Isolation and properties of ion-stimulated ATPase activity associated with cauliflower plasma membranes

The association of K⁺-stimulated, Mg²⁺-dependent ATPase activity with plasma membranes from higher plants has been used as a marker for the isolation and purification of a plasma membrane-enriched fraction from cauliflower (Brassica oleraceae L.) buds. Plasma membranes were isolated by differential centrifugation followed by density gradient centrifugation of the microsomal fraction. The degree of purity of plasma membranes was determined by increased sensitivity of Mg²⁺-ATPase activity to stimulation by K⁺ and by assay of approximate marker enzymes. In the purified plasma membrane fraction, Mg²⁺-ATPase activity was stimulated up to 700% by addition of K⁺. Other monovalent cations also markedly stimulated the enzyme, but only in the presence of the divalent cation Mg²⁺. Ca²⁺ was inhibitory to enzyme activity. ATPase was the preferred substrate for hydrolysis, there being little hydrolysis in the presence of ADP, GTP, or p-nitrophenylphosphate. Monovalent cation-stimulated activity was optimum at alkaline pH. Enzyme activity was inhibited nearly 100% by AgNO₃ and about 40% by diethylstilbestrol.     

Characterization of ATPase activity associated with corn leaf plasma membranes

A Mg²⁺-dependent, cation-stimulated ATPase was associated with plasma membranes isolated from corn leaf mesophyll protoplasts. Potassium was the preferred monovalent cation for stimulating the ATPase above the Mg²⁺-activated level. The enzyme was substrate-specific for ATP, was inhibited by N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, p-chloromercuribenzoate, and orthovanadate, but was insensitive to oligomycin or sodium azide. A K_m of 0.28 millimolar Mg²⁺-ATP was determined for the K⁺-ATPase, and the principal effect of potassium was on the V_max for ATP hydrolysis. Since potassium stimulation was not saturated at high concentrations, a nonspecific role was proposed for potassium stimulation. A nonspecific phosphatase was also found to be associated with corn leaf plasma membranes. However, it could not be determined positively whether this activity represented a separate enzyme.     

Calcium-binding properties and ATPase activities of rat liver plasma membranes

Plasma membranes from rat liver purified according to the procedure of Neville bind calcium ions by a concentration-dependent, saturable process with at least two classes of binding sites. The higher affinity sites bind 45 nmol calcium/mg membrane protein with a K_D of 3 µM. Adrenalectomy increases the number of the higher affinity sites and the corresponding K_D. Plasma membranes exhibit a (Na⁺-K⁺)-independent-Mg²⁺-ATPase activity which is not activated by calcium between 0.1 µM and 10 mM CaCl₂. Calcium can, with less efficiency, substitute for magnesium as a cofactor for the (Na⁺-K⁺)-independent ATPase. Both Mg²⁺- and Ca²⁺-ATPase activities are identical with respect to pH dependence, nucleotide specificity and sensitivity to inhibitors. But when calcium is substituted for magnesium, there is no detectable membrane phosphorylation from [γ-³²P] ATP as it is found in the presence of magnesium. The existence of high affinity binding sites for calcium in liver plasma membranes is compatible with a regulatory role of this ion in membrane enzymic mechanisms or in hormone actions. Plasma membranes obtained by the procedure of Neville are devoid of any Ca²⁺-activated-Mg²⁺-ATPase activity indicating the absence of the classical energy-dependent calcium ion transport. These results would suggest that the overall calcium-extruding activity of the liver cell is mediated by a mechanism involving no direct ATP hydrolysis at the membrane level.     

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.     

Fluorescence properties of plasma membranes from oats and cauliflower in relation to blue light physiology

Fluorescence properties of plasma membranes from dark-grown oat shoots ( Avena saliva L. cv. Sol II) and from cauliflower inflorescences ( Brassica oleracea L.) were investigated. Along with a flavin (with a possible connection to blue light physiology), a blue fluorescing component was present. The effect of NaN₃, phenyl acetic acid (PAA), KI (flavin inhibitors) and salicylhydroxamic acid (SHAM; inhibitor of e.g. the blue light-induced cytochrome b reduction) were followed with regard to the fluorescence properties of the two components as well as with regard to the light-induced cytochrome b reduction (LIAC). A change in flavin fluorescence and LIAC occurred at about the same concentration of PAA and SHAM, while LIAC was much more sensitive to KI and NaN₃ than was the fluorescence. Rapid freezing and thawing did not change the relative fluorescence emission from the flavin and blue fluorescing component, respectively, but storage at -20°C for one or two days increased the fluorescence, especially from the latter. There did not seem to be a tight coupling between the fluorescence properties of the blue fluorescing component (spectrally similar to a pteridine) and the flavin. Therefore, no conclusions could be drawn concerning their connection in blue light physiology, i.e. in processes such as phototropism.     

Characterization and localization of the ATPase associated with pea chloroplast envelope membranes

Chloroplast envelope membranes isolated from Pisum sativum seedlings have been found to contain a Mg-ATPase activity (specific activity 50-175 nanomoles per minute per milligram protein). The ATPase had a broad pH optimum between 7.0 and 9.5. The activity was not inhibited by oligomycin, N,N′-dicyclohexylcarbodiimide, ouabain, or antibodies directed against chloroplast coupling factor 1; nor was the activity stimulated by monovalent cations. However, the ATPase was inhibited by vanadate, molybdate, and adenylyl imidodiphosphate.

The ATPase hydrolyzed a broad range of nucleoside triphosphates, but did not hydrolyze ADP, AMP, or pyrophosphate. The K_m for Mg-ATP was determined to be 0.2 millimolar. The ATPase was found to be distinct from ADPase and pyrophosphatase activities also present in pea envelope membranes.

The ATPase was determined to be located on the inner membrane of the envelope after resolution of inner and outer membranes by sucrose density gradient centrifugation.

     

Phosphorylated intermediate of the ATPase of plant plasma membranes

A partially purified preparation of the plant plasma membrane ATPase was phosphorylated when incubated with [gamma-32P]ATP. The phosphoprotein formed has the characteristics of an enzyme intermediate because of its rapidity of phosphorylation and dephosphorylation. The sensitivity of the phosphoenzyme bond to alkaline pH and to hydroxylamine indicates that it is an acylphosphate. Both the ATPase activity and the phosphorylation of the enzyme exhibited an apparent Km value of 0.3 mM ATP. When the phosphorylated enzyme was analyzed by electrophoresis in sodium dodecyl sulfate, only one major band with a molecular weight of about 105,000 contained radioactivity. These results indicate that the plant plasma membrane ATPase has a subunit composition and reaction mechanism similar to the cation-pumping ATPases of animal and fungal plasma membranes.     

Variable ATPase composition of human tumor plasma membranes

Purified plasma membranes from several transplantable human tumors exhibit very high Mg²⁺-dependent ATPase activities. Three types of Mg²⁺-dependent ATPases can be demonstrated: (1) an ouabain sensitive Na⁺, K⁺-ATPase, which is a minor component of the tumor plasma membrane ATPase, (2) a Mg²⁺-activated ATPase, which is a non-specific nucleoside triphosphatase, and (3) an ATPase activity stimulated by Na⁺ (or K⁺) alone. In three human melanomas, only the first two activities are found. In an astrocytoma and an oat cell carcinoma, all three activities are found. In the same two tumors, the plasma membrane Mg²⁺-ATPase is also stimulated by Con A. The relationship of these ATPases are discussed.     

A unique trypsin-like protease associated with plasma membranes of rat liver

One way in which serum promotes survival of primary cultured hepatocytes is by inhibiting plasma membrane protease (Nakamura, T., Asami, O., Tanaka, K., and Ichihara, A. (1984) Exp. Cell Res. 155, 81-91). One of these proteases was solubilized from the plasma membranes of rat liver with 4% octyl glucoside and purified to a homogeneous state by affinity chromatography on bovine pancreatic trypsin inhibitor linked to Sepharose 4B. The protease had an apparent Mr = 120,000 by Sephacryl S-200 gel filtration and the Mr of its subunits was 30,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It appeared to be a glycoprotein. A high concentration of detergent was necessary to keep the protein soluble. The purified enzyme readily hydrolyzed synthetic tripeptide nitroanilides at sites adjacent to Arg or Lys residues, but did not degrade synthetic substrates of chymotrypsin, elastase, or aminopeptidase. It showed endopeptidase activity, hydrolyzing various proteins such as casein, hemoglobin, and denatured albumin. The enzyme was strongly inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, bovine pancreatic trypsin inhibitor, leupeptin, antipain, and alpha 1-antitrypsin, but not by chymostatin, elastatinal, or inhibitors of carboxyl, thiol, or metallo proteases, suggesting that it is a seryl trypsin-like protease. This protease was found in plasma membranes of rat and mouse liver and in small amounts in those of kidney, but not in those of brain, red cells, Ehrlich ascites tumor, or two Morris hepatomas, suggesting that it may be involved in differentiated functions of normal hepatocytes.     

Purification and reconstitution of the proton-pumping ATPase of fungal and plant plasma membranes

The 11S storage protein (glycinin) of soybean [Glycine max (L.) Merr., cv. Raiden] was studied by polyacrylamide gel electrophoresis and amino acid sequence analysis. It contained the following subunits composed of acidic (A) and basic (B) polypeptides: A1aB2, A1bB1b, A2B1a, and A3B4. However, it lacked polypeptides A4, A5, and B3 which are present in many other cultivars. A new acidic polypeptide called A6 was present in a low amount and was characterized by amino acid sequence analysis. It was homologous to A4, although of a smaller apparent molecular weight. Since Raiden has an average protein content of about 40% and its glycinin fraction can be purified as a 350,000 D complex which is typical of other cultivars, the results imply polymorphism with respect to glycinin subunit composition. Because there is a wide variation in the methionine content of the various subunits, these findings suggest the possibility of genetically manipulating the nutritional quality of soybean seed protein by altering glycinin subunit composition.     

Calmodulin-stimulated Ca(2+)-ATPases in the vacuolar and plasma membranes in cauliflower.

The subcellular locations of Ca(2+)-ATPases in the membranes of cauliflower (Brassica oleracea L.) inflorescences were investigated. After continuous sucrose gradient centrifugation a 111-kD calmodulin (CaM)-stimulated and caM-binding Ca(2+)-ATPase (BCA1; P. Askerlund [1996] Plant Physiol 110: 913-922; S. Malmström, P. Askerlund, M.G. Plamgren [1997] FEBS Lett 400: 324-328) comigrated with vacuolar membrane markers, whereas a 116-kD caM-binding Ca(2+)-ATPase co-migrated with a marker for the plasma membrane. The 116 kD Ca(2+)-ATPase was enriched in plasma membranes obtained by aqueous two-phase partitioning, which is in agreement with a plasma membrane location of this Ca(2+)-ATPase. Countercurrent distribution of a low-density intracellular membrane fraction in an aqueous two-phase system resulted in the separation of the endoplasmic reticulum and vacuolar membranes. The 111-kD Ca(2+)-ATPase co-migrated with a vacuolar membrane marker after countercurrent distribution but not with markers for the endoplasmic reticulum. A vacuolar membrane location of the 111-kD Ca(2+)-AtPase was further supported by experiments with isolated vacuoles from cauliflower: (a) Immunoblotting with an antibody against the 111-kD Ca(2+)-ATPase showed that it was associated with the vacuoles, and (b) ATP-dependent Ca2+ uptake by the intact vacuoles was found to be CaM stimulated and partly protonophore insensitive.     

Diacylglycerol kinase in plasma membranes from wheat

Diacylglycerol kinase activity was demonstrated in highly purified plasma membranes isolated from shoots and roots of dark-grown wheat (Triticum aestivum L.) by aqueous polymer two-phase partitioning. The active site of the diacylglycerol kinase was localized to the inner cytoplasmic surface of the plasma membrane using isolated inside-out and right-side-out plasma membrane vesicles from roots. The enzyme activity in plasma membrane vesicles from shoots showed a broad pH optimum around pH 7. The reaction was Mg²⁺ and ATP dependent, and maximal activity was observed around 0.5 mM ATP and 3 mM MgCl₂. The Mg²⁺ requirement could be substituted only partially by Mn²⁺ and not at all by Ca²⁺. The phosphorylation of endogenous diacylglycerol was strongly inhibited by detergents indicating an extreme dependence of the lipid environment. Inositol phospholipids stimulated the activity of diacylglycerol kinase in plasma membranes from shoots and roots, whereas the activity was inhibited by R59022, a putative inhibitor of several diacylglycerol kinase isoenzymes involved in uncoupling diacylglycerol activation of mammalian protein kinase C.     

Actin-like protein associated with plasma membranes fromEuglena gracilis

Summary Microtubules are characteristic components of the membrane skeleton ofEuglena gracilis, but whether microfilaments are present has been controversial. We here present evidence that an actin-like protein may indeed be associated with the plasma membrane (PM) ofE. gracilis. Firstly, a 47 kDa, PM-associated, polypeptide was recognized by an anti-amoeba actin antibody. Secondly, this 47 kDa protein seemed to be peripherally attached to PM in much the same way as -tubulin, since both could be released from PM by treatment with 150 mM NaOH but not with ethylene glycol, NaCl, or formamide. Thirdly, the 47 kDa polypeptide and -tubulin were found mainly in the Triton X-1 14-insoluble fraction, indicating that they were part of a protein complex resistant to detergents, such as the cytoskeleton. Finally, DNase I activity was inhibited by a fraction enriched in the 47 kDa polypeptide, a property typical of actin.Abbreviations CP-medium cytoskeleton preparation medium - BNSP-skatole 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine - ECL enhanced chemiluminescence - HEPES N-[2-hydroxyethyl]-piperazine-N[2-ethane sulfonic acid] - ICM intracellular membranes - MF mitochondrial and microsomal fraction - PM plasma membrane - PPB potassium phosphate buffer - PVDF polyvinylidene difluoride - SDS sodium dodecyl sulphate - TBS Tris-buffered saline - TBST Tris-buffered saline with Tween 20     

A membrane-bound protein inhibitor of the high affinity Ca ATPase in rat liver plasma membranes

The Ca ATPase from rat liver plasma membranes has been recently characterized and partially purified in our laboratory and was shown to depend on a membrane-bound protein activator (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215). In the present study, we report that a factor derived from ammonium sulfate washings of rat liver plasma membranes inhibits the partially purified enzyme activity measured in the presence of activator. This factor is a protein as judged by its sensitivity to heat and trypsin. A molecular weight of 29,000 was determined by sucrose gradient centrifugation and gel chromatography. The action of the inhibitor is due to a decrease in the maximal velocity of the enzyme reaction and is reversed by an excess of the activator associated with the enzyme. An important point in the mode of action of this inhibitor is its absolute dependence on magnesium, which most probably explains the difficulty in detecting the plasma membrane Ca ATPase when MgCl2 is added to the assay medium.     

Properties of anion-sensitive ATPase from plasma membranes of rat liver

The effects of bivalent cations, pH, anions, 2,4-dinitrophenol, dicyclohexylcarbodiimide and roseofungine on the anion-sensitive ATPase from rat liver plasma membranes were studied. It was found that ATPase from plasma membranes is similar to the anion-sensitive ATPase of rat liver nuclei and differs in some features from mitochondrial ATPase.