Showing papers on "Vanadate published in 1986"

The insulin-like effect of sodium vanadate on adipocyte glucose transport is mediated at a post-insulin-receptor level.

Abstract: Sodium vanadate has several insulin-like effects. To determine whether vanadate acts via the insulin receptor, I investigated the effect of vanadate on glucose transport (2-deoxyglucose uptake) in adipocytes that had been treated to decrease the number of insulin receptors. Trypsin (100 micrograms/ml) caused greater than 95% loss of 125I-insulin binding and rendered glucose transport resistant to both insulin and an anti-insulin-receptor antibody. However, vanadate caused an 8-fold increase in the transport rate [EC50 (concn. giving 50% of maximum effect) 0.2 mM] in both control and trypsin-treated cells, demonstrating that the insulin receptor does not have to be intact for vanadate to stimulate glucose transport. Insulin receptors were depleted by treatment of adipocytes with insulin (100 ng/ml) in the presence of Tris (which blocks receptor recycling). A 2 h treatment caused 60% loss of receptors, and a shift to the right in the dose-response curve for insulin stimulation of glucose transport (EC50 0.3 ng of insulin/ml in controls, 1.2 ng/ml in treated cells). The response to vanadate was again unaffected. Treatment with insulin for 4 h caused a 67% decrease in insulin binding and, in addition to the rightward shift in the insulin dose-response curve, a decrease in basal and maximal transport rates (which cannot be explained by decreased insulin receptor number). The EC50 of vanadate was again equal in control and treated cells, but glucose transport in the presence of a maximally effective concentration of vanadate (1 mM) was decreased. I conclude that the effect of vanadate on glucose transport is independent of the insulin receptor. Induction of a post-receptor defect (which may be a decrease in the total number of cellular glucose transporters) by prolonged exposure to insulin decreases the potency of a maximally effective concentration of vanadate. The findings demonstrate that vanadate stimulates glucose transport by an effect at a level distal to the insulin receptor.

H-pumping driven by the vanadate-sensitive ATPase in membrane vesicles from corn roots.

Abstract: The initial rate of quenching of quinacrine fluorescence was used to monitor Mg:ATP-dependent H+-pumping in membrane vesicles from corn (Zea mays L. cv WF9 × MO17) roots and obtain a preparation in which vanadate-sensitive H+-pumping could be observed. Separation of membranes on a linear sucrose density gradient resulted in two distinct peaks of H+-pumping activity: a major one, at density 1.11 grams per cubic centimeter, was sensitive to NO3− and resistant to vanadate, while a minor one, at density 1.17 grams per cubic centimeter, was substantially resistant to NO3− and sensitive to vanadate. A membrane fraction enriched in the vanadate-sensitive H+-pump could be obtained by washing microsomes prepared in the presence of 10% glycerol with 0.25 molar KI. The kinetics of inhibition of H+-pumping by vanadate in this membrane preparation indicated that most of the H+-pumping activity in this fraction is sensitive to inhibition by vanadate, 50% inhibition being reached at about 60 micromolar vanadate. This value is fairly close to that observed for inhibition by vanadate of the ATPase activity in similar experimental conditions (40 micromolar). The inhibitor sensitivity, divalent cation dependence, pH optimum (6.5), and Km for ATP (0.7 millimolar) of the H+-pumping activity match quite closely those reported for the plasma membrane ATPase of corn roots and other plant materials.

Electrogenic h-pumping pyrophosphatase in tonoplast vesicles of oat roots.

Abstract: A H+-translocating inorganic pyrophosphatase (H+-PPase) was associated with low density membranes enriched in tonoplast vesicles of oat roots. The H+-PPase catalyzed the electrogenic transport of H+ into the vesicles, generating a pH gradient, inside acid (quinacrine fluorescence quenching), and a membrane potential, inside positive (Oxonol V fluorescence quenching). Transport activity was dependent on cations with a selectivity sequence of Rb+ = K+ > Cs+; but it was inhibited by Na+ or Li+. Maximum rates of transport required at least 20 millimolar K+ and the Km for this ion was 4 millimolar. Fluoride inhibited both ΔpH formation and K+-dependent PPase activity with an I50 of 1 to 2 millimolar. Inhibitors of the anion-sensitive, tonoplast-type H+-ATPase (e.g. a disulfonic stilbene or NO3−) had no effect on the PPase activity. Vanadate and azide were also ineffective. H+-pumping PPase was inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide, but its sensitivity to N,N′-dicyclohexylcarbodiimide was variable. The sensitivity to ions and inhibitors suggests that the tonoplast H+-PPase and the H+-ATPase are distinct activities and this was confirmed when they were physically separated after Triton X-100 solubilization and Sepharose CL-6B chromatography. H+ pumping activity was strongly affected by Mg2+ and pyrophosphate (PPi) concentrations. At 5 millimolar Mg2+, H+ pumping showed a KmaPP for PPi of 15 micromolar. The rate of H+ pumping at 60 micromolar PPi was often equivalent to that at 1.5 millimolar ATP. The results suggest PPi hydrolysis could provide another source of a proton motive force used for solute transport and other energy-requiring processes across the tonoplast and other membranes with H+-PPase.

Relationship of Transplasmalemma Redox Activity to Proton and Solute Transport by Roots of Zea mays

Abstract: Transplasmalemma redox activity, monitored in the presence of exogenous ferricyanide stimulates net H+ excretion and inhibits the uptake of K+ and α-aminoisobutyric acid by freshly cut or washed, apical and subapical root segments of corn (Zea mays L. cv “Seneca Chief”). H+ excretion is seen only following a lag of about 5 minutes after ferricyanide addition, even though the reduction of ferricyanide occurs before 5 minutes and continues linearly. Once detected, the enhanced rate of H+ excretion is retarded by the ATPase inhibitors N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and vanadate. A model is presented in which plasmalemma redox activity in the presence of ferricyanide involves the transport only of electrons across the plasmalemma, resulting in a depolarization of the membrane potential and activation of an H+-ATPase. Such a model implies that this class of redox activity does not provide an additional and independent pathway for H+ transport, but that the activity may be an important regulator of H+ excretion. The 90% inhibition of K+ (86Rb+) uptake within 2 minutes after ferricyanide addition can be contrasted with the 5 to 15% inhibition of uptake of α-aminoisobutyric acid. The possibility exists that a portion of the K+ and most of the α-aminoisobutyric acid uptake inhibitions are related to the ferricyanide-induced depolarization of the membrane potential, but that the redox state of some component of the K+ uptake system may also regulate K+ fluxes.

Interaction of vanadate with phenol and tyrosine: implications for the effects of vanadate on systems regulated by tyrosine phosphorylation

Abstract: The interaction of vanadate with phenol and N-acetyltyrosine ethyl ester in aqueous solution has been studied by using 51V nuclear magnetic resonance spectroscopy. On the basis of these studies, it has been concluded that vanadate rapidly esterifies the hydroxyl group of the aromatic ring to yield a phenyl vanadate. For phenol, the equilibrium constant for this reaction in terms of the convention that the activity of liquid water is 1.0 is K1 = [phenyl vanadate]/[phenol][vanadate] = 0.97 +/- 0.02. This value is well over 4 orders of magnitude larger than estimates from the literature for the corresponding equilibrium constant for the esterification of phenol by phosphate. The equilibrium constant for esterification of the phenol moiety of N-acetyltyrosine ethyl ester is similar to that for esterification of phenol. The relevance of these observations to processes that are regulated by reversible phosphorylation/dephosphorylation of tyrosine residues is discussed, in particular the insulin-like effect of vanadate.

Calcium transport in tonoplast and endoplasmic reticulum vesicles isolated from cultured carrot cells.

Abstract: Two active calcium (Ca2+) transport systems have been identified and partially characterized in membrane vesicles isolated from cultured carrot cells (Daucus carota Danvers). Both transport systems required MgATP for activity and were enhanced by 10 millimolar oxalate. Ca2+ transport in membrane vesicles derived from isolated vacuoles equilibrated at 1.10 grams per cubic centimeter and comigrated with Cl−-stimulated, NO3−-inhibited ATPase activity on sucrose density gradients. Ca2+ transport in this system was insensitive to vanadate, but was inhibited by nitrate, carbonyl cyanide-m-chlorophenylhydrazone (CCCP), N,N′-dicyclohexylcarbodiimide (DCCD), and 4,4-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS). The Km for MgATP and Ca2+ were 0.1 mm and 21 micromolar, respectively. The predominant Ca2+ transport system detectable in microsomal membrane preparations equilibrated at a density of 1.13 grams per cubic centimeter and comigrated with the endoplasmic reticulum (ER) marker, antimycin A-insensitive NADH-dependent cytochrome c reductase. Ca2+ transport activity and the ER marker also shifted in parallel in ER shifting experiments. This transport system was inhibited by vanadate (I50 = 12 micromolar) and was insensitive to nitrate, CCCP, DCCD, and DIDS. Transport exhibited cooperative MgATP dependent kinetics. Ca2+ dependent kinetics were complex with an apparent Km ranging from 0.7 to 2 micromolar. We conclude that the vacuolar-derived system is a Ca2+/H+ antiport located on the tonoplast and that the microsomal transport system is a Ca,Mg-ATPase enriched on the ER. These two Ca2+ transport systems are proposed to restore and maintain cytoplasmic Ca2+ homeostasis under changing cellular and environmental conditions.

Calcium transport in tonoplast and endoplasmic reticulum vesicles isolated from cultured carrot cells. [Daucus carota Danvers]

Abstract: Two active calcium (Ca/sup 2 +/) transport systems have been identified and partially characterized in membrane vesicles isolated from cultured carrot cells (Daucus carota Danvers) Both transport systems required MgATP for activity and were enhanced by 10 millimolar oxalate Ca/sup 2 +/ transport in membrane vesicles derived from isolated vacuoles equilibrated at 110 grams per cubic centimeter and comigrated with Cl/sup -/-stimulated, NO/sub 3//sup -/-inhibited ATPase activity on sucrose density gradients Ca/sup 2 +/ transport in this system was insensitive to vanadate, but was inhibited by nitrate, carbonyl cyanide-m-chlorophenylhydrazone (CCCP), N,N'-dicyclohexylcarbodiimide (DCCD), and 4,4-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) The K/sub m/ for MgATP and Ca/sup 2 +/ were 01 mM and 21 micromolar, respectively The predominant Ca/sup 2 +/ transport system detectable in microsomal membrane preparations equilibrated at a density of 113 grams per cubic centimeter and comigrated with the endoplasmic reticulum (ER) marker, antimycin A-insensitive NADH-dependent cytochrome c reductase Ca/sup 2 +/ transport activity and the ER marker also shifted in parallel in ER shifting experiments This transport system was inhibited by vanadate (I/sub 50/ = 12 micromolar) and was insensitive to nitrate, CCCP, DCCD, and DIDS Transport exhibited cooperative MgATP dependent kinetics Ca/sup 2 +/ dependent kinetics were complexmore » with an apparent K/sub m/ ranging from 07 to 2 micromolar We conclude that the vacuolar-derived system is a Ca/sup 2 +//H/sup +/ antiport located on the tonoplast and that the microsomal transport system is a Ca,Mg-ATPase enriched on the ER These two Ca/sup 2 +/ transport systems are proposed to restore and maintain cytoplasmic Ca/sup 2 +/ homeostasis under changing cellular and environmental conditions« less

Vanadium(V) oxyanions: the interaction of vanadate with pyrophosphate, phosphate, and arsenate

Abstract: Interactions etudiees par spectroscopie RMN. Formation d'anhydrides mixtes avec le vanadate analogues a pyrophosphate et triphosphate

Inhibition of Potassium Uptake and Regulation of Membrane-Associated Mg2+-ATPase Activity of Pea Roots by Aluminium

Abstract: The uptake of K+ by pea decreased markedly by the treatment with AICl3. The decrease was alleviated by the co-treatment with Ca2+. The membrane associated with ATPase which was isolated from pea roots, was considered to be the plasma membrane enriched by the natures of associated ATPase. The activity was highly dependent on the presence of Mg2+, but not Ca2+ and the optimum activity was observed at neutral pH. The activity was inhibited by diethylstilbesterol, N,N’ -dicyclohexyl carbodiimide. and vanadate. Rather specific requirement of ATP was observed when ATP acted as a substrate. The membrane-associated ATPase was competitively inhibited by AICl3 added to the assay medium with respect to ATP. The rate of inhibition by Al at various pHs was parallel with that of the activity in the absence of AI. Various chemicals were tested for the alleviation of the inhibition of membrane-associated ATPase by AI. Tripolyphosphate, citric acid, glucose 6-phosphate, pyrophosphate, glutamic acid, and malic acid restore...

Shape control in the human red cell

Abstract: When the human red cell consumes its ATP, the cell loses its discoid character in favour of a spiculated and eventually a spherical form. This discocyte-echinocyte transformation parallels both degradation of phosphatidylinositol 4,5-bisphosphate and phosphatidic acid but not dephosphorylation of cytoskeletal proteins. Dephosphorylation of both spectrin and band 3 lags behind metabolic crenation. Exogenous vanadate accelerates both shape changes and lipid dephosphorylation in a parallel manner during metabolic depletion. In contrast to its effect on lipids, vanadate reduces the rate of protein dephosphorylation. These observations strongly support a shape control mechanism in the red cell, based on phosphoinositide metabolism and compatible with a bilayer-couple model.

myo-Inositol 1,4,5-trisphosphate mobilizes Ca2+ from isolated adipocyte endoplasmic reticulum but not from plasma membranes

Abstract: The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.

Studies on H-Translocating ATPases in Plants of Varying Resistance to Salinity : II. K Strongly Promotes Development of Membrane Potential in Vesicles from Cotton Roots.

Abstract: Mg2+-ATP-dependent H+-translocation has been studied in membrane vesicles derived from the roots of Gossypium hirsutum L. var. Acala San Jose 2. Establishment of a positive membrane potential was followed by measuring SCN− accumulation; establishment of ΔpH across the vesicle membranes by measuring quinacrine fluorescence quenching. High specificity for ATP was shown, and H+-translocation was oligomycin stable. The pH profile for H+-translocation showed an optimum at 5.5. The relationship between SCN− accumulation and ATP concentration was approximately Michaelian; the apparent Km was 0.7 millimolar. K-2-(N-morpholino)ethanesulfonic acid strongly promoted ATP-dependent SCN− uptake (up to 180% stimulation). The effect was not given by Na-Mes. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone totally inhibited SCN− accumulation, both in the presence and absence of K-2(N-morpholino)ethanesulfonic acid. Vanadate at 200 micromolar inhibited SCN− uptake by about 10 to 40% in the absence of K+, but more strongly in its presence (about 60%). NO3− at 100 millimolar inhibited initial rate of quinacrine quenching by about 25%. The NO3− insensitive fraction was activated by K+; and inhibited by 200 micromolar vanadate to about 40%, provided K+ was present. Saline conditions during the growth of the plants had no appreciable effect on the observed characteristics of H+-translocation.

Fluoride and beryllium interact with the (Na + K)-dependent ATPase as analogs of phosphate

Abstract: Fluoride irreversibly inhibits the (Na + K)-ATPase, and this inactivation requires divalent cations (Mg2+, Mn2+, or Ca2+), is augmented by K+, but is diminished by Na+ and by ATP. Prior incubation with the aluminum chelator deferoxamine markedly slows inactivation, whereas adding 1 µM AlCl3 speeds it, consistent with AlF − 4 being the active species. Prior incubation of the enzyme with vanadate also blocks inactivation by fluoride added subsequently. Fluoride stimulates ouabain binding to the enzyme, and thus the analogy between AlF − 4 and both orthophosphate and orthovanadate is reflected not only in the similar dependence on specific ligands for their enzyme interactions and their apparent competition for the same sites, but also in their common ability to promote ouabain binding. Beryllium also irreversibly inhibits the enzyme, and this inactivation again requires divalent cations, is augmented by K+, but is diminished by Na+ and by ATP. Similarly, prior incubation of the enzyme with vanadate blocks inactivation by beryllium added subsequently. Inactivation by beryllium, however, does not require a halide, and, unlike inactivation by fluoride, increases at basic pHs. These observations suggest that beryllium, as beryllium hydroxide complexes, acts as a phosphate analog, similar to AlF − 4 and vanadate.

Characterization of detergent-solubilized sarcoplasmic reticulum Ca2+-ATPase by high-performance liquid chromatography.

Abstract: Sarcoplasmic reticulum Ca/sup 2 +/-ATPase solubilized by the nonionic detergent octaethylene glycol monododecyl ether was studied by molecular sieve high-performance liquid chromatography (HPLC) and analytical ultracentrifugation. Significant irreversible aggregation of soluble Ca/sup 2 +/-ATPase occurred within a few hours in the presence of less than or equal to 50 ..mu..M Ca/sup 2 +/. The aggregates were inactive and were primarily held together by hydrophobic forces. In the absence of reducing agent, secondary formation of disulfide bonds occurred. The stability of the inactive dimer upon dilution permitted unambiguous assignment of its elution position and sedimentation coefficient. At high /sup 45/Ca/sup 2 +/ concentration (500 ..mu..M), monomeric Ca/sup 2 +/-ATPase was stable for several house. Reversible self-association induced by variation in protein, detergent, and lipid concentrations was studied by large-zone HPLC. The association constant for dimerization of active Ca/sup 2 +/-ATPase was found to be 10/sup 5/-10/sup 6/ M/sup -1/ depending on the detergent concentration. More detergent was bound to monomeric than to dimeric Ca/sup 2 +/-ATPase, even above the critical micellar concentration of the detergent. Binding of Ca/sup 2 +/ and /sup 48/V vanadate as well as ATP-dependent phosphorylation was studied in monomeric and in reversibly associated dimeric preparations. In bothmore » forms, two high-affinity Ca/sup 2 +/ binding sites per phosphorylation site existed. The delipidated monomer purified by HPLC was able to form ADP-insensitive phosphoenzyme and to bind ATP and vanadate simultaneously. The results suggest that formation of Ca/sup 2 +/-ATPase oligomers in the membrane is governed by nonspecific forces (low affinity) and that each polypeptide chain constitutes a functional unit.« less

Ultracytochemical evidence for a proton-pump adenosine triphosphatase in chick osteoclasts.

Abstract: The distribution of Mg+ +-ATPase in osteoclasts along the endosteal surface of the chick tibia was investigated by neutral and alkaline pH cytochemical methods at the electron-microscopic level. Reaction product was observed in mitochondria, cytoplasmic vesicles, and ruffled-border membrane. Levamisole, ouabain, and vanadate did not affect the enzymatic activity. Para-chloromercuribenzoic acid (PCMB) prevented staining of mitochondria, ruffled border, and most cytoplasmic vesicles. Tri-n-butyltin decreased the amount of reaction product in cytoplasmic vesicles and ruffled-border membrane, but did not inhibit reaction product formation within mitochondria. Duramycin, which is a potent inhibitor for proton-pump ATPase, blocked reaction-product formation along the ruffled-border membrane, in mitochondria, and in cytoplasmic vesicles at alkaline pH, but not at neutral pH. It is concluded that the alkaline pH method for Mg+ +-ATPase appears to demonstrate sites of proton-pump ATPase activity.

Characterization of nucleoside triphosphatase activity in isolated pea nuclei and its photoreversible regulation by light.

Abstract: A nucleoside triphosphatase (NTPase) present in highly purified preparations of pea nuclei was partially characterized. The activity of this enzyme was stimulated by divalent cations (Mg2+ = Mn2+ > Ca2+), but was not affected by the monovalent cations, Na+ and K+. The Mg(2+)-dependent activity was further stimulated by concentrations of Ca2+ in the low micromolar range. It could catalyze the hydrolysis of ATP, GTP, UTP, and CTP, all with a pH optimum of 7.5. The nuclear NTPase activity was not inhibited by vanadate, oligomycin, or nitrate, but was inhibited by relatively low concentrations of quercetin and the calmodulin inhibitor, compound 48/80. The NTPase was stimulated more than 50% by red light, and this effect was reversed by subsequent irradiation with far-red light. The photoreversibility of the stimulation indicated that the photoreceptor for this response was phytochrome, an important regulator of photomorphogenesis and gene expression in plants.

Characteristics of the proton pump in rat renal cortical endocytotic vesicles.

Abstract: The characteristics of the H+ pump in isolated rat renal endocytotic vesicles were studied by the delta pH-sensitive dye acridine orange, the voltage-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide, and by a coupled optical ATPase assay. Intravesicular acidification depended on ATP and Mg2+ concentrations with half-maximal activations at 73 and 77 microM, respectively. CTP, GTP, UTP, and ITP partially supported acidification, but ADP and AMP did not. Ouabain, ethoxzolamide, levamisole, and vanadate did not inhibit H+ uptake into endocytotic vesicles. Oligomycin inhibited partially. Depending on concentration and preincubation time, Dio-9, filipin, N-ethylmaleimide (NEM), and dicyclohexylcarbodiimide (DCCD) inhibited H+ uptake completely. Filipin and, partially, DCCD acted nonspecifically by dissipating pH gradients. A specific cation was not required for the H+ pump; Zn2+ inhibited. Compared with mannitol, ATP-driven H+ uptake was stimulated by SCN- greater than Cl- greater than Br- greater than I- much greater than HPO4(2-) = gluconate = HCO3- = F-, but not by SO4(2-), NO3-, CH3COO-, S2O3(2-), and S4O6(2-). Chloride stimulated H+ uptake from the outside of the vesicles with an apparent Km of 27 mM. In the absence of Cl-, ATP-driven proton uptake was increased by intravesicular K+ and valinomycin, suggesting that the pump is electrogenic. The electrogenicity, however, could not be demonstrated with voltage-sensitive dyes. The vesicle membrane contains no significant K+ and Cl- conductances; only a conductance for H+ was found. The vesicles exhibited an ouabain-, oligomycin-, and vanadate-insensitive ATPase activity that was inhibited by DCCD and NEM. Our data indicate the presence of an electrogenic H+ pump in endocytotic vesicles from rat renal proximal tubules with similar characteristics as H+ pumps present in various intracellular (nonmitochondrial) membranes.

Effects of vanadate on the rotational dynamics of spin-labeled calcium adenosinetriphosphatase in sarcoplasmic reticulum membranes.

Abstract: We have studied the effects of vanadate on the rotational motion of the calcium adenosine- triphosphatase (Ca-ATPase) from sarcoplasmic reticulum (SR), using saturation-transfer electron para- magnetic resonance (ST-EPR). Vanadate has been proposed to act as a phosphate analogue and produce a stable intermediate state similar to the phosphoenzyme. This study provides evidence about the physical state of this intermediate. In particular, since ST-EPR provides a sensitive measure of microsecond protein rotational mobility, and hence of protein-protein association, these studies allowed us to ask (a) whether the vanadate-induced protein association observed in electron micrographs of SR vesicles also occurs under physiological (as opposed to fixed, stained, or frozen) conditions and (b) whether vanadate-induced changes in protein association also occur under conditions sufficient for enzyme inhibition but not for the production of large arrays detectable by electron microscopy (EM). At 5 mM decavanadate, a concentration sufficient to crystallize the ATPase on greater than 90% of the membrane surface area in EM, ST-EPR showed substantial immobilization of the spinllabeled protein, indicating protein-protein association in the unstained vesicles. Conventional EPR spectra of lipid probes showed that lipid hydrocarbon chain mobility is unaffected by decavanadate-induced protein crystallization in SR, suggesting that changes in protein-protein contacts do not involve the lipid hydrocarbon region. At 5 mM monovanadate, a concentration sufficient to inhibit the ATPase but not to form crystals detectable by EM, no changes were observed in ST-EPR or conventional EPR spectra of either protein or lipid. In summary, these results indicate that decavanadate induces extensive self-association of the Ca-ATPase but that monovanadate inhibits without a change in protein mobility, thus indicating no change in oligomeric state. If monovanadate produces an analogue of a phosphoenzyme state that is important in the Ca-ATPase cycle, this phosphoenzyme has the same oligomeric state as the unphosphorylated enzyme. Alternatively, if decavanadate produces a phosphoenzyme analogue, the present results suggest that protein association is increased in the phosphoenzyme state.