High Concentrations Of Vanadate Research Articles

The interactions of vanadium with Phycomyces blakesleeanus mycelium: enzymatic reduction, transport and metabolic effects

The biological and chemical basis of vanadium action and transport in fungi is relatively poorly understood. In this study we investigated the interactions of vanadium in physiologically-relevant redox states: vanadate (+5) and vanadyl (+4), with mycelium of fungus Phycomyces blakesleeanus using EPR and 31P NMR spectroscopy and biochemical assays. We determined that P. blakesleeanus reduces V5+ to V4+ in the extracellular compartment by the means of cell surface enzyme with ferricyanide reductase activity, which contains molybdenum–molybdopterin as a cofactor. Both, V5+ and V4+ bind to cell wall. They enter the cytoplasm via phosphate transporter and cation channels, respectively, and exhibit different metabolic effects. Vanadate provokes increased biomass production, the effects being inverted to toxic at higher V5+ concentrations. In addition, V5+ activates the synthesis of sugar phosphates and oligophosphates. On the other hand, V4+ exhibits toxic effects even at low concentrations. The V4+ detoxification route involves binding to vacuolar polyphosphates. Altogether our results imply that the mechanism of interaction of vanadium with P. blakesleeanus involves three major steps: extracellular enzymatic V5+/V4+ reduction, V4+ influx, and vacuolar storage, with an additional step – V5+ import occurring at higher vanadate concentrations.

Early signalling pathways in rice roots under vanadate stress

Vanadate is beneficial to plant growth at low concentration. However, plant exposure to high concentrations of vanadate has been shown to arrest cell growth and lead to cell death. We are interested in understanding the signalling pathways of rice roots in response to vanadate stress. In this study, we demonstrated that vanadate induced rice root cell death and suppressed root growth. In addition, we found that vanadate induced ROS accumulation, increased lipid peroxidation and elicited a remarkable increase of MAPKs and CDPKs activities in rice roots. In contrast, pre-treatment of rice roots with ROS scavenger (sodium benzoate), serine/threonine protein phosphatase inhibitor (endothall), and CDPK antagonist (W7), reduced the vanadate-induced MAPKs activation. Furthermore, the expression of a MAPK gene (OsMPK3) and four tyrosine phosphatase genes (OsDSP3, OsDSP5, OsDSP6, and OsDSP10) were regulated by vanadate in rice roots. Collectively, these results strongly suggest that ROS, protein phosphatase, and CDPK may function in the vanadate-triggered MAPK signalling pathway cause cell death and retarded growth in rice roots.

Vacuolar Acidification in Citrus Fruit: Comparison between Acid Lime (Citrus aurantifolia) and Sweet Lime (Citrus limmetioides) Juice Cells

Vacuolar acidification was investigated in `Palestine' sweet (Citrus limmetioides Tanaka) and `Persian' acid lime [(Citrus aurantifolia (Christm.) Swingle] (vacuolar pHs of 5.0 and 2.1, respectively) using tonoplast vesicles isolated from juice cells. The ATPase activity of tonoplast-enriched vesicles from sweet limes was strongly inhibited by bafilomycin A1 and NO3-, but was unaffected by vanadate. In contrast, the ATPase activity in acid lime membranes was only slightly inhibited by bafilomycin A1 and NO3- and was strongly inhibited by high concentrations of vanadate. The vacuolar origin of the acid lime vesicles was confirmed by immunoblotting. After solubilization and partial purification of the two enzymes by gel filtration, their inhibitor profiles were largely unchanged. Based on equal ATPase activities, vesicles from sweet and acid limes were able to generate similar pH gradients. However, in tonoplast vesicles from sweet limes, the maximum ΔpH was reached four times faster than in those from acid limes. Addition of ethylenediamine tetraacetic acid (EDTA) to chelate Mg+2 after the maximal ΔpH was attained resulted in collapse of the pH gradient in vesicles from sweet limes, whereas no change in ΔpH was observed in vesicles from acid limes, indicating a less H+ permeable membrane. Vacuolar ATPases from both cultivars exhibited identical pH optima and showed similar Mg+2 dependence, but only the acid lime ATPase activity was inhibited by Ca+2. These data confirm that the vanadate-sensitive form of the V-ATPase found in lemon and acid limes is specific to hyperacidifying tissues rather than to citrus juice cells. Sweet lime vacuoles bear the classical V-ATPase also found in vegetative plant tissues.

Dynein-ADP as a Force-Generating Intermediate Revealed by a Rapid Reactivation of Flagellar Axoneme

Fragmented flagellar axonemes of sand dollar spermatozoa were reactivated by rapid photolysis of caged ATP. After a time lag of 10 ms, axonemes treated with protease started sliding disintegration. Axonemes without protease digestion started nanometer-scale high-frequency oscillation after a similar time lag. Force development in the sliding disintegration was measured with a flexible glass needle and its time course was corresponded well to that of the dynein-ADP intermediate production estimated using kinetic rates previously reported. However, with a high concentration (∼80 μM) of vanadate, which binds to the dynein-ADP intermediate and forms a stable complex of dynein-ADP-vanadate, the time course of force development in sliding disintegration was not affected at all. In the case of high frequency oscillation, the time lag to start the oscillation, the initial amplitude, and the initial frequency were not affected by vanadate, though the oscillation once started was damped more quickly at higher concentrations of vanadate. These results suggest that during the initial turnover of ATP hydrolysis, force generation of dynein is not blocked by vanadate. A vanadate-insensitive dynein-ADP is postulated as a force-generating intermediate.

Vanadate oxidation activates contraction in skinned smooth muscle without myosin light chain phosphorylation.

Phosphorylation of the myosin regulatory light chain (LC20-P1) is the major route of smooth muscle activation. However, after prior exposure to vanadate, permeabilized guinea pig taenia coli smooth muscle contracts in the absence of LC20-P1. We characterized the vanadate-induced contraction and investigated the mechanism of this novel activation pathway. Addition of vanadate to a control contracture (6.6 microM Ca2+) inhibits force (effective dose for 50% response was approximately 100 microM). In contrast, preincubation with high concentrations of vanadate (threshold at 1-2 mM) elicited a contraction on subsequent transfer of the fiber to a vanadate-free, Ca(2+)-free solution. Maximum isometric force of approximately 60% of control was obtained in fibers preincubated in 4 mM vanadate for 10 min. Addition of Ca2+ to a vanadate-induced contracture increased force, but the total force never exceeded the initial control. After maximal thiophosphorylation of LC20 with adenosine 5'-O-(3-thiotriphosphate), treatment with vanadate did not increase force. Unloaded shortening velocity (Vmax) was similar in Ca2+ and vanadate contractures and was additive. After thiophosphorylation, preincubation in vanadate had no effect on Vmax, suggesting that vanadate affected the number of activated bridges and not cycle rate. Vanadate mechanisms likely involve oxidation, since preincubation with 4 mM vanadate and 25 mM dithiothreitol (DTT) did not produce force. DTT could reverse a vanadate-induced contracture in 30-60 min. Subsequently, fibers demonstrated control contraction/relaxation cycles. Thus vanadate treatment did not cause irreversible damage, such as the extraction of proteins. Potential oxidation sites are proteins at 17 kDa and between 30 and 40 kDa, which were not alkylated by N-ethylmaleimide if they were treated in the presence of vanadate or in the rigor state. Vanadate-induced contractures are likely mediated by a reversible oxidation that activates cross bridges similarly to that of LC20-Pi and may play an important role in oxidant injury.

Lack of Conventional ATPase Properties in CFTR Chloride Channel Gating

CFTR shares structural homology with the ABC transporter superfamily of proteins which hydrolyze ATP to effect the transport of compounds across cell membranes. Some superfamily members are characterized as P-type ATPases because ATP-dependent transport is sensitive to the presence of vanadate. It has been widely postulated that CFTR hydrolyzes ATP to gate its chloride channel. However, direct evidence of CFTR hydrolytic activity in channel gating is lacking and existing circumstantial evidence is contradictory. Therefore, we evaluated CFTR chloride channel activity under conditions known to inhibit the activity of ATPases; i.e., in the absence of divalent cations and in the presence of a variety of ATPase inhibitors. Removal of the cytosolic cofactor, Mg2+, reduced both the opening and closing rates of CFTR suggesting that Mg2+ plays a modulatory role in channel gating. However, channels continued to both open and close showing that Mg2+ is not an absolute requirement for channel activity. The nonselective P-type ATPase inhibitor, vanadate, did not alter the gating of CFTR when used at concentrations which completely inhibit the activity of other ABC transporters (1 mM). Higher concentrations of vanadate (10 mM) blocked the closing of CFTR, but did not affect the opening of the channel. As expected, more selective P-type (Sch28080, ouabain), V-type (bafilomycin A1, SCN-) and F-type (oligomycin) ATPase inhibitors did not affect either the opening or closing of CFTR. Thus, CFTR does not share a pharmacological inhibition profile with other ATPases and channel gating occurs in the apparent absence of hydrolysis, although with altered kinetics. Vanadate inhibition of channel closure might suggest that a hydrolytic step is involved although the requirement for a high concentration raises the possibility of previously uncharacterized effects of this compound. Most conservatively, the requirement for high concentrations of vanadate demonstrates that the binding site for this transition state analogue is considerably different than that of other ABC transporters.

Copper and Silver Transport by CopB-ATPase in Membrane Vesicles of Enterococcus hirae

The P-type ATPase, CopB, of Enterococcus hirae is required for the copper resistance displayed by this organism and thus was postulated to be a copper pump. Using 64Cu+ and 110mAg+, we here show ATP-driven copper and silver accumulation catalyzed by CopB in native inside-out membrane vesicles of E. hirae. CopB ATPase exhibited an apparent Km for Cu+ and Ag+ of 1 microM and for ATP of 10 microM. Transport was maximal at pH 6 and had an apparent Vmax of 0.07 nmol.min-1.mg-1 for both copper and silver transport. Vanadate displayed a biphasic effect on transport: maximal inhibition was observed at 40 microM vanadate for copper transport and 60 microM for silver transport, respectively. At higher vanadate concentrations, these inhibitions were reversed. The CopB ATPase of E. hirae is thus a pump for the extrusion of monovalent copper and silver ions, with copper probably being the natural substrate.

Vanadate increases cytosolic free calcium in rat aortic smooth muscle cells

Although several studies have shown that vanadate evokes vasoconstriction whether it elevates cytosolic free calcium, [Ca2+]i, in vascular smooth muscle (VSM) cells has not been investigated. The present study shows that acute additions of low concentrations of vanadate (10-200 microM) to cultured aortic smooth muscle cells (ASMC) produced a rapid and a concentration-dependent increase in [Ca2+]i with an EC50 (mean +/- SEM) value of 42 +/- 11 microM. Inclusion of vanadate (200 microM) led to a significant increase (p < 0.05) in the peak [Ca2+]i level to 190 +/- 23 nM from a basal level of 102 +/- 2 nM. At concentrations > 200 microM, vanadate caused quenching of fura-2 fluorescence. For example, addition of 1 mM vanadate led to an apparent decrease in fluorescence by about 50% (due to a quenching effect), followed by a transient rise. H2O2, which is used in the preparation of peroxide forms of vanadate, pervanadate (PV), also produced a rise in [Ca2+]i. These data suggest that vanadate promotes vascular tone by elevating [Ca2+]i in ASMC. However, [Ca2+]i measurements made with higher concentrations of vanadate and PV, using the fura-2 method, must be interpreted with caution.

Vanadate influence on calcium (45Ca2+) absorption by sorghum root tips

Abstract One‐cm root‐tips of Sorghum bicolor (L.) Moench cv Funk G522DR, SC574, SC283, or GP‐10 were exposed to calcium (45Ca2+) for 2‐hr at pH 5.5 in the presence of vanadate (0,0.1, 1.0, or 10.0 mM). Genetic variation in vanadate‐untreated absorption of 45Ca2+ was observed. Vanadate inhibited 45Ca2+ uptake in Funk G522DR, SC574, and GP‐10 linearly with the log concentration. In SC283, 0.1 mM vanadate significantly decreased 45Ca2+ absorption; but, 45Ca2+ absorption increased at higher vanadate concentrations.

Interaction of vanadate with isolated rat parotid acini

1. In rat parotid acini, vanadate affects amylase secretion and intracellular messengers according to its concentration. 2. Low concentrations (in the micromolar range) concentrations of vanadate stimulate amylase secretion and potentiate the secretory effect of carbamylcholine, but inhibit the amylase release in response to isoproterenol. 3. Vanadate increases inositol phosphates (mono-, bis- and trisphosphate) and potentiate the stimulatory effect of carbachol on inositol bis- and trisphosphate. 4. Vanadate also decreases the intracellular cyclic AMP concentration and the increase in response to isoproterenol or forskolin. 5. High concentrations (in the millimolar range) of vanadate inhibit the increase in inositol phosphate induced by carbachol. 6. It is concluded that low concentrations of vanadate activate regulatory proteins coupled to phospholipase C and adenylate cyclase while high concentrations of vanadate inhibit inositol bisphosphatase.

Vanadate and insulin stimulate gene 33 expression

Vanadate and insulin stimulated gene 33 expression in rat H4 hepatoma cells. Vanadate (10 −3 M) maximally increased gene 33 transcription 4-fold at 15 min. The maximal insulin induction (3–4-fold) was observed using 5×10 −9 M insulin for 30 min. Both vanadate and insulin increased cytoplasmic mRNA levels in a dose-dependent manner, with maximal effects observed by 60 min. These effects on gene 33 mRNA accumulation were greater than those on transcription. Vanadate (10 −3 M) maximally stimulated gene 33 mRNA levels 10–12-fold. The vanadate and insulin effects were not synergistic. Thus, high concentrations of vanadate regulate gene 33 expression in an insulin-like manner and, like insulin, vanadate probably controls transcriptional as well as post-transcriptional events.

Stimulation of endothelial cell proliferation by vanadate is specific for microvascular endothelial cells.

Micromolar concentrations of sodium orthovanadate stimulated the proliferation of bovine capillary endothelial cells, but not bovine aortic endothelial cells. Vanadate was equally potent at inducing protein tyrosine phosphorylation and changes in morphology in both types of cells. However, vanadate treatment lead to an inhibition of protein tyrosine kinase activity in the aortic endothelial cells, but not the capillary endothelial cells. In capillary endothelial cells, the effect of vanadate was additive with basic FGF (bFGF) at low concentrations of bFGF. There was no interaction between bFGF and vanadate in aortic endothelial cells. TGF-beta, which inhibits the induction of endothelial cell proliferation by bFGF, appeared to shift the dose response curve to vanadate in capillary endothelial cells, increasing the proliferative effect of vanadate at low vanadate concentrations, but decreasing the proliferative effect at higher vanadate concentrations.

Ethylene synthesis and growth of tomato hypocotyls: Induction by auxin and fusicoccin and inhibition by vanadate

The effects of fusicoccin (FC) on growth and ethylene synthesis of tomato (Lycopersicon esculentum Mill.) hypocotyls were compared to those of indole-3-acetic acid (IAA). Fusicoccin promoted both growth and ethylene production maximally at <2μM. Growth was stimulated to a slightly greater extent by FC as compared to IAA, while ethylene synthesis rates in response to FC were about 50% less than those induced by IAA. Cycloheximide (0.5 μM) inhibited auxin-induced growth by 80% but had no effect on FC-induced growth; ethylene production was inhibited to the same extent (58%) when induced by either IAA or FC. Both IAA and FC caused tissue contents of 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl-ACC to increase, indicating that like IAA, FC induces ethylene synthesis by stimulating the formation of ACC. Orthovanadate, a potent inhibitor of proton-translocating plasma membrane ATPases, reduced both IAA- and FC-induced growth and ethylene synthesis at concentrations less than 1 mM, with ethylene synthesis being approximately 10 times more sensitive to inhibition than growth. Vanadate did not affect tissue ACC levels, slightly reduced total ACC production, and inhibited conversion of ACC to ethylene. However, significant inhibition of in vivo ethylene-forming enzyme activity required high concentrations of vanadate (1 mM) and was less effective than inhibition by cobaltous ion. The site of action of vanadate in inhibiting ethylene synthesis remains unclear, but the ion did not prevent the elevation of tissue ACC levels in response to IAA or FC. It is unlikely, therefore, that stimulation of plasma membrane H+-ATPase activity is required for the induction of ACC synthase by IAA and FC.

Volume-sensitive K influx in human red cell ghosts.

K influx into resealed human red cell ghosts increases when the ghosts are swollen. The influx demonstrates properties similar to volume-sensitive K fluxes present in other cells. The influx is, for the most part, insensitive to the nature of the major intracellular cation and therefore is not a K-K exchange. The influx is much greater when the major anion is Cl than when the major anion is NO3; Cl stimulates the flux and, at constant Cl, NO3 inhibits it. Increase in the influx rate is rapid when shrunken ghosts are swollen or when NO3 is replaced by Cl. The volume-sensitive K influx requires intracellular MgATP at low concentrations, and ATP cannot be replaced by nonhydrolyzable ATP analogues. The volume-sensitive influx is inhibited by Mg2+ and by high concentrations of vanadate, but is stimulated by low concentrations of vanadate. It is not modified by cAMP, the removal of Ca2+ by EGTA, substances that activate protein kinase C, or by inhibition of phosphatidylinositol kinase. The influx is inhibited by neomycin and by trifluoperazine.

Studies on Mg2+-dependent ATPase in bovine adrenal chromaffin granules. With special reference to the effect of inhibitors and energy coupling.

The Mg2+-ATPase activities of bovine adrenal chromaffin granules were studied in highly purified preparations of granule ghosts and in intact organelles. The overall ATPase activity (150-250 nmol ADP min-1 mg-1) of the granule ghost preparations was inhibited less than 5% by the bathophenanthroline chelate of Fe(II), a potent inhibitor of mitochondrial F1-ATPase. This small inhibition can be accounted for by a very minor contamination with mitochondria or mitochondrial fragments. The overall ATPase activity of native granule ghosts was inhibited about 75% by N-ethylmaleimide, with half-maximal inhibition at about 20 microM. The titration curve was slightly shifted towards higher concentrations as compared to the inhibition curve for the proton pump activity, which was completely inhibited at 25 microM. N,N'-Dicyclohexylcarbodiimide inhibited the overall ATPase activity by 75-80% at 1.1 mumol/mg protein, a concentration that completely abolished the proton pump activity. Low concentrations (10 microM) of vanadate inhibited the overall ATPase activity by about 15% but had no effect on the proton pump activity, which was partly inhibited only at higher vanadate concentrations. Our attempts to assign a function to the vanadate-sensitive and N-ethylmaleimide-insensitive ATPase have so far been unsuccessful. In particular, our assay for ATP diphosphohydrolase activity was negative, although the chromaffin granule ghosts revealed a low Mg2+-ADPase activity (11.8 nmol AMP min-1 mg-1 protein). In intact chromaffin granules the specific Mg2+-ATPase activity (50-70 nmol ADP min-1 mg-1) was stimulated 2-fold by uncouplers, as compared to 1.6-1.7-fold in granule ghosts. The degree of energy coupling was rather independent of the external pH (6.5 less than pH less than 8.0) and temperature (20-45 degrees C). As expected, partial inhibition (about 15%) of the overall ATPase activity by 10 microM vanadate increased the ATPase control ratio. ADP was found to be a potent inhibitor of the proton pump activity with MgATP as the substrate, and the effect can partly be explained by a competitive type of inhibition of the hydrolytic reaction. This effect of ADP explains some of the kinetic data reported for MgATP-dependent (H+-ATPase-dependent) reactions in this organelle, notably the energy-dependent accumulation and storage of catecholamines.

Polarized microtubule gliding and particle saltations produced by soluble factors from sea urchin eggs and embryos.

In this report, we describe an in vitro system for analyzing microtubule-based movements in supernatants of sea urchin egg and embryo homogenates. Using video enhanced DIC microscopy, we have observed bidirectional saltatory particle movements on native taxol-stabilized microtubules assembled in low speed supernatants of Lytechinus egg homogenates, and gliding of these microtubules across a glass surface. A high speed supernatant of soluble proteins, depleted of organelles, microtubules, and their associated proteins supports the gliding of exogenous microtubules and translocation of polystyrene beads along these microtubules. The direction of microtubule gliding has been determined directly by observation of the gliding of flagellar axonemes in which the (+) and (-) ends could be distinguished by biased polar growth of microtubules off the ends. Microtubule gliding is toward the (-) end of the microtubule, is ATP sensitive, and inhibited only by high concentrations of vanadate. These characteristics suggest that the transport complex responsible for microtubule gliding in S2 is kinesin-like. The implications of these molecular interactions for mitosis and other motile events are discussed.

Vanadate Inhibition of ATPases of Dunaliella parva in vitro and in vivo

The effect of orthovanadate on both isolated ATPases and reactions of intact cells of the halotolerant unicellular green alga Dunaliella parva has been studied. Orthovanadate strongly inhibits isolated ATPases associated with the plasmalemma or the flagella, but does not affect the activity of the ATPases of mitochondria or thylakoids. In vivo, orthovanadate strongly inhibits motility of the cells and photosynthetic CO2-fixation. Much higher vanadate concentrations are required for inhibition of photosynthesis than for the inhibition of the isolated plasmalemma ATPase. Inhibition of photosynthesis by vanadate is favoured by low phosphate and high Cl- concentrations in the medium. However, high K+ concentrations in the medium decrease the inhibition. It is demonstrated that inhibition of photosynthesis by vanadate is caused indirectly by vanadate-induced changes of internal Na+, K+ and Cl- concentrations. The results suggest that two primary targets exist for vanadate in intact cells: a plasmalemma ATPase which energizes ion uptake and extrusion in Dunaliella and a flagellar ATPase responsible for motility.

The effects of vanadate on rabbit ventricular muscle adenylate cyclase and sodium pump activities

Vanadate in the +5 oxidation state has been reported to have a positive inotropic action on cardiac ventricular muscle. We have investigated the biochemical actions of vanadate on ventricular muscle adenylate cyclase and sodium pump activities in both intact or disrupted cell systems in an attempt to elucidate the mechanism(s) responsible for the physiological response. Vanadate at concentrations up to 100 μM ( K a = 2 μM) stimulated adenylate cyclase activity in sarcolemmal membrane preparations or disrupted myocytes isolated from rabbit ventricular muscle by 2–3-fold. Increasing the vandate concentrations above 100 μM resulted in a progressive inhibition of basal or hormone-stimulated adenylate cyclase activity ( K i = 5 mM) which was similar to that found by the reaction product, pyrophosphate ( K i = 0.5 mM). Both activation and inhibition by vanadate was fully reversible. Maximum activation of adenylate cyclase by vanadate and isoprenaline were not additive whereas maximum fluoride activation was decreased (18%) and the forskolin-stimulated response was slightly potentiated. Vanadate reversibly inhibited ouabain-sensitive p-nitrophenylphosphatase activity ( K i = 60 nM) in sarcolemmal membrane preparations and disrupted myocytes. Complete inactivation was found at 1 μM vanadate. Acute or chronic incubation of intact myocytes with vanadate at concentrations up to 0.5 mM had no measurable affect on ouabain-sensitive 86Rb influx or isobutylmethylxanthine, isoprenaline or forskolin-stimulated accumulation of intracellular cAMP concentration. Inhibition of 86Rb influx and cAMP accumulation was found at higher concentrations of vanadate; however, this accompanied the progressive decrease in cell viability as measured by the decrease in percentage of rod-shaped cells. It is concluded that vanadate, at concentrations which have been reported to induce a positive inotropic action on mammalian ventricular muscle, does not increase adenylate cyclase activity or inhibit the sodium pump activity in intact myocytes. These results show that caution must be applied when extrapolating the actions found with vanadate in broken cell systems to intact tissues.

Isolation of pepsinogen granules from rabbit gastric mucosa.

Pepsinogen granules were isolated from rabbit stomachs by a combination of differential and isopycnic gradient centrifugation. The isolation procedure utilized 1 M sucrose and alkaline pH to stabilize the granules. The isolated granules were shown to be 8.4-fold enriched in pepsinogen and free of mitochondria and microsome enzyme markers. In addition to pepsinogen, a cation-insensitive but anion-sensitive Mg2+-ATPase co-purified with the zymogen. The enzyme was unaffected by aurovertin, oligomycin, and ouabain, but inhibited by high concentrations of vanadate, N,N'-dicyclohexylcarbodiimide, and azide. The enzyme activity was stimulated by tetrachlorosalicylanilide and the combination of valinomycin and nigericin in K+-containing media. The similarities between this enzyme and other secretory granule ATPases are discussed.

Vanadate inhibits the ATP-dependent degradation of proteins in reticulocytes without affecting ubiquitin conjugation.

Reticulocytes contain a nonlysosomal, ATP-dependent system for degrading abnormal proteins and normal proteins during cell maturation. Vanadate, which inhibits several ATPases including the ATP-dependent proteases in Escherichia coli and liver mitochondria, also markedly reduced the ATP-dependent degradation of proteins in reticulocyte extracts. At low concentrations (K1 = 50 microM), vanadate inhibited the ATP-dependent hydrolysis of [3H]methylcasein and denatured 125I-labeled bovine serum albumin, but it did not reduce the low amount of proteolysis seen in the absence of ATP. This inhibition by vanadate was rapid in onset, reversed by dialysis, and was not mimicked by molybdate. Vanadate inhibits proteolysis at an ATP-stimulated step which is independent of the ATP requirement for ubiquitin conjugation to protein substrates. When the amino groups on casein and bovine serum albumin were covalently modified so as to prevent their conjugation to ubiquitin, the derivatized proteins were still degraded by an ATP-stimulated process that was inhibited by vanadate. In addition, vanadate did not reduce the ATP-dependent conjugation of 125I-ubiquitin to endogenous reticulocyte proteins, although it markedly inhibited their degradation. In intact reticulocytes vanadate also inhibited the degradation of endogenous proteins and of abnormal proteins containing amino acid analogs. This effect was rapid and reversible; however, vanadate also reduced protein synthesis and eventually lowered ATP levels in the intact cells. Vanadate (10 mM) has also been reported to decrease intralysosomal proteolysis in hepatocytes. However, in liver extracts this effect on lysosomal proteases required high concentrations of vanadate (K1 = 500 microM) and was also observed with molybdate, unlike the inhibition of ATP-dependent proteolysis in reticulocytes.