Enzymic and protein character of tonoplast from hippeastrum vacuoles

The membrane of anthocyanin containing Hippeatrum petal vacuoles was examined for protein and enzyme content after purification by equilibrium density centrifugation. Light scattering, protein, and a Mg²⁺-dependent nucleotide specific ATPase were associated with membrane having a density of 1.08 to 1.12 grams per cubic centimeter. A small amount of acid phosphatase was also present in this region of the gradient, but this activity peaked at about 1.12 grams per cubic centimeter. A component of yeast tonoplast, α-mannosidase, was not significantly present. UDP-glucose, anthocyanidin-3-O-glucosyltransferase, thought to be a cytosol enzyme in Hippeastrum, was absent from tonoplast of vacuoles isolated by osmotic shock in 0.2 molar K₂HPO₄ or 0.35 molar mannitol. Vacuolar acid phosphatase was insensitive to ethylenediaminetetraacetate but was 80% inhibited by 10 millimolar KF, while ATPase was inactivated by 2 millimolar ethylenediaminetetraacetate and only 50% inhibited by 10 millimolar KF. Five major and about 9 minor polypeptides were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane protein on 5 to 30 and 6 to 16% gradient gels.     

Variable Effects of Nitrate on ATP-Dependent Proton Transport by Barley Root Membranes

The effects of NO₃⁻ and assay temperature on proton translocating ATPases in membranes of barley (Hordeum vulgare L. cv California Mariout 72) roots were examined. The membranes were fractionated on continuous and discontinuous sucrose gradients and proton transport was assayed by monitoring the fluorescence of acridine orange. A peak of H⁺-ATPase at 1.11 grams per cubic centimeter was inhibited by 50 millimolar KNO₃ when assayed at 24°C or above and was tentatively identified as the tonoplast H⁺-ATPase. A smaller peak of H⁺-ATPase at 1.16 grams per cubic centimeter, which was not inhibited by KNO₃ and was partially inhibited by vanadate, was tentatively identified as the plasma membrane H⁺-ATPase. A step gradient gave three fractions enriched, respectively, in endoplasmic reticulum, tonoplast ATPase, and plasma membrane ATPase. There was a delay before 50 millimolar KNO₃ inhibited ATP hydrolysis by the tonoplast ATPase at 12°C and the initial rate of proton transport was stimulated by 50 millimolar KNO₃. The time course for fluorescence quench indicated that addition of ATP in the presence of KNO₃ caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KNO₃ was explained by the temperature-dependent delay of the inhibition by KNO₃. The plasma membrane H⁺-ATPase maintained a pH gradient in the presence of KNO₃ for up to 30 minutes at 24°C.     

Properties of proton and sugar transport at the tonoplast of tomato (Lycopersicon esculentum) fruit

Tonoplast vesicles were isolated from tomato (Lycopersicon esculentum Mill.) fruit pericarp and purified on a discontinuous sucrose gradient. ATPase activity was inhibited by nitrate and bafilomycin A₁ but was insensitive to vanadate and azide. PPase hydrolytic activity was inhibited by NaF but was insensitive to nitrate, bafilomycin A₁ vanadate and azide. Kimetic studies of PPase activity gave an apparent K_m, for PP₃ of 18 μM. Identical distributions of bafilomycin- and NO₃-sensitive ATPase activities within continuous sucrose density gradients, confirmed that bafilomycin-sensitive ATPase activity is a suitable marker for the tonoplast. By comparing the distribution of bafilomycin-sensitive ATPase activity with that of PPase activity, it was possible to locate the PPase enzyme exclusively at the tonoplast. The apparent density of the tonoplast did not change during fruit development. Measurements of tonoplast PPase and ATPase activities during fruit development over a 35-day period revealed an 80% reduction in PPase specific activity and a small decrease in ATPase specific activity. ATP- and PP₁-dependent ΔpH generation was measured by the quenching of quinacrine fluorescence in tonoplast vesicles prepared on a discontinuous Dextran gradient. No H⁺ efflux was detected on the addition of sucrose to energized vesicles. Therefore a H⁺/sucrose antiport may not be the mechanism of sucrose uptake at the tomato fruit tonoplast. Similar results were obtained with glucose, fructose and sorbitol. The lack of ATP (or PP₁) stimulation of [¹⁴C]-sucrose uptake also suggested that an antiport was not involved. Initial uptake rates of radiolabelled glucose and fructose were almost double that for sucrose. The inhibition of hexose uptake by p-chloromercuribenzene sulphonate (PCMBS) implicated the involvement of a carrier. Therefore storage of hexose in the tomato fruit vacuole and maintenance of a downhill sucrose concentration gradient into sink cells is likely to be regulated by the activity of sucrose metabolizing enzymes, rather than by energy-requiring uptake mechanisms at the tonoplast.     

Isolation and partial characterization of vacuoles from tobacco protoplasts

Protoplasts from suspension-cultured cells of Nicotiana glutinosa L. were lysed in 0.3 molar sorbitol in 2 millimolar ethylenediaminetetraacetate-tris(hydroxymethyl) aminomethane (pH 7.5) to release intact vacuoles. The vacuoles were purified by centrifugation in a Ficoll step gradient. About 11% of the vacuoles and 13% of the acid phosphatase activity was recovered in the purified vacuole fraction, suggesting that the vacuole is the major site for acid phosphatase in these cells. NADH-cytochrome c reductase, malate dehydrogenase, and cytochrome c oxidase activities were reduced during vacuole purification. The majority of the adenosine 5′-triphosphate (ATP) hydrolytic activity of purified vacuoles was associated with nonspecific acid phosphatase and not with a transport ATPase. As judged by acid phosphatase distribution and electron microscopy, the effective density of vacuoles in a sucrose gradient was low (less than 1.1 grams per cubic centimeter), although an unequivocal estimate of the vacuole or tonoplast density was not possible from the experiments conducted.     

Purification of an h-translocating inorganic pyrophosphatase from vacuole membranes of red beet

An H⁺-translocating inorganic pyrophosphatase (PPase) was isolated and purified from red beet (Beta vulgaris L.) tonoplast. One major polypeptide of molecular weight 67 kilodalton copurified with fluoride-inhibitable PPase activity when subjected to one-dimensional polyacrylamide gel electrophoresis. Overall, a 150-fold purification of the PPase was obtained, from the tonoplast fraction, through anion exchange chromatography of the detergent-solubilized membranes followed by ammonium sulfate precipitation and gel filtration chromatography. The purified polypeptide showed no cross-reactivity with antibodies raised against the 67 kilodalton subunit of the tonoplast ATPase.     

Preparation of Membrane Vesicles Enriched in ATP-Dependent Proton Transport from Suspension Cultures of Tomato Cells

Membranes enriched in ATP-dependent proton transport were prepared from suspension cultures of tomato cells (Lycopersicon esculentum Mill cv VF36). Suspension cultures were a source of large quantities of membranes from rapidly growing, undifferentiated cells. Proton transport activity was assayed as quench of acridine orange fluorescence. The activity of the proton translocating ATPase and of several other membrane enzymes was measured as a function of the cell culture cycle. The relative distribution of the enzymes between the 3,000, 10,000, and 100,000g pellets remained the same throughout the cell culture cycle, but yield of total activity and activity per gram fresh weight with time had a unique profile for each enzyme tested. Maximal yield of the proton translocating ATPase activity was obtained from cells in the middle logarithmic phase of growth, and from 50 to 90% of the activity was found in the 10,000g pellet. The proton translocating ATPase activity was separable from NADPH cytochrome c reductase and cytochrome c oxidase on a sucrose gradient. Proton transport activity had a broad pH optimum (7.0-8.0), was stimulated by KCl with a K_m of 5 to 10 millimolar, stimulation being due to the anion, Cl⁻, and not the cation, K⁺, and was not inhibited by vanadate, but was inhibited by NO₃⁻. The activity is tentatively identified as the tonoplast ATPase.     

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.     

Proton transport and phosphorylation of tonoplast polypeptides from zucchini are stimulated by the phospholipid platelet-activating factor

The ether phospholipid platelet-activating factor and certain similar phospholipids, including lysophosphatidylcholine, are known to stimulate both H⁺ transport and protein phosphorylation in plant microsomal membranes. In the present work, several polypeptides in highly purified tonoplast membranes from zucchini (Cucurbita pepo L.) showed platelet-activating factor-dependent phosphorylation. Comparison of protein phosphorylation in different membrane fractions separated by sucrose step density gradient centrifugation indicated that some of the phosphoproteins were contaminants or were common to several membrane fractions, but platelet-activating factor-dependent phosphorylation of peptides at 30, 53, and perhaps 100 kilodaltons was tonoplast specific. The phosphoprotein of 53 kilodaltons was shown by three different approaches (one- and two-dimensional polyacrylamide gel electrophoresis, western blots, and immunoprecipitation) to cross-react with antibody raised against the B subunit of the tonoplast ATPase from red beet (Beta vulgaris L.).     

Purification and characterization of tonoplast ATPase from etiolated mung bean seedlings

The tonoplast ATPase from etiolated seedlings of Vigna radiata L. (mung bean) was isolated using a two-step detergent solubilization modified from Mandala and Taiz (S Mandala, L Taiz [1985] Plant Physiol 78: 327-333). After ultracentrifugation on 10 to 28% sucrose gradient, the ATPase showed a 31.6-fold purification over the initial specific activity of the starting tonoplast-enriched membranes. The purified ATPase used Mg²⁺-ATP as the preferred substrate. The tonoplast ATPase was isolated in a form with characteristics similar to that on its native membrane environment. Analysis by SDS-PAGE revealed two prominent bands with molecular weights of 78,000 (α subunit) and 64,000 (β subunit). The intensity of Coomassie blue staining showed a 1:1 stoichiometry for α and β subunits. The amino acid composition of α and β subunits also confirmed the suggested stoichiometry of the subunit composition of the tonoplast ATPase. Moreover, radiation inactivation analysis yielded a functional size of 414 ± 24 and 405 ± 25 kilodaltons for soluble and membrane bound tonoplast ATPases, respectively. It is possible that the functioning tonoplast ATPase may be in a form of αβ-heteromultimer.     

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.

     

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot : II. Evidence for a Sucrose/H-Antiport

The process of sucrose transport was investigated in sealed putative tonoplast vesicles isolated from sugarbeet (Beta vulgaris L.) taproot. If the vesicles were allowed to develop a steady state pH gradient by the associated transport ATPase and 10 millimolar sucrose was added, a transient flux of protons out of the vesicles was observed. The presence of an ATPase produced pH gradient allowed [¹⁴C]sucrose transport into the vesicles to occur at a rate 10-fold higher than the rate observed in the absence of an imposed pH gradient. Labeled sucrose accumulated into the sealed vesicles could be released back to the external medium if the pH gradient was dissipated with carbonylcyanide-m-chlorophenyl hydrazone (CCCP). When the kinetics of ATP dependent [¹⁴C]sucrose uptake were examined, the kinetic profile followed the simple Michaelis-Menten relationship and a Michaelis constant of 12.1 millimolar was found. When a transient, inwardly directed sucrose gradient was imposed on the vesicles in the absence of charge compensating ions, a transient interior negative membrane potential was observed. This membrane potential could be prevented by the addition of CCCP prior to sucrose or dissipated by the addition of CCCP after sucrose was added. These results suggest that an electrogenic H⁺/sucrose antiport may be operating on the vesicle membrane.     

NaCl Induces a Na/H Antiport in Tonoplast Vesicles from Barley Roots

Evidence was found for a Na⁺/H⁺ antiport in tonoplast vesicles isolated from barley (Hordeum vulgare L. cv California Mariout 72) roots. The activity of the antiport was observed only in membranes from roots that were grown in NaCl. Measurements of acridine orange fluorescence were used to estimate relative proton influx and efflux from the vesicles. Addition of MgATP to vesicles from a tonoplast-enriched fraction caused the formation of a pH gradient, interior acid, across the vesicle membranes. EDTA was added to inhibit the ATPase, by chelating Mg²⁺, and the pH gradient gradually dissipated. When 50 millimolar K⁺ or Na⁺ was added along with the EDTA to vesicles from control roots, the salts caused a slight increase in the rate of dissipation of the pH gradient, as did the addition of 50 millimolar K⁺ to vesicles from salt-grown roots. However, when 50 millimolar Na⁺ was added to vesicles from salt-grown roots it caused a 7-fold increase in the proton efflux. Inclusion of 20 millimolar K⁺ and 1 micromolar valinomycin in the assay buffer did not affect this rapid Na⁺/H⁺ exchange. The Na⁺/H⁺ exchange rate for vesicles from salt-grown roots showed saturation kinetics with respect to Na⁺ concentration, with an apparent K_m for Na⁺ of 9 millimolar. The rate of Na⁺/H⁺ exchange with 10 millimolar Na⁺ was inhibited 97% by 0.1 millimolar dodecyltriethylammonium.     

Purification and Properties of the Plasma Membrane H-Translocating Adenosine Triphosphatase of Phaseolus mungo L. Roots

The plasma membrane ATPase of mung bean (Phaseolus mungo L.) roots has been solubilized with a two-step procedure using the anionic detergent, deoxycholate (DOC) and the zwitterionic detergent, zwittergent 3-14 as follows: (a) loosely bound membrane proteins are removed by treatment with 0.1% DOC; (b) The ATPase is solubilized with 0.1% zwittergent in the presence of 1% DOC; (c) the solubilized material is further purified by centrifugation through a glycerol gradient (45-70%). Typically, about 10% of the ATPase activity is recovered, and the specific activity increases about 11-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the peak fraction from the glycerol gradient contains three major polypeptides of M_r = 105,000, 67,000, and 57,000 daltons. The properties of the purified ATPase are essentially the same as those of membrane-bound ATPase, with respect to pH optimum, substrate specificity, inhibitor sensitivity, and ion stimulation.     

ATPase in lipid body membranes of castor bean endosperm

Lipid body membranes purified from castor seed endosperm of dry seeds and 4 d old seedlings were found to have an ATPase activity associated with them. This was confirmed by equilibrium density centrifugation of the membranes using acid lipase as a marker enzyme. The specific activity ranged from 45 to 200 nanomoles per milligram protein per minute. The pH optimum was 9.0 but at pH 7.5 nearly 40% of the maximum activity was retained. The apparent K_m for Mg-ATP was 0.5 millimolar. A divalent cation was required for activity and Mg²⁺ was the most effective. Other nucleoside triphosphates were also hydrolyzed but there was no hydrolysis of pyrophosphate or p-nitrophenylphosphate. The ATPase was not inhibited by oligomycin, vanadate, dicyclohexylcarbodiimide, or molybdate but was inhibited by sodium azide. Washing the membranes with increasing concentrations of NaCl removed up to 60% of the ATPase activity but none was removed by 3 millimolar ethylene-diaminetetraacetate.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: I. Identification and Characterization of an Anion-Sensitive H-ATPase

Microsomal membranes isolated from red beet (Beta vulgaris L.) storage tissue were found to contain high levels of ionophore-stimulated ATPase activity. The distribution of this ATPase activity on a continuous sucrose gradient showed a low density peak (1.09 grams per cubic centimeter) that was stimulated over 400% by gramicidin and coincided with a peak of NO₃⁻-sensitive ATPase activity. At higher densities (1.16-1.18 grams per cubic centimeter) a shoulder of gramicidin-stimulated ATPase that coincided with a peak of vanadate-sensitive ATPase was apparent. A discontinuous sucrose gradient of 16/26/34/40% sucrose (w/w) was effective in routinely separating the NO₃⁻-sensitive ATPase (16/26% interface) from the vanadate-sensitive ATPase (34/40% interface). Both membrane fractions were shown to catalyze ATP-dependent H⁺ transport, with the transport process showing the same differential sensitivity to NO₃⁻ and vanadate as the ATPase activity.

Characterization of the lower density ATPase (16/26% interface) indicated that it was highly stimulated by gramicidin, inhibited by KNO₃, stimulated by anions (Cl⁻ > Br⁻ > acetate > HCO₃⁻ > SO₄²⁻), and largely insensitive to monovalent cations. These characteristics are very similar to those reported for tonoplast ATPase activity and a tonoplast origin for the low density membrane vesicles was supported by comparison with isolated red beet vacuoles. The membranes isolated from the vacuole preparation were found to possess an ATPase with characteristics identical to those of the low density membrane vesicles, and were shown to have a peak density of 1.09 grams per cubic centimeter. Furthermore, following osmotic lysis the vacuolar membranes apparently resealed and ATP-dependent H⁺ transport could be demonstrated in these vacuole-derived membrane vesicles. This report, thus, strongly supports a tonoplast origin for the low density, anion-sensitive H⁺-ATPase and further indicates the presence of a higher density, vanadate-sensitive, H⁺-ATPase in the red beet microsomal membrane fraction, which is presumably of plasma membrane origin.

     

Protein kinase activities in tonoplast and plasmalemma membranes from corn roots

Protein kinase and phosphatase activities were studied in plasmalemma and tonoplast membrane fractions from corn (Zea mays L.) roots in order to test the hypothesis that the tonoplast H⁺-ATPase is regulated by intrinsic protein phosphorylation (G Zocchi, SA Rogers, JB Hanson 1983 Plant Sci Lett 31: 215-221), and to facilitate future purification of kinase activities from these membranes. Kinase activity in the plasmalemma was about three-fold higher than in the tonoplast, and displayed Michaelis Menten-type behavior with a K_m value for MgATP²⁻ of about 50 micromolar. Both activities were optimal at 3 millimolar free Mg²⁺ and had pH optima at 6.6 and 7.0 for the plasmalemma and tonoplast, respectively. Kinase activities in both fractions were stimulated by 1 micromolar free Ca²⁺, but calmodulin had no stimulatory effect, and chlorpromazine was inhibitory only at high concentrations. The pattern of phosphopeptides on SDS polyacrylamide gel electrophoresis was similar in both fractions except for one band of 50 kilodaltons that was present only in the tonoplast. A partially purified H⁺-ATPase fraction was prepared from tonoplast membranes, incubated under conditions optimal for protein phosphorylation. The three polypeptides (of 67, 57, and 36 kilodaltons), enriched in this fraction, did not become phosphorylated, suggesting that this protein is not regulated by endogenous protein phosphorylation. Protein phosphatase activity was detected only in the plasmalemma fraction. These results indicate that a regulatory cycle of protein phosphorylation and dephosphorylation may operate in the plasmalemma. The activity in the tonoplast appears to originate from plasmalemma contamination.     

Solubilization by lysolecithin and purification of the plasma membrane ATPase of the yeast Schizosaccharomyces pombe.

Purified plasma membranes of Schizosaccharomyces pombe were obtained by precipitation at pH 5.2 of a crude particulate fraction, followed by differential centrifugations and isopycnic centrifugation in a discontinuous sucrose gradient. The specific activity of the Mg2+-requiring plasma membrane ATPase activity (EC 3.6.1.3) was enriched from 0.3 mumol min-1 x mg-1 of protein in the homogenate to 26 in the purified membranes. The optimal conditions for solubilization of the ATPase activity by lysolecithin were found to be: 2 mg/ml of lysolecithin, a lysolecithin to protein ratio of 8 at pH 7.5, and 15 degrees C in the presence of 1 mM ATP and 1 mM ethylenediaminetetraacetic acid. A 6- to 7-fold purification of the solubilized ATPase activity was obtained by centrifugation of the lysolecithin extract in sucrose gradient. Part of the ATPase activity which was inactivated during the centrifugation in the sucrose gradient could be restored by addition of a micellar solution of 50 microgram of lysolecithin/ml during the assay. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the purified enzyme showed only one band of Mr = 105,000 stained with Coomassie blue. Another ATPase component of apparent molecular weight lower than 10,000 was stained by periodic Schiff reagent but not colored by Coomassie blue. The purified enzyme was 85% inhibited by 50 micrometer N,N'-dicyclohexylcarbodiimide and 94% inhibited by 53 microgram of Dio-9/ml.     

Purification and properties of a mannosyltransferase solubilized from mitochondrial outer membranes

The enzyme GDPmannose: dolichyl monophosphate mannosyltransferase has been solubilized and purified from mice liver mitochondrial outer membranes. The purification combines detergent extraction of purified outer membranes using Nonidet P-40, with subsequent ion-exchange chromatography on DEAE-cellulose. At this stage, a 400-fold purification is obtained. The partially purified mannosyltransferase is activated choline-containing lipids such as phosphatidylcholine, lysophatidylcholine and sphingomyelin. The reaction is dependent upon the addition of exogenous dolichyl monophosphate. The sole reaction product has been identified as dolichyl posphate-mannose. The partially purified mannosyltrasnferase exhibits a K_m of 1.33 μM for GDPmannose. Enzyme activity, eluted from DEAE-cellulose, could be further purified after incorporation into sphingomyelin vesicles containing dolichyl monophosphate followed by a sucrose density gradient certrifugation. The mannosyltransferase activity is completely associated with the liposomes at the top of the gradient. Significant stabilization and purification (approx. 1600-fold) of enzyme activity associated with these liposomes is obtained. Furthermore, the reconstitution of this purified enzyme within specific liposomes provides a good model membrane to investigate the molecular requirement of this mitochondrial mannosyltransferase.     

Association of H-Translocating ATPase in the Golgi Membrane System from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

The Golgi complex and the disrupted vesicular membranes were prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) using protoplasts as the starting material and employing linear sucrose density gradient centrifugation followed by osmolysis (Ali et al. [1985] Plant Cell Physiol 26: 1119-1133). The isolated Golgi fraction was found to be enriched with marker enzyme activities and depleted of the activity of a typical mitochondrial marker enzyme, cytochrome c oxidase. Golgi complex, and vesicular membranes derived thereof were found to contain the specific ATPase (specific activity of about 0.5 to 0.7 micromoles per minute per milligram protein). Inhibitor studies suggested that the ATPase of Golgi was different from plasma membrane, tonoplast and mitochondrial ATPases as it was not inhibited by sodium vanadate, potassium nitrate, oligomycin and sodium azide. The sensitivity to N-ethylmaleimide further distinguished the Golgi ATPase from F₀ to F₁ ATPase of mitochondria. The internal acidification was measured by monitoring the difference in absorbance at 550 nanometers minus 600 nanometers using neutral red as a probe. The maximum rate detected with Golgi and disrupted membrane system was 0.49 and 0.61 optical density unit per minute per milligram protein, at pH 7.5, respectively, indicating that the proton pump activity was tightly associated with the Golgi membranes. In both cases, the acidification was inhibited 70 to 90% by various ionophores, indicating that the proton pump was electrogenic in nature. Both the Golgi ATPase activity and ATP-dependent acidification were profoundly inhibited by N,N′-dicyclohexylcarbodiimide, which also indicate that the two activities are catalyzed by the same enzyme.     

Purification and properties of membrane-bound coupling factor-latent ATPase from mycobacterium phlei

The membrane bound coupling factor-latent ATPase was solubilized from the membrane vesicles of Mycobacterium phlei by using 0.25 M sucrose or low ionic strength buffer. Purification of the solubilized enzyme by use of Sepharose-ADP conjugate gel yielded a homogenous preparation of latent ATPase which was purified about 216-fold in a single step with an 84% yield. The enzyme exhibits a specific activity of 39 μmoles of ATP hydrolyzed per min per mg protein. The purified enzyme exhibits coupling factor activity. Electrophoresis in two dissociating solvent systems indicates that the enzyme contains at least three major polypeptides of molecular weights 56,000, 51,000 and 46,000 daltons, and two minor polypeptides of 30,000 and 17,000 daltons. Equilibrium binding studies of ADP with purified coupling factor-latent ATPase reveal the presence of two nucleotide binding sites per molecule with an apparent Ka of 8.1 × 10⁻⁵ M. By use of affinity chromatography, another latent ATPase has been isolated from the solubilized enzyme, which does not exhibit coupling factor activity.