Global organization and function of mammalian cytosolic proteasome pools: Implications for PA28 and 19S regulatory complexes

Proteolytic activity of the 20S proteasome is regulated by activators that govern substrate movement into and out of the catalytic chamber. However, the physiological relationship between activators, and hence the relative role of different proteasome species, remains poorly understood. To address this problem, we characterized the total pool of cytosolic proteasomes in intact and functional form using a single-step method that bypasses the need for antibodies, proteasome modification, or column purification. Two-dimensional Blue Native(BN)/SDS-PAGE and tandem mass spectrometry simultaneously identified six native proteasome populations in untreated cytosol: 20S, singly and doubly PA28-capped, singly 19S-capped, hybrid, and doubly 19S-capped proteasomes. All proteasome species were highly dynamic as evidenced by recruitment and exchange of regulatory caps. In particular, proteasome inhibition with MG132 markedly stimulated PA28 binding to exposed 20S alpha-subunits and generated doubly PA28-capped and hybrid proteasomes. PA28 recruitment virtually eliminated free 20S particles and was blocked by ATP depletion. Moreover, inhibited proteasomes remained stably associated with distinct cohorts of partially degraded fragments derived from cytosolic and ER substrates. These data establish a versatile platform for analyzing substrate-specific proteasome function and indicate that PA28 and 19S activators cooperatively regulate global protein turnover while functioning at different stages of the degradation cycle.     

Subcomplexes of PA700, the 19 S Regulator of the 26 S Proteasome, Reveal Relative Roles of AAA Subunits in 26 S Proteasome Assembly and Activation and ATPase Activity

We have identified, purified, and characterized three subcomplexes of PA700, the 19 S regulatory complex of the 26 S proteasome. These subcomplexes (denoted PS-1, PS-2, and PS-3) collectively account for all subunits present in purified PA700 but contain no overlapping components or significant levels of non-PA700 proteins. Each subcomplex contained two of the six AAA subunits (Rpt1–6) that form the binding interface of PA700 with the 20 S proteasome, the protease component of the 26 S proteasome. Unlike intact PA700, no individual PA700 subcomplex displayed ATPase activity or proteasome activating activity. However, both activities were manifested by ATP-dependent in vitro reconstitution of PA700 from the subcomplexes. We exploited functional reconstitution to define and distinguish roles of different PA700 subunits in PA700 function by selective alteration of subunits within individual subcomplexes prior to reconstitution. Carboxypeptidase treatment of either PS-2 or PS-3, subcomplexes containing specific Rpt subunits previously shown to have important roles in 26 S proteasome assembly and activation, inhibited these processes but did not affect PA700 reconstitution or ATPase activity. Thus, the intact C termini of both subunits are required for 26 S proteasome assembly and activation but not for PA700 reconstitution. Surprisingly, carboxypeptidase treatment of PS-1 also inhibited 26 S proteasome assembly and activation upon reconstitution with untreated PS-2 and PS-3. These results suggest a previously unidentified role for other PA700 subunits in 26 S proteasome assembly and activation. Our results reveal relative structural and functional relationships among the AAA subunits of PA700 and new insights about mechanisms of 26 S proteasome assembly and activation.The 26 S proteasome is a 2,500,000-Da protease complex that degrades polyubiquitylated proteins by an ATP-dependent mechanism (1, 2). The biochemical processes required for this function are divided between two subcomplexes that compose the holoenzyme (3, 4). The first, called 20 S proteasome or core particle, is a 700,000-Da complex that catalyzes peptide bond hydrolysis (5). The second, called PA700 or 19 S regulatory particle, is a 700,000-Da complex that mediates multiple aspects of proteasome function related to initial binding and subsequent delivery of substrates to the catalytic sites of the 20 S proteasome (6). The 20 S proteasome is composed of 28 subunits representing the products of 14 genes arranged in four axially stacked heteroheptameric rings (7, 8). Each of the two center β rings contains three different protease subunits that utilize N-terminal threonine residues as catalytic nucleophiles (5, 8, 9). These residues line an interior lumen formed by the stacked rings and thus are sequestered from interaction with substrates by a shell of 20 S proteasome subunits.PA700 is composed of 20 different subunits. Six of these subunits, termed Rpt1–6, are AAA² (ATPases Associated with various cellular Activities) family members that confer ATPase activity to the complex and mediate energy-dependent proteolysis by the 26 S proteasome (2, 10). 26 S proteasome assembly from PA700 and 20 S proteasome requires ATP binding to Rpt subunits (11–15). Binding of PA700 to the 20 S proteasome occurs at an axial interface between a heterohexameric ring of the PA700 Rpt subunits and the heteroheptameric outer ring of α-type 20 S proteasome subunits (16). Substrates enter the proteasome through a pore in the center of the α subunit ring that is reversibly gated by conformationally variable N-terminal residues of certain α subunits in response to PA700 binding (12, 17–19). Although the degradation of polyubiquitylated proteins requires additional ATP hydrolysis-dependent actions by PA700, the assembled 26 S proteasome displays greatly increased rates of energy-independent degradation of short peptides by virtue of their increased access to catalytic sites via diffusion through the open pore (15, 18, 20).Recently, specific interactions between Rpt and α subunits that determine PA700-20 S proteasome binding and gate opening have been defined. These findings established nonequivalent roles among the six different Rpt subunits for these processes (12, 19). For example, carboxypeptidase A treatment of PA700 selectively cleaves the C termini of two Rpt subunits (Rpt2 and Rpt5) and renders PA700 incompetent for proteasome binding and activation (19). Remarkably, short peptides corresponding to the C terminus of either Rpt2 or Rpt5, but none of the other Rpt subunits, were sufficient to bind to the 20 S proteasome and activate peptide substrate hydrolysis by inducing gate opening (12, 15, 18). The C-terminal peptides of Rpt2 and Rpt5 appear to bind to different and distinct sites on the proteasome and produce additive effects on rates of peptide substrate hydrolysis, suggesting that pore size or another feature of gating can be variably modulated (19). These various results, however, do not specify whether the action of one or the other or both C-terminal peptides is essential for function of intact PA700.In addition to its role in activation, PA700 plays other essential roles in 26 S proteasome function related to substrate selection and processing. For example, PA700 captures polyubiquitylated proteins via multiple subunits that bind polyubiquitin chains (21–23). Moreover, to ensure translocation of the bound ubiquitylated protein through the narrow opened substrate access pore for proteolysis, PA700 destabilizes the tertiary structure of the protein via chaperone-like activity and removes polyubiquitin chains via deubiquitylating activities of several different subunits (24–30). These various functions appear to be highly coordinated and may be mechanistically linked to one another and to the hydrolysis of ATP by Rpt subunits during substrate processing.Despite support for this general model of PA700 action, there is a lack of detailed knowledge about how PA700 subunits are structurally organized and functionally linked. Previously, we identified and characterized a subcomplex of PA700 called “modulator” that contained two ATPase subunits, Rpt4 and Rpt5, and one non-ATPase subunit, p27 (31). Although this protein was identified by an assay that measured increased PA700-dependent proteasome activation, the mechanistic basis of this effect was not clear. Moreover, the modulator lacked detectable ATPase activity and proteasome activating activity. The latter feature is surprising in retrospect because of the newly identified capacity of Rpt5 to activate the proteasome directly (12, 19). This disparity suggests that specific interactions among multiple PA700 subunits determine the manifestation and regulation of various activities.This study extends our recent findings regarding relative roles of Rpt subunits in the regulation of proteasome function. It also provides new insights and significance to older work that identified and characterized the modulator as a subcomplex of PA700. Our findings unite two different lines of investigation to offer new information about the structure, function, and regulation of 26 S proteasome. They also offer insights about alternative models for assembly of PA700 and 26 S proteasome in intact cells.     

Identification, purification, and characterization of a protein activator (PA28) of the 20 S proteasome (macropain).

A protein that greatly stimulates the multiple peptidase activities of the 20 S proteasome (also known as macropain, the multicatalytic protease complex, and 20 S protease) has been purified from bovine red blood cells and from bovine heart. The activator protein was a single polypeptide with an apparent molecular weight of 28,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and had a native molecular weight of approximately 180,000. This protein, which we have termed PA28, regulated all three of the putatively distinct peptidase activities displayed by each of two functionally different forms of the proteasome. This regulation usually included both an increase in the maximal reaction velocity and a decrease in the concentration of substrate required for half-maximal velocity and indicated that PA28 acted as a positive allosteric effector of the proteasome. PA28 failed, however, to stimulate the hydrolysis of large protein substrates such as casein and lysozyme. These results suggested that the hydrolysis of protein substrates occurred at a site or sites distinct from those that hydrolyzed small peptides and that the regulation of the two processes could be uncoupled. Evidence for direct binding of PA28 to the proteasome was obtained by glycerol density gradient centrifugation. PA28 may play an important regulatory role in intracellular proteolytic pathways mediated by the proteasome.     

l-Asparaginase from Escherichia coli

Crystalline l-asparaginase from Escherichia coli A-I-3 hydrolyzed d-asparagine, l- and d-glutamine but at much slower rates than the rate at which it hydrolyzed l-asparagine. Inhibitions by these substrates and related compounds were revealed to be competitive.

d-Asparagine showed the same affinity for the enzyme both in its hydrolysis and inhibition of l-asparagine hydrolysis. l-Aspartate, d-aspartate and α-N-ethylasparagine inhibited various hydrolysis reactions with the respective inhibitor constants. The enzyme was found to hydrolyze β-methylaspartate as well as β-aspartohydroxamate. These data strongly suggest that the hydrolysis occurred at the same active site of the enzyme molecule with relatively low specificity for the configuration of the substrate molecule and the kind of bonding which it hydrolyzes.     

High yield bacterial expression and purification of active recombinant PA28αβ complex

The PA28 complexes (also termed REG or 11S complexes) are described as activators of the 20S proteasome, a major intracellular protease in eukaryotic cells. They bind to the ends of the barrel-shaped 20S proteasome, and activate its peptidase activities. The interferon γ inducible PA28αβ, made of the two related subunits PA28α and β, is under sustained investigation as it plays important roles in the production by the proteasome of class I antigen peptides. However, in vitro studies of this complex have been impaired by the difficulty of producing large amount of this protein, mainly due to the poor solubility of its β subunit when expressed in Escherichia coli. Here we describe the construction of a bicistronic vector, allowing simultaneous production of functional human PA28α and β subunits in E. coli. Co-expression of the two proteins allows efficient formation of active PA28αβ complexes, that remain soluble and can be easily purified by regular chromatographic procedures.     

Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors

Proteasomes perform the majority of proteolysis that occurs in the cytosol and nucleus of eukaryotic cells and, thereby, perform crucial roles in cellular regulation and homeostasis. Isolated proteasomes are inactive because substrates cannot access the proteolytic sites. PA28 and PA200 are activators that bind to proteasomes and stimulate the hydrolysis of peptides. Several protein inhibitors of the proteasome have also been identified, and the properties of these activators and inhibitors have been characterized biochemically. By contrast, their physiological roles--which have been reported to include production of antigenic peptides, proteasome assembly and DNA repair--are controversial. In this article, we briefly review the biochemical data and discuss the possible biological roles of PA28, PA200 and proteasome inhibitors.     

Proteasome activator 28γ (PA28γ) allosterically activates trypsin-like proteolysis by binding to the α-ring of the 20S proteasome

Proteasome activator 28γ (PA28γ/REGγ) is a member of the 11S family of proteasomal regulators that is constitutively expressed in the nucleus and implicated in various diseases, including certain cancers and systemic lupus erythematosus. Despite years of investigation, how PA28γ functions to stimulate proteasomal protein degradation remains unclear. Alternative hypotheses have been proposed for the molecular mechanism of PA28γ, including the following: (1) substrate selection, (2) allosteric upregulation of the trypsin-like (T-L) site, (3) allosteric inhibition of the chymotrypsin-like (CT-L) and caspase-like (C-L) sites, (4) conversion of the CT-L or C-L sites to new T-L sites, and (5) gate opening alone or in combination with a previous hypothesis. Here, by mechanistically decoupling gating effects from active site effects, we unambiguously demonstrate that WT PA28γ allosterically activates the T-L site. We show PA28γ binding increases the Kcat/Km by 13-fold for T-L peptide substrates while having little-to-no effect on hydrolysis kinetics for CT-L or C-L substrates. Furthermore, mutagenesis and domain swaps of PA28γ reveal that it does not select for T-L peptide substrates through either the substrate entry pore or the distal intrinsically disordered region. We also show that a previously reported point mutation can functionally switch PA28γ from a T-L activating to a gate-opening activator in a mutually exclusive fashion. Finally, using cryogenic electron microscopy, we visualized the PA28γ-proteasome complex at 4.3 Å and confirmed its expected quaternary structure. The results of this study provide unambiguous evidence that PA28γ can function by binding the 20S proteasome to allosterically activate the T-L proteolytic site.     

The dependency of the stereoselectivity of penicillin amidases-enzymes with R-specific S1- and S-specific S′1-subsites- on temperature and primary structure

Summary The stereoselectivity of penicillin amidase (PA, EC 3.5.1.11) from E coli and homologeous enzymes from other sources has been determined as a function of temperature and substrate for hydrolysis and kinetically controlled synthesis. The stereoselectivity of these reactions decreased almost by one order of magnitude from 5 to 45°C. It increased with the substrate (k _cat/K _m) and nucleophile (k _T/k _H) specificity, and was found to differ in the S₁- (R-specific) and S₁-(S-specific)-binding subsites of the active site. The S₁-stereoselectivity was determined mainly by differences in the activation energy, i.e. the turnover number. The stereoselectivity of PA from different sources differed by almost an order of magnitude for the same substrate.     

Endoproteinase Activity of Type A Botulinum Neurotoxin: Substrate Requirements and Activation by Serum Albumin

Type A botulinum neurotoxin, a zinc-dependent endoproteinase that selectively cleaves the neuronal protein SNAP-25, can also cleave relatively short peptides. We found that bovine and other serum albumins stimulated the type A-catalyzed hydrolysis of synthetic peptide substrates, through a direct effect on the kinetic constants of the reaction. Furthermore, with bovine serum albumin in the assays, the optimum substrate size was 16 residues (11 on the amino-terminal side of the cleavage site and 5 on the carboxy-terminal side). To further investigate the catalytic requirements of the neurotoxin, peptides were synthesized with various amino acid substitutions at the P5 through P5 substrate sites. Changes at all of these locations affected values for both k _cat and K _m. Substitutions at the P2, P1, and P2 sites had more pronounced effects on hydrolysis rates than did substitutions at the P1 site. Enzyme–substrate interactions at the P3 threonine probably involved the side-chain methyl group rather than the hydroxyl group. Replacing the P2 alanine with leucine eliminated detectable hydrolysis, but not binding, since this peptide was an inhibitor. A negatively charged residue was preferred at P5, but not at P4. The data indicate that type A botulinum neurotoxin has an extended substrate recognition region and a requirement for arginine as the P1 residue.     

Structural and functional properties of proteasome activator PA28

The proteasome activator PA28 or 11S regulator is a protein complex composed of two different but homologous polypeptides, termed PA28 and PA28. The purified activator protein (_200 kDa) is a ring-shaped heteromultimer containing the two polypeptides, possibly with an _{3 3} stoichiometry. The activator, which by itself shows no hydrolytic activity elicits activation of the proteasome's multiple peptidase activities by binding to the terminal rings of the proteinase. In vitro, active PA28 can be reconstituted from isolated and subunits, yielding two different oligomers: with the single subunit, PA28 homomultimers with moderate stimulatory activity toward 20S proteasomes are obtained whereas isolated -subunits are unable to form oligomers and are devoid of stimulatory activity. However, in the presence of both subunits, heteromultimers form, concomitant with restoration of full stimulatory activity. The recent finding that PA28 modulates the proteasome-catalyzed production of antigenic peptides presented to the immune system on MHC class I molecules indicates a cellular function of the activator in antigen processing. Abbreviations: IFN – interferon; LMP – low molecular weight peptide; MHC – major histocompatibility complex.     

Interaction of α-Chymotrypsin with Dimethyl Sulfoxide: A Change of Substrate Could “Change” the Interaction Mechanism

The kinetic behavior of -chymotrypsin was studied in water–DMSO mixtures at concentrations of the organic solvent that do not cause irreversible denaturation of the enzyme. Various substrates (N-substituted derivatives of L-tyrosine) were found to display substantially different kinetic patterns of interaction with -chymotrypsin, which can be described by totally different kinetic schemes. The differences were ascribed to competition between the N-acyl group of the substrate and the DMSO molecule at the S ₂ site of substrate binding to the active site of the enzyme.     

T lymphocytes export proteasomes by way of microparticles: a possible mechanism for generation of extracellular proteasomes

The 20S proteasome is almost exclusively localized within cells. High levels of extracellular proteasomes are also found circulating in the blood plasma of patients suffering from a variety of inflammatory, autoimmune and neoplastic diseases. However, the origin of these proteasomes remained enigmatic. Since the proteome of microparticles, small membrane enclosed vesicles released from cells, was shown to contain proteasomal subunits, we studied whether intact proteasomes are actively released into the extracellular space. Using human primary T lymphocytes stimulated with CaCl₂ and the calcium ionophore A23187 to induce membrane blebbing we demonstrate that microparticles contain proteolytically active 20S proteasomes as well as the proteasome activator PA28 and subunits of the 19S proteasome regulator. Furthermore, our experiments reveal that incubation of in vitro generated T lymphocyte‐microparticles with sphingomyelinase results in the hydrolysis of the microparticle membranes and subsequent release of proteasomes from the vesicles. Thus, we here show for the first time that functional proteasomes can be exported from activated immune cells by way of microparticles, the dissolution of which may finally lead to the generation of extracellular proteasomes.     

Mass spectrometric analysis of affinity-purified proteasomes from the human myelogenous leukemia K562 cell line

Proteasomes carry out regulated proteolysis of most proteins in a cell and thereby play a crucial role in the regulation of various cellular processes. Determination of the subunit composition and posttranslational modifications of proteasomes is one of the important stages in understanding of proteasomes functions in the cell and mechanisms of their regulation. To solve this problem a strategy of affinity purification of proteasomes with the subsequent mass spectrometric analysis has been implemented, using human myelogenous leukemia cells. Proteasomes have been purified from the stable K562 cell line expressing β7 (PSMB4) subunit of the 20S proteasome tagged with C-terminal HTBH peptide containing two His₆ fragments, the specific site of cleavage by Tobacco Etch Virus (TEV) protease, and signal sequence for biotinylation in vivo, using method of noncovalent binding through formation of biotin complex with streptavidin with the subsequent elution with TEV protease. All known subunits of the 26S proteasome, as well as PA200 and PA28γ regulators have been identified using MALDI FT-ICR mass spectrometry. We have demonstrated that the heat shock proteins, components of the ubiquitin-proteasome system, and some cytoskeleton proteins are associated with proteasomes. A number of new sites of phosphorylation, ubiquitination, and N-terminal modification have been found for 16 proteasome subunits. The presented mass spectrometric analysis will be useful for the further proteomic studies of proteasomes under cellular stress.     

Trimming of Ubiquitin Chains by Proteasome-associated Deubiquitinating Enzymes

The proteasome generally recognizes substrate via its multiubiquitin chain followed by ATP-dependent unfolding and translocation of the substrate from the regulatory particle into the proteolytic core particle to be degraded. Substrate-bound ubiquitin groups are for the most part not delivered to the core particle and broken down together with substrate but instead recovered as intact free ubiquitin and ubiquitin chains. Substrate deubiquitination on the proteasome is mediated by three distinct deubiquitinating enzymes associated with the regulatory particle: RPN11, UCH37, and USP14. RPN11 cleaves at the base of the ubiquitin chain where it is linked to the substrate, whereas UCH37 and apparently USP14 mediate a stepwise removal of ubiquitin from the substrate by disassembling the chain from its distal tip. In contrast to UCH37 and USP14, RPN11 shows degradation-coupled activity; RPN11-mediated deubiquitination is apparently delayed until the proteasome is committed to degrade the substrate. Accordingly, RPN11-mediated deubiquitination promotes substrate degradation. In contrast, removal of ubiquitin prior to commitment could antagonize substrate degradation by promoting substrate dissociation from the proteasome. Emerging evidence suggests that USP14 and UCH37 can both suppress substrate degradation in this way. One line of study has shown that small molecule USP14 inhibitors can enhance proteasome function in cells, which is consistent with this model. Enhancing protein degradation could potentially have therapeutic applications for diseases involving toxic proteins that are proteasome substrates. However, the responsiveness of substrates to inhibition of proteasomal deubiquitinating enzymes may vary substantially. This substrate specificity and its mechanistic basis should be addressed in future studies.The eukaryotic proteasome is dedicated primarily to the degradation of proteins tagged by ubiquitin (1). Proteasomes strongly prefer multiubiquitinated protein substrates. The successive addition of ubiquitin groups to the substrate by ubiquitin ligases is usually accomplished through the formation of ubiquitin chains. The proteasome has much in common with the simple ATP-dependent proteases of prokaryotes and mitochondria (2, 3), although only the proteasome recognizes the ubiquitin modification. In all cases, the ATPases form a hexameric ring complex. These rings are homomeric in the case of the prokaryotic and mitochondrial proteases, whereas in eukaryotic proteasomes, the ATPase ring is heteromeric. Proteasomes and the simple ATP-dependent proteases are fundamentally similar in that they all have an ATPase ring (found within the regulatory particle [RP]1 in proteasomes, also known as the 19S particle and PA700) abutting a proteolytic complex (the core particle [CP] in proteasomes, also known as the 20S particle), although in some cases, the ATPase and protease domains are present on the same polypeptide chain (Fig. 1). Furthermore, this ancient organization of ATP-dependent proteases involves stacked ring complexes. Substrates are translocated from one ring to the next via the central pore within each ring. For most substrates, movement from ring to ring is driven by ATP hydrolysis. Thus, the substrate is captured by the ATPase ring of the RP and then translocated into the central cavity of the CP where it is hydrolyzed.Open in a separate windowFig. 1.Deubiquitinating enzymes of proteasome. In metazoans, three DUBs associate with the proteasome as shown. Each is associated with the 19-subunit RP. The detailed positioning of these enzymes on the RP is not known and is represented here schematically. RPN11 cuts at the base of the chain to release the chain en bloc. As shown, this is coupled (by an unknown mechanism) to translocation of the substrate from the RP to the CP to be degraded. In contrast, the action of USP14 and UCH37 is thought to promote substrate release from the proteasome rather than degradation. However, it should be noted that the attack of these enzymes on a substrate does not guarantee release, especially as their action on the chain is gradual, proceeding stepwise over time from the distal tip of the ubiquitin chain. Some substrates may carry more than one ubiquitin chain and thus be processed in a more complex manner. Moreover, more than one DUB might act on a given chain. The proteasome icon, adapted from Ref. 30 with permission, is based on cryo-EM imaging.The pathway of translocation contains a series of narrow constrictions through which folded proteins cannot pass. The inability of a typical folded protein to pass through these “filters” defines in part the selectivity of such proteases. However, the ATPases can exert a pulling force on the substrate that is strong enough to unfold the protein, which allows for passage through the series of constrictions. This force is exerted within the central channel of the ATPase complex. Thus, translocation and unfolding of the substrate are generally coupled events (1–3).Although not departing from this paradigm, the eukaryotic proteasome interacts with substrate in a more complex manner as a result of interactions involving the ubiquitin tag. Thus, many of the 13 subunits that were added to the evolutionarily ancient ATPase complex to form the RP in the eukaryotic lineage participate in recognition and processing of the ubiquitin tag (1). For example, the yeast proteasome has five and probably more distinct ubiquitin receptors, two that are integral subunits and three that are reversibly proteasome-associated (4). In addition, proteasomes of mammals have three distinct deubiquitinating enzymes (DUBs). The multiplicity of DUBs points to a surprisingly complex role of deubiquitination in proteasome function.     

Vanadate binding to the (Na + K)-ATPase

A particulate (Na + K)-ATPase preparation from dog kidney bound [⁴⁸V]-ortho-vanadate rapidly at 37°C through a divalent cation-dependent process. In the presence of 3 mM MgCl₂ theK _d was 96 nM; substituting MnCl₂ decreased theK _d to 12 nM but the maximal binding remained the same, 2.8 nmol per mg protein, consistent with 1 mol vanadate per functional enzyme complex. Adding KCl in the presence of MgCl₂ increased binding, with aK _0.5 for KCl near 0.5 mM; the increased binding was associated with a drop inK _d for vanadate to 11 nM but with no change in maximal binding. Adding NaCl in the presence of MgCl₂ decreased binding markedly, with anI ₅₀ for NaCl of 7 mM. However, in the presence of MnCl₂ neither KCl nor NaCl affected vanadate binding appreciably. Both the nonhydrolyzable, ,-imido analog of ATP and nitrophenyl phosphate, a substrate for the K-phosphatase reaction that this enzyme also catalyzes, decreased vanadate binding at concentrations consistent with their acting at the low-affinity substrate site of the enzyme; the presence of KCl increased the concentration of each required to decrease vanadate binding. Oligomycin decreased vanadate binding in the presence of MgCl₂, whereas dimethyl sulfoxide and ouabain increased it. With inside-out membrane vesicles from red blood cells vanadate inhibited both the K-phosphatase and (Na + K)-ATPase reactions; however, with the K-phosphatase reaction extravesicular K⁺ (corresponding to intracellular K⁺) both stimulated catalysis and augmented vanadate inhibition, whereas with the (Na + K)-ATPase reaction intravesicular K⁺ (corresponding to extracellular K⁺) both stimulated catalysis and augmented vanadate binding.