V- AND P-TYPE Ca2+-STIMULATED ATPases IN A CALCIFYING STRAIN OF PLEUROCHRYSIS SP. (HAPTOPHYCEAE)

Coccolithophorids are marine unicellular algae characterized by their ability to carry out controlled, subcellular calcification. The biochemical and kinetic features of membrane-bound Ca²⁺-stimulated ATPases have been examined. Membranes and organelles from axenic cultures of Pleurochrysis sp. (CCMP299) were isolated by means of sucrose density centrifugation. High levels of Ca²⁺-stimulated ATPase were detected in chloroplasts, Golgi apparatus, plasma membrane, and coccolith vesicles. The sensitivity of the enzyme activity in the organelles and membranes was assessed with pharmacologic agents that are known to be specific for the several isoforms of Ca²⁺-stimulated ATPase. The Ca²⁺-stimulated ATPase activity in the Golgi and coccolith vesicle preparations was sensitive to nitrate, thiocyanate, and sodium azide and insensitive to vanadate, cyclopiazonic acid, and thapsigargin. ATP-dependent H⁺ movement, but not ⁴⁵Ca²⁺ transport, across the coccolith vesicle was demonstrated. The Ca²⁺-stimulated ATPase in the plasma membrane preparation was sensitive to vanadate. ATP-dependent, vanadate-sensitive efflux of ⁴⁵Ca²⁺ was demonstrated for microsomal material derived from gradient-isolated plasma membrane. Polypeptides from isolated Golgi and coccolith vesicle preparations cross-reacted to an antibody raised against a subunit of the oat root proton pump, whereas polypeptides from the chloroplast preparations did not cross-react. These findings show that a V-type Ca²⁺-stimulated ATPase is located on the coccolith vesicle membrane and a P-type Ca²⁺-stimulated ATPase is located on the plasma membrane.     

Partial purification of plant plasma membrane K+, Mg2+-ATPase by ion exchange chromatography

A purification procedure is presented which differs in three respects from other procedures for the purification of plant plasma membrane H⁺-pumping ATPase (EC 3.6.1.35) from various plants. Soybean ( Glycine max L. cv. Williams) hypocotyls were homogenized in the presence of physiological ionic strength and plasma membrane vesicles were purified by aqueous polymer two-phase partitioning. Plasma membrane vesicles were then solubilized in one step by using non-ionic detergent (either Triton X-100 or C₁₂E₈). The Mg-ATPase was separated by ion exchange chromatography from other solubilized membrane proteins. ATPase molecules bound to phosphocellulose fibers were eluted by a 0–1 M gradient of NaCl. The NaCl-eluted fractions contained a Mg-ATPase which showed the characteristics of Mg-ATPase present in the plasma membranes. The specific activity of the partially purified enzyme was 2–5 μmol mg⁻¹ min⁻¹ when it was reconstituted into proteoliposomes. This value is in good agreement with data obtained by other purification methods in the literature.     

Plasma membrane H-ATPase activity is involved in adaptation of tomato calli to NaCl

A tomato ( Lycopersicon esculentum Mill. cv. Pera) callus culture tolerant to NaCl was obtained by successive subcultures of NaCl-sensitive calli in medium supplemented with 50 m M NaCl. NaCl-tolerant calli grew better than NaCl-sensitive calli in media supplemented with 50 and 100 m M NaCl. Analysis of callus ion content showed a strong increase in Na⁺ and Cl⁻ both in NaCl-tolerant and -sensitive calli grown in media containing NaCl for one subculture. Cells from NaCl-tolerant calli showed a higher H⁺ extrusion activity than those from NaCl-sensitive calli grown for one subculture in the presence of NaCl. The inhibition of H⁺ extrusion by NaCl-sensitive cells was correlated with an inhibition of microsomal vanadate-sensitive H⁺-ATPase (EC 3.6.1.35) and ATP-dependent H⁺ transport, while the stimulation of H⁺ extrusion by cells tolerant to 50 m M NaCl was correlated with an increase in plasma membrane ATP-dependent H⁺ transport. The increase of ATP-dependent H⁺ extrusion in plasma membranes isolated from 50 m M NaCl-tolerant calli was not a result of stimulation of a vanadate-sensitive ATP hydrolytic activity or an increase in passive permeability to H⁺. Relative to NaCl-sensitive calli, plasma membrane H⁺-ATPase from calli tolerant to 50 m M NaCl showed a lower K_m for Mg²⁺-ATP. Our results indicate that tolerance of tomato calli to 50 m M NaCl increases the affinity of plasma membrane H⁺-ATPase for the substrate ATP and stimulates the H⁺-pumping activity of this enzyme without modifying its phosphohydrolytic activity.     

Kinetics of the purified plasma membrane H+-ATPase from red beet (Beta vulgaris)

The plasma membrane H⁺-ATPase (EC 3.6.1.35) was purified by washing red beet ( Beta vulgaris L.) plasma membranes with sodium deoxycholate and separating the ATPase, solubilized with lysophosphatidylcholine, by centrifugation in a glycerol gradient. The purified H⁺-ATPase had a sedimentation coefficient of about 8S. In the absence of exogenous protein substrates, the purified ATPase preparation did not present protein kinase activity. Compared with the H⁺-ATPase in the plasma membrane, the purified ATPase presented a higher affinity for adenosine 5'-triphosphate (ATP) and a lower sensitivity to the inhibitors vanadate and inorganic phosphate. These changes in the kinetics of the ATPase could also be observed by treating the membranes with lysophosphatidylcholine, without purifying the enzyme. These results can be explained assuming that lysophosphatidylcholine interacts with the ATPase altering its kinetics probably by stimulating the transformation from the inhibitor-binding conformation E2 into the ATP-binding conformation E1.     

Characterization of reconstituted plasma membrane H+-ATPase from maize roots

Corn ( Zea mays L.) plasma membranes from KI-washed microsomal fractions were further purified by isopycnic sucrose density centrifugation. An examination of separated fractions indicated that vesicles with nitrate-insensitive proton transport copurified with fractions containing vanadate-sensitive ATPase activity. The ATPase in purified plasma membrane was reconstituted into liposomes by a detergent dilution technique using deoxycholate. The reconstituted ATPase exhibited characteristics similar to those of the native enzyme. However, reconstituted preparations showed an enhanced sensitivity to vanadate, a diminished phosphatase activity and a high specific rate of ATP-dependent H⁺-transport. Apparent K_i values of reconstituted and native enzymes with respect to vanadate were 20 and 50 μ M , respectively; the KJ value of the H+-pumping of reconstituted ATPase was 30 μ M. The proton pumping of reconstituted vesicles could be discharged rapidly by p -trifluoromethoxyphenyl hydrazone (FCCP), hexokinase and vanadate. The hydrolysis of Mg-ATP by both native and reconstituted ATPases obeyed simple Michaelis-Menten plots with a K_m between 0.5 and 0.6 m M. The reconstituted ATPase retained a pH profile similar to that of native enzyme with a maximum of pH 6.5.     

Brij 58, a polyoxyethylene acyl ether, creates membrane vesicles of uniform sidedness. A new tool to obtain inside-out (cytoplasmic side-out) plasma membrane vesicles

Most of the plasma membrane vesicles formed upon homogenization of plant tissue have a right-side-out (cytoplasmic side-in) orientation. Subsequent purification of plasma membrane vesicles using aqueous two-phase partitioning leads to a further enrichment in right-side-out vesicles resulting in preparations with 80–90% of the vesicles in this orientation. Thus, to be able to assay, e.g. the ion-pumping activities of the H⁺-ATPase and the Ca²⁺-ATPase, which expose their active sites towards the cytoplasm, the vesicles have to be inverted. This is very efficiently achieved by including 0.05% of the detergent Brij 58 (C₁₆E₂₀) in the assay medium, which produces 100% sealed, inside-out (cytoplasmic side-out) vesicles from preparations of 80–90% right-side-out vesicles. This was shown by assaying ATP-dependent H⁺ pumping using the ΔpH probe acridine orange and dissipating the H⁺ gradient with nigericin, and by assaying ATP-dependent Ca²⁺ transport using ⁴⁵CA²⁺ and dissipating the Ca²⁺ gradient with the ionophore A23187. The presence of intact vesicles was confirmed by electronmicroscopy. The detergent Brij 58 is a polyoxyethylene acyl ether and a survey among some other members of this series revealed that those with a head group of relatively large size (E_20–23) showed this 'non-detergent behavior', whereas those with smaller head groups (E_8–10) behaved as normal detergents and permeabilized the membranes. Thus, a very convenient system for studies on ion-pumping activities and other vectorial properties of the plasma membrane is obtained by simply including the detergent Brij 58 in the assay medium.     

Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2

Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P₁ (mg protein)⁻¹ min⁻¹ at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H⁺ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H⁺-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H⁺-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A₂ (EC 3.1.1.4) action, and addition of phospholipase A₂ from animal sources increased the H⁺-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H⁺-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H⁺-ATPase activity in the plasma membranne.     

Aluminum impairs morphogenic transition and stimulates H(+) transport mediated by the plasma membrane ATPase of Yarrowia lipolytica

The effect of aluminum on dimorphic fungi Yarrowia lipolytica was investigated. High aluminum (0.5–1.0 mM AlK(SO₄)₂) inhibits yeast–hypha transition. Both vanadate-sensitive H⁺ transport and ATPase activities were increased in total membranes isolated from aluminum-treated cells, indicating that a plasma membrane H⁺ pump was stimulated by aluminum. Furthermore, Al-treated cells showed a stronger H⁺ efflux in solid medium. The present results suggest that alterations in the plasma membrane H⁺ transport might underline a pH signaling required for yeast/hyphal development. The data point to the cell surface pH as a determinant of morphogenesis of Y. lipolytica and the plasma membrane H⁺-ATPase as a key factor of this process.     

Adaptation of plasma membrane H+-ATPase of rice roots to low pH as related to ammonium nutrition

The preference of paddy rice for NH₄⁺ rather than NO₃^- is associated with its tolerance to low pH since a rhizosphere acidification occurs during NH₄⁺ absorption. However, the adaptation of rice root to low pH has not been fully elucidated. This study investigated the acclimation of plasma membrane H⁺-ATPase of rice root to low pH. Rice seedlings were grown either with NH₄⁺ or NO₃^-. For both nitrogen forms, the pH value of nutrient solutions was gradually adjusted to pH 6.5 or 3.0. After 4 d cultivation, hydrolytic H⁺-ATPase activity, V _max, K _m, H⁺-pumping activity, H⁺ permeability and pH gradient across the plasma membrane were significantly higher in rice roots grown at pH 3.0 than at 6.5, irrespective of the nitrogen forms supplied. The higher activity of plasma membrane H⁺-ATPase of adapted rice roots was attributed to the increase in expression of OSA1, OSA3, OSA7, OSA8 and OSA9 genes, which resulted in an increase of H⁺-ATPase protein concentration. In conclusion, a high regulation of various plasma membrane H⁺-ATPase genes is responsible for the adaptation of rice roots to low pH. This mechanism may be partly responsible for the preference of rice plants to NH₄⁺ nutrition.     

H+/PPi stoichiometry of a membrane‐bound pyrophosphatase of plant mitochondria

The H⁺/PP_i stoichiometry of the mitochondrial H⁺‐PP_iase from pea ( Pisum sativum L.) stem was determined by two kinetic approaches, and compared with the H⁺/substrate stoichiometries of the mitochondrial H⁺‐ATPase, and the vacuolar H⁺‐PP_iase and H⁺‐ATPase. Using sub‐mitochondrial particles or preparations enriched in vacuolar membranes, the rates of substrate‐dependent H⁺‐transport were evaluated: by a mathematical model, describing the time‐course of H⁺‐gradient (ΔpH) formation; or by determining the rate of H⁺‐leakage following H⁺‐pumping inhibition by EDTA at the steady‐state ΔpH. When the H⁺‐transport rates were divided by those of PP_i or ATP hydrolysis, measured under identical conditions, apparent stoichiometries of ca 2 were determined for the mitochondrial H⁺‐PP_iase and H⁺‐ATPase, and for the vacuolar H⁺‐ATPase. The stoichiometry of the vacuolar H⁺‐PP_iase was found to be ca 1. From these results, it is suggested that the mitochondrial H⁺‐PP_iase may, in theory, function as a primary H⁺‐pump poised towards synthesis of PP_i and, therefore, acting in parallel with the main H⁺‐ATPase.     

Plasmalemma from the roots of cucumber: Isolation by two-phase partitioning and characterization

Plasmalemma was isolated from the roots of 2-week-old cucumber plants ( Cucumis sativus L. cv. Rhensk druv) by utilizing an aqueous polymer two-phase system with 6.5%:6.5% (w/w) Dextran T500 and polyethylene glycol (PEG) 3350 at pH 7.8. The plasmalemma fraction comprised ca 6% of the membrane proteins contained in the microsomal fraction. The specific activity of the plasma membrane marker enzyme (K⁺, Mg²⁺-ATPase) was 14- to 17-times higher in the upper (PEG-rich) than in the lower (Dextran-rich) phase, and the reverse was true for marker enzymes (cytochrome c oxidase, EC 1.9.3.1, and antimycin A-resistant NADPH cytochrome c reductase) of intracellular membranes. The ATPase was highly stimulated by the addition of detergent (Triton X-100), so that the isolated plasmalemma vesicles appear tightly sealed and in a right-side-out orientation. Further characterization of the ATPase activities showed a pH optimum at 6.0 in the presence of Mg²⁺. This optimum was shifted to pH 5.8 after addition of K⁺. K⁺ stimulated the ATPase activity below pH 6 and inhibited above pH 6. The ATPase activity was specific for ATP and sensitive to N,N-dicyclohexylcarbodiimide and sodium vanadate, with K⁺ enhancing the vanadate inhibition. The enzyme was insensitive to sodium molybdate, NO⁻₃, azide and oligomycin. No Ca²⁺-ATPase was detected, and even as little as 0.05 m M Ca²⁺ inhibited the Mg²⁺-ATPase activity.     

Probable Role of Allylisothiocyanate-Sensitive H+, K+-ATPase in Spicule Calcification in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

In embryos of the sea urchin, Hemicentrotus pulcherrimus , as well as in cultured cells derived from isolated micromeres, spicule formation was inhibited by allylisothiocyanate, an inhibitor of H⁺, K⁺-ATPase, at above 0.5 μM and was almost completely blocked at above 10 μM. Amiloride, an inhibitor of Na⁺, H⁺ antiporter, at above 100 μM exerted only slight inhibitory effect, if any, on spicule formation. Intravesicular acidification, determined using [ dimethylamine -¹⁴C]-aminopyrine as a pH probe, was observed in the presence of ATP and 200 mM KCl in microsome fraction obtained from embryos at the post gastrula stage, at which embryos underwent spicule calcification. Intravesicular acidification and K⁺-dependent ATPase activity were almost completely inhibited by allylisothiocyanate at 10 μM. Allylisothiocyanate-sensitive ATPase activity was found mainly in the mesenchyme cells with spicules isolated from prisms. H⁺, K⁺-ATPase, an H⁺ pump, probably mediates H⁺ release to accelerate CaCO₃ deposition from Ca²⁺, CO₂ and H₂O in the primary mesenchyme cells. Intravesicular acidification was stimulated by valinomycin at the late gastrula and the prism stages but not at the pluteus stage. K⁺ permeability probably increases after the prism stage to activate H⁺ release.     

Dual effect of ADP on the activity of the plasma membrane H+‐ATPase of red beet

The effect of ADP on the activity of the plasma membrane (PM) H⁺‐ATPase of red beet ( Beta vulgaris L.) parenchyma discs was evaluated by analyzing the effect of increasing concentrations of ADP on the kinetics of the reaction. When the PM H⁺‐ATPase activity was assayed at pH 6.3, ADP behaved as a simple competitive inhibitor. When the activity was assayed at pH 7.1, ADP not only increased the apparent K_m for MgATP but also decreased the V_max of the reaction. When the C‐terminal domain of the PM H⁺‐ATPase was cleaved by controlled trypsin treatment or displaced by addition of lysophosphatidylcholine, only the competitive component of inhibition by ADP of the activity assayed at pH 7.1 was evident. The results are discussed in relation to the physiological relevance of the activation of the PM H⁺‐ATPase by displacement of the autoinhibitory C‐terminal domain.     

Effects of salt stress on H+- ATPase and H+-PPase activities of tonoplast-enriched vesicles isolated from sunflower roots

The control of ion concentration in the cytosol and the accumulation of ions in vacuoles are thought to be key factors in salt tolerance. These processes depend on the establishment in vacuolar membranes of an electrochemical H⁺ gradient generated by two distinct H⁺-translocating enzymes: a H⁺-PPase and a H⁺-ATPase. H⁺-lrans locating activities were characterized in tonoplast-enriched membrane fractions isolated by sucrose gradient centrifugation from sunflower ( Helianthus annuus L.) roots exposed for 3 days to different NaCl regimes. The 15/32% sucrose interface was enriched in membrane vesicles possessing a vacuolar-type H⁺-ATPase and a H⁺-PPase, as indicated by inhibitor sensitivity, pH optimum, substrate specificity, ion effects kinetic data and immunolabelling with specific antibodies. Mild and severe stress did not alter the pH profile, ion dependence, apparent K_m nor the amount of antigenic protein of either enzyme. Saline treatments slightly increased K⁺-stimulaied PPase activity with no change in ATPase activity, while both PP_i-dependent and NO₃-sensitive ATP-dependent H⁺ transport activities were strongly stimulated. These results are discussed in terms of an adaptative mechanism of the moderately tolerant sunflower plants to salt stress.     

Functional analysis of chimerical plasma membrane H+-ATPases from Saccharomyces cerevisiae and Schizosaccharomyces pombe

The plasma membrane H⁺-ATPase from the fission yeast Schizosaccharomyces pombe does not support growth of H⁺-ATPase-depleted cells of the budding yeast Saccharomyces cerevisiae , even after deletion of the enzyme's carboxy terminus. Functional chimerical H⁺-ATPase proteins in which appropriate regions of the S. pombe enzyme were replaced with their S. cerevisiae counterparts were generated by in vivo gene recombination. Site-directed mutagenesis of the H⁺-ATPase chimeras showed that a single amino acid replacement, tyrosine residue 596 by alanine, resulted in functional expression of the S. pombe H⁺-ATPase. The reverse Ala-598 →Tyr substitution was introduced into the S. cerevisiae enzyme to better understand the role of this alanine residue. However, no obvious effect on ATPase activity could be detected. The S. cerevisiae cells expressing the S. pombe H⁺-ATPase substituted with alanine were enlarged and grew more slowly than wild-type cells. ATPase activity showed a more alkaline pH optimum, lower K _m values for MgATP and decreased V _max compared with wild-type S. cerevisiae activity. None of these kinetic parameters was found to be modified in glucose-starved cells, indicating that the S. pombe H⁺-ATPase remained fully active. Interestingly, regulation of ATPase activity by glucose was restored to a chimera in which the S. cerevisiae sequence spans most of the catalytic site.     

Aquaporin functionality in relation to H+-ATPase activity in root cells of Capsicum annuum grown under salinity

As water and nutrient uptake should be related in the response of plants to salinity, the aim of this paper is to establish whether or not aquaporin functionality is related to H⁺-ATPase activity in root cells of pepper ( Capsicum annuum L.) plants. Thus, H⁺-ATPase activity was measured in plasma membrane vesicles isolated from roots and aquaporin functionality was measured using a cell pressure probe in intact roots. Salinity was applied as 60 m M NaCl or 60 m M KCl, to determine which ion (Na⁺, K⁺ or Cl⁻) is producing the effects. We also investigated whether the effects of both salts were ameliorated by Ca²⁺. Similar results were obtained for cell hydraulic conductivity, Lp_c, and H⁺-ATPase activity, large reductions in the presence at NaCl or KCl and an ameliorative effect of Ca²⁺. However, fusicoccin (an activator of H⁺-ATPase) did not alter osmotic water permeability of protoplasts isolated from roots. Addition of Hg²⁺ inhibited both ATPase and aquaporins, but ATPase also contains Hg-binding sites. Therefore, the results indicate that H⁺-ATPase and aquaporin activities may not be related in pepper plants.     

The cytochrome b6f complex. Novel aspects

The Cyt b ₆ f complex from plant chloroplasts, the green alga Chlamydomonas reinhardtii , and the thermophilic cyanobacterium, Mastigocladus laminosus , can be isolated in a highly active state, in which it is dimeric and contains one bound chlorophyll a molecule per monomeric unit. The latter feature is a distinguishing trait compared to the b ₆ f complex of bacterial photosynthesis and the respiratory chain. In contrast to the trans-membrane domains of the b ₆ f complex, and of most other integral membrane proteins, which are characterized by an a -helical structure, the p -side peripheral domains, consisting of Cyt f and the Rieske protein, have a predominantly β-strand secondary structure motif. One consequence of this motif is an extension of these polypeptides from the membrane surface. For example, the length of Cyt f is 75 Å. The heme Fe is 45 Å from the α-carbon of Arg250 at the membrane bilayer interface and, even though Cyt f may be tilted relative to the membrane plane, the heme electron transfer reactions are carried out far from the membrane surface. The presence of an internal 5 water chain, which has the properties of a proton wire, with one water H-bonded to the histidine-25 heme ligand, also suggests that the pathway of long distance H⁺ translocation traverses the extended p -side protein domain of the b ₆ f complex. A mechanism of H⁺ transfer in the chain that is coupled to the redox state of the heme, in which a proton is transferred into the chain to compensate the extra electron in the ferro-heme, is proposed.     

Substrate stabilization of lysophosphatidylcholine-solubilized plasma membrane H+-ATPase from oat roots

Plasma membrane vesicles with H⁺-ATPase activity were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots using an aqueous polymer two-phase system. Of several detergents tested, only lysophosphatidylcholine solubilized the H⁺-ATPase in an active form. Solubilization of the H⁺-ATPase with lysophosphatidylcholine was possible in the absence of glycerol, but the ATPase activity decreased about 4–5 times as rapidly in the absence as in the presence of 30% (w/v) glycerol. The solubilized enzyme was further stabilized by ATP and protons. Addition of 1 m M ATP to the plasma membranes halted inactivation of the H⁺-ATPase. Even in the absence of polyol compounds and ATP, the enzyme was stable for hours at relatively low pH with an optimum around pH 6.7 at room temperature. The curve for the stability of soluble H⁺-ATPase as a function of pH closely resembles the pH curve for the activity of the H⁺-ATPase. This suggests that binding of protons to transport sites may stabilize the soluble H⁺-ATPase in an enzymatically active form.     

Purification of plasma membranes from dry maize embryos

Isolation of subcellular fractions from dry structures such as seeds or their tissues is difficult. In the present work, plasma membranes were isolated from dry maize ( Zea mays L.) embryos with an enrichment of 11-fold as estimated by glucan synthase II (GSII, EC 2.4.1.34) activity and a purity of 78 to 90% as judged by the sensitivity of ATP hydrolysis to vanadate, a specific inhibitor of the plasma membrane H⁺-ATPase (EC 3.6.1.35). The procedure involved a double homogenization of the dry embryos and the addition of a 1500- g supernatant to an aqueous polyethyleneglycol-dextran two-phase partitioning system; the optimal ratio of polyethyleneglycol-dextran for purification of plasma membranes from dry seeds was 6.8/6.8% (w/w). In the isolated membranes a trace of a tonoplast enzyme marker (tonoplast H⁺-ATPase, EC 3.6.1.3) could be detected, but there were negligible amounts of mitochondrial and rough endoplasmic reticulum markers, H⁺-ATPase (EC 3.6.1.34) and diacylglycerol acyltrans-ferase (EC 2.3.1.20), respectively. The technique could also be used in hydrated embryos. The entire procedure can be carried out in 5 to 6 h. The resulting preparation is stable for at least 2 months at −70°C. The membranes of dry and hydrated embryos exhibited a high level of vanadate-sensitive ATPase activity that was increased by lysophosphatidylcholine.     

ATP-dependent H+-pumping of a low-density fraction of pea stem microsomes

A low-density fraction of pea ( Pisum sativum L. cv. Alaska) stem microsomes, obtained from a discontinuous sucrose gradient, possessed an H⁺-ATPase able to generate a proton gradient and an electrical potential. The proton pumping was insensitive to monovalent cations, to vanadate and oligomycin, required a permeant anion and was inhibited by nitrate, N, N'-dicyclohexylcarbodiimide and diethylstilbestrol. The H⁺-ATPase had a pH optimum around 6.0–6.5 and was saturable with respect to the substrate Tris-ATP (K_m≅ 0.4 m M ). Ca²⁺ (0.05–1 m M ) induced a dissipation of the ATP-generated δpH without affecting ATPase activity. At physiological concentrations (1–5 m M ), nitrate caused an initial slight increase of the ATP-generated proton gradient followed by a complete dissipation after 2–3 min. The dissipating effect was not caused by inhibition of ATPase activity, since ATP prevented the nitrate-induced collapse of δpH. On the other hand, ATPase activity, evaluated as release of P_i, was not inhibited by concentrations lower than 20 m M KNO₃. These results indicate that nitrate entered the vesicles in response to an electrical potential and then could exit in symport with protons, while Ca²⁺ entered in exchange for protons (antiport).