Characterization of a nuclear phosphatidylinositol 4-kinase in carrot suspension culture cells

We have shown previously that a nuclear phosphatidylinositol (PI) 4-kinase activity was present in intact nuclei isolated from carrot suspension culture cells (Daucus carota L.). Here, we further characterized the enzyme activity of the nuclear enzyme. We found that the pH optimum of the nuclear-associated PI kinase varied with assay conditions. The enzyme had a broad pH optimum between 6.5–7.5 in the presence of endogenous substrate. When the substrate was added in the form of phosphatidylinositol/phosphatidylserine (PI/PS) mixed micelles (1 mM PI and 400 μM PS), the enzyme had an optimum of pH 6.5. In comparison, the pH optimum was 7.0 when PI/Triton X-100 mixed micelles (1 mM PI in 0.025 %, v/v final concentration of Triton X-100) were used. The nuclear-associated PI kinase activity increased 5-fold in the presence of low concentrations of Triton X-100 (0.05 to 0.3 %, v/v); however, the activity decreased by 30 % at Triton X-100 concentrations greater than 0.3 % (v/v). Calcium at 10 μM inhibited 100 % of the nuclear-associated enzyme activity. The K_m for ATP was estimated to be between 36 and 40 μM. The nuclear-associated PI kinase activity was inhibited by both 50 μM ADP and 10 μM adenosine. Treatment of intact nuclei with DNase, RNase, phospholipase A₂ and Triton X-100 did not solubilize the enzyme activity. Based on sensitivity to calcium, ADP, detergent, pH optimum and the product analysis, the nuclear-associated PI 4-kinase was compared with previously reported PI kinases from plants, animals and yeast.     

Pyruvate Kinase of Streptococcus lactis

The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5′-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent V_max values and decreased the K_m values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5′-triphosphate also caused inhibition. The K_m values for phosphoenolpyruvate and adenosine 5′-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5′-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5′-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed.     

Immunochemical Analysis of Triton X-100-Insoluble Residues from Micrococcus lysodeikticus Membranes

Triton X-100-insoluble residues from Micrococcus lysodeikticus membranes were analyzed by crossed immunoelectrophoresis after dispersal of the residues in sodium dodecyl sulfate (SDS). Conditions which produce no obvious distortion of the immunoprecipitate profile and which allow qualitative and quantitative analyses of the antigens present in the extracts are described. Two main antigens were detected; these were identified as succinate dehydrogenase (EC 1.3.99.1) and adenosine triphosphatase (EC 3.6.1.3). As determined by peak area estimations, the maximal release of succinate dehydrogenase and of adenosine triphosphatase from Triton X-100-insoluble membrane residues occurred at protein/SDS ratios of about 4.3:1 (0.2% SDS) and 6.8:1 (0.13% SDS), respectively. A comparison of enzyme activities of SDS extracts with those of untreated, control Triton X-100-insoluble membrane residues indicated that both the succinate dehydrogenase and the adenosine triphosphatase antigens were released with a full (or enhanced) catalytic potential at or below concentrations of SDS required to effect maximal solubilization of the enzyme in question. Evidence is also presented to suggest that the more acidic of the two components detected by crossed immunoelectrophoresis for the heterogeneous adenosine triphosphatase antigen is more sensitive to SDS than is the other. Both succinate dehydrogenase and adenosine triphosphatase lost catalytic activity and were denatured at protein/SDS ratios lower than 3.4:1.     

Properties of particulate, membrane-associated and soluble guanylate cyclase from cardiac muscle, skeletal muscle, cerebral cortex and liver.

1. Guanylate cyclase of washed particles and plasma membranes showed S-shaped progress curves when titrated with either GTP or Mn2+ ions; similar results were obtained with Triton X-100-solubilized enzyme preparation from washed particles. Hill plots of these data revealed multiple metal-nucleotide and free-metal binding sites. 2. Guanylate cyclase of supernatant fractions displayed typical Michaelis-Menten properties when enzyme required excess of (free) Mn2+ (over GTP) for maximal activities; Ka (free Mn2+) was about 0.15-0.25 mM at subsaturating concentrations of GTP. 4 MnATP, MnADP, and MnGDP were found to increase the activities of both particulate and superantant enzyme, when MnGTP concentration was below saturation and free Mn2+ ion concentration was low (less than 100 muM); MnATP (50muM-1 mM) inhibited both these activities at high free Mn2+ concentration (1.5 mM) and inhibition of the particulate enzyme was greater than that of supernatant enzyme. 5. Ca2+ ions stimulated supernatant-enzyme activity; the stimulatory concentration of Ca2+ ions depended on the concentration of Mn2+ and GTP. 6. A modest stimulation of particulate guanylate cyclase by pyrophosphate (0.02-1 mM) was observed; the pyrophosphate effect appeared to be competitive with respect to GTP. At a higher concentration (2 mM), pyrophosphate produced a marked inhibition of particulate enzyme; the nature of inhibitory effect appeared complex. 7. Inorganic salts (e.g. NaCl, KCl, LiBr, NaF) produced inhibition of particulate enzyme; the degree of inhibition of Triton X-100-stimulated activity was less than that of unstimulated activity. 9. Treatment of sarcolemmal or microsomal membranes with either phospholipase C or trypsin decreased, whereas phospholipase A increased, the activity of guanylate cyclase.     

Purification and chemical modification of a phosphatidylinositol kinase from sheep brain.

A membrane-bound phosphatidylinositol (PtdIns) kinase has been purified approximately 9500-fold to apparent homogeneity from sheep brains. The purification procedure involves: solubilisation of the membrane fraction with Triton X-100, ammonium sulphate fractionation and a number of ion-exchange and gel-filtration chromatography steps. The purified enzyme exhibited a final specific activity of 1149 nmol.min-1.mg-1. The molecular mass of the enzyme was estimated to be 55 kDa by SDS/PAGE and 150 +/- 10 kDa by HPLC gel filtration in the presence of Triton X-100. Kinetic measurements have shown that the apparent Km value of PtdIns kinase for the utilisation of PtdIns is 22 microM and for ATP 67 microM. Mg2+ was the most effective divalent cation activator of PtdIns kinase, with maximal enzymatic activity reached at a concentration of 10 mM Mg2+. In addition to adenosine and ADP, the 2'(3')-O-(2,4,6-trinitrophenyl) derivative of ATP was found to be a strong competitive inhibitor of the enzyme, with a Ki of 32 microM. Enzymatic activity was found to be stimulated by Triton X-100 but inhibited by deoxycholate.     

Properties of a Partially Purified Nucleoside Triphosphatase (NTPase) from the Chloroplast Envelope of Pea

The Mg-nucleoside triphosphatase activity associated with the inner envelope membrane of the pea chloroplast is comprised of at least two components, a major activity that is sensitive to vanadate and sodium fluoride and a minor insensitive activity. The vanadate/fluoride sensitive activity has been partially purified (about 35-fold) from Triton X-100 solubilized membranes by DEAE-Sephadex chromatography and sucrose density gradient centrifugation. The partially purified enzyme resembles the membrane-bound activity in requiring either Mg²⁺ or Mn²⁺, having a broad specificity for nucleoside triphosphates, having a K_m for ATP of 0.18 millimolar, and being inhibited by N-ethylmaleimide, but insensitive to sodium azide and dicyclohexylcarbodiimide. The partially purified enzyme obtained after sucrose gradient centrifugation has a markedly increased sensitivity to inhibition by inorganic pyrophosphate compared with the less pure enzyme. Pyrophosphate is not a substrate of either the membrane-bound or partially purified enzyme.     

Subcellular localization and topology of homogalacturonan methyltransferase in suspension-cultured Nicotiana tabacum cells

Pectin is a complex polysaccharide in the primary walls of all plant cells that is thought to be synthesized in the cellular endomembrane system and inserted into the wall via exocytosis. The most abundant pectic polysaccharide, homogalacturonan, is partially methylesterified within the cell by the pectin methyltransferase homogalacturonan methyltransferase (HGA-MT). The subcellular location of HGA-MT activity was determined in tobacco (Nicotiana tabacum L. cv. Samsun) cell membranes separated on linear sucrose gradients. The activity of HGA-MT and two enzymatic markers of the Golgi apparatus, IDPase and UDPase, were found to be located in the same membrane fraction. No NADH cytochrome c reductase activity, a marker for the endoplasmic reticulum, was detected in the Golgi fraction. Homogalacturonan methyltransferase activity was not reduced by protease treatment of intact membranes or membranes treated with 0.01% Triton X-100. In contrast, HGA-MT activity was reduced by protease treatment of membranes permeabilized with 0.02% Triton X-100. The sensitivity of HGA-MT in detergent-permeabilized membranes, and the lack of inhibition of HGA-MT activity by protease-treatment of intact membranes, provides evidence that the catalytic site of HGA-MT is located on the lumenal side of the Golgi. Received: 2 December 1998 / Accepted: 9 February 1999     

Purification and properties of the NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum

Summary NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum was purified 9300 fold with a yield of 4.6%. The enzyme is a hexamer of apparent molecular weight 294 kDa on Sephacryl S400 and a subunit molecular weight of 52 kDa as determined by SDS gel electrophoresis. The apparent K_mS for -ketoglutarate, NADPH and NH _inf4 ^sup+ are 1.2 mM, 9.7 µM and 2.2 mM respectively, and the purified enzyme has a broad pH optimum with a peak at pH 7.75. GTP has a slight stimulatory effect (22% at 83 µM) as does ADP (11% at 1 mM), and AMP is slightly inhibitory (9% at 1 mM) whereas adenosine, ATP and cAMP have little or no effect. Neither the Zn²⁺ chelating compound 1,10-phenanthroline nor EDTA have any effect on the enzyme while p-hydroxymercuribenzoic acid inhibits enzyme activity (50% at 80 µM) yet N-ethylmaleimide does not.In addition, the NADP-GDH activity varies little during the various stages of morphogenesis.Abbreviations EDTA Ethylenediamine Tetraacetic Acid - Tris Tris(hydroxymethyl)aminomethane - Bis-tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - TRITON X-100 iso-octylphenoxypoly-ethoxyethanol - pHMB p-Hydroxymercuribenzoic acid     

Characterization of the Mn2+-stimulated (di)adenosine polyphosphate hydrolase encoded by the Deinococcus radiodurans DR2356 nudix gene

The DR2356 nudix hydrolase gene from Deinococcus radiodurans has been cloned and the product expressed as an 18 kDa histidine-tagged protein. The enzyme hydrolysed adenosine and diadenosine polyphosphates, always generating ATP as one of the initial products. ATP and other (deoxy)nucleoside triphosphates were also substrates, yielding (d)NDP and Pi as products. The DR2356 protein was most active at pH 8.6–9.0 and showed a strong preference for Mn²⁺ as activating cation. Mg²⁺ ions at 15 mM supported only 5% of the activity achieved with 2 mM Mn²⁺. K _m and k _cat values for diadenosine tetra-, penta- and hexaphosphates were 2.0, 2.4 and 1.1 μM and 11.4, 28.6 and 12.0 s⁻¹, respectively, while for GTP they were 20.3 μM and 1.8 s⁻¹, respectively. The K _m for adenosine 5′-pentaphosphate was <1 μM. Expression analysis showed the DR2356 gene to be induced eight- to ninefold in stationary phase and in cells subjected to slow dehydration plus rehydration. Superoxide (but not peroxide) treatment and rapid dehydration caused a two-to threefold induction. The Mn-requirement and induction in stationary phase suggest that DR2356 may have a specific role in maintenance mode metabolism in stationary phase as Mn²⁺ accumulates.     

Phytoene synthase from tomato (Lycopersicon esculentum) chloroplasts – partial purification and biochemical properties

 Phytoene synthase activity in tomato chloroplasts is membrane-associated, requiring treatment with high ionic strength buffer or mild non-ionic detergent for solubilisation. Using a combination of ammonium sulphate precipitation, cation and anion exchange, dye-ligand and hydrophobic interaction chromatography, phytoene synthase has been purified 600-fold from tomato (Lycopersicon esculentum Mill.) chloroplasts. The native molecular mass of the enzyme was 43 kDa, with an isoelectric point of 4.6. Although phytoene synthase was functional in a monomeric state, under optimal native conditions it was associated with a large (at least 200 kDa) protein complex which contained other terpenoid enzymes such as isopentenyl diphosphate isomerase and geranylgeranyl diphosphate (GGPP) synthase. Both Mn²⁺ and ATP, in combination, were essential for catalytic activity; their effect was stochiometric from 0.5 to 2 mM, with K _m values for Mn²⁺, ATP and the substrate GGPP of 0.4 mM, 2.0 mM and 5 μM, respectively. The detergents Tween 60 and Triton X-100 (0.1 w/v) stimulated (5-fold) enzyme activity, but lipids (crude chloroplast lipids and phospholipids) had no such effect and could not compensate for the absence of detergent. A number of metabolites with possible regulatory effects were investigated, including β-carotene, which reduced enzyme activity in vitro some 2-fold. A comparison of phytoene synthase activity from partially purified chloroplast and chromoplast preparations indicated biochemical differences. Received: 20 January 2000 / Accepted: 16 February 2000     

Adaptation of intestinal cell membrane enzymes to low temperatures in the Antarctic teleost Pagothenia bernacchii

The enzymatic activity (expressed as milliunits per milligram total proteins) of three intestinal brush-border membrane enzymes, leucine aminopeptidase, alkaline phosphatase and maltase, measured over a range of temperatures between 1.5 and 37 °C, has been found to be much higher in the Antarctic fish Pagothenia bernacchii than in the temperate fish Anguilla anguilla. To explain this experimental observation the apparent Michaelis-Menten constant, the maximal velocity, the activation energy values and the thermal stability of these three enzymes were measured. The apparent Michaelis-Menten constant values of leucine amino peptidase and alkaline phosphatase were different in the intestine mucosal homogenate of the two fish at each measured temperature (from a minimum of 2.5 to a maximum of 37 °C). However, the values found at 2.5 °C for the Antarctic species and 15 °C for the eel where comparable. Furthermore, its value was unchanged in eel intestine apical membranes, both in the presence and without enzyme lipid microenvironment. While the maximal enzymatic activities of the leucine aminopeptidase and maltase did not decrease without their enzyme lipid microenvironment, produced by treatment with Triton X-100, the impairment of alkaline phosphatase maximal activity cannot be significantly differentiated from a non-specific inhibitory effect of the detergent. The activation energy values of leucine amino peptidase, alkaline phosphatase and maltase were lower in the Antarctic fish (11.7, 5.6 and 11.8 kcal·mol^-1, respectively) than in the eel (13.6, 7.6 and 13.1 kcal·mol^-1, respectively). The thermal stability of alkaline phosphatase and maltase is different in Pagothenia bernacchii and Anguilla anguilla intestinal homogenate.Abbreviations BBM brush border membrane - E _a activation energy - EGTA ethyleneglycol-bis-(-amino ethylether)N, N-tetraacetic acid - HEPES 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethane sulphonic acid - Km_app apparent Michaelis-Menten constant - PMSF phenylmethyl-sulphonyl fluoride - TRIS TRIS (hydroxymethyl)-aminomethane     

Removal of bound Triton X-100 from purified bovine heart cytochrome bc1

Cytochrome bc₁ isolated from Triton X-100-solubilized mitochondrial membranes contains up to 120 nmol of Triton X-100 bound per nanomole of the enzyme. Purified cytochrome bc₁ is fully active; however, protein-bound Triton X-100 significantly interferes with structural studies of the enzyme. Removal of Triton X-100 bound to bovine cytochrome bc₁ was accomplished by incubation with Bio-Beads SM-2 in the presence of sodium cholate. Sodium cholate is critical because it does not interfere with the adsorption of protein on the hydrophobic surface of the beads. The resulting Triton X-100-free cytochrome bc₁ retained nearly full activity, absorption spectra, subunit, and phospholipid composition.     

Characterization of phosphoenolpyruvate carboxykinase from Corynebacterium glutamicum

Abstract Phosphoenolpyruvate (PEP) carboxykinase is present in crude extracts of Corynebacterium glutamicum grown on both glucose and lactate. Preparation of PEP carboxykinase free from interfering PEP carboxylase and oxaloacetate decarboxylase showed an absolute dependence on divalent manganese and IDP for activity in the oxaloacetate (OAA) formation. Other diphosphate nucleotides could not substitute for IDP. The enzyme activity displayed Michaelis-Menten kinetics for the substrates PEP, IDP, KHCO₃, OAA and ITP with a K _m of 0.7 mM, 0.4 mM, 12 mM, 1.0 mM, and 0.5 mM, respectively. At the optimum pH of 6.6, 850 nmol of OAA were formed per min per mg of protein. ATP inhibited PEP carboxykinase in the OAA forming reaction for 60% at 0.1 mM, indicating that the enzyme mainly functions in gluconeogenesis.     

Purification and characterization of acetyl-CoA carboxylase from developing soybean seeds

Acetyl-CoA carboxylase from two lines of soybean (Glycine max) seeds has been purified to apparent homogeneity. The procedure included affinity chromatography of the enzyme on avidin-monomer-Sepharose 4B. The enzyme from both lines showed a single band on polyacrylamide gel electrophoresis. On sodium dodecyl sulphatepolyacrylamide gel electrophoresis, the enzyme from experimental line 9686 showed a single protein band having the M, 240 000. The enzyme from the commercial line Wayne, however, showed three protein bands having the M, s 240 000, 65 000 and 58 000, respectively. High concentrations of the enzyme were required for stability as well as the presence of dithiothreitol, glycerol and Triton X-100. The enzyme was active over a wide pH range, with an optimum at 8.2 for 9686 and 7.5 for Wayne. The enzyme from both 9686 and Wayne showed absolute specificity for acetyl-CoA as a substrate and this could not be replaced by propionyl-CoA, butyryl-CoA, hexanoyl-CoA or S-methylerotonyl-CoA. At the optimum pH the apparent K_m values for the substrates were: bicarbonate, 1.13 mM; acetyl-CoA, 0.32 mM; ATP, 0.46 mM for the Wayne carboxylase and bicarbonate, 1.56 mM; acetyl-CoA, 0.17 mM; ATP, 0.14 mM for the 9686 enzyme. Citrate, at higher concentrations, was strongly inhibitory. Both ADP and AMP inhibited the enzyme from 9686 and Wayne. The enzyme from both 9686 and Wayne did not appear to be highly regulated by cellular metabolites.     

The Non-Ionic Detergent Brij 58 Conserves the Structure of the Tonoplast H+-ATPase of Mesembryanthemum crystallinum L. During Solubilization and Partial Purification

The effects of solubilization with Triton X-100 or Brij 58 on the polypeptide composition and the substrate affinity of the tonoplast H⁺-ATPase of plants of Mesembryanthemum crystallinum performing C₃ photosynthesis or crassulacean acid metabolism (CAM) have been compared. Although all known subunits of the tonoplast H⁺-ATPase were present in the fraction of solubilized proteins after treatment with Brij 58 or Triton X-100, with Triton X-100 the apparent K_M value for ATP hydrolysis was increased by a factor of 1.8 and 1.5 in preparations from C₃ and CAM plants, respectively, even at low concentrations in contrast to treatment with Brij 58. This is explained by structural changes of the tonoplast H⁺-ATPase due to the Triton X-100 treatment. After solubilization with Brij 58 the tonoplast H⁺-ATPase was partially purified by Superose-6 size-exclusion FPLC. When Brij 58 was present, addition of lipids to the chromatography buffer was not necessary to conserve enzyme activity in contrast to previously described purification methods using Triton X-100. The substrate affinity of the partial purified H⁺-ATPase was similar to the substrate affinity obtained for ATP-hydrolysis of native tonoplast vesicles, indicating that the enzyme structure during partial purification was conserved by using Brij 58. The results underline that the lipid environment of the tonoplast H⁺-ATPase is important for enzyme structure and function.     

Pyruvate kinase in the bovine adrenal cortex

Pyruvate kinase from bovine adrenal cortex was purified to an electrophoretically homogeneous state. The molecular weight of the native enzyme is about 230 000, that of one subunit is 57 000. The maximal values of the pyruvate kinase initial reaction rate were obtained in 50 mM imidazole-acetate buffer within the pH range of 6.8 to 7.0. The curve of the initial pyruvate kinase reaction rate versus phosphoenolpyruvate (PEP) and ADP concentrations is hyperbolic and obeys the Michaelis-Menten kinetics with Km for PEP and ADP of 0.055 X 10(-3) M and 0.25 X 10(-3) M, respectively. The enzyme is activated by Mn2+ and Co2+ by 43 and 38%, respectively. IDP, GDP, and UDP may be used as analogs of ADP. The enzyme is not activated by fructose-1.6-diphosphate and is inhibited by L-phenylalanine and ATP.     

Unidirectional inhibition of phosphoenolpyruvate carboxykinase from Rhodospirillum rubrum by ATP

The kinetic and regulatory properties of partially purified phosphoenolpyruvate (PEP) carboxykinase (EC 4.1.1.32) from Rhodospirillum rubrum were studied. The enzyme was active with guanosine-and inosinephosphates and must thus be classified as GTP (ITP): oxaloacetate carboxylyase (transphosphorylating). In the direction of oxaloacetate-formation, the enzyme was strongly inhibited by ATP (K_i=0.03 mM). ITP, UTP, CTP and GTP were less inhibitory. The inhibition was competitive with respect to GDP or IDP, but not with respect to PEP. In the direction of PEP-synthesis, the enzyme was not inhibited, but rather activated by ATP.     

Purification and characterization of membrane-bound phosphatidylinositol kinase from rat brain

A membrane-bound phosphatidylinositol (PI) kinase was purified from rat brain. The enzyme was solubilized with Triton X-100 from salt-washed membrane and purified 11,183-fold, with a final specific activity of 150 nmol/min/mg of protein. Purification steps included several chromatography using Q-Sepharose Fast Flow, cellulose phosphate, Toyopearl HW 55 and Affi-Gel Blue. The purified PI kinase had an estimated molecular weight of 80,000 by gel filtration and 76,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified kinase phosphorylated only PI and did not phosphorylate phosphatidylinositol 4-phosphate or diacylglycerol. Km values for PI and ATP were found to be 115 and 150 microM, respectively. The enzyme required Mg2+ (5-20 mM) or Mn2+ (1-2 mM) for activity, was stimulated by 0.1-1.0% (w/v) Triton X-100, and completely inhibited by 0.05% sodium dodecyl sulfate. The enzyme activity showed a broad pH optimum at around 7.4. The enzyme utilized ATP and not GTP as phosphate donor. Nucleoside triphosphates other than ATP and diphosphates significantly inhibited the kinase activity. However, inhibitory effects of adenosine, cAMP, and quercetin were weak.     

Activation and Deactivation of F0F1-ATPase in Yeast Mitochrondia

The regulation of membrane-bound proton F₀F₁ATPase by the protonmotive force and nucleotides was studied in yeastmitochondria. Activation occurred in whole mitochondria and the ATPaseactivity was measured just after disrupting the membranes with Triton X-100.Deactivation occurred either in whole mitochondria uncoupled with FCCP, or indisrupted membranes. No effect of Triton X-100 on the ATPase was observed,except a slow reactivation observed only in the absence of MgADP. BothAMPPNP and ATP increased the ATPase deactivation rate, thus indicating thatoccupancy of nucleotidic sites by ATP is more decisive than catalyticturnover for this process. ADP was found to stimulate the energy-dependentATPase activation. ATPase deactivated at the same rate in uncoupled anddisrupted mitochondria. This suggests that deactivation is not controlled byrebinding of some soluble factor, like IF1, but rather by the conversion ofthe F₁.IF1 complex into an inactive form.     

Effect of Mg2+, tritorn X-100 and temperature on basal and FC- stimulated plasma membrane ATPase activity

H⁺-pumping driven by the plasma membrane H⁺-ATPase in membrane vesicles from 24-hour-old radish seedlings is stimulated by pretreatment of the membranes with fusicoccin (FC) (Rasi-Caldogno et al., Plant Physiol., 82 (1986) 121).FC-pretreatment stimulates also the ATPase activity, but to a lesser extentthan H⁺-pumping. More than 80% of the ATPase activity is inhibited by 100 μM vanadate or by 3 mM Ca²⁺.Preincubation of diluted membranes in the presence of 5 mM MgSO₄ without ATP lowers both ATPase and H⁺-pumping activity by 20—30% without affecting FC-stimulated activities (i.e. the differences between FC-treated samples and the controls).After preincubation with MgSO₄, ATPase activity of membranes pretreatedwith or without FC is delivery affected by Triton X-100 and by temperature: Triton X-100 activates FC-stimulated ATPase more than that of the controls and an increase of temperature (between 13 and 33°C) enhances ATPase activity of the controls more than the FC-stimulated one.These results have been interpreted as suggesting that, while H⁺-pumping in this membrane fraction is driven only by the plasma membrane H⁺-ATPase, ATP-hydrolysis is catalyzed by two different enzymes (or forms of the same enzxxyme) diversely sensitive to FC, Triton X-100 and temperature and possibly diversely involved in H⁺-pumping.