Properties and function of clostridial membrane ATPase.

ATPase (ATP phosphohydrolase, EC 3.6.1.3) was detected in the membrane fraction of the strict anaerobic bacterium, Clostridium pasteurianum. About 70% of the total activity was found in the particulate fraction. The enzyme was Mg2+ dependent; Co2+ and Mn2+ but not Ca2+ could replace Mg2+ to some extent; the activation by Mg2+ was slightly antagonized by Ca2+. Even in the presence of Mg2+, Na+ or K+ had no stimulatory effect. The ATPase reaction was effectively inhibited by one of its products, ADP, and only slightly by the other product, inorganic phosphate. Of the nucleoside triphosphates tested ATP was hydrolyzed with highest affinity ([S]0.5 v = 1.3 mM) and maximal activity (120 U/g). The ATPase activity could be nearly completely solubilized by treatment of the membranes with 2 M LiCl in the absence of Mg2+. Solubilization, however, led to instability of the enzyme. The clostridial solubilized and membrane-bound ATPase showed different properties similar to the "allotopic" properties of mitochondrial and other bacterial ATPases. The membrane-bound ATPase in contrast to the soluble ATPase was sensitive to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). DCCD, at 10(-4) M, led to 80% inhibition of the membrane-bound enzyme; oligomycin ouabain, or NaN3 had no effect. The membrane-bound ATPase could not be stimulated by trypsin pretreatment. Since none of the mono- or divalent cations had any truly stimulatory effect, and since a pH gradient (interior alkaline), which was sensitive to the ATPase inhibitor DCCD, was maintained during growth of C. pasteurianum, it was concluded that the function of the clostridial ATPase was the same as that of the rather similar mitochondrial enzyme, namely H+ translocation. A H+-translocating, ATP-consuming ATPase appears to be intrinsic equipment of all prolaryotic cells and as such to be phylogenetically very old; in the course of evolution the enzyme might have been developed to a H+-(re)translocating, ATP-forming ATPase as probably realized in aerobic bacteria, mitochondria and chloroplasts.     

Phospholipids of Azotobacter vinelandii

Analyses of resting cells of Azotobacter vinelandii revealed that numerous phospholipids were present that did not concentrate in the membranous R(3) fraction which carried out electron transport function.     

Cytochrome c oxidation by the electron transport fraction of Azotobacter vinelandii

The spectrophotometric oxidation of horse heart ferrocytochrome c was examined by use of the particulate electron transport fraction (R(3)) of Azotobacter vinelandii strain O. Unlike cytochrome c, purified preparations of native Azotobacter cytochromes c(4) + c(5) were oxidized only slowly by the electron transport fraction. The oxidation of mammalian cytochrome c proceeded at an appreciable rate and displayed "apparent" first-order kinetics at a pH optimum of 9.0 with tris(hydroxymethyl)aminomethane-chloride buffer. The calculated V(max) value was 0.22 mumole of cytochrome c oxidized per min per mg of protein (25 C) and a K(m) value for cytochrome c of 2.3 x 10(-5)m was obtained. Ferricytochrome c was a "strict" competitive inhibitor for this oxidation. Cytochrome c oxidation by the Azotobacter electron transport system was markedly sensitive to cyanide, azide, and hydroxylamine, although carbon monoxide inhibition could not be demonstrated. It was sensitive also to high concentrations of phosphate, ethylenediaminetetraacetate, and some metal cations. "Aging" or prolonged storage of the Azotobacter R(3) fraction, at 4 C for 10 days, resulted in a threefold increase in specific activity. The cytochrome c peroxidase type of reaction did not occur with the R(3) electron transport fraction.     

Membrane bound and soluble adenosine triphosphatase of Escherichia coli K 12. kinetic properties of the basal and trypsin-stimulated activities

Basal and trypsin-stimulated adenosine triphosphatase activities of Escherichia coli K 12 have been characterized at pH 7.5 in the membrane-bound state and in a soluble form of the enzyme. The saturation curve for Mg2+/ATP = 1/2 was hyperbolic with the membrane-bound enzyme and sigmoidal with the soluble enzyme. Trypsin did not modify the shape of the curves. The kinetic parameters were for the membrane-bound ATPase: apparent Km = 2.5 mM, Vmax (minus trypsin) = 1.6 mumol-min-1-mg protein-1, Vmax (plus trypsin) = 2.44 mumol-min-1-mg protein-1; for the soluble ATPase: [S0.5] = 1.2 mM, Vmax (-trypsin) = 4 mumol-min-1-mg protein-1; Vmax (+ trypsin) = 6.6 mumol-min-1-mg protein-1. Hill plot analysis showed a single slope for the membrane-bound ATPase (n = 0.92) but two slopes were obtained for the soluble enzyme (n = 0.98 and 1.87). It may suggest the existence of an initial positive cooperativity at low substrate concentrations followed by a lack of cooperativity at high ATP concentrations. Excess of free ATP and Mg2+ inhibited the ATPase but excess of Mg/ATP (1/2) did not. Saturation for ATP at constant Mg2+ concentration (4 mM) showed two sites (groups) with different Kms: at low ATP the values were 0.38 and 1.4 mM for the membrane-bound and soluble enzyme; at high ATP concentrations they were 17 and 20 mM, respectively. Mg2+ saturation at constant ATP (8 mM) revealed michealian kinetics for the membrane-bound ATPase and sigmoid one for the protein in soluble state. When the ATPase was assayed in presence of trypsin we obtained higher Km values for Mg2+. These results might suggest that trypsin stimulates E. coli ATPase by acting on some site(s) involved in Mg2+ binding. Adenosine diphosphate and inorganic phosphate (Pi) act as competitive inhibitors of Escherichia coli ATPase. The Ki values for Pi were 1.6 +/- 0.1 mM for the membrane-bound ATPase and 1.3 +/- 0.1 mM for the enzyme in soluble form, the Ki values for ADP being 1.7 mM and 0.75 mM for the membrane-bound and soluble ATPase, respectively. Hill plots of the activity of the soluble enzyme in presence of ADP showed that ADP decreased the interaction coefficient at ATP concentrations below its Km value. Trypsin did not modify the mechanism of inhibition or the inhibition constants. Dicyclohexylcarbodiimide (0.4 mM) inhibited the membrane-bound enzyme by 60-70% but concentrations 100 times higher did not affect the residual activity nor the soluble ATPase. This inhibition was independent of trypsin. Sodium azide (20 muM) inhibited both states of E. coli ATPase by 50%. Concentrations 25-fold higher were required for complete inhibition. Ouabain, atebrin and oligomycin did not affect the bacterial ATPase.     

L-malate oxidation by the electron transport fraction of Azotobacter vinelandii

The membrane-bound l-malate oxidoreductase of Azotobacter vinelandii strain O was found to be a flavoprotein-dependent enzyme associated with the electron transport system (R(3)) of this organism. The particulate R(3) fraction, which possessed the l-malate oxidoreductase, carried out the cyanide-sensitive oxidation of l-malate, d-lactate, reduced nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, succinate, cytochrome c, tetramethyl-p-phenylenediamine, and p-phenylenediamine, with molecular O(2) as the terminal electron acceptor. d-Malate was not oxidized, but l-malate was oxidized to oxalacetate. Phenazine methosulfate (PMS), vitamin K(3), K(3)Fe(CN)(6), nitro blue tetrazolium, and dichloroindophenol all served as good terminal electron acceptors for the l-malate oxidoreductase. Cytochrome c was a poor electron acceptor. Extensive studies on the l-malate oxidase and PMS and K(3) reductases revealed that all were stimulated specifically by flavine adenine dinucleotide and nonspecifically by di- or trivalent cations, i.e., Ca(++), Ba(++), Mn(++), Mg(++), Fe(+++), Ni(++), and Al(+++). All these activities were markedly sensitive to ethylenediaminetetraacetate (EDTA). The V(max) values for the l-malate oxidase, PMS, and vitamin K(3) reductases were, respectively, 3.4, 15.1, and 45.5 mumoles of substrate oxidized per min per mg of protein at 37 C. Spectral studies revealed that the Azotobacter R(3) flavoprotein and cytochromes (a(2), a(1), b(1), c(4), and c(5)) were reduced by l-malate. l-Malate oxidase activity was sensitive to various inhibitors of the electron transport system, namely, p-chloromercuriphenylsulfonic acid, chlorpromazine, 2-n-heptyl-4-hydroxyquinoline-N-oxide, antimycin A, and KCN. Minor inhibitory effects were noted with the inhibitors 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione, rotenone, and Amytal.     

Membrane adenosine triphosphatase of Micrococcus lysodeikticus: effect of millimolar Mg2+ in modulating the properties of the membrane-bound enzyme

The latency of Micrococcus lysodeikticus membrane-bound Mg(2+)-adenosine triphosphatase (ATPase) is expressed by the ratio of its activity assayed in the presence of trypsin ("total") versus the activity assayed in absence of the protease ("basal"). By isolating membranes in the presence of variable concentrations of Mg(2+) (50 mM, 10 mM, or none) and by washing them with different Mg(2+)- and ethylenediaminetetraacetic acid-containing tris(hydroxymethyl)aminomethane-hydrochloride buffers (pH 7.5), we showed that the enzyme latency was dependent on the environmental concentration of this divalent metal ion. Mg(2+) bound to at least two classes of sites. The binding of Mg(2+) to low-affinity sites (saturation at approximately 40 mM external Mg(2+)) induced a high basal ATPase activity, whereas its binding to medium-affinity sites (saturation at about 2 mM Mg(2+)) correlated with low basal activity and a very high stimulation by trypsin. Membranes with tightly bound Mg(2+) (high affinity?) revealed an intermediate behavior for the latency of M. lysodeikticus ATPase. The Mg(2+)/Ca(2+) antagonism as activators of the membrane ATPase was not directly related to Mg(2+) binding by the membranes. The efficiency of the ATPase release from M. lysodeikticus membrane by 3 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.5) was inversely proportional to the concentration of external and/or bound Mg(2+). Deoxycholate (DOC) (1%) solubilized the ATPase from all types of membrane. All the soluble ATPases behaved as Ca(2+)-ATPases, but the DOC-soluble fractions showed degrees of latency like those of the original membranes. The DOC-soluble ATPase preparation revealed a vesicular structure and complex protein patterns by sodium dodecyl sulfate gel electrophoresis. We propose that ATPase latency is modulated via a Mg(2+)-ATPase-membrane complex.     

Characterization of rat heart plasma membrane Ca2+/Mg2+ ATPase

The Ca2+/Mg2+ ATPase of rat heart plasma membrane was activated by millimolar concentrations of Ca2+ or Mg2+; other divalent cations also activated the enzyme but to a lesser extent. Sodium azide at high concentrations inhibited the enzyme by about 20%; oligomycin at high concentrations also inhibited the enzyme slightly. Trifluoperazine at high concentrations was found inhibitory whereas trypsin treatment had no significant influence on the enzyme. The rate of ATP hydrolysis by the Ca2+/Mg2+ ATPase decayed exponentially; the first-order rate constants were 0.14-0.18 min-1 for Ca2+ ATPase activity and 0.15-0.30 min-1 for Mg2+ ATPase at 37 degrees C. The inactivation of the enzyme depended upon the presence of ATP or other high energy nucleotides but was not due to the accumulation of products of ATP hydrolysis. Furthermore, the inactivation of the enzyme was independent of temperature below 37 degrees C. Con A when added into the incubation medium before ATP blocked the ATP-dependent inactivation; this effect was prevented by alpha-methylmannoside. In the presence of low concentrations of detergent, the rate of ATP hydrolysis was reduced while the ATP-dependent inactivation was accelerated markedly. Both Con A and glutaraldehyde decreased the susceptibility of Ca2+/Mg2+ ATPase to the detergent. These results suggest that the Ca2+/Mg2+ ATPase is an intrinsic membrane protein which may be regulated by ATP.     

Oxidative phosphorylation in Azotobacter vinelandii. Effect of inhibitors and uncouplers on P/O ratio, trypsin-induced ATPase and ADP-stimulated respiration

1. The effect of various inhibitors and uncouplers of mitochondrial oxidative phosphorylation on phosphorylation by Azotobacter particles is reported.

2. The ATPase activity of Azotobacter particles and of the soluble factor involved in oxidative phosphorylation is stimulated 10–20-fold by incubation with trypsin. The trypsin-induced ATPase is Mg²⁺ or Ca²⁺ dependent, Mg²⁺ being the more effective cation. The ATPase is stimulated somewhat by 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole, desaspidin and carbonyl cyandie m-chlorophenylhydrazone, and inhibited by atebrin and Dio-9.

3. In previous work ADP was shown to stimulate the oxidation of NADH but not of the Site-II substrates malate, lactate or succinate. Stimulation by ADP of oxidation of malate has now been observed by inhibiting electron transport by 2-heptyl-4-hydroxyquinoline-N-oxide or by increasing the electron flow by adding lactate. The stimulation is, therefore, not confined to phosphorylation Site I.     

Properties and function of clostridial membrane ATPase

ATPase (ATP phosphohydrolase, EC 3.6.1.3) was detected in the membrane fraction of the strict anaerobic bacterium, Clostridium pasteurianum. About 70% of the total activity was found in the particulate fraction. The enzyme was Mg²⁺ dependent; Co²⁺ and Mn²⁺ but not Ca²⁺ could replace Mg²⁺ to some extent; the activation by Mg²⁺ was slightly antagonized by Ca²⁺. Even in the presence of Mg²⁺, Na⁺ or K⁺ had no stimulatory effect. The ATPase reaction was effectively inhibited by one of its products, ADP, and only slightly by the other product, inorganic phosphate. Of the nucleoside triphosphates tested ATP was hydrolyzed with highest affinity ([S]_{0.5 V} = 1.3 mM) and maximal activity (120 U/g). The ATPase activity could be nearly completely solubilized by treatment of the membranes with 2 M LiCl in the absence of Mg²⁺. Solubilization, however, led to instability of the enzyme.

The clostridial solubilized and membrane-bound ATPase showed different properties similar to the “allotopic” properties of mitochondrial and other bacterial ATPases. The membrane-bound ATPase in contrast to the soluble ATPase was sensitive to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). DCCD, at 10^-4 M, led to 80% inhibition of the membrane-bound enzyme; oligomycin, ouabain, or NaN₃ had no effect. The membrane-bound ATPase could not be stimulated by trypsin pretreatment.

Since none of the mono- or divalent cations had any truly stimulatory effect, and since a pH gradient (interior alkaline), which was sensitive to the ATPase inhibitor DCCD, was maintained during growth of C. pasteurianum, it was concluded that the function of the clostridial ATPase was the same as that of the rather similar mitochondrial enzyme, namely H⁺ translocation. A H⁺-translocating, ATP-consuming ATPase appears to be intrinsic equipment of all prokaryotic cells and as such to be phylogenetically very old; in the course of evolution the enzyme might have been developed to a H⁺-(re)translocating, ATP-forming ATPase as probably realized in aerobic bacteria, mitochondria and chloroplasts.     

A reinvestigation of the pre-steady-state ATPase activity of the nitrogenase from Azotobacter vinelandii.

The pre-steady-state ATPase activity of nitrogenase has been reinvestigated. The exceptionally high burst in the hydrolysis of MgATP by the nitrogenase from Azotobacter vinelandii communicated by Cordewener et al. (1987) [Cordewener J., ten Asbroek A., Wassink H., Eady R. R., Haaker H. & Veeger C. (1987) Eur. J. Biochem. 162, 265-270] was found to be caused by an apparatus artefact. A second possible artefact in the determination of the stoichiometry of the pre-steady-state ATPase activity of nitrogenase was observed. Acid-quenched mixtures of dithionite-reduced MoFe or Fe protein of Azotobacter vinelandii nitrogenase and MgATP contained phosphate above the background level. It is proposed that due to this reaction, quenched reaction mixtures of nitrogenase and MgATP may contain phosphate in addition to the phosphate released by the ATPase activity of the nitrogenase complex. It was feasible to monitor MgATP-dependent pre-steady-state proton production by the absorbance change at 572 nm of the pH indicator o-cresolsulfonaphthalein in a weakly buffered solution. At 5.6 degrees C, a pre-steady-state phase of H+ production was observed, with a first-order rate constant of 2.2 s-1, whereas electron transfer occurred with a first-order rate constant of 4.9 s-1. At 20.0 degrees C, MgATP-dependent H+ production and electron transfer in the pre-steady-state phase were characterized by observed rate constants of 9.4 s-1 and 104 s-1, respectively. The stopped-flow technique failed to detect a burst in the release of protons by the dye-oxidized nitrogenase complex. It is concluded that the hydrolysis rate of MgATP, as judged by proton release, is lower than the rate of electron transfer from the Fe protein to the MoFe protein.     

Ca2+-Mg2+-ATPase activity in brain coated microvesicles purified on immunosorbents

Coated microvesicle fractions isolated from ox forebrain cortex by the ultracentrifugation procedure of Pearse (1) and by the modified, less time consuming method of Keen et al (2) had comparable Ca2+ +Mg2+ dependent ATPase activities (about 9 mumol/h per mg protein). The Na+ +K+ +Mg2+ dependent ATPase activity was 3.2 mumol/h per mg (+/- 1.0, S.D., n = 3) when microvesicles were prepared according to (1) and 1.5 mumol/h per mg (+/- 1.0, S.D., n = 3) when prepared according to (2). Oligomycin, ruthenium red, and trifluoperazine, inhibitors of Ca2+ transport in mitochondria and erythrocyte membranes had no effect on Ca2+ +Mg2+ dependent ATPase from any of the preparations. As demonstrated both by ATPase assays and electron microscopy, coated microvesicles could be bound to immunosorbents prepared with poly-specific antibodies against a coated microvesicle fraction obtained by the method of Pearse (1). The binding could be inhibited by dissolved coat protein using partially purified clathrin. The fraction of coated vesicles eluted from the immunosorbent was purified relative to the starting material as judged by electron microscopy. The Ca2+ +Mg2+ ATPase activity and calmodulin content was copurified with the coated microvesicles and the specific activity of Na+ +K+ +Mg2+ ATPase was decreased. Na+ +K+ +Mg2+ dependent ATPase activity in the coated microvesicle fraction could be ascribed to membranes with the appearance of microsomes. These membranes were also bound to the immunosorbents, but the binding was not influenced by clathrin. The capacity of the immunosorbents for these membranes was less than for the coated microvesicles, resulting in a decrease of Na+ +K+ +Mg2+ dependent ATPase activity in the eluted coated microvesicle fraction. It was concluded that Ca2+ +Mg2+ ATPase activity is not a contamination from plasma membrane vesicles or mitochondrial membranes but seems to be an integral part of the coated vesicle membrane.     

Freezing injury and uncoupling of phosphorylation from electron transport in chloroplasts

Freezing of chloroplast membranes uncouples photophosphorylation from electron transport and inactivates the light-dependent and thiol-requiring ATPase, conformational changes and the light-dependent proton uptake. All of these energy requiring activities can be protected against inactivation by addition of sucrose prior to freezing. The direct relation to photophosphorylation is demonstrated by the quantitatively similar response of photophosphorylation and the other activities to sucrose protection. Salts interfere with the protection afforded by sucrose.

In contrast to the light-dependent ATPase, the ATPase activities which are unmasked by digestion with trypsin show no significant response to freezing. Similarly, the chloroplast coupling factor, which is released from the membranes by ethylenediamine tetraacetic acid treatment, survives freezing. The membranes, which are depleted of the factor, are damaged by freezing.

The results suggest that uncoupling of phosphorylation from electron transport is caused by interference of freezing with a structure involved in the formation of a non-phosphorylated high energy state of chloroplasts. They are best explained on the basis of Mitchell's theory of phosphorylation. Since freezing alters the permeability properties of chloroplast membranes—frozen membrane vesicles no longer function as osmometers—it may be assumed that freezing uncouples phosphorylation from electron transport by preventing the formation of a pH gradient across the vesicle membranes owing to proton leakage through the membranes. From the results, the basic injury caused by freezing appears to consist in the alteration of permeability properties of biological membranes due to the dehydration which accompanies freezing.

     

Properties of detergent-solubilized and membranous (Ca2+ + Mg2+)-activated ATPase from sarcoplasmic reticulum as studied by sulfhydryl reactivity and ESR spectroscopy. Effect of protein-protein interactions

(1) Sulfhydryl reactivity and electron spin resonance spectra of nitroxide maleimide spin labels, covalently attached to sarcoplasmic reticulum ATPase, were examined on both detergent-solubilized and membranous material. Monomeric and oligomeric ATPases were prepared by the use of dodecyloctaethylene glycol monoether as a solubilizing detergent. (2) Immediately after solubilization, the reaction curve of nonomeric ATPase with 5,5'-dithiobis(2-nitrobenzoate) was characterized by positive cooperativity (S-shaped as a function of time). In contrast, the SH reactivity of both oligomeric and membranous ATPases obeyed usual first-order kinetics and could be analyzed in terms of three classes of reactive site. All enzymatically active ATPase preparations responded to addition of ADP with a decrease in SH reactivity. During enzymatic inactivation of monomeric ATPase, the SH-modification rate was dramatically enhanced with loss of cooperative features. Ca2+ removal from the high-affinity sites stimulated SH reactivity before inactivation had taken place. (3) ESR spectroscopy indicated less motional constraints on monomeric than on oligomeric and membranous ATPases. Arrhenius plots of ESR spectral parameters suggest a conformational transition in both membranous and solubilized ATPases at about 22 degrees C. The transition was also present in EGTA-, but not in heat-inactivated ATPase. Although SH reactivity of monomeric ATPase was dramatically enhanced by EGTA inactivation, the results of ESR, circular dichroism and analytical ultracentrifugation experiments indicate limited conformational changes induced by EGTA treatment. (4) The data indicate marked differences in the properties of monomeric ATPase on the one hand and oligomeric and membranous enzymes on the other hand. They are consistent with previous functional evidence for the presence of ATPase in an associated state in the membrane (M?ller, J.V., Lind, K.E. and Andersen, J.P. (1980) J. Biol. Chem. 255, 1912-1920).     

Isolation and characterization of an inhibitory subunit of the Mg2+--Ca2+-ATPase of Escherichia coli.

1. Stimulation of the Escherichia coli ATPase activity by urea and trypsin shows that the ATPase activity both in the membrane-bound and the solubilized form is partly masked. 2. A protein, inhibiting the ATPase activity of Escherichia coli, can be isolated by sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified ATPase. The inhibitor was identified with the smallest of the subunits of E. coli ATPase. 3. The molecular weight of the ATPase inhibitor is about 10,000, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and deduced from the amino acid composition. 4. The inhibitory action is independent of pH, ionic strength or the presence of Mg2+ or ATP. 5. The ATPase inhibitor is heat-stable, insensitive to urea but very sensitive to trypsin degradation. 6. The Escherichia coli ATPase inhibitor does not inhibit the mitochondrial or the chloroplast ATPase.     

Lipid environment of gastric potassium ion-stimulated adenosine triphosphatase.

The K+-stimulated ATPase associated with the purified gastric microsomal fraction can be completely inactivated by treatment with 15% (v/v) ethanol for 60s at 37 degrees C, but not at 25 degrees C. Sequential exposure of the microsomal fraction to 15% ethanol at 25 degrees C and 37 degrees C caused release of 2.5% and 2.9% of the total membrane phospholipids respectively. Restoration of the enzyme activity was achieved by sonication with phosphatidylcholine in the presence of Mg2+, K+ and ATP, which were essential for the reconstitution. Our data suggest that the phospholipids extracted by 15% ethanol at 37 degrees C are derived primarily from the immediate lipid environment of the enzyme, and ATP, together with the metal ions, helps the partially delipidated enzyme to retain the appropriate configuration for the subsequent reconstitution.     

Partial purification of (Ca2+ + Mg2+)-dependent ATPase from pig smooth muscle and reconstitution of an ATP-dependent Ca2+-transport system.

(CaMg)ATPase [(Ca2+ + Mg2+)-dependent ATPase] was partially purified from a microsomal fraction of the smooth muscle of the pig stomach (antrum). Membranes were solubilized with deoxycholate, followed by removal of the detergent by dialysis. The purified (CaMg)ATPase has a specific activity (at 37 degrees C) of 157 +/- 12.1 (7)nmol.min-1.mg-1 of protein, and it is stimulated by calmodulin to 255 +/- 20.9 (7)nmol.min.mg-1. This purification of the (CaMg)ATPase resulted in an increase of the specific activity by approx. 18-fold and in a recovery of the total enzyme activity of 55% compared with the microsomal fraction. The partially purified (CaMg)ATPase still contains some Mg2+-and (Na+ + K+)-dependent ATPase activities, but their specific activities are increased relatively less than that of the (CaMg)ATPase. The ratios of the (CaMg)ATPase to Mg2+- and (Na+ + K+)-dependent ATPase activities increase from respectively 0.14 and 0.81 in the crude microsomal fraction to 1.39 and 9.07 in the purified preparation. During removal of the deoxycholate by dialysis, vesicles were reconstituted which were capable of ATP-dependent Ca2+ transport.     

A new, smaller actin-activatable myosin subfragment 1 which lacks the 20-kDa, SH1 and SH2 peptide

The actin-dependent ATPase activity of myosin is retained in the separated heads (S1) which contain the NH2-terminal 95-kDa heavy chain fragment and one or two light chains. The S1 heavy chain can be degraded further by limited trypsin treatment into characteristic 25-, 50-, and 20-kDa peptides, in this order from the NH2-terminal end. The 20-kDa peptide contains an actin-binding site and SH1 and SH2, two thiols whose modification dramatically affects ATPase activity. By treating myosin filaments with trypsin at 4 degrees C in the presence of 2 mM MgCl2, we have now obtained preferential cleavage at the 50-20-kDa heavy chain site without any cleavage at the head-rod junction and hinge region in the rod. Incubation of these trypsinized filaments at 37 degrees C in the presence of MgATP released a new S1 fraction which lacked the COOH-terminal 20-kDa heavy chain peptide region. This fraction, termed S1'(75K), has more than 50% of the actin-activated Mg2+-ATPase activity of S1 and the characteristic Ca2+-ATPase and K+-EDTA ATPase activities of myosin. These results show that SH1 and SH2 are not essential for ATPase activity and that binding of actin to the 20-kDa region is not essential for the enhancement of the Mg2+-ATPase activity.     

Thermal behavior and lipid composition of cauliflower plasma membranes in relation to ATPase activity and chilling sensitivity

A plasma-membrane fraction rich in ion-stimulated ATPase activity was isolated from cauliflower (Brassica oleracea L.) buds. The activity of the ATPase was dependent on Mg(2+) and stimulated 4-fold by K(+). The lipids of the membrane fraction contained 57% by weight of phospholipid, 16% glycolipid including sterol glycosides, and 27% neutral lipids. Sterols and sterol esters comprised 9% by weight of the total lipid fraction, and the m ratio of total sterol to phospholipid was 0.5. Fatty acid unsaturation of the membrane lipids was 75%. Arrhenius plots of the Mg(2+) and Mg(2+) + K(+) stimulated ATPase activity were biphasic with an increase in activation energy occurring below about 12 degrees C, a response typical of some membrane-associated enzymes of chilling-sensitive plants. No thermal transitions were detected in the membranes or membrane lipids between 0 and 30 degrees C using differential scanning calorimetry and electron spin resonance spectroscopy. This type of thermal behavior is typical of membranes of chilling-resistant plants. It was concluded that the low temperature increase in activation energy of the ion-stimulated, membrane-associated ATPase is an intrinsic property of the enzyme system and not the result of a transition in the bulk membrane lipid.     

Regulation of the (Ca2+ + Mg2+)-ATPase activity and calcium transport in reconstituted cardiac sarcolemmal vesicles. Effect of sodium and potassium

A reconstitution procedure for a cardiac sarcolemmal enriched fraction is described. In the reconstituted cardiac sarcolemmal inside-out vesicles, a difference in calcium transport and (Ca2+ + Mg2+)-ATPase activity was found depending on the side of the membrane at which sodium and potassium were placed. Having inhibited the (Na+ + K+)- ATPase activity with ouabain, the active transport of calcium was increased when potassium was located outside and sodium inside the reconstituted vesicles. Nevertheless, this activity was maximal having potassium present on both sides. During calcium transport it was also shown that 86Rb moves opposite to calcium. When the experiment was carried out having 22Na located at the inside, there was no movement of this cation despite the low calcium transport observed. The present study supports the possibility of potassium having a stimulatory effect upon the sarcolemmal (Ca2+ + Mg2+)-ATPase activity and suggests the existence of a Ca2+, K+ co-transport carried out by this enzyme.     

Effect of concanavalin A on the activity of membrane-bound and detergent-solubilized Mg2+ -ATPase

Plasma membranes were isolated after binding liver and hepatoma cells to polylysine-coated polyacrylamide beads, and the effect of concanavalin A on the membrane-bound Mg2+ -ATPase and the Mg2+ -ATPase solubilized by octaethylene glycol monododecyl ether (C12E8) was studied. In the experiment of membrane-bound Mg2+ -ATPase, plasma membranes were pretreated with Concanavalin A and the activity was assayed. Concanavalin A stimulated the activity of both liver and hepatoma enzymes assayed above 20 degrees C. Concanavalin A abolished the negative temperature dependency characteristic of liver plasma membrane Mg2+ -ATPase. On the other hand, Concanavalin A prevented the rapid inactivation due to storage at -20 degrees C, which was characteristic of hepatoma plasma membrane Mg2+ -ATPase. With solubilized Mg2+ -ATPase from liver plasma membranes, the negative temperature dependency was not observed. Concanavalin A, which was added to the assay medium, stimulated the activity of the enzyme solubilized in C12E8 at a high ionic strength. However, Concanavalin A failed to show any effect on the enzyme solubilized in C12E8 at a low ionic strength. With solubilized Mg2+ -ATPase from hepatoma plasma membranes, Concanavalin A could not prevent the inactivation of the enzyme during incubation at -20 degrees C.