Communication Vol. 267, No. 21, lasue of July 25, pp. 14539-14542.1992 THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U. S. A. A Cytosolic Inhibitor of Vacuolar H+-ATPases from Mammalian Kidney* (Received for publication, January 8, 1992) Kun Zhang$$, Zhi-Qiang Wang$$, and Stephen GluckS$l(l From the Departments of $Medicine and llCell Biologyand Physiology, Washington University School of Medicine and St. Louis, Missouri 631 IO the §Renal Diuision, Jewish Hospital of St. Louis, Regulation of the vacuolar H+-ATPase in organellar and transepithelial acidification has been attributed to the effects of the proton electrochemical gradient across the membrane or to changes in the number of proton pumps. We now report the identification and purification of a protein from bovine kidney cytosol that inhibits both ATPase activity and proton translo- cating activity of vacuolar H+-ATPases. Its relative molecular weight (Mr) is 6300, similar to that for protein inhibitors of the mitochondrial FoF1-ATPase. The newly identified cytosolic inhibitor protein may participate in the physiologic regulation of the vacu- olar H+-ATPase by suppressing activity directly. ~ ~~ ~ ~~~~~ Vacuolar H+-ATPases acidify the intracellularcompart- ments of all eukaryotic cells, participating in diverse endocytic and secretory functions (1). In specialized hydrogen ion- secreting cells, such as the renal collecting duct intercalated cell, vacuolar H+-ATPases reside in the plasma membrane, effecting transcellular proton transport (2,3). The regulation of acidification of intracellular compartments and of distal urinary acidification has been attributed to the transmem- brane proton electrochemical gradient(4-6) or to changes in the number of plasma membrane proton pumps (3, 7). We report here the isolation of aprotein from bovine kidney cytosol that inhibits vacuolar H+-ATPases directly and may represent a new H+-ATPaseregulatory protein. EXPERIMENTAL PROCEDURES Mannheim. All other reagents were from Sigma. Mouse monoclonal Materials-O-Glycanase and N-glycanase were from Boehringer antibody MOPC21' (Sigma) was used as the source of immunoglob- ulin G. * This work was supported by National Institutesof Health Grants DK38848, DK09976, and AR32087, by a grant from the Monsanto- Washington University Fund, and by Washington University George M. O'Brien Kidney and Urological Diseases Center Grant P50- DK45181. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 11 Sandoz Pharmaceutical Corporation Established Investigator of the American Heart Association. To whom correspondence should be addressed Renal Div., Jewish Hospital, Washington University School of Medicine, 216 S. Kingshighway Blvd., St. Louis, MO 63110. Tel.: 314-454-7729 or 314-454-7771; Fax: 314-454-5126. The abbreviations used are: MOPC21, mouse immunoglobulin G; PMSF, phenylmethanesulfonyl fluoride; BTP, bis-tris-propane; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-l-propane sul- fonic acid; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; MES, 4-morpholineethanesulfonic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. Inhibitor Activity Assay-Immunoprecipitation of H+-ATPase and assay of immunoprecipitated ATPase activity were performed as described previously (8). Bovine kidney microsomes solubilized with nonyl glucopyranoside and CHAPS (8) were incubated with staphy- lococcal beads coupled to purified anti-H'-ATPase monoclonal anti- body H6.1 at 4 "C for 2 h, then washed twice with 20 mM Tris-C1,5 mM sodium azide, pH 7.5 (TA) containing 1% polyethylene glycol (M, 20,000), and once with TA buffer. For assay of inhibitor activity, 90 pl of inhibitor from different purification steps was preincubated with 30 pl of immunoprecipitated ATPase (a 10% suspension of staphylococcal beads) in a final volume of 150 pl containing 20 mM Tris-C1, 2 mM MgCl,, 50 mM KCl, pH 6.5 for 10 min, then 50 pl of the mixture was added to 450 pl of ATPase activity assay solution (3 mM ATP, 3 mM MgSO,, 1 mM NaN3, 1 mM Na3V04, 20 mM Tris- MES, pH 6.5), and incubated at 37 "C for 20 min. Protein was measured with the Micro-BCA protein assay reagent using bovine serum albumin as a standard (9). Phosphate was determined colori- metrically (8). Each assay contained 6.5-10.0 nmol of P;.min" im- munoprecipitated H+-ATPase activity. Isolation of Vacuolar H+-ATPase Inhibitor-Bovine kidney was homogenized in a blender (8), and the cytosol was centrifuged at 55,000 X g for 1 h. A final concentration of 80% (NHJZSO4 was added tothe supernatant; the sample was stirred at 4 "C for 30 min, centrifuged at 20,000 X g for 20 min, and the precipitate was dialyzed against 20 mM Tris-HC1, 5 mM NaN3, 1 mM phenylmethanesulfonyl fluoride (PMSF), and 1 mM EDTA, pH 7.5. The sample pH was then adjusted to 4.75 with citric acid it was stirred at 4 "C for 10 min, and centrifuged at 20,000 X g for 20 min. The supernatant was applied to a 2.5 X 7.5-cm CM-52 column equilibrated with 50 mM sodium citrate, 2 mM NaN3, pH 4.75. The flow-through fraction from CM-52 column was collected, adjusted to pH 6.0 with solid bis-tris-propane base (BTP), and was applied to a 2.5 X 7.5-cm DEAE-Sepharose column equilibrated with 20 mM BTP-Cl, 2 mM NaN3, pH 6.0. The column was washed with 100 mM NaCl in equilibration buffer, and the inhibitor was recoveredfrom the column by step elution with 300 mM NaCl in equilibration buffer. The eluate was concentrated with an Amicon ultrafiltration cell to 1.1 ml, and applied to a 1.5 X 55-cm Sephacryl FS-200 gel filtration column equilibrated with 20 mM BTP- C1, 300 mM NaCl, 2 mM NaN3, 1 mM PMSF, pH 6.0, and 1.5-ml fractions were collected. The fractions containing inhibitor were collected, concentrated, dialyzed against BTP buffer (20 mM BTP- C1, 2 mM NaN3, 1 mM EDTA, 1 mM PMSF, pH 7.6), and applied to a 10 X 100-mm Protein-Pak DEAE column (Millipore) equilibrated with BTP buffer. The inhibitor was eluted from the column using an exponential 75-600 mM NaCl gradient in BTP buffer, and 1.0-ml fractions were collected. The peak of eluted activity was recoveredat approximately 250 mM NaC1. Effect of Enzymes, Protease Inhibitors, and Proteins on Inhibitor Activity-Inhibitor was incubated at 22 "C for 20 min with the agents indicated in the figure legends, and was then added to the H+-ATPase for a 20-min preincubation period prior to assay for ATPase activity. The phospholipase A, and trypsin used were covalently coupled to agarose beads (insoluble enzyme) and were removedby centrifugation prior to thepreincubation with H+-ATPase. For each agent tested, a control ATPase assay was performed under identical preincubation and assay conditions except that inhibitor was omitted. None of the agents tested affected the control H+-ATPase activity significantly, although phosphate in some of the agent buffers elevated the back- ground slightly in some experiments. For the experiment examining the effect of EDTA, the final concentration of EDTA in the ATPase assay was 0.5 mM and the M P concentration was 3 mM, and base- line H+-ATPase activity was the same as in control incubations. Effect of Inhibitor on Transport ATPases-Transport ATPases were assayed in different membrane fractions of bovine kidney. Plasma membrane, mitochondria, and endoplasmic reticulum were purified using sucrose gradients, and were used for assays of Na,K- ATPase (lo), mitochondrial FaFl H+-ATPase (ll), and Ca2'-ATPase (12), respectively. For Na+,K+-ATPase, the activity was assayed in a total volume of 0.5 ml in 20 mM HEPES-Tris, pH 7.0,3 mM MgSO,, 20 mM KCl, 100 mm NaCl, and 3 mM ATP with and without 1 mM ouabain, a t 37 "C for 20 min; the base-line ouabain-sensitive ATPase 14539
Transcript
Communication Vol. 267, No. 21, lasue of July 25, pp. 14539-14542.1992 THE JOURNAL OF BIOLOGICAL CHEMISTRY
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U. S. A.
A Cytosolic Inhibitor of Vacuolar H+-ATPases from Mammalian Kidney*
(Received for publication, January 8, 1992) Kun Zhang$$, Zhi-Qiang Wang$$, and Stephen GluckS$l(l From the Departments of $Medicine and llCell Biology and Physiology, Washington University School of Medicine and
St. Louis, Missouri 631 IO the §Renal Diuision, Jewish Hospital of St. Louis,
Regulation of the vacuolar H+-ATPase in organellar and transepithelial acidification has been attributed to the effects of the proton electrochemical gradient across the membrane or to changes in the number of proton pumps. We now report the identification and purification of a protein from bovine kidney cytosol that inhibits both ATPase activity and proton translo- cating activity of vacuolar H+-ATPases. Its relative molecular weight (Mr) is 6300, similar to that for protein inhibitors of the mitochondrial FoF1-ATPase. The newly identified cytosolic inhibitor protein may participate in the physiologic regulation of the vacu- olar H+-ATPase by suppressing activity directly.
~ ~~ ~ ~~~~~
Vacuolar H+-ATPases acidify the intracellular compart- ments of all eukaryotic cells, participating in diverse endocytic and secretory functions (1). In specialized hydrogen ion- secreting cells, such as the renal collecting duct intercalated cell, vacuolar H+-ATPases reside in the plasma membrane, effecting transcellular proton transport (2,3). The regulation of acidification of intracellular compartments and of distal urinary acidification has been attributed to the transmem- brane proton electrochemical gradient (4-6) or to changes in the number of plasma membrane proton pumps (3, 7). We report here the isolation of a protein from bovine kidney cytosol that inhibits vacuolar H+-ATPases directly and may represent a new H+-ATPase regulatory protein.
EXPERIMENTAL PROCEDURES
Mannheim. All other reagents were from Sigma. Mouse monoclonal Materials-O-Glycanase and N-glycanase were from Boehringer
antibody MOPC21' (Sigma) was used as the source of immunoglob- ulin G.
* This work was supported by National Institutes of Health Grants DK38848, DK09976, and AR32087, by a grant from the Monsanto- Washington University Fund, and by Washington University George M. O'Brien Kidney and Urological Diseases Center Grant P50- DK45181. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
11 Sandoz Pharmaceutical Corporation Established Investigator of the American Heart Association. To whom correspondence should be addressed Renal Div., Jewish Hospital, Washington University School of Medicine, 216 S. Kingshighway Blvd., St. Louis, MO 63110. Tel.: 314-454-7729 or 314-454-7771; Fax: 314-454-5126.
Inhibitor Activity Assay-Immunoprecipitation of H+-ATPase and assay of immunoprecipitated ATPase activity were performed as described previously (8). Bovine kidney microsomes solubilized with nonyl glucopyranoside and CHAPS (8) were incubated with staphy- lococcal beads coupled to purified anti-H'-ATPase monoclonal anti- body H6.1 at 4 "C for 2 h, then washed twice with 20 mM Tris-C1,5 mM sodium azide, pH 7.5 (TA) containing 1% polyethylene glycol (M, 20,000), and once with TA buffer. For assay of inhibitor activity, 90 pl of inhibitor from different purification steps was preincubated with 30 pl of immunoprecipitated ATPase (a 10% suspension of staphylococcal beads) in a final volume of 150 pl containing 20 mM Tris-C1, 2 mM MgCl,, 50 mM KCl, pH 6.5 for 10 min, then 50 pl of the mixture was added to 450 pl of ATPase activity assay solution (3 mM ATP, 3 mM MgSO,, 1 mM NaN3, 1 mM Na3V04, 20 mM Tris- MES, pH 6.5), and incubated at 37 "C for 20 min. Protein was measured with the Micro-BCA protein assay reagent using bovine serum albumin as a standard (9). Phosphate was determined colori- metrically (8). Each assay contained 6.5-10.0 nmol of P;.min" im- munoprecipitated H+-ATPase activity.
Isolation of Vacuolar H+-ATPase Inhibitor-Bovine kidney was homogenized in a blender (8), and the cytosol was centrifuged at 55,000 X g for 1 h. A final concentration of 80% (NHJZSO4 was added to the supernatant; the sample was stirred at 4 "C for 30 min, centrifuged at 20,000 X g for 20 min, and the precipitate was dialyzed against 20 mM Tris-HC1, 5 mM NaN3, 1 mM phenylmethanesulfonyl fluoride (PMSF), and 1 mM EDTA, pH 7.5. The sample pH was then adjusted to 4.75 with citric acid it was stirred at 4 "C for 10 min, and centrifuged at 20,000 X g for 20 min. The supernatant was applied to a 2.5 X 7.5-cm CM-52 column equilibrated with 50 mM sodium citrate, 2 mM NaN3, pH 4.75. The flow-through fraction from CM-52 column was collected, adjusted to pH 6.0 with solid bis-tris-propane base (BTP), and was applied to a 2.5 X 7.5-cm DEAE-Sepharose column equilibrated with 20 mM BTP-Cl, 2 mM NaN3, pH 6.0. The column was washed with 100 mM NaCl in equilibration buffer, and the inhibitor was recovered from the column by step elution with 300 mM NaCl in equilibration buffer. The eluate was concentrated with an Amicon ultrafiltration cell to 1.1 ml, and applied to a 1.5 X 55-cm Sephacryl FS-200 gel filtration column equilibrated with 20 mM BTP- C1, 300 mM NaCl, 2 mM NaN3, 1 mM PMSF, pH 6.0, and 1.5-ml fractions were collected. The fractions containing inhibitor were collected, concentrated, dialyzed against BTP buffer (20 mM BTP- C1, 2 mM NaN3, 1 mM EDTA, 1 mM PMSF, pH 7.6), and applied to a 10 X 100-mm Protein-Pak DEAE column (Millipore) equilibrated with BTP buffer. The inhibitor was eluted from the column using an exponential 75-600 mM NaCl gradient in BTP buffer, and 1.0-ml fractions were collected. The peak of eluted activity was recovered at approximately 250 mM NaC1.
Effect of Enzymes, Protease Inhibitors, and Proteins on Inhibitor Activity-Inhibitor was incubated at 22 "C for 20 min with the agents indicated in the figure legends, and was then added to the H+-ATPase for a 20-min preincubation period prior to assay for ATPase activity. The phospholipase A, and trypsin used were covalently coupled to agarose beads (insoluble enzyme) and were removed by centrifugation prior to the preincubation with H+-ATPase. For each agent tested, a control ATPase assay was performed under identical preincubation and assay conditions except that inhibitor was omitted. None of the agents tested affected the control H+-ATPase activity significantly, although phosphate in some of the agent buffers elevated the back- ground slightly in some experiments. For the experiment examining the effect of EDTA, the final concentration of EDTA in the ATPase assay was 0.5 mM and the M P concentration was 3 mM, and base- line H+-ATPase activity was the same as in control incubations.
Effect of Inhibitor on Transport ATPases-Transport ATPases were assayed in different membrane fractions of bovine kidney. Plasma membrane, mitochondria, and endoplasmic reticulum were purified using sucrose gradients, and were used for assays of Na,K- ATPase (lo), mitochondrial FaFl H+-ATPase ( l l ) , and Ca2'-ATPase (12), respectively. For Na+,K+-ATPase, the activity was assayed in a total volume of 0.5 ml in 20 mM HEPES-Tris, pH 7.0,3 mM MgSO,, 20 mM KCl, 100 mm NaCl, and 3 mM ATP with and without 1 mM ouabain, a t 37 "C for 20 min; the base-line ouabain-sensitive ATPase
14539
14540 Vacuolar H+-ATPase Cytosolic Inhibitor from Bovine Kidney activity was 6.5 nmol of Pi.min". For the mitochondrial FoFl H+- ATPase, submitochondrial particles, prepared from bovine kidney (13), were used as the source of ATPase. Activity was assayed in a total volume of 0.5 ml containing of 20 mM HEPES-Tris, pH 7.0, 50 mM KCl, 0.5 pg of valinomycin/mg of protein, 250 mM sucrose, 3 mM MgSO,, and 3 mM ATP with and without 2 mM NaN3. The base-line NaN3-sensitive activity was 10.4 nmol of Pi.min". For Ca2+-ATPase, activity was assayed in a total volume of 0.5 ml containing 20 mM HEPES-Tris, pH 7.0, 100 mM KCl, 5 mM MgCl,, 100 p~ CaCI2, 85 p M EGTA, 0.3 mM sucrose, 3 mM ATP with and without 4 mM EGTA. The base-line Ca2+-dependent ATPase activity was 8.1 nmol of Pi. rnin". Microsomal and brush border membranes were prepared as described (14); bovine kidney lysosomes were isolated by a modi- fication of procedures (15) using differential centrifugation and two sucrose step gradients. Vacuolar H+-ATPase was purified from each of these membrane fractions by immunoprecipitation as described above.
Effect of Inhibitor on Proton Transport-ATP-dependent proton transport was measured as ATP-induced uptake of acridine orange (8).
RESULTS AND DISCUSSION
Bovine kidney cytosol was subjected to several fractionation steps indicated in Table I. The cytosol contained an activating factor (16) that stimulated vacuolar H+-ATPase activity and masked the inhibitor in the cruder fractions, but inhibitory activity was apparent in more purified fractions. The presence of the activator prevented a precise assessment of the degree of purification; a 118-fold purification of specific activity was obtained from the CM-52 flow-through step, with 34% recov- ery of activity (Table I).
A time course for the effect of the inhibitor on H+-ATPase activity is shown in Fig. la. The maximal H+-ATPase inhi- bition of 48% was reached by 10 min of preincubation with 6.0 pg of inhibitor, but required longer preincubation times with smaller amounts of inhibitor. The inhibitor was unaf- fected by pretreatment with phospholipase Az, 0-glycosidase, N-glycosidase, RNase, or DNase, but inhibition was reduced to 40% of control by pretreatment with trypsin (Fig. lb) , indicating that the inhibitor is probably a protein. 32% of the inhibitor activity was lost by heating it at 60 "C for 10 min, and 72% was lost by heating at 100 "C for 5 min.
Because the kinetics of inhibition were relatively slow, we examined the possibility that the inhibitor is a protease (Fig. IC). Neither inhibitors of several classes of protease nor addition of several types of competing proteins had any effect on inhibition. These results, together with the observation in Fig. l a of a maximal H+-ATPase inhibition of 48% without further changes with time, indicate that inhibition is likely
TABLE I Purification summary for the vacuolar H+-ATPase inhibitor
Fraction Volume Protein activity activity and (recoverv)
umn (NaCl gra- dient)" HPLC, high performance liquid chromatography.
A
B
C
FIG. 1. a, time course for effect of inhibitor on H+-ATPase activ- ity. H6-H+-ATPase beads with varying amounts of inhibitor protein as indicated were incubated for 1, 5, 10, or 20 min and transferred to buffer for ATPase assay as described under "Experimental Proce- dures." Assays contained 12.2 nmol of Pi. min" ATPase activity and 6.0 pg of ATPase and the reaction time was 7 min. b, effect of enzymes on H+-ATPase inhibitor activity. Assays contained 11.5 nmol of Pi. min" H+-ATPase activity and 6.0 pg of H-ATPase. 3.5 pg of inhibitor protein was added to each sample, which suppressed H+-ATPase activity by 45% in control incubations. Inhibitor protein was pre- treated with phospholipase AS (1 unit, insoluble enzyme), O-glycosi- dase (1 unit), N-glycosidase (1 unit), RNase (1 Kunitz unit), DNase (2 Kunitz units), or trypsin (1 unit, insoluble enzyme) as indicated for 20 min at 22 "C, then assayed for inhibitor activity as described under "Experimental Procedures." c, H+-ATPase inhibitor activity is not affected by protease inhibitors or added proteins. Assays con- tained 10.3 nmol of Pi.min" H+-ATPase activity and 6.0 pg of H+- ATPase. 3.5 pg of inhibitor protein was added to each sample, which suppressed H+-ATPase activity by 42% in control incubations. All of the agents indicated were incubated with the inhibitor protein for 20 min at 22 "C prior to ATPase assay as described under "Experimental Procedures."
not the result of protease activity. An SDS-polyacrylamide gel of sequential fractions in the
isolation of the inhibitor is shown in Fig. 2a, demonstrating enrichment of a M, 6300 polypeptide. Fig. 2b shows the inhibitor activity profile and an SDS-polyacrylamide gel of fractions from the Sephacryl FS-200 gel filtration column. The inhibitor activity eluted at an apparent M, of 16,200. An SDS gel of the same fractions revealed a polypeptide at a M, 6300 whose distribution coincided with the inhibitor activity. These results indicate that the inhibitor is likely the polypep- tide of M , 6300, which may exist as a dimer in the active fractions. The concentration dependence of inhibition is shown in Fig. 2c. The inhibition of H+-ATPase activity showed saturation with increasing concentration of inhibitor, suggesting that the inhibitor functions by binding rather than by enzymatic modification of the H+-ATPase. A Hill plot of the data yielded a Hill coefficient (aH) of 1.46, indicating a cooperative interaction in inhibition, and also suggesting that
Vacuolar H+-ATPase Cytosolic Inhibitor from Bovine Kidney 14541 a
~ ~ ~ 1 0 ~ . - E
10.6 - C 6.3
3.5 -
$ 1.0
$ 0 2
-6.3
Rotein- (r0)
FIG. 2. a, SDS-PAGE of vacuolar H'-ATPase inhihitor at succes- sive stages of purification. Lone 1, ammonium sulfate precipitate of cytosol, 75 pg; lane 2, acid precipitate, 75 pg; lane 3 , CM-52 flow- through, 75 pg; lane 4, DEAE-Sepharose eluate, 40 pg; lane 5 , FS-200 eluate, 15 pg; lane 6. high performance liquid chromatography DEAE eluate, 5 pg. Samples were applied to a discontinuous SDS-PAGE system described hy Schagger and von Jagow (M), consisting of a comhination of 10% (2 cm) and 16.5% (10 cm) polyacrylamide; the gel was stained with Coomassie Blue (34). b, protein and H+-ATPase inhihitor activity profiles on a Sephacryl FS-200 gel filtration profile, and SDS-PAGE analysis of column fractions. 7.9 mg of the DEAE- Sepharose fraction from bovine kidney cytosol (Table I ) was applied t o a FS-200 column, equilibrated with 20 mM BTP-CI, 2 mM NaN:3, 300 mM NaCI, and 1 mM PMSF, pH 6.0. H+-ATPase inhibitor activity was assayed as in Table 1. .50-pl aliquots of indicated column fractions (1-50 pg) were applied to the same gel system used in panel a, and the gel was stained with Coomassie Blue. c, concentration dependence o f inhihition of H'-ATPase activity. ATPase activity assay was performed as in Table I following preincuhation with different amounts of purified inhibitor as indicated in the graph. Inset, Hill plot of the data, with a Hill coefficient (a") of 1.46.
the active inhibitor may be a dimer. The experiment in Fig. 3a demonstrates that the inhibitor
is active against the vacuolar H+-ATPase in its native envi-
C
05-s : pH
FIG. 3. a, effect of inhihitor on ATP-dependent proton transport in bovine kidney microsomes. 10 pl of microsomes (40 pg of protein) was used for each assay. Assay huffer contained 2 mM Tris-HCI, 150 mM KCI, 5 mM MgCI,, 1 pM valinomycin, 5 pg of oligomycin, and 10 p~ acridine orange; 1 p~ nigericin was added where indicated. 1 mM N-ethylmaleimide or 10 pg of partially purified inhibitor were added 5 min prior to initiating transport in the indicated tracings. b. activity spectrum of inhihitor on different transport ATPases. lnhihition assay was as described in Tohle I, and preparation of the different memhrane fractions was as described under "Experimental Proce- dures." ATPase assays were 20 min. The hase-line activities were as follows: microsomal, 10.7 nmol of P,.min"; hrush border 10.4 nmol of P, .min": lysosomal, 12.0 nmol of P,.min". The partially purified inhihitor fraction contained 7.5 pg of protein. c, pH dependence of inhihitor. Inhihitor activity was assayed as in Table I , except the huffer contained 60 mM Tris and 60 mM MES, adjusted to the various pH values with HCI or NaOH. Each assay contained 11.0 nmol.min." H+-ATPase and 7 pg of partially purified inhihitor.
ronment in the membrane. Addition of inhibitor to bovine kidney microsomes reduced oligomycin and vanadate resist- ant, N-ethylmaleimide-sensitive ATP-dependent proton transport by 90%. Fig. 36 shows the activity spectrum of the inhibitor on several transport ATPases, assayed in different kidney membrane fractions, and on several purified vacuolar H+-ATPases from bovine kidney. The inhibitor diminished activity of affinity-purified renal microsomal, brush border, and lysosomal vacuolar H+-ATPase by 71.2, 82, and 85% respectively. The effect of the inhibitor was not dependent on binding of the H+-ATPase to the affinity beads, since the inhibitor also abated the activity of unbound, partially puri- fied H+-ATPase (17) by 69% (not shown). In contrast, the inhibitor decreased the total microsomal ATPase activity, Na+,K+-ATPase activity, and Ca'+-ATPase activity by only 0.1, 0.73, and 2.61%. respectively. The inhibitor exerted a partial effect on the mitochondrial F,,F, ATPase, reducing activity by 13.8%. The effect of pH on the inhibitor is shown in Fig. 3c. The percent inhibition was relatively constant at about 45% over the pH range of 6.0-7.5. The percent inhibi-
14542 Vacuolar H+-ATPase Cytosolic Inhibitor from Bovine Kidney
tion rose steadily above pH 7.5, and was greatest at pH 8.1, the highest pH tested.
This is the first reported example, to our knowledge, of a cytosolic inhibitor of the vacuolar H+-ATPases. Inhibitors of both mitochondrial H+-ATPases (18-21) and Ca*+-ATPase (22) have been isolated which have a M, of about 6000 on SDS gels (although the actual molecular mass is larger). The vacuolar H+-ATPase inhibitor described in this report is strongly reminiscent of the inhibitors of the mitochondrial FoFl ATPase. The vacuolar H+-ATPases and the FoFl AT- Pases appear to have diverged from a common ancestral proton pump, and several of their subunits have homologous amino acid sequences (23). The amino acid sequences of bovine and yeast mitochondrial H+-ATPase inhibitors have been remarkably well conserved during the course of evolution (24,25). The vacuolar H+-ATPase inhibitor exerted a partial inhibitory effect on the mitochondrial H+-ATPase, suggesting that the mitochondrial and vacuolar enzymes may have a similar inhibitor binding site. The bovine mitochondrial in- hibitor binds to the FoF1 H+-ATPase with a stoichiometry of 1 per enzyme (26, 27); the Hill coefficient of 1.46 for the concentration dependence of the vacuolar H+-ATPase inhib- itor suggests either that dimerization may be required for activity or that multiple inhibitor binding sites with cooper- ative interactions are present on the H+-ATPase. The mito- chondrial H+-ATPase inhibitor has a sharp pH dependence, with optimal percent inhibition at pH <6.5 (18), attuned to the relatively alkaline environment of the inner mitochondrial matrix. In contrast, the vacuolar H+-ATPase inhibitor showed the largest percent inhibition above pH 7.5. This difference may reflect the possible physiologic role of the vacuolar inhib- itor; as cytoplasmic pH increased, the inhibitor would become increasingly effective. Recent studies have revealed that the regulation of mitochondrial H+-ATPase inhibition is complex and may involve participation of more than one type of inhibitor (21, 28) and two additional low molecular weight proteins (29). Similarly complex interactions of vacuolar H+- ATPase regulatory proteins could provide a means for the cell to regulate acidification independently in different membrane compartments. In support of this concept, we have recently identified a cytosolic activator of the vacuolar H+-ATPase whose effects vary on vacuolar H+-ATPases isolated from different membrane compartments (16).
Mitochondrial inhibitor bound to the FoFl H+-ATPase is released when a proton electrochemical gradient (AfiH+) fa-
voring ATP synthesis is applied across the membrane (30, 31), an effect which appears to be the result of a AfiH+- induced conformational change (30, 32). If a similar mecha- nism exists for the vacuolar H+-ATPase inhibitor, it could provide a new means for sensing and regulating the pH gradient across a membrane; if the binding of the inhibitor were influenced by conformational changes in the vacuolar H+-ATPase occurring in response to the transmembrane pH gradient, the proton pump itself could act as the transmem- brane pH sensor.
Acknowledgments-We thank Edna Major and Debbie Windle for help in preparing the manuscript.
REFERENCES 1. 2. 3.
4.
5.
6. 7.
8. 9.
10. 11. 12.
13.
15. 14.
16.
Forgac, M. (1989) Physiol. Reu. 69,765-796 Brown, D., Hirsch, S., and Gluck, S. (1988) Nature 331 , 622-624 Bastani, B., Purcell, H., Hemken, P., and Gluck, S. (1991) J. Clin. Inuest.
AS. 12Ci-136 Mulberg, A. E., Tulk, B. M., and Forgac, M. (1991) J. Biol. Chem. 266 ,
Fuchs, R., Schmid, S., and Mellman, I. (1989) Proc. Natl. Acad. Sci. U. S. A.
Steinmetz P. R. (1986) Am. J. Physiol. 2 6 1 , F173-F187 Gluck S. ’ Cannon C. and Al-Awqati, Q. (1982) Proc. Natl. Acad. Sci.
Gluck, S., and Caldwell, J. (1987) J. Biol. Chem. 2 6 2 , 15780-15789 Smith, P. Krohn, R., Hermanson, G., Mallia, G., Gartner, F., Provenzano,
M., Fujimoto, E., Goeke, N., Olson, B., and Klenk, D. (1985) Anal. Biochem. 160 , 76-85
--,--- ---
20590-20593
86,539-543
U. 8. A,’ 79,4323-43’31
Jorgensen P. L. (1988) Methods Enzymol. 1 6 6 , 2 9 4 3 Penefsky,’H. S. (1979) Methods Enzymol. 66,304-308 Chu, A., Dixon, M. C., Saito, A., Seiler, S., and Fleischer, S. (1988) Methods
Gluck, S., and AI-Aw ati Q (1984) J. Clin. Inuest. 73,1704. Wanf, 2.-Q, and Glu& 4. (1990) J. Bzol. Chem. 266,21957. Hari umar, P., and Reeves, J. P. (1983) J. Biol. Chem. 268 , Zhf::,p; K., Wang, Z.-Q., and Gluck, S. (1992) J. Bml. Chen
Enzymol. 167,36-46 -1710 -21965
1. 267 , 9701- 10403-10410
17. Gluck S. and Caldwell J. (1988) Am. J. Ph siol. 2 6 4 , F71-F79 18. Pullmk,’M. E., and MAnroy B. C. (1963) YBiol. Chem. 238,3762-3769 19. Satre, M.. de Jerphanion, M.-b., Huet, J., and Vignais, P. V. (1975) Bmchm.
J I”“
20. 21. 22.
23. 24.
25. 26.
27.
Bioph s Acta-387, 241-255 Ebner, &,‘and Maier K. (1977) J. Biol. Chem. 262,671-676 Yamada, E. W. and kuzel, N. J. (1988) J. Biol. Chem. 263,11498-11503 Au, K. S., Cho,’K. L., Lee, K. S., and Lai, K. M. (1985) Biochim. Biophys.
Nelson, N., and Taiz, L. (1989) Trends Biochem. Sci. 14 , 113-116 Frangione B. Rosenwasser E., Penefsky, H. S., and Pullman, M. (1981)
Dianoux A. C. and Ho e J. (1987) Eur. J. Blochem. 163,155-160 Harris h. A., ’Husain,y Jackson, P. J., Lunsdorf, H., Schiifer, G., and
Klein. 8.: Satre. M.. Dianoux. A. C.. andvimais, P. V. (1980) Biochemistrv
Acta 821,348-354
Proc. Nbtl. h a d . Sei. U. $ . A . 78,7403-7407,
Tied e H (1986) Eur. J. Blochem. 167,181-186
19,’ 2919-2925 ’ , . - .
28. Cintrbn, N. M., and Pedersen, P. L. (1979) J. Biol. Chem. 264,3439-3443 29. Hashimoto, T., Yoshida, Y., and Tagawa, K. (1990) J. Biochem. (Tokyo)
30. Husain, I., Jackson, P., and Harris, D. A. (1985) Biochim. Biophys. Acta
31. Rouslin, W. (1987) J. Biol. Chem. 262,3472-3476 32. Power, J., Cross, R. L., and Harris, D. A. (1983) Biochim. Biophys. Acta
33. Schagger H., and von Jagow, G. (1987) A d . Biochem. 166,368-379 34. O’Farrell: P. (1975) J. Biol. Chem. 260,4007-4021
![ABCC Transporters Mediate the Vacuolar Accumulation of ... · ABCC Transporters Mediate the Vacuolar Accumulation of Crocins in Saffron Stigmas[OPEN] Olivia Costantina Demurtas,a](https://static.documents.pub/doc/80x56/5f5f892908d0895e8549ba74/abcc-transporters-mediate-the-vacuolar-accumulation-of-abcc-transporters-mediate.jpg)
![Vacuolar Transporters – Companions on a Longtime … · Vacuolar Transporters – Companions on a Longtime Journey[OPEN] Enrico Martinoia1 Department of Plant and Microbial Biology,](https://static.documents.pub/doc/80x56/603fbba48d3fd353b308f80e/vacuolar-transporters-a-companions-on-a-longtime-vacuolar-transporters-a-companions.jpg)
![Review V-ATPases and osteoclasts: ambiguous future of V ...thno.org/v08p5379.pdfosteoclasts, which is a key factor for bone resorption [2]. The V-ATPases-related regulation of extracellular](https://static.documents.pub/doc/80x56/5ee15f47ad6a402d666c473b/review-v-atpases-and-osteoclasts-ambiguous-future-of-v-thnoorg-osteoclasts.jpg)