Evidence that the Loss of the Voltage-Dependent Mg2+ Block at the N-Methyl-D-Aspartate Receptor Underlies Receptor Activation During Inhibition of Neuronal Metabolism

Both phosphointermediate- and vacuolar-type (P- and V-type, respectively) ATPase activities found in cholinergic synaptic vesicles isolated from electric organ are immunoprecipitated by a monoclonal antibody to the SV2 epitope characteristic of synaptic vesicles. The two activities can be distinguished by assay in the absence and presence of vanadate, an inhibitor of the P-type ATPase. Each ATPase has two overlapping activity maxima between pH 5.5 and 9.5 and is inhibited by fluoride and fluorescein isothiocyanate. The P-type ATPase hydrolyzes ATP and dATP best among common nucleotides, and activity is supported well by Mg2+, Mn2+, or Co2+ but not by Ca2+, Cd2+, or Zn2+. It is stimulated by hyposmotic lysis, detergent solubilization, and some mitochondrial uncouplers. Kinetic analysis revealed two Michaelis constants for MgATP of 28 microM and 3.1 mM, and the native enzyme is proposed to be a dimer of 110-kDa subunits. The V-type ATPase hydrolyzes all common nucleoside triphosphates, and Mg2+, Ca2+, Cd2+, Mn2+, and Zn2+ all support activity effectively. Active transport of acetylcholine (ACh) also is supported by various nucleoside triphosphates in the presence of Ca2+ or Mg2+, and the Km for MgATP is 170 microM. The V-type ATPase is stimulated by mitochondrial uncouplers, but only at concentrations significantly above those required to inhibit ACh active uptake. Kinetic analysis of the V-type ATPase revealed two Michaelis constants for MgATP of approximately 26 microM and 2.0 mM. The V-type ATPase and ACh active transport were inhibited by 84 and 160 pmol of bafilomycin A1/mg of vesicle protein, respectively, from which it is estimated that only one or two V-type ATPase proton pumps are present per synaptic vesicle. The presence of presumably contaminating Na+,K(+)-ATPase in the synaptic vesicle preparation is demonstrated.     

The ATPase activity of phosphorylase kinase is regulated in parallel with its protein kinase activity.

Phosphorylase kinase from rabbit skeletal muscle has been found to have an intrinsic ATPase activity that occurs at a rate approximately 0.2% of that of its phosphorylase conversion activity and about three times that of its autophosphorylation activity. The characteristics of this ATPase activity were in all aspects tested essentially the same as the kinase's phosphorylase conversion activity. The ATPase requires Mg2+ and is dramatically stimulated by Ca2+ ions. At neutral pH there is a pronounced lag in the rate of product formation that is not present at alkaline pH, a condition that greatly stimulates both the phosphorylase conversion and ATPase activities. ATP is preferentially hydrolyzed over GTP and the Km for MgATP determined in the ATPase assay is 0.14 mM. ADP, an allosteric activator of phosphorylase conversion, also stimulates the ATPase activity, whereas beta-glycerophosphate, an inhibitor of phosphorylase conversion, is an inhibitor of the ATPase activity. Phosphorylation or partial proteolysis of the kinase, which are known to activate phosphorylase conversion, also activate the ATPase activity. Because the phosphorylase conversion and ATPase activities are regulated in parallel, we conclude that activation of the two catalytic activities must share a common underlying basis, namely an enhanced phosphotransferase activity that is independent of the phosphoryl acceptor.     

Inhibitory effects of phenolic ecotoxicants on photobacteria at various pH values

Kinetic characteristics of light emission by intact cells of the photobacteria Photobacterium phosphoreum and Vibrio harveyi at pH 5.5, 7.0, and 8.0 were studied as well as specific features of inhibitory effects of 2,4-di- and 2,4,5-triphenoxyacetic acids (2,4-D and 2,4,5-T), pentachlorophenol (PCP), and 2,6-dimethylphenol (2,6-DMP) at the same pH values. Nonstationarity of emission kinetics was observed at all the pH values studied. Exponential luminescence decay in a 60-sec range was observed at pH 5.5; a 5-min luminescence activation, at pH 7.0 and 8.0. The cell respiratory activity drops by over one order of magnitude at pH 5.5 compared with the activities at pH 7.0 and 8.0. The inhibitory effects of 2,4-D, 2,4,5-T, and PCP differ by one-two orders of magnitude depending on pH. The maximal cell sensitivity to these compounds appears at pH 5.5; the minimal, at pH 8.0. The effect of 2,6-DMP is independent of pH. As is demonstrated, it is hydrophobicity of the molecule and pK values of the toxicants that determine the inhibitory effect. Characteristic of the substrate-starved photobacterial cells are higher sensitivity to chlorophenolic compounds compared with the cells provided with high energy supply at all the pH values.     

Adenosine triphosphate hydrolysis by purified rubisco activase

Activation of ribulose bisphosphate carboxylase/oxygenase (rubisco) in vivo is mediated by a specific protein, rubisco activase. In vitro, activation of rubisco by rubisco activase is dependent on ATP and is inhibited by ADP. Purified rubisco activase hydrolyzed ATP with a specific activity of 1.5 mumol min-1 mg-1 protein, releasing approximately stoichiometric amounts of ADP and Pi. Hydrolysis was highly specific for ATP-Mg and had a broad pH optimum, with maximum activity at pH 8.0-8.5. ATPase activity was inhibited by ADP but not by molybdate, vanadate, azide, nitrate, or fluoride. Addition of rubisco in either the inactive or activated form had no significant effect on ATPase activity. Incubation of rubisco activase in the absence of ATP resulted in loss of both ATPase and rubisco activation activities. Both activities were also heat labile, with 50% loss in activity after 5 min at 38 degrees C and complete inhibition following treatment at 43 degrees C. Both activities showed a sigmoidal response to ATP concentration, with half-maximal activity at 0.053 mM ATP. Rubisco activation activity was dependent on the concentrations of both ATP and ADP. The results suggest that ATPase activity is an intrinsic property of rubisco activase.     

酸碱胁迫对不同体质量尼罗罗非鱼幼鱼能量代谢的影响

利用6×3析因试验设计,设置6组pH(5.0、6.0、7.0、8.0、9.0和10.0)和3组体质量(1.02、5.13和10.31 9),在试验室条件下研究了酸碱胁迫对不同体质量尼罗罗非鱼幼鱼的呼吸、排泄、乳酸脱氢酶(LDH)和Na+-K+ATPase活力的影响.结果表明:尼罗罗非鱼幼鱼的耗氧率和排氨率随pH值的升高先上升后下降,在pH7.0～8.0时达到最高值.耗氧量与排氨量随体质量的增加而增加并呈幂函数相关.体质量的一次和pH的一次、二次效应对尼罗罗非鱼幼鱼的耗氧率和排氨率有极显著的影响(P＜0.0l);体质量与pH之间无互作效应(P＞0.05);建立了pH与体质量对耗氧率、排氨率之间的回归方程,决定系数分别达到0.942和0.936;体质量对氧氮比(O:N)值有显著影响(P＜0.01),酸碱胁迫使尼罗罗非鱼幼鱼的能源物质利用模式发生改变.高pH条件不利于LDH活力的增加,但有利于Na+-K+ ATPase活力的上升,pH的一次和二次效应对酶活有极显著影响(P＜0.01),体质量对Na+-K+ ATPase活力没有显著影响,且pH与体质量对酶活无互作效应(P＞0.05).     

Biochemical properties of Candida parapsilosis ecto-5'-nucleotidase and the possible role of adenosine in macrophage interaction

Candida parapsilosis is considered to be an emerging fungal pathogen because it is associated with an increasing range of infections. In this work, we biochemically characterized ecto-5'-nucleotidase activity on the surface of living, intact C. parapsilosis cells. At a pH of 4.5, intact cells were able to hydrolyze 5'-AMP at a rate of 52.44 ± 7.01 nmol Pi h(-1) 10(-7) cells. 5'-AMP, 5'-IMP and 5'-UMP were hydrolyzed at similar rates, whereas 5'-GMP and 5'-CMP hydrolyzed at lower rates. Enzyme activity was increased by about 42% with addition of Mg(2+) or Ca(2+), and the optimum pH was in the acidic range. An inhibitor of phosphatase activities, sodium orthovanadate, showed no effect on AMP hydrolysis; however, as expected, ammonium molybdate, a classical nucleotidase inhibitor, inhibited the activity in a dose-dependent manner. The results indicated that the existence of an ecto-5'-nucleotidase could play a role in the control of extracellular nucleotide concentrations.     

Reconstitution of adenosine triphosphatase of thermophilic bacterium from purified individual subunits.

1. Five subunits (alpha, beta, gamma, delta, and epsilon) of an ATPase from a thermophilic bacterium PS3 were purified in the presence of 8 M urea by ion exchange chromatography. Then the ATPase activity was reconstituted by mixing the subunit solutions and incubating them at 20-45 degrees, at pH 6.3 to 7.0. 2. Mixtures containing beta + gamma or alpha + beta + delta regained ATP-hydrolyzing activity, but mixtures of alpha + beta and beta + delta did not. Combinations not including beta were all inactive. 3. The ATPase activity reconstituted from alpha + beta + delta was thermolabile and insensitive to NaN3, whereas the activities obtained from mixtures containing beta and gamma were thermostable and sensitive to NaN3, like the native ATPase. 4. The assemblies containing both beta and gamma subunits had the same mobility as the native ATPase molecule on gel electrophoresis, those without the gamma subunit moved more rapidly toward the anode. 5. Subunits epsilon and delta did not inhibit the ATPase activity of either the assembly (alpha + beta + gamma) or the native ATPase.     

Isolation and characterization of a specific endogenous Na+,K+-ATPase inhibitor from bovine adrenal

In order to identify a specific endogenous Na+,K+-ATPase inhibitor which could possibly be related to salt-dependent hypertension, we looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na+,K+-ATPase; (ii) competitive displacing activity against [3H]ouabain binding to the enzyme; (iii) inhibitory activity for 86Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C18 open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na+,K+-ATPase such as unsaturated free fatty acids, lysophosphatidylcholines, vanadate, dihydroxyeicosatrienoic acid, dehydroepiandrosterone sulfate, dopamine, lignan, ascorbic acid, etc. This substance was further purified by using an additional five steps of high-performance liquid chromatography with five different types of columns. Molecular mass was estimated as below 350 by fast atom bombardment mass spectroscopy and ultrafiltration. Heat treatment at 250 degrees C for 2 h and acid treatment with 6 N HCl at 115 degrees C for 21 h almost completely destroyed the inhibitory activity of the purified substance for Na+ pump activity. Additionally, alkaline treatment with 0.2 N NaOH at 23 degrees C for 2 h destroyed approximately 70% of the inhibitory activity, whereas boiling for 10 min and various enzyme digestion did not destroy the activity. The dose dependency for the four types of the activities for this substance paralleled those of ouabain, spanning 2 orders of magnitude in concentration range. The inhibitory potencies of the purified substance for Na+,K+-ATPase, Na+ pump, and ouabain binding activities were diminished with increasing K+ concentration, exhibiting a characteristic typical of cardiac glycosides. This substance had no effect on the Ca2+-ATPase activity or the Ca2+ loading rate into the vesicle prepared from skeletal muscle sarcoplasmic reticulum. These results strongly suggest that this water-soluble nonpeptidic Na+,K+-ATPase inhibitor may be a specific endogenous regulator for the ATPase.     

Characterization of the plasma membrane Mg2+-ATPase from the yeast, Saccharomyces cerevisiae.

The plasma membrane of Saccharomyces cerevisiae has a Mg2+-dependent ATPase which is distinct from the mitochondrial Mg2+-ATPase and at the pH optimum of 5.5 has a Km for ATP of 1.7 mM and a Vmax of 0.42 mumol of ATP hydrolyzed/mg/min. At least three protein components of the crude membrane (Mr = 210,000, 160,000 and 115,000) are labeled with [gamma"32P]ATP at pH 5.5. These phosphoproteins form rapidly in the presence of Mg2+, rapidly turn over the bound phosphate when unlabeled ATP is added, and dephosphorylate after incubation in the presence of hydroxylamine. Vanadate, an inhibitor of the Mg2+-ATPase activity, blocks the phosphorylation of the 210,000- and 115,000-dalton proteins. At pH 7.0, only the 210,000- and 160,000-dalton proteins are phosphorylated. While these three phosphorylated intermediates have not been unambiguously identified as components of the Mg2+-ATPase, the finding of such phosphorylated components in association with that activity implies that this enzyme differs in mechanism from the mitochondrial proton pump and that it is similar in mechanism to the metal ion pumps ((Na+-K+)-ATPase and Ca2+-ATPase) of the mammalian plasma membrane.     

Mechanism for 3,3',4',5-tetrachlorosalicylanilide-induced activation of sarcoplasmic reticulum ATPase

Effects of 3,3',4',5-tetrachlorosalicylanilide (TCS), a lipophilic weak acid, on Ca2+ uptake and ATP hydrolysis by the sarcoplasmic reticulum calcium pump were characterized to obtain insight into the possible role of hydrophobic portions of the Ca2+-ATPase in the catalytic mechanism of the enzyme. TCS exhibited both the stimulatory and inhibitory effects on the calcium pump activities depending on its concentration. At optimal concentrations, it increased these activities by up to 5-fold at pH 7.0 and 6 degrees C. Analysis of partial reactions of ATP hydrolysis by the purified ATPase revealed that TCS accelerated Ca2+ release from the ADP-sensitive phosphoenzyme up to 6-fold, whereas it affected other reaction steps to a much less extent, indicating that the site of the stimulatory action of TCS is rather specific in terms of the reaction sequence. These effects of TCS became less prominent at higher temperatures, although the enzyme-TCS interactions as detected in the direct binding experiment or by measurement of quenching of protein fluorescence were not affected by a similar change in temperature. The TCS effects were also dependent on pH of the 8.0 suggested that the protonated form of TCS is responsible for both the stimulatory and inhibitory effects of the drug. These results, taken together with those obtained previously with a spin-labeled probe (Barratt, M. D., and Weaver, A. C. (1979) Biochim. Biophys. Acta 555, 337-348), may suggest that TCS stimulates the calcium pump activity through its effect on the lipid bilayer, although its direct action on hydrophobic portion(s) of the ATPase protein cannot be ruled out.     

Volume regulation in Mycoplasma gallisepticum: evidence that Na+ is extruded via a primary Na+ pump.

The primary extrusion of Na+ from Mycoplasma gallisepticum cells was demonstrated by showing that when Na+-loaded cells were incubated with both glucose (10 mM) and the uncoupler SF6847 (0.4 microM), rapid acidification of the cell interior occurred, resulting in the quenching of acridine orange fluorescence. No acidification was obtained with Na+-depleted cells or with cells loaded with either KCl, RbCl, LiCl, or CsCl. Acidification was inhibited by dicyclohexylcarbodiimide (50 microM) and diethylstilbesterol (50 microM), but not by vanadate (100 microM). By collapsing delta chi with tetraphenylphosphonium (200 microM) or KCl (25 mM), the fluorescence was dequenched. The results are consistent with a delta chi-driven uncoupler-dependent proton gradient generated by an electrogenic ion pump specific for Na+. The ATPase activity of M. gallisepticum membranes was found to be Mg2+ dependent over the entire pH range tested (5.5 to 9.5). Na+ (greater than 10 mM) caused a threefold increase in the ATPase activity at pH 8.5, but had only a small effect at pH 5.5. In an Na+-free medium, the enzyme exhibited a pH optimum of 7.0 to 7.5, with a specific activity of 30 +/- 5 mumol of phosphate released per h per mg of membrane protein. In the presence of Na+, the optimum pH was between 8.5 and 9.0, with a specific activity of 52 +/- 6 mumol. The Na+-stimulated ATPase activity at pH 8.5 was much more stable to prolonged storage than the Na+-independent activity. Further evidence that two distinct ATPases exist was obtained by showing that M. gallisepticum membranes possess a 52-kilodalton (kDa) protein that reacts with antibodies raised against the beta-subunit of Escherichia coli ATPase as well as a 68-kDa protein that reacts with the anti-yeast plasma membrane ATPases antibodies. It is postulated that the Na+ -stimulated ATPases functions as the electrogenic Na+ pump.     

Actin-activation of unphosphorylated gizzard myosin

The effect of light chain phosphorylation on the actin-activated ATPase activity and filament stability of gizzard smooth muscle myosin was examined under a variety of conditions. When unphosphorylated and phosphorylated gizzard myosins were monomeric, their MgATPase activities were not activated or only very slightly activated by actin, and when they were filamentous, their MgATPase activities could be stimulated by actin. At pH 7.0, the unphosphorylated myosin in the presence of ATP required 2-3 times as much Mg2+ for filament formation as did the phosphorylated myosin. The amount of stimulation of the unphosphorylated myosin filaments depended upon pH, temperature, and the presence of tropomyosin. At pH 7.0 and 37 degrees C and at pH 6.8 and 25 degrees C, the MgATPase activity of filamentous, unphosphorylated, gizzard myosin was stimulated 10-fold by actin complexed with gizzard tropomyosin. These tropomyosin-actin-activated ATPase activities were 40% of those of the phosphorylated myosin. Under other conditions, pH 7.5 and 37 degrees C and pH 7.0 and 25 degrees C, even though the unphosphorylated myosin was mostly filamentous, its MgATPase activity was stimulated only 4-fold by tropomyosin-actin. Thus, both unphosphorylated and phosphorylated gizzard myosin filaments appear to be active, but the cycling rate of the unphosphorylated myosin is less than that of the phosphorylated myosin. Active unphosphorylated myosin may help explain the ability of smooth muscles to maintain tension in the absence of myosin light chain phosphorylation.     

Myofibril ATPase activity of cardiac and skeletal muscle of exhaustively exercised rats

The activation characteristics of Mg-ATP and Ca2+ on cardiac and skeletal muscle myofibril ATPase activity were studied in rats following a run to exhaustion. In addition, the effect of varying ionic strength was determined on skeletal muscle from exhausted animals. The exhausted group (E) ran at a speed of 25 m min-1 with an 8% incline. Myofibril ATPase activities for control (C) and E were determined with 1, 3 and 5 mM Mg-ATP and 1 and 10 microM Ca2+ at pH 7.0 and 30 degrees C. For control skeletal muscle, at 1 and 10 microM Ca2+, there was an increase in ATPase activity from 1 to 5 mM Mg-ATP (P less than 0.05). For E animals the myofibril ATPase activities at 10 microM Ca2+ and all Mg-ATP concentrations were similar to C (P greater than 0.05). At 1.0 microM Ca2+ and all Mg-ATP concentrations were similar to C (P greater than 0.05). At 1.0 microM Ca2+ the activities at 3 and 5 mM Mg-ATP were greater for the E animals (P less than 0.05). Increasing KCl concentrations resulted in greater inhibition for E animals. With cardiac muscle, the myofibril ATPase activities at 1.0 microM free Ca2+ were lower for E at all Mg-ATP levels (P less than 0.05). In contrast, at 10 microM Ca2+, the E group exhibited an elevated myofibril ATPase activity. The results indicate that Mg-ATP and Ca2+ activation of cardiac and skeletal muscle myofibril ATPase is altered with exhaustive exercise.     

Mitochondrial adenosinetriphosphatase inhibitor protein: reversible interaction with complex V (ATP synthetase complex)

Mitochondrial ATPase inhibitor protein (IF1) reacts reversibly with complex V and inhibits up to 90% of its ATPase activity. Both the rate and extent of inhibition are pH and temperature dependent and increase as the pH is lowered from pH 8 tp 6.7 (the lowest pH examined) or as the temperature is increased from 4 to 36 degrees C. Nucleotide triphosphates plus Mg2+ ions are required for inhibition of complex V ATPase activity by IF1. In the presence of Mg2+ ions, the effectiveness order of nucleotides is ATP greater than ITP greater than GTP greater than UTP. Highly purified complex V, which requires added phospholipids for expressing ATPase and ATP-Pi exchange activities, cannot be inhibited by IF1 plust ATP-Mg2+ unless phospholipids are also added. This indicates that the active state of the enzyme is necessary for the IF1 effect to be manifested, because F1-ATPase, which does not contain nor require phospholipids for catalyzing ATP hydrolysis, can be inhibited by IF1 plus ATP-Mg2+ in the absence of added phospholipids. The IF1-inhibited complex V, but not IF1-inhibited F1-ATPase, can be reactivated by incubation at pH greater than 7.0 in the absence of ATP-Mg2+. The reactivation rate is pH dependent and is influenced by temperature and enzyme concentration. Complex V preparations contain small and variable amounts of IF1. This endogenous IF1 behaves the same as added IF1 with respect to conditions described above for inhibition and reactivation and can result in 25-50% inhibition in different complex V preparations. However, complex V lacking endogenous IF1 can be reconstituted from F0, F1, oligomycin sensitivity conferring protein, and phospholipids. Inhibition of this reconstituted preparation in the presence of ATP-Mg2+ depends entirely on addition of IF1. In general, the ATP-Pi exchange activity of complex V is more sensitive to the chemical inhibitors of F1-AtPase tha its ATPase activity. This is not so, however, for IF1. Under conditions that IF1 caused approximately 75% inhibition of ATPase activity of complex V, no more than 10% of the ATP-Pi exchange activity was inhibited.     

Characterization of the plasmalemma ATPase activity from roots of Plantago major ssp. pleiosperma, purified by the two-phase partitioning method

A purified plasmalemma preparation from roots of Plantago major L. ssp. pleiosperma (Pilger) was obtained by the two-phase partitioning method, using 6.5% (w/w) of Dextran T-500 and polyethylene glycol 3350, respectively. The distribution of murker enzymes proved the purity of the plasmalemma fraction. The ATPase activity was characterized by determining its sensitivity to anions, cations and inhibitors. The Mg²⁺-dependent ATPase activity peaked at pH 7.25, K⁺-stimulation at pH 6.75, and the Cl⁻ -stimulation both at pH 6.75 and 7.5 (all in the presence of 3 m M MgSO₄). The plasmalemma preparations hydrolyzed preferentially ATP (in the presence of Mg²⁺), although they were less specific for ATP at pH 7.5 than at pH 6.75. The Cl⁻ - stimulated ATPase is probably associated with and located on the plasmalemma. The question if the Cl⁻ -stimulated activity is due to an ATPase distinct from the classical K⁺-stimulated ATPase is considered.     

Phosphatase and ATPase activities in isonuclear lines of cytoplasmic male-sterile and male-fertile petunia

Summary Soluble and membrane-bound fractions of plant leaves, cell suspension cultures and seedlings of petunia were examined for phosphohydrolase activity on p-nitrophenyl phosphate (pNPPase) and adenosine triphosphate (ATPase). One cytoplasmic male-sterile (CMS) and one fertile (F) line was examined for each tissue. Both pNPPase and ATPase exhibited a broad optimal activity between pH 5.5–7.0 for the membrane-bound fraction and between 4.5–7.0 for the soluble fractions. The activity of both were inhibited by divalent ions including Mg²⁺. At pH 7.2, the activities on various triphosphonucleotides were similar and they were hydrolyzed by a rate of 20–50% of that of ATP. Significant differences between CMS and F extracts were: (a) higher activities in CMS membranes; (b) lower Ea (energy of activation) values for activities in CMS membrane functions; (c) seedling and cell-culture CMS extracts exhibited a higher sensitivity to high temperature denaturation; (d) the hydrolase activity on monoand triphospho-cytosine compounds was significantly higher in CMS than in F membranes.Contribution from the Agricultural Research Organization, The Volcani Center, Bet-Dagan, Israel No. 355-E, 1992 series     

Structural and functional properties of a Ca2+-ATPase from human platelets

An antibody prepared against highly purified rabbit muscle Ca2+-ATPase from sarcoplasmic reticulum has been observed to cross-react with proteins in human platelet membrane vesicles. The antibody specifically precipitated Ca2+-ATPase activity from solubilized human platelet membranes and recognized two platelet polypeptides denatured in sodium dodecyl sulfate with Mr = 107,000 and 101,000. Ca2+-ATPase activity from Brij 78-solubilized platelet membranes was purified up to 10-fold. The purified preparation consisted mainly of two polypeptides with Mr approximately 100,000, and 40,000. The lower molecular weight protein appeared unrelated to Ca2+-ATPase activity. The Ca2+-ATPase in human platelet membrane vesicles exhibited "negative cooperativity" with respect to the kinetics of ATP hydrolysis. The apparent Km for Ca2+ activation of ATPase activity was 0.1 microM. Ca2+-dependent phosphorylation of platelet vesicles by [gamma-32P]ATP at 0 degrees C yielded a maximum of 0.2-0.4 nmol of PO4/mg of protein that was labile at pH 7.0 and 20 degrees C. This result suggests that only about 2-4% of the total protein in platelet membrane vesicles is the Ca2+-ATPase, which agrees with an estimate based on the specific activity of the Ca2+-ATPase in platelet membranes (20-50 nmol of ATP hydrolyzed/min/mg of protein at 30 degrees C). Calmodulin resulted in only a 1.6-fold stimulation of Ca2+-ATPase activity even after extensive washing of membranes with a calcium chelator or chlorpromazine. It is concluded that human platelets contain a Ca2+-ATPase immunochemically related to the Ca2+ pump from rabbit sarcoplasmic reticulum and that the enzymatic characteristics and molecular weight of the platelet ATPase are quite similar to those of the muscle ATPase.     

Proton transport catalyzed by the sodium pump. Ouabain-sensitive ATPase activity and the phosphorylation of Na,K-ATPase in the absence of sodium ions

The possibility that H+ might substitute for Na+ at Na+ sites of Na+,K+-ATPase was studied. Na+,K+-ATPase purified from pig kidney showed ouabain-sensitive K+-dependent ATPase activity in the absence of Na+ at acid pH (H+,K+-ATPase). The specific activity was 1.1 mumol Pi/mg/min at pH 5.7, whereas the specific activity of Na+,K+-ATPase was 14 mumol Pi/mg/min at pH 7.5. The enzyme was phosphorylated from ATP in the absence of Na+ at the acid pH. The initial rate of the phosphorylation was also accelerated at the acid pH in the absence of Na+, and the maximal rate obtained at pH 5.5 without Na+ was 9% of the rate at pH 7.0 with Na+. The phosphoenzyme was sensitive to K+ but almost insensitive to ADP. The phosphoenzyme was sensitive to hydroxylamine treatment and the alpha-subunit of the enzyme was found to be phosphorylated. H+,K+-ATPase was inhibited as effectively as Na+,K+-ATPase by N-ethylmaleimide but was less inhibited by oligomycin or dimethyl sulfoxide. These results indicate that protons have an Na+-like effect on the Na+ sites of Na+,K+-ATPase and suggest that protons can be transported by the sodium pump in place of Na+.     

Isolation and characteristics of scallop sarcoplasmic reticulum with calcium transport activity.

Sarcoplasmic reticulum with calcium transport activity has been isolated from the cross-striated adductor muscle of the scallop, which lives in cold (< or = 20 degrees C) sea water, by using pH 7.0 buffer solution both to homogenize the tissue and to sediment the membrane fraction. The yield of the preparation was 60-100 mg protein from 100 g of the scallop muscle. Ca(2+)-activated ATPase protein of about 100 kDa accounted for 40-50% of the protein preparation. The maximum activities of ATP-dependent, oxalate-facilitated calcium accumulation and Ca(2+)-ATPase were observed at a pH of about 7.0 and temperature of 20-30 degrees C, and their values were about 2 mumol Ca2+/mg of protein/min and about 3 mumol ATP hydrolysis/mg of protein/min, respectively. At 0 degree C, 10-20% of these activities was maintained, while at 37 degrees C, the activities were irreversibly lost. The Ca(2+)-ATPase activity was half-maximally activated at about 0.3 microM [Ca2+]. The ATPase activity exhibited non-Michaelian behavior with respect to ATP, with two different Km values of approximately 10 microM and 0.1-0.3 mM. GTP, CTP, and ITP were also hydrolyzed by the preparation at a rate of 10-30% of that of ATP. The preparation was stored at -80 degrees C with retention of function for about a year.     

The production of hemicellulases by aerobic fungi on medium containing residues of banana plant as substrate

Trichoderma harzianum strains T4 and T6, Acrophialophora nainiana, and Humicola grisea var. thermoidea were screened for their ability to produce carbohydrate-degrading enzyme activities in a medium containing banana plant residue as the carbon source. The best balance of enzyme activities was obtained from cultures of H. grisea var. thermoidea. Xylanase activity from crude extract of A. nainiana had a maximum activity at pH 5.5-7.0 and a temperature range of 50-55 degrees C. It was stable up to 55 degrees C at pH 7.0 for at least 2 h. The fungi were also able to produce xylanase and pectinase activities when grown on extractives as substrate.