Differentiation between Several Types of Phosphohydrolases in Light Microsomes of Corn Roots

The phosphohydrolase activity of a light microsomal fraction isolated from corn roots (Zea mays L. cv LG 55) was investigated. The fraction, which appears to be enriched in endoplasmic reticulum and Golgi membranes, has ATPase and pyrophosphatase activities that hydrolyze ATP and pyrophosphate at an optimum pH of 7.0, with K_m values of about 160 and 240 micromolar and with V_max values of about 200 and 50 nanomoles substrate hydrolyzed per milligram protein per minute, respectively. These enzymes differ in their sensitivity to anions and inhibitors. The ATPase is stimulated by sulfate anions, whereas pyrophosphatase is inhibited by molybdate. Furthermore, the simultaneous addition of ATP and pyrophosphate to the reaction medium increases phosphohydrolysis, suggesting that separate enzymes are operating in the membranes. We also observed that pyrophosphate competitively inhibits the ATPase, whereas ATP has no significant effect on the pyrophosphatase. On the other hand, we observed a detergent-stimulated, molybdate-insensitive inosine diphosphatase activity which, in the native state, hydrolyzes inosine diphosphate with a K_m of about 700 micromolar and a V_max of about 450 nanomoles inosine diphosphate hydrolyzed per milligram protein per minute. In the solubilized form, the enzyme appears to be fully active, exhibiting lower K_m values to hydrolyze inosine diphosphate. Furthermore, we found that native inosine diphosphatase is inhibited either by ATP or pyrophosphate, whereas inosine diphosphate inhibits the ATPase, but has no significant effect on the pyrophosphatase. It appears that inosine diphosphate is a positive modulator of the inosine diphosphatase, whereas ATP and pyrophosphate act as negative modulators of this enzyme.     

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.     

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.

     

Characterization of an ATPase Associated with the Inner Envelope Membrane of Amyloplasts from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Amyloplast envelope membranes isolated from cultured, white-wild cells of sycamore (Acer pseudoplatanus L.) have been found to contain a Mg²⁺-ATPase, ranging in specific activity from 5 to 30 nanomoles per minute per milligram protein. This ATPase hydrolyzes a broad range of nucleoside triphosphates, whereas it hydrolyzes nucleoside mono- and diphosphates poorly, if at all. The ATPase activity was stimulated by several divalent cations, including Mg²⁺, Mn²⁺ and Ca²⁺, whereas it was not affected by Sr²⁺, K⁺, or Na⁺. The K_m for total ATP was 0.6 millimolar, and the activity showed a broad pH optimum between 7.5 and 8.0. The ATPase was insensitive to N,N′-dicyclohexylcarbodiimide and oligomycin, but it was inhibited by vanadate. All these characteristics are basically similar to those reported previously for the Mg²⁺-ATPase of the chloroplast inner-envelope membrane. Likewise, the amyloplast envelope enzyme was shown to be located specifically on the inner envelope membrane. The amyloplast envelope membranes were chemically modified with a series of unique affinity labeling reagents, the adenosine polyphosphopyridoxals (M Tagaya, T Fukui 1986 Biochemistry 25: 2958-2964). About 90% of the ATPase activity was lost when the envelope membranes were preincubated with 0.1 millimolar adenosine triphosphopyridoxal. Notably, the enzyme was protected completely from inactivation in the presence of its substrate, ATP. In contrast, both adenosine diphosphopyridoxal and pyridoxal phosphate caused much less of an inhibitory effect. This greater relative reactivity of the triphosphopyridoxal analog is similar to that reported previously with Escherichia coli F₁ ATPase (T Noumi et al. 1987 J Biol Chem 262: 7686-7692).     

Properties of a Membrane-bound Phosphatase from the Thylakoids of Spinach Chloroplasts

A 3-phosphoglycerate phosphatase activity of about 2 micromoles per minute per milligram chlorophyll is associated with the thylakoid membranes of spinach chloroplasts. The K_m for 3-phosphoglycerate is 3 millimolar. The enzyme can be solubilized from thylakoid membranes by treatment with 0.33 molar MgCl₂ or sodium deoxycholate. The activity is not stimulated by sulfhydryl reagents or the addition of 10 millimolar MgCl₂. The enzymic activity is insensitive to ethylenediaminetetraacetate. The pH optimum is broad, between 5.5 to 7.5. Although the substrate specificity is broad, 3-phosphoglycerate is the best substrate of those tested at neutral pH. However, p-nitrophenyl phosphate was a more effective substrate at pH 5.5. The enzyme exhibits the general characteristics of an acid phosphatase.     

Calcium Transport in the Green Alga Mesotaenium caldariorum: Preliminary Characterization and Subcellular Distribution

The subcellular localization and biochemical characterization of calcium transport were studied in the unicellular green alga Mesotaenium caldariorum. Membrane fractions prepared by osmotic lysis of Mesotaenium protoplasts exhibit high rates of ATP-dependent calcium uptake. Sucrose gradient centrifugation separates two pools of activity, which display specific activities for calcium transport as high as 15 nanomoles Ca²⁺ per minute per milligram of protein. Marker enzyme analysis shows that this dual distribution of calcium transport activity is similar to that of vanadate-insensitive ATPase and pyrophosphatase, activities considered to be associated with the tonoplast. Plasma membranes, endoplasmic reticulum vesicles, mitochondrial membranes, and thylakoids band at higher densities than either calcium transport fraction. Both pools of ATP-dependent calcium uptake contain two components which are not separable on sucrose gradients but can be distinguished on the basis of inhibitor sensitivity. One component is inhibited by nigericin or trimethyltin chloride (I₅₀ values of 3 nanomolar and 4 micromolar, respectively), while the other component is vanadate sensitive (I₅₀ of 25 micromolar). These results suggest that direct Ca²⁺ transport and Ca²⁺/H⁺ antiport activities are present in both sucrose gradient fractions.     

Role of alphaPhe-291 residue in the phosphate-binding subdomain of catalytic sites of Escherichia coli ATP synthase

The role of αPhe-291 residue in phosphate binding by Escherichia coli F₁F₀-ATP synthase was examined. X-ray structures of bovine mitochondrial enzyme suggest that this residue resides in close proximity to the conserved βR246 residue. Herein, we show that mutations αF291D and αF291E in E. coli reduce the ATPase activity of F₁F₀ membranes by 350-fold. Yet, significant oxidative phosphorylation activity is retained. In contrast to wild-type, ATPase activities of mutants were not inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium. Whereas, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by ∼75%, although reaction still occurred at residue βTyr-297, proximal to αPhe-291 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in βE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild-type but not in mutants. In addition, our data suggest that the interaction of αPhe-291 with phosphate during ATP hydrolysis or synthesis may be distinct.     

Functional Importance of Arginine 64 in Chlamydomonas reinhardtii Phosphoribulokinase

Phosphoribulokinase (EC 2.7.1.19) was investigated in wild-type Chlamydomonas reinhardtii and in mutant strains deficient in this enzyme activity. Immunoblot analysis revealed substantial amounts of phosphoribulokinase in mutant 12-2B but none in mutant F-60. The pH optimum of the wild-type enzyme was 8.0 and that of the 12-2B enzyme was 6.5. The mutant kinase possessed a K_m value for ribulose 5-phosphate of about 45 millimolar, nearly three orders of magnitude greater than the wild-type value of 56 micromolar. K_m values for ATP in the range of 36 to 72 micromolar were observed with both wild-type and mutant enzymes. The V_max of the wild-type enzyme was about 450 micromoles per minute per milligram of protein, and values for the mutant enzyme were 140 micromoles per minute per milligram at pH 6.5 and 36 micromoles per minute per milligram at pH 7.8. Thermal stabilities of the wild-type and mutant kinases were similar. Sequence analysis of the 12-2B phosphoribulokinase gene revealed a C to T transition that caused an arginine to cysteine change at position 64 of the enzyme. This arginine residue is conserved in phosphoribulokinases from vascular plants, algae, and photosynthetic bacteria and appears to function in binding ribulose 5-phosphate.     

Isolation and Characterization of Phosphoenolpyruvate Phosphatase from Germinating Mung Beans (Vigna radiata)

A phosphoenolpyruvate (PEP) phosphatase was purified to homogeneity from germinating mung beans (Vigna radiata). It was found to be a tetrameric protein (molecular mass 240,000 daltons) made up of apparently identical subunits (subunit molecular mass 60,000 daltons). It was free from bound nucleotides. It did not show pyruvate kinase activity. The enzyme showed high specificity for PEP. Pyrophosphate and some esters (nucleoside di- and triphosphates) were hydrolyzed slowly and phosphoric acid monoesters were not hydrolyzed. The enzyme showed maximum activity at pH 8.5. At this pH, the K_m of PEP was 0.14 millimolar and the V_max was equal to 1.05 micromoles pyruvate formed per minute per milligram enzyme protein. Dialysis of the enzyme against 10 millimolar triethanolamine buffer (pH 6.5), led to loss of the catalytic activity, which was restored on addition of Mg²⁺ ions (K_m = 0.12 millimolar). Other divalent metal ions inhibited the Mg²⁺ -activated enzyme. PEP-phosphatase was inhibited by ATP and several other metabolites.     

Proton-transporting urinary epithelia. Reactivity with N,N′-dicyclohexylcarbodiimide

Dicyclohexylcarbodiimide (DCCD), a potent inhibitor of the F₀F₁-type H⁺-translocating ATPase, was employed to determine the possible involvement of such an ATPase in urinary acidification. Two methods were used in this approach: (1) the reaction of [¹⁴C]DCCD with tissues involved in urinary acidification and (2) the inhibition of ATPase activity by DCCD. Membrane components from epithelial cells of toad and turtle urinary bladder and brush borders of rabbit kidney were reacted with [¹⁴C]DCCD and analyzed by polyacrylamide gel electrophoresis both before and after extraction with organic solvents. Although a DCCD-binding component was extracted from toad and turtle bladder membranes by chloroform/methanol (2:1, $\text{v}\text{v}$), the binding was not saturable. Analysis of this DCCD-binding component by thin-layer chromatography indicated that there was no ninhydrin reactivity associated with the [¹⁴C]DCCD binding. Moreover, all attempts to precipitate a DCCD-binding protein were unsuccessful. This and other evidence suggested that the observed DCCD binding was to phospholipid. In the second type of experiments, the ATPase activity present in brush borders from rabbit kidney was partially inhibited by DCCD, but at a concentration that is over two orders of magnitude greater than that required for typical DCCD-sensitive ATPase. We conclude from our failure to find positive evidence of a DCCD-reactive protein and from the relative insensitivity of the ATPase to DCCD that either urinary acidification is not accomplished by a typical F₀F₁-type translocating ATPase, or the F₀ has been modified so that the sensitivity to DCCD has been altered or lost.     

Latency of Plasma Membrane H-ATPase in Vesicles Isolated by Aqueous Phase Partitioning : Increased substrate Accessibility or Enzyme Activation

The properties of the plasma membrane H⁺-ATPase and the cause of its latency have been studied using a highly purified plasma membrane fraction from oat (Avena sativa L., cv Victory) roots, prepared by aqueous two-phase partitioning. The ATPase has a maximum specific activity (at 37°C) in excess of 4 micromoles inorganic phosphate per milligram protein per minute in the presence of nondenaturing surfactants. It is inhibited by more than 90% by vanadate, is specific for ATP, has a pH optimum of 6.5, and is stimulated more than 4-fold by 50 millimolar K⁺ in the presence of low levels of the nondenaturing surfactants Triton X-100 and lysolecithin. This `latent' activity is usually explained as being a result of the inability of ATP to reach the ATPase in right-side out, sealed vesicles, until they are disrupted by surfactants. Consistent with this idea, trypsin digestion significantly inhibited the ATPase only in the presence of the surfactants. Electron spin resonance spectroscopy volume measurements confirmed that surfactant-free vesicles were mostly sealed to molecules similar to ATP. However, the Triton to protein ratio required to disrupt vesicle integrity completely is 10-fold less than that needed to promote maximum ATPase activity. We propose that plasma membrane ATPase activation is due not solely to vesicle disruption and accessibility of ATP to the ATPase but to the surfactants activating the ATPase by altering the lipid environment in its vicinity or by removing an inhibitory subunit.     

Purification and Characterization of Acetyl-CoA Carboxylase from the Diatom Cyclotella cryptica

Acetyl-CoA carboxylase from the diatom Cyclotella cryptica has been purified to near homogeneity by the use of ammonium sulfate fractionation, gel filtration chromatography, and affinity chromatography with monomeric avidin-agarose. The specific activity of the final preparation was as high as 14.6 micromoles malonyl-CoA formed per milligram protein per minute, indicating a 600-fold purification. Native acetyl-CoA carboxylase has a molecular weight of approximately 740 kilodaltons and appears to be composed of four identical biotin-containing subunits. The enzyme has maximal activity at pH 8.2, but enzyme stability is greater at pH 6.5. K_m values for MgATP, acetyl-CoA, and HCO₃- were determined to be 65, 233, and 750 micromolar, respectively. The purified enzyme is strongly inhibited by palmitoyl-CoA, and is inhibited to a lesser extent by malonyl-CoA, ADP, and phosphate. Pyruvate stimulates enzymatic activity to a slight extent. Acetyl-CoA carboxylase from Cyclotella cryptica is not inhibited by cyclohexanedione or aryloxyphenoxypropionic acid herbicides as strongly as monocot acetyl-CoA carboxylases; 50% and 0% inhibition was observed in the presence of 23 micromolar clethodim and 100 micromolar haloxyfop, respectively.     

Characterization of a proton-translocating ATPase in microsomal vesicles from corn roots

Sealed microsomal vesicles were prepared from corn (Zea mays, Crow Single Cross Hybrid WF9-Mo17) roots by centrifugation of a 10,000 to 80,000g microsomal fraction onto a 10% dextran T-70 cushion. The Mg²⁺-ATPase activity of the sealed vesicles was stimulated by Cl⁻ and NH₄⁺ and by ionophores and protonophores such as 2 micromolar gramicidin or 10 micromolar carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). The ionophore-stimulated ATPase activity had a broad pH optimum with a maximum at pH 6.5. The ATPase was inhibited by NO₃⁻, was insensitive to K⁺, and was not inhibited by 100 micromolar vanadate or by 1 millimolar azide.

Quenching of quinacrine fluorescence was used to measure ATP-dependent acidification of the intravesicular volume. Quenching required Mg²⁺, was stimulated by Cl⁻, inhibited by NO₃⁻, was insensitive to monovalent cations, was unaffected by 200 micromolar vanadate, and was abolished by 2 micromolar gramicidin or 10 micromolar FCCP. Activity was highly specific for ATP. The ionophore-stimulated ATPase and ATP-dependent fluorescence quench both required a divalent cation (Mg²⁺ ≥ Mn²⁺ > Co²⁺) and were inhibited by high concentrations of Ca²⁺. The similarity of the ionophore-stimulated ATPase and quinacrine quench and the responses of the two to ions suggest that both represent the activity of the same ATP-dependent proton pump. The characteristics of the proton-translocating ATPase differed from those of the mitochondrial F₁F₀-ATPase and from those of the K⁺-stimulated ATPase of corn root plasma membranes, and resembled those of the tonoplast ATPase.

     

Solubilization of a phospholipid-stimulated adenosine triphosphatase complex from membranes of Escherichia coli.

A phospholipid-stimulated adenosine triphosphatase (ATPase) complex was solubilized and partially purified from membrane particles of Escherichia coli ML308-225. The complex was of large molecular size and contained 16 polypeptides, five of which were subunits of the F₁-type ATPase of E. coli. Components of the respiratory chain were absent. Enzyme activity was stimulated by lysophosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, and cardiolipin but not by phosphatidylethanolamine. The ATPase activity of the complex was inhibited by N,N′-dicyclohexylcarbodiimide and by Dio-9 at lower inhibitor:protein ratios than required for inhibition of the F₁-type ATPase of E. coli. However, the ATPase complex was less sensitive than the membrane-bound enzyme to inhibition by these compounds.     

Partial characterization of the plasma membrane ATPase from arho 0 petite strain ofSaccharomyces cerevisiae

Crude membrane preparations of arho ⁰ mutant ofSaccharomyces cerevisiae exhibit Mg²⁺-dependent ATPase activity. Over the optimal pH range, 5.0–6.75, the apparentV _max of the enzyme equals 590 nmoles of ATP hydrolyzed per minute per milligram protein, with an apparentK _m for ATP of 1.3 mM. ATP hydrolysis is insensitive to ouabain, venturicidin, aurovertin, and the protein inhibitor described by Pullman and Monroy; inhibited by oligomycin (at high concentrations) and sodium orthovandate, and it is sensitive to dicyclohexylcarbodiimide,p-hydroxymercuribenzoate, hydroxylamine, sodium fluoride, and sodium iodoacetate. The pH optimum and the inhibitor pattern distinguish the plasma membrane enzyme from the mitochondrial F₁ ATPase still present in these cells (this activity is sensitive to efrapeptin, aurovertin, and the protein inhibitor, but resistant to DCCD). In addition, the activity of the plasma membrane enzyme and its affinity for ATP are responsive to changes in the composition of the growth medium, with the highest activity observed in cells grown on methyl--d-glucoside, a sugar which results not only in partial release from catabolite repression but also requires the induction of an active transport system for growth.Author to whom correspondence should be addressed; recipient of a Research Career Award No. K06 05060 from the Institute of General Medical Sciences.     

Characterization and Effect of Light on the Plasma Membrane H+-ATPase of Bean Leaves

Proton excretion from bean (Phaseolus vulgaris L.) leaf cells is increased by bright white light. To test whether this could be due, at least in part, to an increase in plasma membrane (PM) ATPase activity, PM vesicles were isolated from primary leaves by phase partitioning and used to characterize PM ATPase activity and changes in response to light. ATPase activity was characterized as magnesium ion dependent, vanadate sensitive, and slightly stimulated by potassium chloride. The pH optimum was 6.5, the K_m was approximately 0.30 millimolar ATP, and the activity was about 60% latent. PM vesicles were prepared from leaves of plants grown for 11 days in dim red light (growing slowly) or grown for 10 days in dim red light and then transferred to bright white-light for 1 day (growing rapidly). For both light treatments, ATPase specific activity was approximately 600 to 700 nanomoles per milligram protein per minute, and the latency, K_m, and sensitivity to potassium chloride were also similar. PM vesicles from plants grown in complete darkness, however, exhibited a twofold greater specific activity. We conclude that the promotion of leaf growth and proton excretion by bright white light is not due to an increase in ATPase specific activity. Light does influence ATPase activity, however; both dim red light and bright white light decreased the ATPase specific activity by nearly 50% as compared with dark-grown leaves.     

An Enzymatic Conversion of Lipoxygenase Products by a Hydroperoxide Lyase in Blue-Green Algae (Oscillatoria sp.)

An enzyme has been isolated from blue-green algae Oscillatoria sp. which utilizes the product, 13-hydroperoxy-9, 11-octadecadienoic acid (13-HPOD), of lipoxygenase for its substrate. This enzyme, termed hydroperoxide lyase, converts the conjugated diene 13-hydroperoxide of linoleic acid to 13-oxotrideca-9, 11-dienoic acid. The structure of the latter has been determined by ultraviolet spectroscopy and mass spectrometry. 9-HPOD is not a substrate for this enzyme. The hydroperoxide lyase from Oscillatoria sp. has a maximum of activity at pH 6.4 and 30°C. The molecular weight of the enzyme was estimated at 56,000. The enzyme was not inhibited by BW 755C, but was inhibited by molecules containing more than one hydroxyl group. Quercetin was found to be the best inhibitor of the enzyme activity. The purified hydroperoxide lyase from Oscillatoria sp. showed an apparent K_m of 7.4 micromolar and a V_max of 35 nanomoles per minute per milligram of protein for 13-HPOD. An enzymatic pathway for the biogenesis of oxodienoic acid from linoleic acid is proposed. This involves the sequential activity of lipoxygenase and hydroperoxide lyase enzymes.     

Elicitor-Induced l-Tyrosine Decarboxylase from Plant Cell Suspension Cultures : II. Partial Characterization

l-Tyrosine decarboxylase (EC 4.1.1.25) activity was induced in cell suspension cultures of Thalictrum rugosum Ait. and Eschscholtzia californica Cham. with a yeast polysaccharide preparation (elicitor). The highest l-tyrosine decarboxylase activity in extracts from 7-day-old cell cultures of E. californica was observed 5 hours after addition of 30 to 40 micrograms elicitor per gram cell fresh weight. The enzyme extracted from cells of E. californica was purified 1540-fold to a specific activity of 2.6 micromoles CO₂ produced per minute per milligram protein at pH 8.4 and 30°C. Purified enzyme from T. rugosum showed a specific activity of 0.18 micromoles per minute per milligram protein. The purification procedure involved ammonium sulfate fractionation, anion-exchange fast protein liquid chromatography, ultrafiltration, and hydrophobic interaction chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme from the two plant cell cultures had subunits of identical molecular weight (56,300 ± 300 daltons.     

Partial purification of a tonoplast ATPase from corn coleoptiles

The tonoplast ATPase from corn coleoptile membranes was solubilized using a two-step procedure consisting of a pretreatment with 0.15% (w/v) deoxycholate to remove 60% of the protein, and 40 millimolar octyl-glucoside to solubilize the ATPase. During ultracentrifugation, the solublized ATPase entered a linear sucrose gradient faster than the majority of the protein, resulting in an 11-fold purification over the initial specific activity. The partially purified ATPase was almost completely inhibited by KNO₃ with an estimated K_i of 10 millimolar. The specific activity of the KNO₃-sensitive ATPase was increased 29-fold during purification. N,N′-Dicyclohexylcarbodiimide also completely inhibited the ATPase with half-maximal effects at a concentration of 4 micromolar. Neither vanadate nor azide inhibited enzyme activity. The purified ATPase was stimulated by Cl⁻ and preferred Mg-ATP as substrate. Analysis of frations from the sucrose gradient by sodium dodecyl sulfate-polyacrylamide gel electrophoresis led to the identification of two major polypeptides at 72,000 and 62,000 daltons which were best correlated with ATPase activity. Several minor bands also appeared to copurify with enzyme activity, but were less consistent. Radiation inactivation experiments with intact membranes indicated that the functional molecular size of the tonoplast ATPase was nearly 400,000 daltons. This suggests that the ATPase is composed of several polypeptides, possibly including the 72,000- and 62,000-dalton proteins.     

Solubilization and partial purification of n,n'-dicyclohexylcarbodiimide-sensitive ATPase from pea cotyledon mitochondria

The N,N′-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase of pea (Pisum sativum L.) cotyledon mitochondria was solubilized from submitochondrial particle membranes with sodium cholate and ammonium sulfate. Ammonium sulfate precipitation of the enzyme resulted in an increase in specific activity. At between 38% and 45% saturated ammonium sulfate, 20% of the ATPase activity was precipitated, with a specific activity 4 to 5 times higher than that of the crude enzyme. The precipitate was highly sensitive to DCCD.

The properties of the ammonium sulfate preparation were investigated. It contained levels of cytochrome and NADH dehydrogenase contamination comparable to those of the highly purified F₀F₁ preparations from animal tissue. The high degree of purification was corroborated by sodium dodecyl sulfate electrophoresis.