Effects of Mg2+ ions on the plasma membrane [H+]-ATPase of Neurospora crassa. II. Kinetic studies

The rate of ATP hydrolysis by the Neurospora plasma membrane [H+]-ATPase has been measured over a wide range of Mg2+ and ATP concentrations, and on the basis of the results, a kinetic model for the enzyme has been developed. The model includes the following three binding sites: 1) a catalytic site at which MgATP serves as the true substrate, with free ATP as a weak competitive inhibitor; 2) a high affinity site for free Mg2+, which serves to activate the enzyme with an apparent K1/2 (termed KMgA) of about 15 microM; and 3) a separate low affinity site at which Mg2+ causes mixed type inhibition, lowering the Vmax while raising the KS for MgATP at the catalytic site. The Ki for Mg2+ at the low affinity site (termed KMgI) is about 3.5 mM. The model satisfactorily explains the activity of the enzyme as Mg2+ and ATP are varied, separately and together, over a wide range. It can also account for the previously reported effects of Mg2+ and ATP on the inhibition of the Neurospora [H+]-ATPase by N-ethylmaleimide (Brooker, R. J., and Slayman, C. W. (1982) J. Biol. Chem. 257, 12051-12055; Brooker, R. J., and Slayman, C. W. (1983) J. Biol. Chem. 258, 8827-8832).     

Modification of the Neurospora crassa plasma membrane [H+]-ATPase with N,N'-dicyclohexylcarbodiimide

The [H+]-ATPase of the Neurospora plasma membrane is composed of a single Mr = 104,000 polypeptide (B. J. Bowman, F. Blasco, and C. W. Slayman, J. Biol. Chem. (1981) 256, 12343-12349). The carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) inactivates the ATPase with pseudo-first order kinetics, suggesting that one site on the enzyme is involved. The rate constant for inactivation at pH 7.5 and 30 degrees C is approximately 1000 M-1 min-1, similar to values reported for the DCCD-binding proteolipid of F0-F1-type [H+]-ATPases and for the sarcoplasmic reticulum [Ca+2]-ATPase. Although hydrophobic carbodiimides are inhibitory at micromolar concentrations, a hydrophilic analogue, 1-ethyl-3-(dimethylaminopropyl)-carbodiimide, is completely inactive even at millimolar concentrations. This result implies that the DCCD-reactive site is located in a lipophilic environment. [14C]DCCD is incorporated into the Mr = 104,000 polypeptide at a rate similar to the rate of inactivation. There is no evidence for a separate low molecular weight DCCD-binding proteolipid. Using quantitative amino acid analysis, we established that complete inhibition occurs at a stoichiometry of 0.4 mol of DCCD/mol of polypeptide. Overall, the results are consistent with the idea that DCCD reacts with a single amino acid residue of the Neurospora [H+]-ATPase, thereby blocking ATP hydrolysis and proton translocation.     

Identification of tryptic cleavage sites for two conformational states of the Neurospora plasma membrane H+-ATPase

Previous work has shown that the tryptic degradation pattern of the Neurospora plasma membrane H+-ATPase varies with the presence and absence of ligands, thus providing information about conformational states of the enzyme (Addison, R., and Scarborough, G. A. (1982) J. Biol. Chem. 257, 10421-10426; Brooker, R. J., and Slayman, C. W. (1983) J. Biol. Chem. 258, 8827-8832). In the present study, sites of tryptic cleavage have been mapped by immunoblotting with N- and C-terminal specific antibodies and by direct sequencing of proteolytic products after electro-transfer to polyvinylidene difluoride filters. In the absence of ligands (likely to represent the E1 conformation), trypsin cleaved the 100-kDa ATPase polypeptide at three sites very near the N terminus: Lys-24, Lys-36, and Arg-73. Removal of the first 36 amino acid residues only slightly affected ATPase activity, but removal of the subsequent 37 residues inactivated the enzyme completely. In the presence of vanadate and Mg2+ (E2 conformation), the rate of trypsinolysis at Arg-73 was greatly reduced, and enzyme activity was protected. In addition, a new cleavage site near the C terminus (Arg-900) became accessible to trypsin. Both effects of vanadate occurred at micromolar concentrations, well within the range previously measured for vanadate inhibition of ATPase activity. Taken together, these results suggest that the Neurospora ATPase undergoes significant conformational changes at both termini of the polypeptide during its reaction cycle.     

Cysteine 532 and cysteine 545 are the N-ethylmaleimide-reactive residues of the Neurospora plasma membrane H+-ATPase

Previous studies from this laboratory (Brooker, R. J., and Slayman, C. W. (1983) J. Biol. Chem. 258, 222-226; Davenport, J. W., and Slayman, C. W. (1988) J. Biol. Chem. 263, 16007-16013) have used the sulfhydryl reagent N-ethylmaleimide (NEM) to define two sites on the Neurospora plasma membrane H+-ATPase: a "fast" site which reacts in several minutes with no loss of enzymatic activity and a "slow" site which reacts in tens of minutes to produce complete inactivation of the enzyme. The slow site is protected when MgATP or MgADP is bound to the catalytic site of the ATPase. The present study demonstrates that the fluorescent reagent 5-[2-iodoacetamido)ethyl)-1-aminonaphthalenesulfonic acid (IAEDANS) can be used to label five of the eight cysteine residues of the Neurospora ATPase (Cys376, Cys409, Cys472, Cys532, Cys545). Tryptic peptides bearing those residues have been purified by high performance liquid chromatography and located within the known primary structure of the ATPase by amino acid analysis and/or sequencing. By pretreating the enzyme with NEM in the presence or absence of MgADP before incubation with IAEDANS, it has been possible to identify the fast NEM site as Cys545 and the slow MgADP-protectable NEM site as Cys532. Both residues lie within the central hydrophilic domain of the protein, close to a highly conserved stretch of amino acids that may be involved in nucleotide binding. However, all five IAEDANS-reactive cysteines can be nearly completely modified by the less bulky sulfhydryl reagent methyl methanethiosulfonate with less than 20% inhibition of enzyme activity; thus, none of the five cysteines can be considered to play a direct role in the reaction cycle of the ATPase.     

A structural change in the Neurospora plasma membrane [H+]ATPase induced by N-ethylmaleimide

The reaction of N-ethylmaleimide (NEM) with Cys-532 of the Neurospora plasma membrane [H+]ATPase results in inhibition of ATP hydrolysis which is protected by MgADP (Pardo, J. P., and Slayman, C. W. (1989) J. Biol. Chem. 264, 9373-9379). To examine the conformational state of the ATPase upon NEM modification, we have used limited trypsinolysis and domain-specific antibodies. The NEM-reacted ATPase shows increased sensitivity to trypsin, particularly in the central hydrophilic region of the polypeptide thought to contain the ATP binding and phosphorylation sites. In addition, competitive enzyme-linked immunosorbent assays indicate that the C-terminal domain of the ATPase becomes more accessible to antibody binding while the N-terminal region becomes more protected. The NEM-induced structural change is accompanied by loss of the ability to form a phosphoenzyme intermediate. The change in tertiary conformation occurs specifically upon NEM reaction with Cys-532 since neither NEM modification of Cys-545 nor fluorescein 5'-isothiocyanate modification of Lys-474 alters the tryptic digestion pattern of the ATPase. Furthermore, modification of Cys-532 with the less bulky sulfhydryl reagent methyl methanethiosulfonate does not result in a detectable structural change or loss of enzymatic activity. Thus, the introduction of a relatively bulky maleimide group at Cys-532 has specific and far-reaching effects upon the structure and function of the ATPase.     

Ca2+-activated ATPase in microsomes from human liver

Human liver microsomal fractions exhibit ATP-supported Ca2+ uptake which is half-maximal at 7 X 10(-7) M free Ca2+ in the presence of oxalate. Ca2+ uptake is coupled to a Ca2+-stimulated ATPase activity, which is half-maximal at 4 X 10(-7) M free Ca2+. Catalysis involves formation of an Mr = 116,000 phosphoprotein with stability characteristics of an acylphosphate compound suggested to represent a phosphoryl protein intermediate of the Ca2+-ATPase. Phosphorylation is half-maximal at about 10(-6) M free Ca2+. The Mr = 116,000 protein is highly susceptible to proteolysis with trypsin. The phosphorylated active site was localized in an Mr = 58,000 primary tryptic fragment and in an Mr = 34,000 subfragment. Analyses on the mechanism of the Ca2+-ATPase suggest the following reaction sequence: formation of an ADP-reactive phosphoenzyme (Mr = 116,000) with bound Ca2+, which can transphosphorylate its Pi to ADP, giving rise to synthesis of ATP; reversible transformation of the ADP-reactive phosphoenzyme into an isomer without bound Ca2+, which cannot further react with ADP; hydrolytical cleavage, probably catalyzed by Mg2+, of the ADP-unreactive phosphoenzyme with liberation of Pi. Comparison with the Ca2+-transport ATPase in sarcoplasmic reticulum of skeletal muscle led us to suggest that the Mr = 116,000 Ca2+-ATPase belongs to the class of E1P . E2P-ATPases and might be operative as a Ca2+-transport ATPase at the level of the endoplasmic reticulum in human liver.     

Location of a dicyclohexylcarbodiimide-reactive glutamate residue in the Neurospora crassa plasma membrane H+-ATPase

The proton pump (H+-ATPase) found in the plasma membrane of the fungus Neurospora crassa is inactivated by dicyclohexylcarbodiimide (DCCD). Kinetic and labeling experiments have suggested that inactivation at 0 degrees C results from the covalent attachment of DCCD to a single site in the Mr = 100,000 catalytic subunit (Sussman, M. R., and Slayman, C. W. (1983) J. Biol. Chem. 258, 1839-1843). In the present study, when [14C]DCCD-labeled enzyme was treated with the cleavage reagent, N-bromosuccinimide, a single major radioactive peptide fragment migrating at about Mr = 5,300 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was produced. The fragment was coupled to glass beads and partially sequenced by automated solid-phase Edman degradation at the amino terminus and at an internal tryptic cleavage site. By comparison to the DNA-derived amino acid sequence for the entire Mr = 100,000 polypeptide (Hager, K., and Slayman, C. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7693-7697), the fragment has been identified as arising by cleavage at tyrosine 100 and tryptophan 141. Covalently incorporated [14C]DCCD was released at a position corresponding to glutamate 129. The DCCD-reactive glutamate is located in the middle of the first of eight predicted transmembrane sequences. When the sequence surrounding the DCCD site is compared to that surrounding the DCCD-reactive residue of two other proton pumps, the F0F1-ATPase and cytochrome c oxidase, no homology is apparent apart from an abundance of hydrophobic amino acids.     

The amino and carboxyl termini of the Neurospora plasma membrane H+-ATPase are cytoplasmically located

Based on hydropathy analysis, the P-type cation translocating ATPases are believed to have similar topological arrangements in the membrane, but little independent evidence exists for their precise pattern of transmembrane folding. As a first step toward defining the topology of the Neurospora plasma membrane H+-ATPase, we have mapped the orientation of the amino and carboxyl termini. In three different types of experiments, both termini of the H+-ATPase were shown to be exposed at the cytoplasmic surface of the plasma membrane: 1) antibodies specific for the amino and carboxyl termini bound to permeabilized but not intact cells; 2) inside-out plasma membrane vesicles were approximately 100-fold more effective than intact cells in competing for antibody binding; and 3) trypsin, which is known to proteolyze three sites at the amino terminus and one site at the carboxyl terminus of the purified Neurospora H+-ATPase (Mandala, S. M., and Slayman, C. W. (1988) J. Biol. Chem. 263, 15122-15128), was found in the present study to cleave the same sites in inside-out plasma membrane vesicles but not in intact cells. These results indicate that the ATPase polypeptide traverses the membrane an even number of times, in support of a previously published topological model (Hager, K. M., Mandala, S. M., Davenport, J. W., Speicher, D. W., Benz, E. J., Jr., and Slayman, C. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7693-7697).     

Purification and characterization of the plasma membrane ATPase of Neurospora crassa

The plasma membrane of Neurospora crassa contains a proton-translocating ATPase, which functions to generate a large membrane potential and thereby to drive a variety of H+-dependent co-transport systems. We have purified this ATPase by a three-step procedure in which 1) loosely bound membrane proteins are removed by treatment with 0.1% deoxycholate; 2) the ATPase is solubilized with 0.6% deoxycholate in the presence of 45% glycerol; and 3) the solubilized enzyme is purified by centrifugation through a glycerol gradient. This procedure typically yields approximately 30% of the starting ATPase activity in a nearly homogeneous enzyme preparation of high specific activity, 61-98 mumol/min/mg of protein. The membrane-bound and purified forms of the ATPase are very similar with respect to kinetic properties (pH optimum, nucleotide and divalent cation specificity, sigmoid dependence upon Mg-ATP concentration) and sensitivity to inhibitors (including N,N'-dicyclohexylcarbodiimide and vanadate). Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified ATPase displays a single major polypeptide band of Mr = 104,000, which is essentially identical in its electrophoretic mobility with the large subunit of [Na+, K+]-ATPase of animal cell membranes and [Ca2+]-ATPase of sarcoplasmic reticulum. The structural similarity of the fungal and animal cell ATPases, together with the fact that both are known to form acyl phosphate intermediates, suggests that they may share a common reaction mechanism.     

Characterization of plasma membrane adenosine triphosphatase of Neurospora crassa.

It has been proposed (Slayman, C.L., Long W.S., and Lu, C.Y.-H. (1973) J. Membr. Biol. 14, 305--338) that in Neurospora crassa, a plasma membrane ATPase functions to pump H+ ions out of the cell, thereby generating an electrochemical gradient that can drive transport processes. Using the concanavalin A method of Scarborough (Scarborough G.A. (1975)J. Biol. Chem. 250, 1106--1111), we have prepared plasma membranes of Neurospora and have deomonstrated that they do contain a distinct ATPase activity with the following properties. It has a pH optimum of 6.0, is highly specific for ATP (hydrolyzing other nucleoside triphosphates less than 6% as rapidly), requires Mg2+ at concentrations approximately equimolar to the concentration of ATP, is weakly stimulated by certain monovalent cations (K+ and NH4+) and anions (SCN- and acetate), is inhibited by N,N'-dicyclohexylcarbodiimide, but is not affected by oligomycin or ouabain. The plasma membrane fraction also contains residual mitochondrial contamination, which can be determined quantitatively by assaying oligomycin-sensitive ATP-ase activity, at pH 8.25, and succinic dehydrogenase activity.     

Proteolytic digestion of Cl(-)-ATPase in rat brain synaptosomes

Upon tryptic digestion of synaptosomes, ATPase activities decreased in the order of Cl(-)-ATPase greater than or equal to Na+,K+-ATPase greater than anion-insensitive Mg2+-ATPase. Upon synaptosome treatment with hypotonic solution, Cl(-)-ATPase or anion-insensitive Mg2+-ATPase was slightly inactivated, while Na+,K+-ATPase underwent a much larger degree of inactivation. ATP-Mg inhibited the ATPase digestion in the hypotonic-solution-treated synaptosomes in a concentration-dependent manner, but not in the untreated synaptosomes. These results suggest that trypsin-digestible site of Cl(-)-ATPase are present on both sides of the synaptosomal plasma membrane, and the ATP-Mg binding site of the enzyme is located on the inner surface of the membrane.     

Inhibition by lead ion of Electrophorus electroplax (Na+ + K+)-adenosine triphosphatase and K+-p-nitrophenylphosphatase.

Inorganic lead ion in micromolar concentrations inhibits Electrophorus electroplax microsomal (Na+ + K+)-adenosine triphosphatase ((Na+ + K+)-ATPase) and K+-p-nitrophenylphosphatase (NPPase). Under the same conditions, the same concentrations of PbCl2 that inhibit ATPase activity also stimulate the phosphorylation of electroplax microsomes in the absence of added Na+. Enzyme activity is protected from inhibition by increasing concentrations of microsomes, ATP, and other metal ion chelators. The kinetics follow the pattern of a reversible noncompetitive inhibitor. No kinetic evidence is elicited for interactions of Pb2+ with Na+, K+, Mg2+, ATP, or p-nitrophenylphosphate. Na+- ATPase, in the absence of K+, and (Na+ + K+)-NPPase activity at low [K+] are also inhibited. ATP inhibition of NPPase is not reversed by Pb2+. The calculated concentrations of free [Pb2+] that produce 50% inhibition are similar for ATPase and NPPase activities. Pb2+ may act at a single independent binding site to produce both stimulation of the kinase and inhibition of the phosphatase activities.     

Interaction of a K(+)-competitive inhibitor, a substituted imidazo[1,2a] pyridine, with the phospho- and dephosphoenzyme forms of H+, K(+)-ATPase

Defining the structural and catalytic properties of the ion transport site(s) of enzyme-phosphorylating ATPases is of key importance in understanding the mechanism of ion transport by these enzymes. In the case of the H+, K(+)-ATPase, SCH 28080 (3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2a]-pyridine) has been shown to act as a high affinity, extracytosolic, K(+)-competitive inhibitor of Mg2+, K(+)-ATPase activity (Wallmark, B., Briving, C., Fryklund, J., Munson, K., Jackson, R., Mendlein, J., Rabon, E., and Sachs, G. (1987) J. Biol. Chem. 262, 2077-2084). To define the nature of the SCH 28080-binding site in relation to the catalytic cycle of the enzyme, we have investigated the effects of this potential K+ transport site probe on the steady-state and partial reactions of the H+, K(+)-ATPase. In the absence of K+, SCH 28080 inhibits Mg2(+)-ATPase activity with high affinity (apparent Ki = 30 nM). Inhibition is due to K(+)-like prevention of phosphoenzyme formation. SCH 28080 has no effect on Mg2(+)-catalyzed dephosphorylation. SCH 28080, at concentrations less than 0.5 microM, increases the apparent Km for K+ for Mg2+, K(+)-ATPase activity with little effect on the maximum velocity. At higher concentrations of SCH 28080, reversal of inhibition by higher K+ concentrations is not complete, due to inhibition of ATPase activity by high K+. In contrast, SCH 28080 inhibits K(+)-stimulated dephosphorylation by competitively displacing K+ from phosphoenzyme with an extracytosolic conformation of the monovalent cation site (E2P) at low concentrations of SCH 28080 and K+. At higher concentrations, 10 microM SCH 28080 and 50 mM K+, a slowly dephosphorylating complex with both SCH 28080 and K+ bound to E2P may form which represents a small fraction of the total E2P (15-25%). Preincubation of SCH 28080 with E2P completely blocks K(+)-stimulated dephosphorylation, and K+ is unable to reverse this preincubation effect, indicating that the SCH 28080 dissociation rate is at least as slow as K(+)-independent dephosphorylation of E2P. These findings indicate that SCH 28080 inhibits K(+)-stimulated ATPase activity by competing with K+ for binding to E2P and blocking K(+)-stimulated dephosphorylation. In the absence of K+, SCH 28080 has a higher apparent affinity for E2P, but it permits K(+)-independent dephosphorylation. Since the dissociation rate of SCH 28080 from the enzyme is slow, phosphoenzyme formation is prevented by SCH 28080 remaining bound to the extracytosolic conformation of the monovalent cation site, thereby reducing the steady-state level of phosphoenzyme.     

Size of the plasma membrane H+-ATPase from Neurospora crassa determined by radiation inactivation and comparison with the sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle

Using radiation inactivation, we have measured the size of the H+-ATPase in Neurospora crassa plasma membranes. Membranes were exposed to either high energy electrons from a Van de Graaff generator or to gamma irradiation from 60Co. Both forms of radiation caused an exponential loss of ATPase activity in parallel with the physical destruction of the Mr = 104,000 polypeptide of which this enzyme is composed. By applying target theory, the size of the H+-ATPase in situ was found to be approximately 2.3 X 10(5) daltons. We also used radiation inactivation to measure the size of the Ca2+-ATPase of sarcoplasmic reticulum and got a value of approximately 2.4 X 10(5) daltons, in agreement with previous reports. By irradiating a mixture of Neurospora plasma membranes and rabbit sarcoplasmic reticulum, we directly compared the sizes of these two ATPases and found them to be essentially the same. We conclude that both H+-ATPase and Ca2+-ATPase are oligomeric enzymes, most likely composed of two approximately 100,000-dalton polypeptides.     

The rat liver plasma membrane high affinity (Ca2+-Mg2+)-ATPase is not a calcium pump. Comparison with ATP-dependent calcium transporter

The high affinity (Ca2+-Mg2+)-ATPase purified from rat liver plasma membrane (Lin, S.-H., and Fain, J. N. (1984) J. Biol. Chem. 259, 3016-3020) has been further characterized. This enzyme also possesses Mg2+-stimulated ATPase activity with K0.5 of 0.16 microM free Mg2+. However, the Vm of the Mg2+-stimulated activity is only half that of the Ca2+-stimulated ATPase activity. The effects of Ca2+ and Mg2+ on this enzyme are not additive. Both the Ca2+-stimulated ATPase and Mg2+-stimulated ATPase activities have similar affinities for ATP (0.21 mM and 0.13 mM, respectively) and similar substrate specificities (they are able to utilize ATP, GTP, UTP, CTP, ADP, and GDP as substrates); both activities are not inhibited by vanadate, p-chloromercuribenzoate, ouabain, dicyclohexylcarbodiimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, oligomycin, F-, N-ethylmaleimide, La3+, and oxidized glutathione. These properties of the Mg2+- and Ca2+-ATPases indicate that both activities reside on the same protein. A comparison of the properties of this high affinity (Ca2+-Mg2+)-ATPase with those of the liver plasma membrane ATP-dependent Ca2+ transport activity reconstituted into artificial liposomes (Lin, S.-H. (1985) J. Biol. Chem. 260, 7850-7856) suggests that this high affinity (Ca2+-Mg2+)-ATPase is not the biochemical expression of the liver plasma membrane Ca2+ pump. The function of this high affinity (Ca2+-Mg2+)-ATPase remains unknown.     

Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum

The mycotoxin, cyclopiazonic acid (CPA), inhibits the Ca2+-stimulated ATPase (EC 3.6.1.38) and Ca2+ transport activity of sarcoplasmic reticulum (Goeger, D. E., Riley, R. T., Dorner, J. W., and Cole, R. J. (1988) Biochem. Pharmacol. 37, 978-981). We found that at low ATP concentrations (0.5-2 microM) the inhibition of ATPase activity was essentially complete at a CPA concentration of 6-8 nmol/mg protein, indicating stoichiometric reaction of CPA with the Ca2+-ATPase. Cyclopiazonic acid caused similar inhibition of the Ca2+-stimulated ATP hydrolysis in intact sarcoplasmic reticulum and in a purified preparation of Ca2+-ATPase. Cyclopiazonic acid also inhibited the Ca2+-dependent acetylphosphate, p-nitrophenylphosphate and carbamylphosphate hydrolysis by sarcoplasmic reticulum. ATP protected the enzyme in a competitive manner against inhibition by CPA, while a 10(5)-fold change in free Ca2+ concentration had only moderate effect on the extent of inhibition. CPA did not influence the crystallization of Ca2+-ATPase by vanadate or the reaction of fluorescein-5'-isothiocyanate with the Ca2+-ATPase, but it completely blocked at concentrations as low as 1-2 mol of CPA/mol of ATPase the fluorescence changes induced by Ca2+ and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) in FITC-labeled sarcoplasmic reticulum and inhibited the cleavage of Ca2+-ATPase by trypsin at the T2 cleavage site in the presence of EGTA. These observations suggest that CPA interferes with the ATP-induced conformational changes related to Ca2+ transport. The effect of CPA on the sarcoplasmic reticulum Ca2+-ATPase appears to be fairly specific, since the kidney and brain Na+,K+-ATPase (EC 3.6.1.37), the gastric H+,K+-ATPase (EC 3.6.1.36), the mitochondrial F1-ATPase (EC 3.6.1.34), the Ca2+-ATPase of erythrocytes, and the Mg2+-activated ATPase of T-tubules and surface membranes of rat skeletal muscle were not inhibited by CPA, even at concentrations as high as 1000 nmol/mg protein.     

Phosphorylated intermediate of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase in endoplasmic reticulum from rat pancreatic acinar cells

Formation and decomposition of the phosphorylated intermediate of endoplasmic reticulum (Ca2+ + Mg2+)-ATPase from pancreatic acinar cells have been studied using lithium dodecyl sulfate- and tetradecyltrimethylammonium bromide-polyacrylamide gel electrophoresis. Incorporation of 32P from [gamma-32P]ATP is Ca2+-dependent (approximate Km for free [Ca2+] = 2-3 X 10(-8) mol/liter). Formation of the 100-kDa phosphoprotein is rapid, reaching maximal 32Pi incorporation within 1 s at room temperature. At 4 degrees C, phosphorylation is slower and dephosphorylation is drastically decreased. For dephosphorylation, Mg2+ and monovalent cations such as K+ or Na+ are necessary. Vanadate inhibits both 32P incorporation and 32P liberation dose dependently (Km = 3 X 10(-6) mol/liter), whereas mitochondrial inhibitors and ouabain have no effect. The phosphoprotein is stable at pH 2 and destabilizes with increasing pH being completely decomposed at pH 9. Reduction of 32P incorporation in the presence of high concentrations of cold ATP and hydroxylamine suggests formation of acylphosphate present in the ATPase intermediate. The characteristics of Ca2+, cation, and pH dependencies of the ATPase activity are similar to those previously described for MgATP-dependent Ca2+ transport into rough endoplasmic reticulum from pancreatic acinar cells (Bayerd?rffer, E., Streb, H., Eckhardt, L., Haase, W., and Schulz, I. (1984) J. Membr. Biol. 81, 69-82). The data suggest that the 100-kDa phosphoprotein as described in this study is the intermediate of this Ca2+ transport ATPase.     

The sulfhydryl groups involved in the active site of myosin B adenosinetriphosphatase. I. Relantionship of the sulfhydryl group responsible for Mg2+-ATPase activation to the S1 and S2 groups.

The reactivity of the sulfhydryl groups in myosin B to N-ethylmaleimide (NEM) was investigated under various conditions. Under the conditions where actin and myosin associate, i.e. at low ionic strength, only Mg2+-ATPase [EC 3.6.1.3] activity was markedly activated by NEM treatment, whereas coupling of EDTA-ATPase inhibition with Ca2+-ATPase activation, which was seen on blocking S1 of myosin A with NEM, was observed under conditions at which the dissociation of actomyosin occurs, i.e. at high ionic strength, suggesting the covering with actin of the S1 region of myosin. Nevertheless, APT accelerated the reactivity of S1 and S2 much more in the myosin B system than in myosin alone. NEM-modified myosin B ATPase exhibited a shift of the KCL dependence curve to high concentration, a shift of the maximum activation of ATPase activity to high Mg ion concentration and a suppression of substrate inhibition at high substrate concentrations. These all indicate that the blocking by NEM of Sa, the sulfhydryl group related to the activation of Mg2+-ATPase of myosin B, brings about an increase in the association of myosin and actin in the myosin B system, resulting in an activation of Mg2+-ATPase activity. In addition, the relationship between Sa and a sulfhydryl group(s) essential for Ca2+ sensitivity was discussed.     

Limited tryptic digestion of Acanthamoeba myosin IA abolishes regulation of actin-activated ATPase activity by heavy chain phosphorylation

Acanthamoeba myosin IA is a globular protein composed of a 140-kDa heavy chain and a 17-kDa light chain. It expresses high actin-activated Mg2+-ATPase activity when one serine on the heavy chain is phosphorylated. We previously showed that chymotrypsin cleaves the heavy chain into a COOH-terminal 27-kDa peptide that can bind to F-actin but has no ATPase activity and a complex containing the NH2-terminal 112-kDa peptide and the light chain. The complex also binds F-actin and has full actin-activated Mg2+-ATPase activity when the regulatory site is phosphorylated. We have now localized the ATP binding site to within 27 kDa of the NH2 terminus and the regulatory phosphorylatable serine to a 20-kDa region between 38 and 58 kDa of the NH2 terminus. Under controlled conditions, trypsin cleaves the heavy chain at two sites, 38 and 112 kDa from the NH2 terminus, producing a COOH-terminal 27-kDa peptide similar to that produced by chymotrypsin and a complex consisting of an NH2-terminal kDa peptide, a central 74-kDa peptide, and the light chain. This complex is similar to the chymotryptic complex but for the cleavage which separates the 38- and 74-kDa peptides. The tryptic complex has full (K+, EDTA)-ATPase activity (the catalytic site is functional) and normal ATP-sensitive actin-binding properties. However, the actin-activated Mg2+-ATPase activity and the F-actin-binding characteristics of the tryptic complex are no longer sensitive to phosphorylation of the regulatory serine. Therefore, cleavage between the phosphorylation site and the ATP-binding site inhibits the effects of phosphorylation on actin binding and actin-activated Mg2+-ATPase activity without abolishing the interactions between the ATP- and actin-binding sites.     

Effect of SH-reagents on ATPase systems of rabbit skeletal muscle nuclei]

The influence of sulfhydryl reagents on ATPase systems of rabbit sceletal muscles nuclei was studied. It is found that p-ChMB at low concentration similarly inhibits both Mg2+- and Mg2+, Ca2+-ATPases. p-ChMB at higher concentrations inhibits completely Mg2+, Ca2+-ATPase, while Mg2+- ATPase--only by 60%. N-EM is lesser specific inhibitor of SH-groups, than p-ChMB. The degree of nuclear ATPases inhibition by N-EM is practically identical. Using inhibitory analysis, two hypes of skeletal muscles nuclei SH-groups are found: easily reacting with N-EM, and those reacting with N-EM at more high concentrations, which are essential for ATPase ATP-hydrolysing activity. ATP defends Mg2+, Ca2+-ATPase, but not the Mg2+-ATPase from N-EM inhibitory action. Cysteine completely eliminates the inhibitory effect of p-ChMB on Mg2+-ATPase but only 40% on MG2+, Ca2+-ATPase. Mg2+, Ca2+-ATPase of nuclei is more sensitive to the sulfhydryl venoms action than Mg2+-ATPase.