The enzyme beta-galactosidase was purified from a cold-adapted organism isolated from Antarctica. The organism was identified as a psychotrophic Pseudoalteromonas sp. The enzyme was purified with high yields by a rapid purification scheme involving extraction in an aqueous two-phase system followed by hydrophobic interaction chromatography and ultrafiltration. The beta-galactosidase was optimally active at pH 9 and at 26 degrees C when assayed with o-nitrophenyl-beta-D-galactopyranoside as substrate for 2 min. The enzyme activity was highly sensitive to temperature above 30 degrees C and was undetectable at 40 degrees C. The cations Na+, K+, Mg2+ and Mn2+ activated the enzyme while Ca2+, Hg2+, Cu2+ and Zn2+ inhibited activity. The shelf life of the pure enzyme at 4 degrees C was significantly enhanced in the presence of 0.1% (w/v) polyethyleneimine. The pure beta-galactosidase was also evaluated for lactose hydrolysis. More than 50% lactose hydrolysis was achieved in 8 h in buffer at an enzyme concentration of 1 U/ml, and was increased to 70% in the presence of 0.1% (w/v) polyethyleneimine. The extent of lactose hydrolysis was 40-50% in milk. The enzyme could be immobilized to Sepharose via different chemistries with 60-70% retention of activity. The immobilized enzyme was more stable and its ability to hydrolyze lactose was similar to that of the soluble enzyme.