Effect of abscisic acid on membrane-bound epidermal ATPase from tobacco leaves

Membrane-bound Mg⁺⁺-activated ATPase activity in epidermal stripsfrom tobacco leaves (Nicotiana tabacum L. Samsun NN) was stimulatedby abscisic acid (ABA) when the strips were floated on ABA solutionin light or in darkness. The optimum ABA concentrations in lightand in darkness were 10^–5 M and 10^–6 M, respectively.Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and N, N'-dicyclohexylcarbodiimide(DCCD) completely blocked the basal level membrane-bound epidermalATPase activity. ABAinduced membrane-bound epidermal ATPaseactivity was completely inhibited by CCCP, but only partly byDCCD. H⁺-influx into epidermal strips on a solution in light was lowerthan that in darkness. ABA stimulated H⁺-influx into epidermalstrips in light and in darkness. CCCP suppressed basal levelH⁺-influx, whereas DCCD did not. CCCP also suppressed ABA-inducedH⁺-influx, whereas DCCD did not. Interaction between H⁺-influxand membranebound epidermal ATPase activity is discussed. (Received May 23, 1978; )     

In vitro binding of IAA to plasma membrane-rich fractions containing Mg++-activated ATPase from mung bean hypocotyls

Cell homogenates of dark-grown mung bean hypocotyls were fractionatedinto six fractions (L-0, L-l to L-5) by stepwise sucrose density-gradientcentrifugation. The majority (ca. 84%) of Mg⁺⁺-activated ATPase activity ofthe 10,000 x g pellet was localized in the L-0 (1.03 d 1.14)and L-l (1.14 d 1.16) fractions. Over 40% of the vesicularmembrane in the L-0 fraction and 60% of the L-l fraction couldbe stained with phosphotungstic acid (PTA)-chromic acid, a selectivestaining for the plant plasma membrane. In vitro binding of ¹⁴C-IAA to the fraction components was thegreatest in the L-l fraction among the six. The binding of ¹⁴C-IAAto the L-l fraction in vitro was markedly interfered with bythe presence of a high concentration of cold IAA (2 x 10^–4M).However, it was not affected by the IAA analogues IPA, IBA andIAN. This indicates that IAA highly specifically binds to theL-l fraction. In vitro specific binding of ¹⁴C-IAA to L-l andL-0 was decreased with an increasing acidity from pH 8.0 to5.0. In vitro binding of ¹⁴C-IAA to L-l and L-5 was furtherenhanced when these fractions were isolated from sections pretreatedwith 10^–5M cold IAA for 60 min ¹Present address: Institute for Plant Virus Research, 959 Aobacho,Chiba 280, Japan. (Received August 14, 1975; )     

Mg++ -activated and -inhibited ATPases from mung bean hypocotyls

Mg⁺⁺-activated and inhibited ATPases were isolated from dark-grownmung bean hypocotyls. The enzymes hydrolyzed nucleoside tri-,di- and monophosphates and ß-glycerophosphate. Theeffect of Mg⁺⁺ was most marked when ATP and other nucleosidetriphosphates were used as substrates. Mg⁺⁺-activated ATPases: The activity of enzyme-I was localizedin the membranes and was not released by treatment with 0.1%deoxycholate. Enzyme-II was released and separated by CM-cellulosecolumn chromatography. Enzyme-V was separated from the solublefraction of the cell homogenate by DEAE-cellulose column chromatography.The rates of activivation by Mg⁺⁺ of enzyme-II and enzyme-Vwere very small compared to that of enzyme-I. Mg⁺⁺-inhibited ATPases: Enzyme-II and -IV were precipitatedwith 50–80% ammonium sulfate from the soluble fractionof the cell homogenate and were separated by successive columnchromatographies on Sepharose 6B and DEAE-cellulose. The activitiesof enzyme-III and -IV were inhibited by Mg⁺⁺, when ATP, UTPand GTP were used as substrates. Enzyme-III was purified approximately38-fold, and was more remarkably inhibited by Mg⁺⁺ than wasenzyme-IV. ¹Present address: Institute for Plant Virus Research, 959 Aobacho,Chiba 280, Japan. (Received January 7, 1974; )     

Characterization of the epidermis from barley primary leaves

A method is described for isolating epidermal protoplasts from the primary leaves of barley (Hordeum vulgare L.). Epidermal protoplasts are lighter than mesophyll protoplasts because of their smaller ratio of cytoplasm to vacuole, and can be separated from the latter by density-gradient centrifugation after complete digestion of the leaves. We have started a basic characterization of the epidermal protoplast fraction in comparison with mesophyll protoplasts. Epidermal protoplasts had a mean diameter of 63.5 m, whereas that of mesophyll protoplasts was 35.7 m. Their respiratory oxygen consumption was not influenced by light. They contained acid hydrolases and cytoplasmic enzymes in relative activities different from those of mesophyll protoplasts. Their polypeptide pattern as judged from two-dimensional separations was, in principle, similar to that of mesophyll cells after elimination of the plastids from the latter by the preparation of vacuoplasts. However, in addition, a considerable number of epidermis-specific polypeptides were observed. Isolated epidermal protoplasts were viable and efficiently incorporated [³⁵S]methionine into newly synthesized proteins. The results show that epidermal protoplasts are suitable for the investigation of the physiological and molecular properties of epidermal cells in leaves.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We are grateful to Professor U. Heber (Lehrstuhl Botanik 1, Würzburg) for his continuous support. This work was supported by the DFG and the University of Würzburg within the Sonderforschungsbereich 176.     

Characterization of the membrane-bound ATPase from a facultatively anaerobic alkalophile

We have studied the properties of membrane-bound ATPase of a facultatively anaerobic alkalophile. The enzyme could not be solubilized without detergent, suggesting an integral membrane protein. The activity was accelerated by NH4+ and acetate anion, and inhibited by NH3-. The enzyme required Mg2+ or Mn2+ as a divalent cation for the maximal activity. In addition to ATP, the enzyme utilized other triphosphates of nucleosides as a substrate, but not di- nor monophosphates. The enzyme was suggested to crossreact with an antibody against the alpha-subunit of Na+/K+-ATPase from dog kidney.     

Properties of the Mg2+-induced low-affinity nucleotide binding site of (Na++ K+)-activated ATPase

(1) The Mg²⁺-induced low-affinity nucleotide binding by (Na⁺ + K⁺)-ATPase has been further investigated. Both heat treatment (50–65°C) and treatment with N-ethylmaleimide reduce the binding capacity irreversibly without altering the K_d value. The rate constant of inactivation is about one-third of that for the high-affinity site and for the (Na⁺ + K⁺)-ATPase activity. (2) Thermodynamic parameters (ΔH° and ΔS°) for the apparent affinity in the ATPase reaction (K_m ATP) and for the true affinity in the binding of AdoPP[NH]P (K_d and K_i) differ greatly in sign and magnitude, indicating that one or more reaction steps following binding significantly contribute to the K_m value, which thus is smaller than the K_d value. (3) Ouabain does not affect the capacity of low-affinity nucleotide binding, but only increases the K_d value to an extent depending on the nucleotide used. GTP and CTP appear to be most sensitive, ATP and ADP intermediately sensitive and AdoPP[NH]P and least sensitive to ouabain. Ouabain reduces the high-affinity nucleotide binding capacity without affecting the K_d value. (4) The nucleotide specificity of low-affinity binding site is the same for binding (competition with AdoPP[NH]P) and for the ATPase activity (competition with ATP): AdoPP[NH]P > ATP > ADP > AMP. (5) The low-affinity nucleotide binding capacity is preserved in the ouabain-stabilized phosphorylated state, and the K_d value is not increased more than by ouabain alone. (6) It is inferred that the low-affinity site is Iocated on the enzyme, more specifically its α-subunit, and not on the surrounding phospholipids. It is situated outside the phosphorylation centre. The possible functional role of the low-affinity binding is discussed.     

Mg2+- or Ca2+-activated ATPase activities of plasma membranes isolated from vascular smooth muscle

Studies of ATP hydrolysis by various subcellular fractions isolated from rat mesenteric arteries and veins indicate that an apparent ATPase activity, which can be activated by Mg2+ or Ca2+, is primarily associated with the plasma membranes. Although both Mg2+-activated and Ca2+-activated ATPase activities under the optimal condition are substantially lower in venous than in arterial plasma membrane fraction, their dependence on the concentration of Mg2+ and Ca2+ are quite similar in arterial as well as venous plasma membrane fractions. No synergistic effect on ATP hydrolysis was observed in the presence of both Mg2+ and Ca2+. In addition, Mg2+-activated and Ca2+-activated ATPase activities show similar pH dependence, inhibition by deoxycholate, stability toward heat inactivation and substrate specificity. Furthermore, Mg2+-activated and Ca2+-activated ATPase activities were similarly reduced in vascular smooth muscles of spontaneously hypertensive rats. These results suggest that the activation of ATP hydrolysis by Mg2+ or Ca2+ may represent a single enzyme moiety in the plasma membrane of vascular smooth muscle. The possible involvement of such ATPase in the Ca2+ transport function of vascular smooth muscle is discussed.     

Polyribosomes in protoplasts isolated from tobacco leaves

Polyribosomes (polysomes), active in an amino acid incorporation system in vitro, were isolated from tobacco leaf protoplasts. A comparison of polysome profiles indicated that the polysome/monosome ratio is greatly decreased in isolated protoplasts as compared to the intact leaf. In isolated protoplasts, a marked accumulation of ribosomal subunits was also found. The division of protoplasts, as investigated in the 8-cell and callus stages, was associated with a(n) (at least) partial regeneration of polysome profiles characteristic for leaves. Plasmolysis of leaves attached to the plant had no great effect on the polysome profile. However, leaf excision per se resulted in a dramatic loss of polysomes, even when the leaf tissue was floated on water. It is concluded that the isolation of the cell from its normal environment, and not the osmotic stress and associated increase in RNase activity, is the most important factor responsible for the loss of polysomes in isolated protoplasts.Abbreviations EGTA ethylene glycol bis (2-aminoethyl ether)-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - mRNA messenger ribonucleic acid - RNase ribonuclease - Tris tris(hydroxymethyl)aminomethane - TCA trichloroacetic acid     

Influence of Ca++ and Mg++ on the vanadate inhibition of the Ca++- ATPase from pig heart sarcoplasmic reticulum

Vanadate inhibits the Ca⁺⁺-ATPase of sarcoplasmic reticulum from pig heart half maximally at about 10^?5 M. Mg⁺⁺ promotes this inhibition by vanadate whereas increasing Ca⁺⁺-concentrations protect the enzyme against vanadate inhibition. Keeping the ratio $\text{Mg}{}^{\mathrm{++}}\text{ATP}$ constant there was no influence of ATP on the vanadate inhibition at concentrations up to 5 × 10^?3 M ATP. Whenever the ratio $\text{Mg}{}^{\mathrm{++}}\text{ATP}$ was higher than 1:1 the inhibitory effect of vanadate on the Ca⁺⁺-ATPase was increased.     

Effect of auxin on Mg++-activated and -inhibited ATPases from mung bean hypocotyls

Mg⁺⁺-activated (A) and -inhibited (B) ATPases were isolatedfrom dark-grown mung bean hypocotyls and the effect of in vivoand in vitro IAA-treatment on these ATPases was observed. The activity of the A-ATPase, which was localized in the plasmamembrane-rich fractions, was enhanced by exogenously added l0^–10M IAA (ca. 35%). The stimulatory effect of in vitro IAA-treatmentwas particularly found in the A ATPase isolated from sectionstreated with 10^–5 M IAA for 60 min (ca. 280%). The activity of the B-ATPase, which was localized in the solublefraction of the cell homogenate, was markedly increased by invivo treatment with 10^–7 M IAA for 60 min (ca. 360%).The stimulatory effect of IAA-treatment was observed even ifprotein synthesis was inhibited by cycloheximide. These results suggest that the enhancement of the activitiesof the A- and B-ATPases caused by in vivo and in vitro IAA-treatmentsoccur as a result of the activation of already present enzymes,rather than of the synthesis of these enzymes. ¹Present address: Institute for Plant Virus Research, 959 Aobacho,Chiba 280, Japan. (Received March 27, 1974; )     

Active Uptake of Ca++ and Ca++-Activated Mg++ ATPase in Red Cell Membrane Fragments

Isolated human red blood cell membrane fragments (RBCMF) were found to take up Ca⁺⁺ in the presence of ATP.¹ This ATP-dependent Ca⁺⁺ uptake by RBCMF appears to be the manifestation of an active Ca⁺⁺ transport mechanism in the red cell membrane reported previously (Schatzmann, 1966; Lee and Shin, 1969). The influences of altering experimental conditions on Ca⁺⁺-stimulated Mg⁺⁺ ATPase (Ca⁺⁺ ATPase) and Ca⁺⁺ uptake of RBCMF were studied. It was found that pretreatment of RBCMF at 50°C abolished both Ca⁺⁺ ATPase and Ca⁺⁺ uptake. Pretreatment of RBCMF with phospholipases A and C decreased both Ca⁺⁺ ATPase and Ca⁺⁺ uptake, whereas pretreatment with phospholipase D did not significantly alter either Ca⁺⁺ ATPase or Ca⁺⁺ uptake. Both Ca⁺⁺ ATPase and Ca⁺⁺ uptake had ATP specificity, similar optimum pH's, and optimum incubation temperatures. From these results, it was concluded that Ca⁺⁺ uptake is intimately linked to Ca⁺⁺ ATPase.     

Sterol distribution in intracellular organelles isolated from tobacco leaves

All membrane-containing fractions isolated from tobacco leaves contained free sterols, sterol glycosides, and sterol esters. The three sterol forms increased, on a dry weight basis, with a decrease in particle size. The supernatant fraction contained only trace amounts of sterol. The major sterols in all cellular fractions, in the order of decreasing amounts, were: stigmasterol, β-sitosterol, campesterol, and cholesterol. The 500g pellet contained the largest percentage of free sterol, while the 46,000g pellet contained the largest percentage of esterified sterol. The individual sterol composition of the free sterol and sterol glycoside fraction was very similar; however, the composition of the sterol ester fraction varied widely among intracellular fraction. The intracellular distribution pattern of cholesterol-¹⁴C added to the isolation medium provided evidence that the intracellular sterol distribution pattern is not an artifact. These results support the suggestion that sterols in plant cells may have a physiological function associated with membranes.     

Binding of tobacco mosaic virus to membrane material isolated from tobacco leaves.

Binding of tobacco mosaic virus (TMV) to disrupted tobacco leaf membrane was studied. Membrane isolated from tobacco leaves was treated successively with (NH4)2SO4, Li-diiodosalicylate and then pronase. TMV-binding substance was thus isolated in a soluble form. From enzymatic digestion experiments, it was suggested that the binding substance was composed of lipid and carbohydrate.     

Characterization and solubilization of the membrane-bound ATPase of Mycoplasma gallisepticum.

The membrane-bound ATPase of Mycoplasma gallisepticum selectively hydrolyzed purine nucleoside triphosphates and dATP. ADP, although not a substrate, inhibited ATP hydrolysis. The enzyme exhibited a pH optimum of 7.0 to 7.5 and an obligatory requirement for divalent cations. Dicyclohexylcarbodiimide at a concentration of 1 mM inhibited 95% of the ATPase activity at 37 degrees C, with 50% inhibition occurring at 22 microM dicyclohexylcarbodiimide. Sodium or potassium (or both) failed to stimulate activity by greater than 37%. Azide (2.6 mM), diethylstilbestrol (100 micrograms/ml), p-chloromercuribenzoate (1 mM), and vanadate (50 microM) inhibited 50, 91, 89, and 60%, respectively. The ATPase activity could not be removed from the membrane without detergent solubilization. Although most detergents inactivated the enzyme, the dipolar ionic detergent N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (0.1%) solubilized approximately 70% of the enzyme with only a minor loss in activity. The extraction led to a twofold increase in specific activity and retention of inhibition by dicyclohexylcarbodiimide and ADP. Glycerol greatly increased the stability of the solubilized enzyme. The properties of the membrane-bound ATPase are not consistent with any known ATPase. We postulate that the ATPase functions as an electrogenic proton pump.     

Evidence for the influence of the protein-phospholipid interface on sarcoplasmic reticulum Ca++ Mg++ ATPase activity.

Sarcoplasmic reticulum from the white hind leg muscle of the rabbit was examined with 31P nuclear magnetic resonance as a nonperturbing probe of phospholipid-protein interactions in the intact membrane. The phospholipids of the sarcoplasmic reticulum appear to inhabit two distinct environments: one very similar in behavior to pure phospholipid lamellar dispersions and the other immobilized by the protein in the membrane. Measurement of the population of the latter environment suggests that it is dependent on salt concentration and probably not due to the Ca++ Mg++ ATPase of the sarcoplasmic reticulum. This immobilization can be removed completely by papain proteolysis of the membrane protein, but only partially by trypsin treatment. The phospholipid composition of recombinants with the Ca++ Mg++ ATPase was varied in order to look for effects of the phospholipid-protein interface on enzymatic activity of the Ca++ Mg++ ATPase. Both transphosphatidylated phosphatidylethanolamine (from egg phosphatidylcholine) and bovine brain phosphatidylserine readily partitioned into the putative boundary layer, whereas under the same conditions soybean phosphatidylethanolamine was excluded. Only phosphatidylserine affected the activity of the enzyme, causing an inhibition that was proportional to the phosphatidylserine content, relative to phosphatidylcholine.