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 membranebound 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°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°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°C.     

Kinetic properties of C12E8-solubilized (Na+ + K+)-ATPase

The properties of the rectal gland (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.8) solubilized in octaethyleneglycol dodecylmonoether ( C12E8 ) have been investigated. The kinetic properties of the solubilized enzyme resemble those of the membrane-bound enzyme to a large extent. The main difference is that Km for ATP for the (Na+ + K+)-ATPase is about 30 microM for the solubilized enzyme and about 100 microM for the membrane-bound enzyme. The Na+-form (E1) and the K+-form (E2) can also be distinguished in the solubilized enzyme, as seen from tryptic digestion, the intrinsic fluorescence and eosin fluorescence responses to Na+ and K+. The number of vanadate-binding sites is unchanged upon solubilization, and it is shown that vanadate binding is much more resistant to detergent inactivation than the enzymatic activities. The number of phosphorylation sites on the 95-100% pure supernatant enzyme is about 3.8 nmol/mg, and is equal to the number of vanadate sites. Inactivation of the enzyme by high concentrations of detergent can be shown to be related to the C12E8 /protein ratio, with a weight ratio of about 4 being a threshold for the onset of inactivation at low ionic strength. At high ionic strength, more C12E8 is required both for solubilization and inactivation. It is observed that the commercially available detergent polyoxyethylene 10-lauryl ether is much less deleterious than C12E8 , and its advantages in the assay of detergent-solubilized (Na+ + K+)-ATPase are discussed. The results show that (Na+ + K+)-ATPase can be solubilized in C12E8 in an active form, and that most of the kinetic and conformational properties of the membrane-bound enzyme are conserved upon solubilization. C12E8 -solubilized (Na+ + K+)-ATPase is therefore a good model system for a solubilized membrane protein.     

The inhibitor of liver plasma membrane (Ca2+-Mg2+)-ATPase. Purification and identification as a mediator of glucagon action

Rat liver plasma membranes contain (Ca2+-Mg2+)-ATPase sensitive to inhibition by both glucagon and Mg2+. We have previously shown that Mg2+ inhibition is mediated by a 30,000-dalton inhibitor, originally identified as a membrane-bound protein. In fact, this inhibitor is also present in the 100,000 X g supernatant of the total liver homogenate. Its purification was achieved from this fraction by a combination of ammonium sulfate washing, gel filtration, and cationic exchange chromatography. N-Ethylmaleimide (NEM) treatment caused the inactivation of the purified inhibitor, which suggested that this protein possesses at least one NEM-sensitive sulfhydryl group essential for its activity. Treatment of the liver plasma membranes with NEM resulted in a 2- and 5-fold decrease in the affinity of the (Ca2+-Mg2+)-ATPase for glucagon and Mg2+, respectively, while the basal enzyme activity remained unchanged. This effect of NEM was concentration-, pH-, and time-dependent, optimal conditions being obtained by a 60-min treatment of plasma membranes with 50 mM NEM, at pH 7 and at 4 degrees C. The presence of 0.5 mM Mg2+ during NEM treatment of the plasma membranes prevented NEM inactivation. Reconstitution experiments showed that addition of the purified inhibitor to NEM-treated plasma membranes restored the inhibitions of the (Ca2+-Mg2+)-ATPase by both magnesium and glucagon. It is proposed that the (Ca2+-Mg2+)-ATPase inhibitor not only confers its sensitivity of the liver (Ca2+-Mg2+)-ATPase to Mg2+, but also mediates the inhibition of this system by glucagon.     

Stability of [3H]ouabain binding to the (Na+ + K+)-ATPase solubilized with C12E8

The (Na+ + K+)-ATPase from dog kidney and partially purified membranes from HK dog erythrocytes were labeled with [3H]ouabain, solubilized with C12E8 and analyzed by HPLC through a TSK-GEL G3000SW column in the presence of C12E8, Mg2+, HPO4(2-) and glycerol at 20-23 degrees C. The peaks of [3H]ouabain bound to the enzyme from dog kidney and HK dog erythrocyte membranes corresponded to each other with apparent molecular weights of 470 000-490 000. In addition, these bindings of [3H]ouabain to the (Na+ + K+)-ATPase were observed to be stable at 20-23 degrees C for at least 18 h after the solubilization.     

Studies of (Na+ + K+)-sensitive ATPase activity in pig lymphocytes. Effects of concanavalin A.

(Na+ + K+)-ATPase activity is demonstrated in plasma membranes from pig mesenteric lymph nodes. After dodecyl sulfate treatment plasma membranes have an 18-fold higher (Na+ + K+)-ATPase activity, while their ouabain-insensitive Mg2+-ATPase is markedly lowered. A solubilized (Na+ +K+)-ATPase fraction, obtained by Lubrol WX treatment of the membranes, has very high specific activity (21 mumol Pi/h per mg protein). Concanavalin A has no effect on these partially purified (Na+ + K+)-ATPase, while inhibits (40%) this activity in less purified fractions which still contain Mg2+-ATPase activity.     

Solubilization of a high affinity CA-ATPase from dog antrum smooth muscle

A Mg-independent high affinity Ca-ATPase has recently been reported to be present in the plasma membranes of smooth muscle. This enzyme has now been solubilized using deoxycholate. The membrane-bound and the solubilized enzymes resemble each other in Km for Ca2+, and inhibition by fluphenazine. The solubilized enzyme is, however, more sensitive to inhibition by Mg2+ than the membrane bound enzyme. Radiation inactivation analysis shows that whereas the membrane-bound enzyme had a target size of 98,000 +/- 4,000 Daltons, the solubilized enzyme was only 70,000, +/- 7,000 Daltons.     

The effect of changes of the content of membrane cholesterol and the effect of benzyl alcohol on the activity of the intrinsic Mg2+-ATPase from the erythrocyte plasma membrane of the flounder (Platichthys flesus L.)

Extraction of membrane cholesterol and incorporation of cholesteryl hemisuccinate into the membrane affect the activity of the membrane-bound Mg2+-ATPase. Increasing the ratio of cholesterol to phospholipid from 0.30 mg/mg in the control membranes to 0.45-0.90 in the enriched membranes results in a slight increase of the activity of about 20%. Diminishing the ratio of cholesterol to phospholipid to about one tenth of the ratio of the control membrane results in a decrease of the activity to about 30% of the untreated control. Benzyl alcohol inactivates the membrane-bound enzyme. Digitonin-solubilized Mg2+-ATPase is also inactivated by benzyl alcohol. For concentrations below 20 mM the dependence of the solubilized and the membrane-bound enzymes are virtually identical, and linearly dependent on alcohol concentration. This linear relationship continues up to 70 mM for the solubilized enzyme, while inhibition of the membrane-bound form shows a slightly steeper dependence on inhibitor concentration. It is suggested that the activity of the native Mg2+-ATPase depends on the organization of the lipid phase of the membrane and that addition of benzyl alcohol or depletion of cholesterol results in a disorganization of the lipid phase which in turn results in diminished activity.     

Ca2+ binding sites in plasma membranes of rat liver and hepatoma cells, and effect of concanavalin A on the Ca2+ binding sites and cellular uptake of Ca2+

1. Plasma membranes isolated from rat livers and ascites hepatoma cells (AH-130, AH-7974) were assayed for specific Ca2+ binding sites using 45Ca2+ and a Millipore filtration technique. The presence of higher (Kd = 1.4--1.5 . 10(-5) M) and lower (Kd = 0.9--1.0 . 10(-4) M) affinity sites in both liver and hepatoma membranes was observed. The hepatoma plasma membranes however, showed 1.4--2.1-fold as many Ca2+ binding sites (higher and lower affinity sites) as the liver plasma membranes on the basis of protein. 2. Concanavalin A stimulated the specific Ca2+ binding to liver and hepatoma plasma membranes, showing a maximal stimulation (3--5-fold) at 100 microgram/ml. Succinyl concanavalin A was less effective, whereas wheat germ agglutinin and ricinus lectin were ineffective. 3. Concanavalin A stimulated the Ca2+ uptake by AH-7974 cells. The concanavalin A-mediated stimulation of Ca2+ uptake showed lectin-concentrations and Ca2+-concentration dependencies similar to those in the concanavalin A-mediated stimulation of Ca2+ binding.     

The functional unit of sarcoplasmic reticulum Ca2+-ATPase. Active site titration and fluorescence measurements

The properties of sarcoplasmic reticulum Ca2+-ATPase have been studied after modification of the ATP high affinity binding site with fluorescein isothiocyanate, both in the membranous state and after solubilization with the nonionic detergent, octaethyleneglycol monododecyl ether. Total inactivation of both membrane-bound and solubilized Ca2+-ATPase requires covalent attachment of 1 mol of fluorescein/mol of enzyme (115,000 g of protein) or per binding site for ATP. Sedimentation velocity studies of soluble enzyme showed that both unlabeled and fluorescein-labeled Ca2+-ATPase were present in a predominantly monomeric form. The phosphorylation level of unlabeled Ca2+-ATPase was unchanged by solubilization. Dephosphorylation measurements at 0 degree C indicated that the phosphorylation is an intermediate in the ATPase reaction catalyzed by solubilized Ca2+-ATPase. Fluorescein labeling of half of the Ca2+-ATPase in the membrane did not influence the enzyme kinetics of the remaining unmodified Ca2+-ATPase. Measurements of both fluorescein and tryptophan fluorescence indicated that the soluble monomer of Ca2+-ATPase like the membrane-bound enzyme exists in a Ca2+-dependent equilibrium between two principal conformations (E and E). E (absence of Ca2+) is unstable in the soluble form, but the pCa dependence of the E - E equilibrium is identical with that of the membranous Ca2+-ATPase (pCa0.5 = 6.7 and Hill coefficient 2). These results suggest that the Ca2+-ATPase polypeptides function with a high degree of independence in the membrane.     

Properties of oligomycin-induced occlusion of Na+ by detergent-solubilized Na,K-ATPase from pig kidney or shark rectal gland.

Oligomycin induces occlusion of Na+ in membrane-bound Na,K-ATPase. Here it is shown that Na,K-ATPase from pig kidney or shark rectal gland solubilized in the nonionic detergent C12E8 is capable of occluding Na+ in the presence of oligomycin. The apparent affinity for Na+ is reduced for both enzymes upon solubilization, and there is an increase in the sigmoidicity of binding curves, which indicates a change in the cooperativity between the occluded ions. A high detergent/protein ratio leads to a decreased occlusion capacity. De-occlusion of Na+ by addition of K+ is slow for solubilized Na,K-ATPase, with a rate constant of about 0.1 s-1 at 6 degrees C. Stopped-flow fluorescence experiments with 6-carboxyeosin, which can be used to monitor the E1Na-form in detergent solution, show that the K(+)-induced de-occlusion of Na+ correlates well with the fluorescence decrease which follows the transition from the E1Na-form to the E2-form. There is a marked increase in the rate of fluorescence change at high detergent/protein ratios, indicating that the properties of solubilized enzyme are subject to modification by detergent in other respects than mere solubilization of the membrane-bound enzyme. The temperature dependence of the rate of de-occlusion in the range 2 degrees C to 12 degrees C is changed slightly upon solubilization, with activation energies in the range 20-23 kcal/mol for membrane-bound enzyme, increasing to 26-30 kcal/mol for solubilized enzyme. Titrations of the rate of transition from E1Na to E2K with oligomycin can be interpreted in a model with oligomycin having an apparent dissociation constant of about 2.5 microM for C12E8-solubilized shark Na,K-ATPase and 0.2 microM for solubilized pig kidney Na,K-ATPase.     

A comparative study of plasma membrane Mg2+-ATPase activities in normal,regenerating and malignant cells

Plasma membranes have been prepared from rat normal liver cells, regenerating liver cells and Yoshida ascites hepatoma 66 cells after intact cells were first bound to polylysine-coated polyacrylamide beads, and the membrane-associated Mg²⁺-ATPase activity was assayed directly on beads with membrane attached. With plasma membranes from normal liver cells, K_m for ATP and V were found to be higher than those in regenerating liver cells and hepatoma cells. Vanadate caused a different sensitivity of the activity, without an effect in normal liver cells and with an inhibition in regenerating liver cells and hepatoma cells. The activity in normal and regenerating liver cells decreased with increasing temperature above 24–30°C, while the activity in hepatoma cells continued to increase linearly to 37°C. Unlike the enzyme in normal and regenerating liver cells, the hepatoma enzyme was shown to have a higher phase transition temperature and lower activation energies. In all three kinds of cells the activity was increased by the dephosphorylation of plasma membranes and unaffected by the phosphorylation. By means of histochemical Mg²⁺-ATPase staining applied on polyacrylamide gels, at least three major bands which show the enzymic activity were visible in normal and regenerating liver and a single band was detected in hepatoma cells.     

Effect of concanavalin A on membrane-bound enzymes from mouse lymphocytes.

The ionic influence and ouabain sensitivity of lymphocyte mg-2+-atpase and Mg-2+-(Na+ +K+)-activated ATPase were studied in intact cells, microsomal fraction and isolated plasma membranes. The active site of 5'-nucleotidase and Mg2+-ATPase seemed to be localized on the external side of the plasma membrane whereas the ATP binding site of (Na+ +K+)-ATPase was located inside the membrane. Concanavalin A induced an early stimulation of Mg2+-APTase and (Na+ +K+)-ATPase both on intact cells and purified plasma membranes. In contrast, 5'-nucleotidase activity was not affected by the mitogen. Although the thymocyte Mg2+-ATPase activity was 3-5 times lower than in spleen lymphocytes, it was much more stimulated in the former cells (about 40 versus 20%). (Na+ +K+)-ATPase activity was undectectable in thymocytes. However, in spleen lymphocytes (Na+ +K+)-ATPase activity can be detected and was 30% increased by concanavalin A. Several aspects of this enzymic stimulation had also characteristic features of blast transformation induced by concanavalin A, suggesting a possible role of these enzymes, especially Mg2+-ATPase, in lymphocyte stimulation.     

A Ca(2+)-ATPase from rat parotid gland plasma membranes has the characteristics of an ecto-ATPase.

A Ca(2+)-ATPase with an apparent Km for free Ca2+ = 0.23 microM and Vmax = 44 nmol Pi/mg/min was detected in a rat parotid plasma membrane-enriched fraction. This Ca(2+)-ATPase could be stimulated without added Mg2+. However, the enzyme may require submicromolar concentrations of Mg2+ for its activation in the presence of Ca2+. On the other hand, Mg2+ could substitute for Ca2+. The lack of a requirement for added Mg2+ distinguished this Ca(2+)-ATPase from the Ca(2+)-transporter ATPase in the plasma membranes and the mitochondrial Ca(2+)-ATPase. The enzyme was not inhibited by several ATPase inhibitors and was not stimulated by calmodulin. An antibody which was raised against the rat liver plasma membrane ecto-ATPase, was able to deplete this Ca(2+)-ATPase activity from detergent solubilized rat parotid plasma membranes, in an antibody concentration-dependent manner. Immunoblotting analysis of the pellet with the ecto-ATPase antibody revealed the presence of a 100,000 molecular weight protein band, in agreement with the reported ecto-ATPase relative molecular mass. These data demonstrate the presence of a Ca(2+)-ATPase, with high affinity for Ca2+, in the rat parotid gland plasma membranes. It is distinct from the Ca(2+)-transporter, and immunologically indistinguishable from the plasma membrane ecto-ATPase.     

Effect of phospholipid, detergent and protein-protein interaction on stability and phosphoenzyme isomerization of soluble sarcoplasmic reticulum Ca-ATPase

The purpose of the present study was to elucidate the separate roles of lipid, detergent and protein-protein interaction for stability and catalytic properties of sarcoplasmic reticulum Ca-ATPase solubilized in the non-ionic detergent octa(ethylene glycol) monododecyl ether (C12E8). The use of large-zone high-performance liquid chromatography permitted us to define the self-association state of Ca-ATPase peptide at various detergent, phospholipid and protein concentrations, and also during enzymatic turnover with ATP. Conditions were established for monomerization of Ca-ATPase in the presence of a high concentration of phospholipid relative to detergent. The lipid-saturated monomeric preparation was relatively resistant to inactivation in the absence of Ca2+, whereas delipidated enzyme in monomeric or in oligomeric form was prone to inactivation. Kinetics of phosphoenzyme turnover were examined in the presence and absence of Mg2+. Dephosphorylation rates were sensitive to Mg2+, irrespective of whether the peptide was present in soluble monomeric form or was membrane-bound. C12E8-solubilized monomer without added phospholipid was, however, characterized by a fast initial phase of dephosphorylation in the absence of Mg2+. This was not observed with monomer saturated with phospholipid or with monomer solubilized in myristoylglycerophosphocholine or deoxycholate. The mechanism underlying this difference was shown to be a C12E8-induced acceleration of conversion of ADP-sensitive phosphoenzyme (E1P) to ADP-insensitive phosphoenzyme (E2P). The phosphoenzyme isomerization rate was also found to be enhanced by low-affinity binding of ATP. This was demonstrated both in membrane-bound and in soluble monomeric Ca-ATPase. Our results indicate that a single peptide chain constitutes the target for modulation of phosphoenzyme turnover by Mg2+ and ATP, and that detergent effects, distinct from those arising from disruption of protein-protein contacts, are the major determinants of kinetic differences between C12E8-solubilized and membrane-bound enzyme preparations.     

Solubilized (Na+ + K+)-ATPase from shark rectal gland and ox kidney--an inactivation study

The bi-exponential time-course of detergent inactivation at 37 degrees C of C12E8-solubilized (Na+ + K+)-ATPase from shark rectal glands and ox kidney was investigated. The data for shark enzyme, obtained at detergent/protein weight ratios between 2 and 16, are interpreted in terms of a simple model where the membrane bound enzyme is solubilized predominantly as (alpha-beta)2 diprotomers at low detergent concentrations and as alpha-beta protomers at high C12E8 (octaethyleneglycoldodecylmonoether) concentrations. It is observed that the protomers are inactivated 15-fold more rapidly than the diprotomers, and that the rate of inactivation of both oligomers is proportional to the detergent/protein ratio. Inactivation of kidney enzyme was biexponential with a very rapid inactivation of up to 40% of the enzyme activity. The observed rate of inactivation of the slower phase varied with the detergent/protein ratio, but the inactivation pattern for the kidney enzyme could not readily be accommodated within the model for inactivation of the shark enzyme. The rates of inactivation at 37 degrees C were about the same in KCl and NaCl, i.e., in the E2(K) and E1 X Na forms, for both enzymes.     

Membrane Mg-(Ca)-Activated Adenosine Triphosphatase of Escherichia coli: Characterization in the Membrane-Bound and Solubilized States

The membrane-associated Mg(2+)-activated and Ca(2+)-activated adenosine 5'-triphosphatase (EC 3.6.1.3; ATPase) activities of Escherichia coli were further characterized. The degree of inhibition of membrane-bound Mg(2+)-(Ca(2+))-ATPase by a series of anions (i.e., sodium salts of nitrate, iodide, chloride, and acetate) was found to correlate with the relative chaotropic, or solubilizing, effectiveness of these anions. The enzyme was solubilized from washed membrane ghosts by treatment with 0.04% sodium lauryl sulfate at pH 9.0 and 37 C. Solubilized Mg(2+)-(Ca(2+))-ATPase exhibited an initial increase in activity, followed by fairly rapid inactivation, both ATPase activities being particularly cold-labile. The combined stabilizing effects of lauryl mercaptan (1-dodecanethiol), 0.01 m tris(hydroxymethyl)amino-methane-hydrochloride buffer (pH 9.0), 0.2 mm MgCl(2), and ambient temperature facilitated partial purification of the enzyme, the molecular weight of which was estimated to be approximately 100,000 by the gel filtration technique. In general, the membrane-associated Mg(2+)-(Ca(2+))-ATPase of E. coli resembles both mitochondrial membrane ATPase and the well-characterized membrane ATPases of Bacillus megaterium and Microcococcus lysodeikticus. It is of particular interest that N,N'-dicyclohexylcarbodiimide (DCCD), a known inhibitor of mitochondrial ATPase, of mitochondrial oxidative phosphorylation, and of the membrane-bound Mg(2+)-ATPase of Streptococcus faecalis was found to inhibit both the membrane-bound and the solubilized forms of E. coli Mg(2+)-(Ca(2+))-ATPase. The sensitivity of the membrane-associated Mg(2+)-(Ca(2+))-ATPase of E. coli to both anions and cations, its allotopic behavior, and its susceptibility to inhibition by DCCD favor the idea that this enzyme plays a key, probably polyfunctional, role in such biological activities of the membrane as oxidative phosphorylation and ion transport.     

Remarkable stability of solubilized and delipidated sarcoplasmic reticulum Ca2+-ATPase with tightly bound fluoride and magnesium against detergent-induced denaturation

Conditions were developed in the absence of Ca(2+) for purification, delipidation, and long term stabilization of octaethylene glycol monododecyl ether (C(12)E(8))-solubilized sarcoplasmic reticulum Ca(2+)-ATPase with tightly bound Mg(2+) and F(-), an analog for the phosphoenzyme intermediate without bound Ca(2+). The Ca(2+)-ATPase activity to monitor denaturation was assessed after treatment with 20 mm Ca(2+) to release tightly bound Mg(2+)/F(-). The purification and delipidation was successfully achieved with Reactive Red-agarose affinity chromatography. The solubilized Mg(2+)/F(-)-bound Ca(2+)-ATPase was very rapidly denatured at pH 8, but was perfectly stabilized at pH 6 against denaturation for over 20 days at 4 degrees C even without exogenously added phospholipid and at a high C(12)E(8)/enzyme weight ratio (10:1). The activity was not restored unless the enzyme was treated with 20 mm Ca(2+), showing that tightly bound Mg(2+)/F(-) was not released during the long term incubation. The perfect stability was attained with or without 0.1 mm dithiothreitol, but inactivation occurred with a half-life of 10 days in the presence of 1 mm dithiothreitol, possibly due to reduction of a specific disulfide bond(s). The remarkable stability is likely conferred by intimate gathering of cytoplasmic domains of Ca(2+)-ATPase molecule induced by tight binding of Mg(2+)/F(-). The present study thus reveals an essential property of the Mg(2+)/F(-)/Ca(2+)-ATPase complex, which will likely provide clues to understanding structure of the Ca(2+)-released form of phosphoenzyme intermediate at an atomic level.     

Long-term stabilization and crystallization of (Ca2+ + Mg2+)-ATPase of detergent-solubilized erythrocyte plasma membrane.

Conditions which were optimal for the stabilization of Ca2(+)-transporting ATPase in solubilized sarcoplasmic reticulum membranes (Piku?la, S., Mullner, N., Dux, L. and Martonosi, A. (1988) J. Biol. Chem. 263, 5277-5286) were also found conducive for preservation of (Ca2+ + Mg2+)-ATPase activity in detergent-solubilized erythrocyte plasma membrane for up to 60 days. Of particular importance for the stabilization of calmodulin-stimulated Ca2(+)-dependent activity of (Ca2+ + Mg2+)-ATPase of solubilized erythrocyte plasma membrane was the presence of Ca2+ (10-20 mM), glycerol, anti-oxidants, proteinase inhibitors and appropriate detergents. Among eight detergents tested octaethylene glycol dodecyl ether, polyoxyethylene glycol(10) lauryl alcohol and polydocanol were found to be promotive in long-term preservation of the enzyme activity. Under these conditions (Ca2+ + Mg2+)-ATPase of erythrocyte ghosts became highly stable and developed microcrystalline arrays after storage for 35 days. Electron micrographs of the negatively stained and thin sectioned material indicated that crystals of purified, detergent-solubilized, lipid-stabilized erythrocyte (Ca2+ + Mg2+)-ATPase differ from those of Ca2(+)-ATPase of detergent-solubilized sarcoplasmic reticulum microsomes.     

Ligand and lipid domain stabilization of a membraneous Ca2+-ATPase during hyperthermia

The susceptibility of the membranous Ca2+-ATPase of sarcoplasmic reticulum to enzymatic inactivation at hyperthermic temperatures was investigated. Inactivation produced a break in the Arrhenius plot at 45-46 degrees C and was accompanied by an increased mobility of spin label, covalently attached to the Ca2+-ATPase. MgADP and MgATP exerted a markedly stabilizing effect on inactivation, both at pH 7.0 and in acidic media. By contrast, high-affinity Ca2+ or Mg2+ binding only moderately stabilized Ca2+-ATPase (inactivation rates were decreased 2-3 times), and this effect was non-additive, i.e., only observed in the absence of the other divalent cation. But withdrawal of K+ and Na+ gave rise to a pronounced destabilization that could be reversed efficiently by high concentrations of Ca2+ or Mg2+. These results are compared with a previous study on detergent solubilized Ca2+-ATPase (M?ller, J.V., Lind, K.E. and Andersen, J.P. (1980) J. Biol. Chem. 255, 1912-1920) which showed the enzyme to be markedly stabilized by Ca2+ as well as by nucleotide. It is concluded that, due to the presence of nucleotide, inactivation of Ca2+-ATPase is not likely to occur during malignant hyperthermia and that the native environment of the lipid bilayer provides stabilization of the membrane-embedded and Ca2+-translocating domain of the Ca2+-ATPase.     

Desensitization to Mg2+ of the phosphoenzyme in sarcoplasmic reticulum Ca2+-ATPase by the addition of a nonionic detergent and the restoration of sensitivity after its removal

Sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle were solubilized with a high concentration of dodecyl octaethyleneglycol monoether (C12E8) and the kinetic properties of the Ca2+,Mg2+-dependent ATPase [EC 3.6.1.3] were studied. The following results were obtained: 1. SR ATPase solubilized in C12E8 retains high ability to form phosphoenzyme ([EP] = 4--5 mol/10(6) g protein) for at least two days in the presence of 5 mM Ca2+, 0.5 M KCl, and 20% glycerol at pH 7.55. 2. The ATPase activity was dependent on both Mg2+ and Ca2+. However, the rate of E32P decay after the addition of unlabeled ATP was independent of Mg2+. 3. Most of the EP formed in the absence of Mg2+ was capable of reacting with ADP to form ATP in the backward reaction. However, in the presence of 5 mM Mg2+, the amount of ATP formed was markedly reduced without loss of the reactivity of the EP with ADP. 4. The removal of C12E8 from the ATPase by the use of Bio-Beads resulted in the full restoration of the Mg2+ dependency of the EP decomposition. 5. These results strongly suggest that in the case of SR solubilized with a high concentration of C12E8 the decomposition of phosphoenzyme is Mg2+ independent and ATP is mainly hydrolyzed through Mg2+-dependent decomposition of an enzyme-ATP complex, which is in equilibrium with phosphoenzyme and ADP.