Conformational transitions of detergent-solubilized Na,K-ATPase are conveniently monitored by the fluorescent probe 6-carboxy-eosin.

6-carboxy-eosin is introduced as a sensitive, non-covalently bound fluorescent probe for monitoring conformational changes in detergent-solubilized Na,K-ATPase. The dissociation constant for 6-carboxy-eosin is about 0.1 microM in 20 mM NaCl at 6 degrees C (pH 7.0) for Na,K-ATPase solubilized in C12E8. It is shown that the slow conformational change from E2 (in K+) to E1 (in Na+) is 4-fold more rapid in the solubilized state than in the membrane-bound state, both for shark rectal gland and pig kidney Na,K-ATPase. The rate of the E1 to E2 transition is rapid and of the same order of magnitude both for the membrane-bound and the solubilized enzyme. All conformational transitions are considerably slower for pig kidney enzyme than for shark enzyme, both in the membrane-bound and in the solubilized state.     

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.     

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.     

Correlation between the activities and the oligomeric forms of pig gastric H/K-ATPase

Membrane-bound H/K-ATPase was solubilized by octaethylene glycol dodecyl ether (C(12)E(8)) or n-octyl glucoside (nOG). H/K-ATPase activity and the distribution of protomeric and oligomeric components were evaluated by high-performance gel chromatography (HPGC) and by single-molecule detection using total internal reflection fluorescence microscopy (TIRFM). As evidenced by HPGC of the C(12)E(8)-solubilized enzyme, the distribution of oligomers was 12% higher oligomeric, 44% diprotomeric, and 44% protomeric species, although solubilization by C(12)E(8) reduced the H/K-ATPase activity to 1.8% of that of the membrane-bound enzyme. The electron microscopic images of the C(12)E(8)-solubilized enzyme showed the presence of protomers and a combination of two and more protomers. While the nOG-solubilized H/K-ATPase retained the same turnover number and 71% of the specific activity as that of the membrane-bound enzyme, 56% higher oligomeric, 34% diprotomeric, and 10% protomeric species were detected. TIRFM analysis of solubilized fluorescein 5'-isothiocyanate (FITC)-modified H/K-ATPase at Lys-518 of the alpha-chain showed a quantized photobleaching of the FITC fluorescence intensity. For the C(12)E(8)-solubilized FITC-enzyme, the fraction of each of the initial relative fluorescence intensity units of 4, 2, and 1 was, respectively, 5%, 44% and 51%. In the case of the nOG-solubilized FITC-enzyme, each fraction of 4 and 2 units was, respectively, 54% and 46% with no detectable 1 unit fraction. This represents the first direct observation of H/K-ATPase in aqueous solution. The correlation between the enzymatic activities and distribution of oligomeric forms of H/K-ATPase by HPGC and the observation of a single molecule of H/K-ATPase and others suggests that the tetraprotomeric form of H/K-ATPase molecules represents the functional species 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.     

ATP binding to solubilized (Na+ + K+)-ATPase. The abolition of subunit-subunit interaction and the maximum weight of the nucleotide-binding unit

Membrane-bound (Na+ + K+)-ATPase from pig kidney outer medulla shows apparent heterogeneity in its ATP-binding site population when assays are carried out in the presence of K+. This finding has been interpreted as being due to interaction between (at least) two subunits, each containing an ATP-binding site. Treating the membrane-bound enzyme with the detergent, C12E8, has been shown to solubilize enzymatically active alpha beta-protomers. We show that in the dissolved enzyme all ATP-binding sites in the population are identical both in the absence and in the presence of K+, which would be consistent with an abolition of identical both in the absence and in the presence of K+, which would be consistent with an abolition of subunit-subunit interaction. This supports previous suggestions that enzyme solubilized by C12E8 is monomeric and that the membrane-bound enzyme is not. Differential extraction of enzyme-containing membranes with C12E8 yielded preparations with an ATP-binding capacity of up to 5.8 nmol per mg protein, measured by the method of Lowry et al. (Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275), with bovine serum albumin as standard. Evidence is presented that makes it likely that preparations with an ATP-binding capacity of 7.5 nmol per mg protein (as determined by the above-mentioned assay) will be obtainable. This corresponds to an alpha beta-protomer molecular weight of 133 000 which approximates closely to the minimum value found in the literature for an alpha beta-protomer (i.e., 126 000).     

In vitro replication of cowpea mosaic virus RNA. II. Solubilization of membrane-bound replicase and the partial purification of the solubilized enzyme.

A method for the solubilization of membrane-bound Cowpea mosaic virus RNA replicase has been developed by bypassing the use of detergents. Solubilization has been achieved by washing the 31,000 x g-pellet containing the bound replicase with a Mg2+-deficient buffer. This procedure had several advantages as compared to treatments with nonionic or ionic detergents: (i) the solubilized enzyme was stable at 4 C, (ii) more than 80% of the replicase could be solubilized without loss of total enzyme activity, (iii) the replicase was rather selectively released resulting in a two- to threefold increase in specific activity per se, and (iv) most of the green color from chloroplast fragments present in the crude replicase fraction remained membrane bound resulting in only slightly colored preparations of solubilized enzyme. The solubilized replicase has been further purified by DEAE-Bio Gel column chromatography. RNA synthesis directed by the DEAE-purified enzyme was template dependent and proceeded at a linear rate for at least 9 h.     

UDP-Glucose: (1,3)-beta-Glucan Synthase from Daucus carota L. : Characterization, Photoaffinity Labeling, and Solubilization

The membrane-bound UDP-glucose-β-(1,3)-glucan synthase from Daucus carota L. was characterized and a solubilization procedure was developed. The enzyme exhibited maximal activity in the presence of 0.75 millimolar Ca²⁺, 0.5 millimolar EGTA, and 5 millimolar cellobiose at pH 7.5 and 30°C at 1 millimolar UDPG. Reaction products were confirmed to be (1,3)-linked glucan. Polypeptides of 150, 57, and 43 kilodaltons were labeled with the photoactivatible affinity label 5-azido-uridine 5′-β-[³²P] diphosphateglucose. Labeling of the 150 and 57 kilodalton polypeptides was completely protected against by 1 millimolar non-radioactive UDPG suggesting that one or both of these polypeptides may represent the UDPG binding subunit of glucan synthase. Carrot glucan synthase was solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in the absence of divalent cations and chelators; however, the percentage of enzyme which could be solubilized showed variability with membrane source. With microsomal membranes, up to 80% of the enzyme was released with 0.7% CHAPS. Solubilized enzyme was stable for at least 9 hours at 4°C. When more highly purified membrane fractions were isolated from sucrose step gradients a slightly different picture emerged. Activity from the 20/30% interface (Golgi and tonoplast enriched) was readily solubilized and expressed. Activity from the 30/40% interface (plasma membrane enriched) was also solubilized; however, it was necessary to add heat inactivated microsomes to assay mixtures for full activity to be expressed. A requirement for endogenous activators is suggested.     

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.     

Minimum enzyme unit for Na+/K+-ATPase is the alpha beta-protomer. Determination by low-angle laser light scattering photometry coupled with high-performance gel chromatography for substantially simultaneous measurement of ATPase activity and molecular weight

The oligomeric state of canine renal NA+/K+ -ATPase solubilized by octaethylene glycol n-dodecyl ether (C12E8) was studied by means of low-angle laser light scattering photometry coupled with high-performance gel chromatography (HPGC). At around 0 degree C the solubilized enzyme was separated into the (alpha beta)2-diprotomeric and alpha beta-protomeric protein components with Mr values of 302,000 +/- 10,000 and 156,000 +/- 4,000, respectively, in approximately equal quantities. As the temperature of chromatography was increased toward 20 degrees C, the two protein components converged into a single major component. The Mr of this component depended on the monovalent cation included in the elution buffer, and was 255,000 or 300,000 in the presence of 0.1 M NaCl or 0.1 M KCl, respectively. A computer simulation technique showed that the solubilized enzyme was in a dissociation-association equilibrium of 2 protomers = diprotomer at 20 degrees C, and the difference in apparent Mr of the solubilized enzyme between the two species of monovalent cation was interpreted by an association constant (Ka) in the presence of 0.1 M KCl that was about 50-fold larger than in the presence of 0.1 M NaCl. In order to measure ATPase activity and Mr of the solubilized enzyme simultaneously, a TSKgel G3000SW column had been equilibrated and was eluted with an elution buffer containing 0.30 mg/ml C12E8 and 60 microgram/ml phosphatidylserine (bovine brain) as well as the ligands necessary for the enzyme to exhibit the activity at pH 7.0 and 20 degrees C. The solubilized enzyme was always eluted as a single protein component irrespective of the the amount of the protein applied to the column, ranging between 240 and 10 microgram. The Mr of the protein component, however, decreased from 214,000 and 158,000 with the decrease of the protein amount. The specific ATPase activity, however, remained constant at a level of 64 +/- 4% of that of the membrane-bound enzyme even in the range of protein concentration sufficiently low as to allow the enzyme to exist only in the protomeric form. Thus, the alpha beta-protomer is concluded to be the minimum functional unit for the ATPase activity. The value of Ka obtained from the concentration-dependent dissociation curve was 5 . 10(5) M-1 for the enzyme turning over, and 1.1 . 10(7) M-1 for the enzyme inhibited with ouabain. It was discussed, based on the values of Ka obtained, that the enzyme would exist as the diprotomer or the higher oligomer in the membrane.     

Na,K-ATPase: radiation inactivation studies

Na,K-ATPase from duck salt gland and ox brain in the membrane-bound or solubilized form was studied by the radiation inactivation technique using ATP, CTP, GTP or p-NPP as substrates. The values of radiation inactivation size (RIS) were compared with the target size (TS) for the alpha-subunit of the enzyme obtained by an independent method as well as with analytical centrifugation data obtained for C12E8-solubilized enzyme. It was concluded that during ATP (CTP) hydrolysis the enzyme operates as an oligomeric structure; the complex formation requires the presence of K+ and adenosine triphosphate binding to the sites with a low affinity for the nucleotide. Specially designed experiments revealed that the degree of enzyme oligomerization increases with an increase in the microviscosity of the membrane lipid environment.     

Energy-Transducing Adenosine Triphosphatase from Escherichia coli: Purification, Properties, and Inhibition by Antibody

The membrane adenosine triphosphatase (E.C. 3.6.1.3) from Escherichia coli has been solubilized with Triton X-100 and purified to near homogeneity. The purified enzyme has a sedimentation coefficient of 12.9S in a sucrose gradient, corresponding to a molecular weight of about 360,000. On electrophoresis in gels containing sodium dodecyl sulfate, it dissociates into subunits with apparent molecular weights of 60,000, 56,000, 35,000, and 13,000. The purified enzyme loses activity and breaks down into subunits when stored in the cold. Guanosine 5'-triphosphate and inosine 5'-triphosphate are alternative substrates. Ca(2+) and, to a small extent, Co(2+) or Ni(2+) will substitute for Mg(2+) in the reaction. The K(m) for Mg-adenosine triphosphate of the membrane-bound enzyme is 0.23 mM, and for the pure enzyme it is 0.29 mM. Azide is a noncompetitive inhibitor of both the membrane-bound enzyme and the pure enzyme. P(i) is a noncompetitive inhibitor of the solubilized enzyme. An antibody to the purified enzyme was obtained from rabbits. The antibody inhibits the solubilized enzyme and virtually all of the adenosine triphosphate hydrolysis by membranes from cells grown aerobically or anaerobically. The antibody also inhibits the adenosine triphosphate-stimulated pyridine nucleotide transhydrogenase (E.C. 1.6.1.1) of the E. coli membrane.     

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.     

Partial characterization and inactivation of membrane-bound phosphofructokinase from Tetrahymena pyriformis

In Tetrahymena pyriformis, 6-phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) is membrane-bound. Enzyme activity is solubilized by treatment of membranes with Triton X-100 or by high ionic strength in the presence of a chelator. The solubilized enzyme has an approximate molecular weight of 300 000. Both the membrane-bound enzyme and the solubilized enzyme exhibit maximum activity over a wide pH range. At low pH, the membrane-bound form of the enzyme is irreversibly inactivated, whereas the solubilized enzyme is not. The membrane-bound enzyme is inactivated by incubation with Mg2+, ATP, fluoride and a soluble factor that is heat labile, nondialysis, (NH4)2SO4 precipitable and sensitive to trypsin. The solubilized enzyme is not inactivated under similar conditions.     

Genetic identification and purification of the respiratory NADH dehydrogenase of Escherichia coli

Escherichia coli membrane particles were solubilized with potassium cholate. An NADH:ubiquinone oxidoreductase was resolved by hydroxylapatite chromatography of the solubilized material. This enzyme has been identified as the respiratory NADH dehydrogenase since it is absent in chromatograms of solubilized material from an ndh mutant strain. Such mutants lack membrane-bound NADH oxidase activity and have previously been shown to have an inactive NADH dehydrogenase complex [Young, I. G., & Wallace, B. J. (1976) Biochim. Biophys. Acta 449, 376-385]. The respiratory NADH dehydrogenase was amplified 50- to 100-fold in vivo by using multicopy plasmid vectors carrying the ndh gene and then purified to homogeneity on hydroxylapatite. Hydroxylapatite chromatography of cholate-solubilized material from genetically amplified strains purified the enzyme approximately 800- to 100-fold relatively to the activity in wild-type membranes. By use of a large-scale purification procedure, 50-100 mg of protein with a specific activity of 500-600 mumol of reduced nicotinamide adenine dinucleotide oxidized min-1 mg-1 at pH 7.5, 30 degrees C, was obtained. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme showed that the enzyme consists of a single polypeptide with an apparent Mr of 45 000.     

Transformation of neutral to alkaline fructose 1,6-bisphosphatase. Converting enzyme activity in the large-particle fraction from rabbit liver

A liver particle fraction containing lysosomes catalyzes the conversion of native rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11), having a neutral pH optimum, to a modified form with an alkaline pH optimum. The “converting enzyme” activity is partially recovered with the membranes from disrupted particles, and is also detected in “intact” particles isolated and maintained in isotonic buffered sucrose. The converting enzyme activity associated with the membrane fraction is expressed at pH 6.5, but not at pH 4.5, although activity at the lower pH appears when the enzyme is released from the membranes with Triton X-100. In contrast, proteolytic activity as measured with peptide and protein substrates is maximal at pH 5.0 or below, and is the same for the membrane-bound or solubilized proteases. The results suggest that a specific converting enzyme, at least partially associated with a particle (possibly lysosomal) membrane, is responsible for the modification of fructose bisphosphatase and the change in its catalytic properties.     

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.     

Secondary structure of the Neurospora crassa plasma membrane H+-ATPase as estimated by circular dichroism

In a previous communication, a water-soluble, hexameric form of the Neurospora crassa plasma membrane H+-ATPase was described (Chadwick, C. C., Goormaghtigh, E., and Scarborough, G. A. (1987) Arch. Biochem. Biophys. 252, 348-356). To facilitate physical studies of the hexamers, the H+-ATPase isolation procedure has been improved, resulting in a structurally and functionally stable hexamer preparation that contains only 5 to 10% non-ATPase protein, approximately 12 mol of enzyme-bound lysophosphatidylcholine/mol of H+-ATPase monomer, and little or no residual plasma membrane phospholipid. Importantly, when activated by lysophosphatidylglycerol, which satisfies the acidic phospholipid requirement of the enzyme, the hexameric quaternary structure of the enzyme is retained, indicating that the functional properties of the water-soluble hexamers are relevant to those of the native, membrane-bound enzyme. The circular dichroism (CD) spectrum of this H+-ATPase preparation has been measured from 184 to 260 nm and used to estimate the secondary structure of the enzyme. The H+-ATPase is estimated to consist of approximately 36% helix, 12% antiparallel beta-sheet, 8% parallel beta-sheet, 11% beta-turn, and 26% other (irregular) structure. There is no change in the CD spectrum when known enzyme ligands are added to the H+-ATPase solution, suggesting that any changes in secondary structure that might occur during ligand binding and/or catalytic cycling are either minor or result in compensatory changes in secondary structure. The CD spectrum of the H+-ATPase is also compared to published spectra of the animal cell Na+/K+- and Ca2+-ATPases and is shown to be quite similar in shape and intensity, suggesting that all of these ATPases, which have significant sequence homology and are mechanistically similar, may have similar secondary structure composition as well.     

Solubilization, purification and reconstitution of Ca(2+)-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: preservation of native structure and function of the enzyme by DHPC

The properties of Ca(2+)-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca(2+)-ATPase with a greater specific activity than solubilization with C(12)E(8) or Triton X-100. DHPC was determined to be superior to C(12)E(8); while that the C(12)E(8) was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca(2+)-ATPase retained the E1Ca-E1*Ca conformational transition as that observed for native microsomes; whereas the C(12)E(8) and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca(2+) transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C(12)E(8) and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca(2+)-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C(12)E(8) and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca(2+) uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca(2+)-ATPase retained more organized and native secondary conformation compared to C(12)E(8) and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C(12)E(8) and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca(2+)-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C(12)E(8) and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of the membrane-bound enzyme.     

Effects of solubilisation on some properties of the membrane-bound respiratory enzyme D-amino acid dehydrogenase of Escherichia coli

Solubilisation, delipidation and partial purification of the membrane-bound enzyme D-amino acid dehydrogenase of Escherichia coli K12 produced significant changes in several of its properties. Solubilised enzyme showed a broader substrate specificity, increased affinity for at least three substrates, and a lower pH optimum with D-alanine as substrate. Solubilised enzyme was more heat-labile than native enzyme, particularly at 37 degrees C, and re-binding to envelope preparations restored protection against heat denaturation. Activity of delipidated enzyme could be increased by addition of pure phospholipids. Native enzyme showed biphasic Arrhenius kinetics associated with phase changes of membrane lipids.