ARCHIVES

OF

BIOCHEMISTRY

Properties

AND

BIOPHYSICS

of the Neurospora

180,

384-393

(1977)

crassa

Plasma Membrane

ATPasel

GENE A. SCARBOROUGH Department

of Biochemistry,

University

of Colorado

School of Medicine,

Received August

Denver,

Colorado

80262

27, 1976

A variety of the biochemical properties of the electrogenic plasma membrane ATPase of Neurosporu crussa are described. The enzyme catalyzes the hydrolysis of ATP, resulting in the formation of ADP and inorganic phosphate. Optimal activity is observed between pH 6 and 6.5. ATP hydrolysis approaches a maximum rate at an Mg-ATP concentration of lo-20 mM with a half-maximal velocity around 2 mM Mg-ATP. The enzyme requires a divalent cation for activity in the following order of preference at 10 mM: MgZ+, Co*+ > Mn2+ > Zn*+ > Fez+, Ca 2+, Cu2+. The enzyme is quite specific for ATP compared to the other nucleotides tested. Treatment of the plasma membranes with sodium deoxycholate inactivates the ATPase and the inactivation can be prevented by the addition of certain acidic phospholipids with the deoxycholate. Other classes of lipids cannot prevent the deoxycholate inhibition. The organic mercurials parachloromercuribenzoate and parachloromercuriphenylsulfonate are potent inhibitors of the ATPase, but N-ethylmaleimide at a similar concentration is not inhibitory. The organic mercurial inhibition is not reversed by mercaptoethanol. Under appropriate conditions, the inhibitory effect of p-chloromercuribenzoate is suppressed in the presence of ATP. Treatment of the plasma membranes with trypsin leads to a marked inhibition of the ATPase activity and this inhibition can be prevented by Mg-ATP. Neither the organic mercurial reactive site(s) nor the trypsin-sensitive site(s) are accessible from the outer surface of the plasma membranes. Some of the implications of the above findings are discussed.

The probable role of ATPases in a variety of energy-transducing processes has long been recognized. It is now widely accepted that certain membrane associated ATPases, such as the Na,K-stimulated, ouabain-sensitive Mg-ATPase in a variety of cells and the Ca-dependent Mg-ATPase in the sarcoplasmic reticulum are directly involved in the active transport of cations. In mitochondria, chloroplasts, and bacteria, membrane-bound ATPases are generally considered to be biological energy transducers which can utilize the energy of ATP hydrolysis to generate proton gradients and/or membrane potentials by effecting the net separation of H+ and OHacross the membrane in which they reside. The plasma membrane of the eukaryotic microorganism Neurospora crassa is now ’ This investigation was supported by grants from the National Institutes of Health (GM19971) and the National Science Foundation (GB38801).

established as a useful membrane system for investigating the role of ATPases in energy transducing processes. In the last few years, primarily on the basis of electrophysiological studies with intact cells, Slayman et al. (l-3) have provided evidence that the membrane potential (ap proximately 200 mV, interior negative) across the Neurospora plasma membrane is generated and maintained via the hydrolysis of ATP. Utilizing an entirely different approach, recent studies in this laboratory with isolated Neurospora plasma membrane vesicles have demonstrated in vitro that the Neurosporu plasma membrane ATPase is an electrogenic pump (4). The Neurosporu plasma membrane is unique among energy-transducing membranes thus far studied in that it is the only system available in which generation of a membrane potential via ATP hydrolysis can be studied with microelectrodes in intact cells and with biochemical tech384

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN 0003-9861

Neurospora

crassa

PLASMA MEMBRANEATPase

niques in isolated plasma membrane vesicles. With the knowledge that the Neurospora plasma membrane ATPase is an electrogenic pump, a major interest in this laboratory is an elucidation of the molecular mechanism by which this enzyme utilizes the energy of ATP hydrolysis to generate a membrane potential. As a necessary first step toward this goal we have characterized a variety of biochemical properties of the Neurospora plasma membrane ATPase. The results of these studies are the topic of this communication. MATERIALSAND METHODS Materials. ATP, GTP,ITP, UTP, CTP,ADP (all as sodium salts), PCMB,*PCMPS,NEM, ergosteriol, triolein, phosphatidylethanolamine, oligomyr tin, DNP, ouabain, and Tris were obtained from Sigma; ITP (sodium salt) from P-L; NaN,, NaAsO,, and NaF from Mallinckrodt; DNase and TPCK trypsin from Worthington; sodium deoxycholate from Nutritional Biochemicals; [“‘C]ATP from New England Nuclear; phosphatidylglycerol and phosphatidylserine (Se) from Serdary; phosphatidic acid and phosphatidylserine (Su) from Supelco; phosphatidylcholine from Fluka-Buchs; phosphatidylserine (Mi) from Miles; Folch fraction I from Mann; CCCP and soybean trypsin inhibitor from Calbiochem; TCS from Eastman; DCCD from Aldrich; and NaCN from Merck. Monophosphosphoinositide was prepared from Folch fraction I by DEAE-cellulose column chromatography according to Hendrickson and Ballou (5). Di- and triphosphoinositide were extracted from bovine brain by the method of Michell et al. (6) and purified by DEAE-cellulose column chromatography. N - 2 - Naphthylmaleimide, N - benzylmaleimide, and N-benzoylmaleimide were the generous gifts of Dr. H. R. Kaback of the Roche Institute of Molecular Biology, Nutley, New Jersey. Growth of Cells. Cells of the sl strain of Neurospora crassa were grown as previously described (7). Isolation of plasma membranes. Plasma membrane ghosts were isolated from 500-ml cultures of Neurospora crassa sl strain as described previously (7) except that Worthington DNase (Code D, 5 mg) 2 Abbreviations used: PCMB, p-chloromercuribenzoic acid; PCMPS, p-chloromercuriphenylsulfonic acid; CCCP, carbonylcyanide-m-chlorophenylhydrazone; DCCD, dicyclohexylcarbodiimide; TCS, tetrachlorosalicylanilide; NEM, N-ethylmaleimide; DNP, 2,4-dinitrophenol; TPCK, N”-tosylphenylalanine chloromethyl ketone; DEAE-, diethylaminoethyl-.

385

was substituted for the Sigma DNase (Code DN25, 50 mg) and tritiated cells were not included in the isolation procedure. The plasma membrane ghost pellets were suspended in 2 to 5 ml of ice-cold 0.01 M Tris-HCl, pH 7.5 and used immediately or frozen and thawed once. Standard ATPase assay. The assay mixtures contained MgSO, (10 mM), disodium ATP (10 mM, adjusted to pH 6 with Tris), Tris-succinate buffer, pH 6.0 (20 mM) and 20 ~1 of membrane suspension (0.022-0.112 mg of protein) in a total volume of 0.1 ml. Incubations were started by the addition of membranes and the reactions carried out at 30°C for the desired time interval. The reactions were stopped by the addition of 0.4 ml of ice-cold 3.75% perchloric acid, mixed, and centrifuged, and the inorganic phosphate in the supernatant fluids was estimated essentially by the method of Stanton (8). Any deviations from this standard protocol are mentioned in the figure and table legends. Specific activities of the enzyme are variable from day to day in the range of 100 to 400 nmol of Pi liberated/mg of proteimmin. Other. Protein was estimated by the method of Lowry et al. (9) with bovine serum albumin as a standard. Radioactivity was determined by liquid scintillation counting in the toluene-Triton X-100 (2:l) mixture described by Patterson and Greene (10).

RESULTSAND DISCUSSION As previously reported, Neurospora plasma membranes can be isolated as open sheets (ghosts) or as closed vesicles (7). In the studies reported here, ghosts have been used exclusively to avoid misinterpretations which might occur as a result of the inaccessibility of substrates, inhibitors, etc., in the plasma membrane vesicle preparation. pH Optimum

Figure 1 shows Mg-ATPase activity as a function of the pH of the assay medium. The enzyme displays optimal activity at pH 6-6.5. In control incubations contain: ing all of the reaction components except membranes there was no ATP hydrolysis at any pH indicated in the figure. Optimum

Substrate

Concentration

Figure 2 shows the rate of ATP hydrolysis as a function of the concentration of Mg-ATP. The enzyme activity approaches maximum levels at an Mg-ATP concentration of lo-20 mM with half-maximum ve-

386

GENE A. SCARBOROUGH

PH

FIG. 1. ATPase activity as a function of pH. Standard ATPase assay (0.049 mg of protein) with the pH of the buffer varied. Incubations were carried out for 30 min. O-O, 0.1 M succinic acid adjusted to the indicated pH with Tris; O-O, 0.1 M Tris adjusted to the indicated pH with succinic acid. Points are the average of duplicate determinations.

I Mg-ATPI

mh.4

FIG. 2. ATPase activity as a function of Mg-ATP concentration. Standard ATPase assay (0.05 mg of protein) with the concentration of Mg-ATP varied. Mg-ATP refers to an equimolar mixture of MgSO, and disodium ATP adjusted to pH 6.0 with Tris. Incubations were carried out for 10 min.

locity at about 2 mM Mg-ATP. This is consistent with the K,,, value reported by Slayman et al. (2) for the ATP-dependent maintenance of the membrane potential in intact cells and the concentration of MgATP required for half-maximal generation of the membrane potential in isolatedNeurospora plasma membrane vesicles (4). Products

tions (0 and 30 min) containing 0.4 &i of [14ClATP (sp act 50 mCi/mmol) were carried out. The [14ClATP-containing reactions were terminated by the addition of an equal volume of ice-cold absolute ETOH and centrifuged, and aliquots of the resulting supernatant fluids were applied to Whatman DE-81 paper and developed in the solvent system (0.6 M ammonium formate, pH 3.1) of Morrison (11). The resulting chromatograms were dried, and the distribution of radioactivity on the chromatograms was estimated with a Packard radiochromatogram scanner. The radioactivity on the chromatogram obtained from the 0-min incubation was confined to the area where ATP migrates. Figure 3 shows the distribution of radioactivity on the chromatogram obtained from the 30-min incubation. It can be seen that the major radioactive product is ADP with a trace of AMP. The ATP area on the chromatogram obtained from the 0-min incubation and the ATP and ADP areas on the chromatogram from the 30-min incubation were excised from the chromatograms and hydrolyzed in 1 N HCl (96X, 1 h), and the radioactivity in these areas was quantitated by liquid scintillation counting. The amount of ADP produced in 30 min, as estimated from the radioactivity on the chromatograms, and the amount of Pi liberated in 30 min, as estimated by chemical analysis, were essentially identical. Thus, the products of the reaction are ADP and Pi. Time Course

Figure 4 shows the rate of ATP hydrolysis by the plasma membrane ATPase as a function of time. It can be seen that the

of the Reaction

To determine the products of the ATP hydrolysis reaction, the standard assay (0.06 mg of protein) was carried out as usual to determine the amount of Pi liberated in 30 min; in addition, similar incuba-

CtNTlMlTtRS

FIG. 3. Products of the ATPase reaction. See text for experimental details.

Neurospora

crassa

PLASMA

minutes FIG. 4. Time course of ATP hydrolysis. Incubations contained plasma membranes (0.260 mg of protein), MgSO, (10 mM), disodium ATP (10 mM, adjusted to pH 6 with Tris), and Tris-succinate buffer, pH 6.0 (20 mM) in a final volume of 1 ml. At the indicated times O.l-ml aliquots were transferred to tubes containing 0.4 ml of ice-cold 3.75% perchloric acid and mixed, and the liberated Pi was estimated as described under Methods. Points are the average of duplicate determinations.

MEMBRANE

ATPase

387

ysis after resuspension of the membranes in fresh assay medium was similar to the rate which was in progress at the 30-min time point, and this rate was markedly lower than the initial rate of ATP hyrolysis. Therefore, it can be concluded that product inhibition is not the primary reason for the nonlinear time course but rather that the enzyme activity decays during the incubation period. With this likelihood, several agents were added to the incubation mixtures in an attempt to obtain a more linear time course. The addition of trypsin inhibitor or albumin (to suppress possible proteolytic activity), monophosphoinositide (to suppress possible lipolytic activity), or P-mercaptoethano1 (to prevent thiol oxidation) did not prevent the decay in the enzyme activity (data not shown). Assays carried out at lower (23, 15°C) and higher (37,43”C!) temperatures were similarly nonlinear. As yet, the reason for the nonlinear time course is unknown.

rate of hydrolysis is not linear with time even in the first few minutes of the reacStability tion. A possible explanation for the nonlinThe ATPase activity in the membranes ear time course is that ADP or Pi, which accumulate as ATP hydrolysis proceeds, is relatively stable to freezing once, but multiple cycles of freezing and thawing act as feedback inhibitors of the reaction. To test this possibility, a time course simi- lead to a marked decline in enzyme activlar to that shown in Figure 4 was carried ity (data not shown). The enzyme is unstaout in the presence or absence of 2 mM ble to overnight dialysis at 4°C and the loss ADP (data not shown). The results demon- of activity (approximately 80%) is not prestrated that 2 mM ADP (in the presence of vented by inclusion of mercaptoethanol in 10 mM ATP) inhibits the reaction only 24% the dialysate nor is it restored by the addiat each time point. In a similar experi- tion of a concentrated dialysate to the diment, Pi at 5 mM had little effect on the alyzed membranes (data not shown). The reason for the marked loss of activity upon rate of ATP hydrolysis. In the experiment shown in Fig. 4, ADP and Pi had accumu- freezing and thawing or dialysis is preslated to approximately 2 mM at the 30-min ently unexplained, but these phenomena may be related to the instability of the time point and the rate of ATP hydrolysis had decreased by about 76%. Thus, prod- enzyme in the assay mixture described in uct inhibition can account for only a small the previous section. The enzyme activity is stable for more than a week when fraction of the decrease in enzyme activity with time. To corroborate these findings in freshly prepared membranes are susanother way, an experiment similar to pended in 60% glycerol in 0.01 M Tris-HCl, that described in Fig. 4 was carried out, pH 7.5 (2-4 mg of protein/ml) and stored at and after 30 min the membranes were pel- -20°C (data not shown). leted, washed, and resuspended in fresh ATPase assay medium to remove accumu- Cation Requirement Table I describes the divalent cation related ADP and Pi, and the time course of ATP hydrolysis was again determined quirement of the Neurospora plasma (data not shown). The rate of ATP hydrolmembrane ATPase. The enzyme is most

388

GENE TABLE

A. SCARBOROUGH

I

DIVALENT CATION REQUIREMENT OF THE Neurospora PLASMA MEMBRANE ATPase” Additions

Nanomoles of Pi liberated per milligram of protein per minute 11 312 319 166 114 43 26 14

None WSO, coso, MnSO, ZnSO, PeSO, CaC& cuso,

a Standard ATPase assay (0.091 mg of protein) with the indicated divalent cation at 10 mM. Incubations were carried out for 15 min. Entries indicate the average of duplicate determinations.

active with Mg2+ or Co2+ but significant activity is obtained with Mn2+ or Zn2+. Fe2+, Ca2+, and Cu2+ are much less effective. In a previous communication (7), an Na,K-stimulated Mg-ATPase activity in the Neurospora plasma membranes was reported. Further investigation of the Na,K-stimulated Mg-ATPase activity (data not shown) indicated that the observed Na,K stimulation is probably due to nonspecific salt stimulation of the MgATPase rather than the presence of a separate, specific Na,K-stimulated Mg-ATPase. Thus, if such an enzyme exists in the Neurospora plasma membranes it has not yet been detected. Nucleotide

Specificity

Table II summarizes the nucleotide specificity of the ATPase. The enzyme catalyzes the hydrolysis of ATP much more rapidly than any other nucleotide tested. Lipid

Requirement

In attempts to solubilize the plasma membrane ATPase with detergents, it was observed that treatment of the plasma membranes with 0.1% sodium deoxycholate led to a marked decline in the activity of the enzyme (60-90% inhibition). It was subsequently discovered that the deoxycholate inhibition could be reversed by the addition of certain acidic phospholipids to deoxycholate-treated membranes after removal of the excess deoxycholate. In ex-

periments of somewhat different design it was found that deoxycholate inhibition could be prevented when certain lipids, but not others, were included with the deoxycholate. Since aqueous dispersions of several of the lipids employed in these studies are easier to prepare in the presence of deoxycholate, the latter type of experiment was chosen to investigate the specificity of the lipid requirement. Table III summarizes the efficiency of various lipids in preventing the inhibitory effect of deoxycholate. Data in the table represent a compilation of the results obtained from 10 different experiments with various lipids. In each experiment, ATPase activity was measured in untreated, deoxycholate-treated, and deoxycholate- plus - lipidtreated membranes. Zero percent efflciency means that the lipid did not stimulate activity above the deoxycholate treated control. One hundred percent efficiency means that the activity was equal to the activity of untreated membranes. Values in excess of 100% indicate a stimulation of the ATPase activity to values greater than the untreated membranes. It can be seen that neutral lipids such as ergosterol and triolein do not prevent deoxycholate inhibition. Similarly, phosphatidylethanolamine and phosphatidylcholine are essentially inert. However, acidic phospholipids effectively prevent the deoxycholate inhibition of the ATPase activity in the following order of effectiveness: phosphatidylglycerol > monophosphoin@ TABLE

II

NUCLEOTIDE SPECIFICITY OF THE Neurospora PLASMA MEMBRANE ATPase” Nucleotide ATP GTP ITP UTP CTP ADP

Nanomoles of P, liberated per milligram of protein per minute 172 45 44 51 25 13

‘l Standard ATPase assay (0.085 mg of prot..eir/ with the indicated nucleotides (sodium salts, ad justed to pH 6.0 with Tris) at 10 mM each. Incub$, tions were carried out for 30 min. Entries indicadc / the average of dunlicate determinations.

Neurospora TABLE

crassa

PLASMA

III

EFFTCIENCY OF VARIOUS LIPIDS IN PREVENTING SODIUM DEOXYCHOLATE INHIBITION OF THE Neurospora PLASMA MEMBRANE ATPase”

Lipid added Folch fraction I Monophosphoinositide Diphosphoinositide Phosphatidylglycerol Phosphatidylserine (Su)’ Phosphatidylserine (Mi)” Phosphatidylserine (Se)’ Triphosphoinositide Phosphatidic acid Phosphatidylcholine Phosphatidylethanolamine Ergosterol Triolein

Percent efficiencyh 84; 123; 109; 94 141; 153; 138 84 166; 160 157; 119 103; 143; 102 79; 96 14; 14 54; 23 9; 3 0; 0 0; 0 0; 1

U Standard ATPase assay except that the order of addition of certain components was changed. Twenty microliters of plasma membrane suspension (approximately 0.05 mg of protein) was mixed with 20 ~1 of 0.05 M MgSO,, 20 ~1 of 0.1 M Tris-succinate, pH 6.0, and 20 ~1 of H20, or 20 ~1 of 0.5% (w/v) sodium deoxycholate, or 20 ,ul of the indicated lipid dispersions (10 mg/ml in 0.5% (w/v) sodium deoxycholate). The resulting mixtures were preincubated for 10 min at 3o”C, and the reactions were started by the addition of 20 ,ul of 0.05 M disodium ATP (adjusted to pH 6 with Tris). Incubations were carried out for 30 min. Subsequent steps were as described under Methods. Individual entries represent the average of duplicate determinations in separate experiments. ” See text. V Obtained from Supelco. ‘! Obtained from Miles. V Obtained from Serdary.

MEMBRANE

ATPase

389

for catalytic activity. Similar acidic phospholipid requirements have been reported for the Na,K-stimulated Mg-ATPases from a variety of cells (12). The acidic phospholipid requirement of the plasma membrane ATPase reported here may be related to the inositol lipid requirement for the function of the glucose active transport system in Neuropsora. It was previously demonstrated (13) that inosit01 deprivation in an inositol-requiring mutant leads to a marked decline in the activity of the glucose active transport system which-can be rapidly reversed by the addition of inositol. The active transport of glucose and glucose analogs by this system is thought to be driven via proton symport in response to the membrane potential (14) which is produced and maintained via ATP hydrolysis by the plasma membrane ATPase (l-4). Thus, inositol deprivation may led to decreased plasma membrane ATPase adtivity and a lower membrane potential, which could result in depression of the activity of the glucose active transport system.

Inhibitors Figure 5 demonstrates the effectiveness of several compounds as inhibitors of the plasma membrane ATPase, plotted as function of the inhibitor concentration. PCMB and PCMPS, the most effective inhibitors thus far tested, inhibit the enzyme about 70% at 10m3M. TCS and DNP inhibit the ATPase moderately at the concentrations tested. DCCD inhibits only 40% at 10e4M (89 nmol/mg of protein), a concensitide > phosphatidylserine > diphosphoi- tration much higher than that required for nositide > phosphatidic acid. Triphosphoimaximal inhibition of the bacterial and nositide is essentailly inactive. Folch frac- mitochondrial ATPases (15, 16). The Neution I is a crude mixture of lipids but does rospora plasma membrane ATPase is thus satisfy the acidic phospholipid require- relatively insensitive to inhibition by ment and thus may serve as an economical DCCD. The enzyme is also insensitive to source of lipid for certain applications. oligomycin, being inhibited only lo-20% at These findings are consistent with the levels of oligomcyin as high as 143 kglmg interpretation that deoxycholate extracts of protein (data not shown). This concenacidic phospholipids from the membranes tration of oligomycin is roughly two orders leading to an inactivation of the enzyme of magnitude higher than that needed to activity. From these results it is reasona- maximally inhibit the mitochondrial ATPble to speculate that the ATPase in situ is ase (17). Other compounds tested, includassociated with acidic phospholipids and ing NEM (10M3M), NaN, (1O-2M), NaCN that these lipids are somehow necessary (lo-” M), NaAsO, (lo-* M), NaF (10e3 M),

390

GENE A. SCARBOROUGH

FIG. 5. Concentration dependence of several inhibitors. Standard ATPase assay (0.112 mg of protein) with the inclusion of the various inhibitors at the indicated concentrations. Incubations were carried out for 15 min. TCS and DCCD were added as ethanolic solutions (1% (v/v) ethanol, final concentration in the incubation) and the values obtained were corrected for the slight inhibition by 1% ethanol alone. When DCCD, PCMB, and PCMPS were used, membranes were preincubated with the inhibitor for 5 min prior to initiating the assay by the addition of ATP. Points are the average of duplicate determinations. O-0, DCCD; O-U, PCMB; A-A, PCMPS; O-O, TCS; W-B, DNP.

CCCP (5 x lo-” M), and ouabain (5 x lo-” M) do not significantly effect the ATPase activity. Effects of Thiol

Reagents

As shown above, the organic mercurials PCMB and PCMPS markedly inhibit the Neurospora plasma membrane ATPase. An interesting feature of the inhibition by organic mercurials is the irreversibility of the reaction. Incubation of PCMPS- or PCMB-treated plasma membranes in the presence of p-mercaptoethanol (10 mM> for periods up to 1 h results in little restoration of ATPase activity (data not shown). Irreversible organic mercurial inhibition has also been observed for the Ca-dependent Mg-ATPase of sarcoplasmic membranes (18). In this case, the irreversible inhibitory effect of mercurials is apparently due to structural changes in the enzyme and/or membrane which occur as a result of the mercurial treatment. It is possible that the irreversible mercurial inhibition of the Neurospora plasma membrane ATPase can be similarly explained.

It is also of interest that although organic mercurials inhibit the Neurospora plasma membrane ATPase, NEM does not. One possible explanation for this finding is that although NEM does react with the mercurial-sensitive site(s), the ethyl succinimide derivative (from the NEM treatment) is not able to exert the same inhibitory effect as the mercurial derivative. If this explanation were correct, then pretreatment of the plasma membranes with NEM should render the enzyme insensitive to inhibition by mercurials. When this experiment was performed (data not shown), it was found that the ATPase activity in membranes pretreated with NEM was just as susceptible to inhibition by PCMPS as was the ATPase activity in control membranes. Thus, it is not likely that NEM is able to react with the same functional group(s) that PCMPS reacts with in the course of its inhibitory action. Another possible explanation for the inability of NBM to inhibit the ATPase is that the mercurial-sensitive site(s) is inaccessible in NEM. To investigate this possiblity, the effects of several other maleimides with different solubility properties were investigated. It was found that neither N-2naphthylmaleimide, N-benzylmaleimide, nor N-benzoylmaleimide significantly affect the enzyme activity at a concentration of lop3 M (data not shown). Thus, since these maleimides are similar in structure (N-benzoylmaleimide in particular) to PCMB and PCMPS, it does not seem likely that the mercurial sensitive site(s) is inaccessible to NEM. The above findings suggest that organic mercurials do not inhibit the Neurospora plasma membrane ATPase by reacting with free SH groups but rather with some other functional group(s). This possibility is presently under investigation. Since thiol reagent inhibition of the Na,K-stimulated Mg-ATPase from several sources (19-22) and the Ca-dependent MgATPase of sarcoplasmic membranes (23) can be partially suppressed in the presence of ATP, it was of interest to determine if ATP could suppress the inhibitory affects of organic mercurials on the Neurospora plasma membrane ATPase. To test this

Neurospora

crassa

PLASMA

possibility, the experiment described in Fig. 6 was carried out. This experiment shows the time course of PCMB inhibition at 10e4and 10e3M PCMB in the presence or absence of ATP. It can be seen that the inhibition brought about by PCMB at 10m4 M is largely suppressed in the presence of ATP. The ATP suppression of PCMB inhibition is obviously dependent upon the relative concentrations of ATP and PCMB since there is no protection by ATP when

5

10

15

minutes

FIG. 6. Protection against PCMB inactivation by ATP. Plasma membrane ghosts derived from a 500ml culture of cells were suspended in 4.2 ml of 0.1 M Tris-HCl, pH 7.5 at RT and divided into five 0.8-ml portions, designated A-E. The membrane suspensions then received the following additions: A, O-O, 0.2 ml of H,O; B, W---m, 0.1 ml of H,O and then 0.1 ml of 10 mM PCMB (adjusted to pH 7.5 with Tris); C, O-O, 0.1 ml of 0.1 M ATP (adjusted to pH 7.5 with Tris) and then 0.1 ml of 10 mM PCMB; D, A-A, 0.1 ml of H,O and then 0.1 ml of 1 mM PCMB; E, O-O, 0.1 ml of 0.1 M ATP and then 0.1 ml of 1 mM PCMB. At 5-min intervals, 0.3-ml aliquots from each incubation were transferred to tubes containing an equal volume of 1 mM p-mercaptoethanol, allowed to stand for 1 min, and chilled. The membranes were then pelleted by centrifugation (7OOg for 3 min), resuspended in 1 ml of ice-cold 0.01 M Tris-HCl, pH 7.5, centrifuged again, resuspended in 0.1 ml of 0.01 M Tris-HCl pH 7.5, and assayed for ATPase activity as described in Methods. Individual assays contained 0.022-0.027 mg of protein. Incubations were carried out for 30 min. Points indicate the average of duplicate determinations. The zero time point was obtained by extrapolation of the control points to zero time.

MEMBRANE

ATPase

391

PCMB is present at a lo-fold higher concentration. ATP protection against PCMB inhibition should be of value in future attempts to covalently label and identify the ATPase . The irreversible PCMPS inhibition of the ATPase and the membrane impermeant nature of PCMPS (24) can be exploited to gain information concerning the orientation of the organic mercurial-sensitive site(s) of the ATPase in the Neurospora plasma membrane. With this in mind, the following experiment was carried out. Intact sl cells were treated with PCMPS (10e3 M) in the presence and absence of lo+ M NaN, (which depresses cellular ATP levels and thus obviates the possible protective effect of intracellular ATP), and the plasma membranes subsequently isolated and assayed for ATPase activity. Plasma membranes were also isolated from untreated cells for comparison. The ATPase activity in the plasma membranes isolated from the PCMPS-treated cells (with or without NaN,) was identical to the ATPase activity in plasma membranes derived from control cells. Thus, it can be concluded that the organic mercurial sensitive site(s) in the ATPase is not accessible to PCMPS from the outer surface of the plasma membrane. The Effect of Trypsin

It has been demonstrated that treatment of the Na,K-stimulated Mg-ATPase from kidney (25) and the Ca-dependent Mg-ATPase from sarcoplasmic reticulum (26) with trypsin leads to an inactivation of these enzymes and that the trypsin inactivation in both cases can be suppressed by ATP. Thus, it was of interest to examine the effect of trypsin on the Neurospora plasma membrane ATPase. The experiment presented in Fig. 7 shows the effect of trypsin treatment as a function of time under several different conditions. The data in the figure demonstrate that trypsin inactivates the enzyme (approximately 70%), that ATP (20 mM> alone partly suppresses the trypsin inactivation, and that ATP (20 mM) plus Mg2+ (20 mM completely suppresses trypsin inactivation of the ATPase activity. Mg2+ alone has no effect

392

GENE

A. SCARBOROUGH

NaN,) had ATPase activity almost identical to that of control cells; thus the trypsinsensitive site(s) of the Neurospora plasma membrane ATPase is not accessible to trypsin from the outer surface of the plasma membrane. Similar results have been reported for the Na,K-dependent MgATPase in erythrocyte plasma membranes cm. minutes

7. Protection by Mg-ATP against trypsin inactivation. Plasma membrane ghosts derived from a 500-ml culture of cells were suspended in 4.2 ml of 0.01 M Tris-HCl, pH 7.5 at RT. A small amount of the membrane suspension was placed on ice for a control and the remainder was divided into five 0.8ml portions, designated A-E. The membrane suspensions then received the following additions: A, O-O, none; B, m---m, 20 ~1 of TPCK trypsin (4 mg/ml); C, A-A, 40 ~1 of 0.4 M ATP (adjusted to pH 7.5 with Tris), then 20 ~1 of TPCK trypsin (4 mgl ml); D, O-0, 40 ~1 of 0.4 M MgS04, then 20 ~1 of TPCK trypsin (4 mg/ml); and E, O-O, 40 ~1 of 0.4 M ATP, 40 ~1 of 0.4 M MgSO,, then 20 ~1 of TPCK trypsin (4 mglml). At 15-min intervals, 0.4-ml aliquots from each incubation were transferred to tubes containing 0.1 ml of trypsin inhibitor solution (1 mg/ml) and chilled, and the resulting membrane preparations were assayed for ATPase activity as described under Methods. Individual assays contained 0.041-0.050 mg of ghost protein. Incubations were carried out for 30 min. Poims indicate the average of duplicate determinations. FIG.

Solubilization

Attempts

A variety of agents have been employed in attempts to solubilize the Neurospora plasma membrane ATPase, including: extraction with several concentrations of the detergents sodium deoxycholate, Triton X100, and cetyltrimethylammonium bromide; Lubrol WX extraction according to the method of Hokin et al. (27); extraction with the chaotropic agents lithium diiodosalicylate (0.3 M), sodium iodide (1 M), and urea (8 M); alkali extraction with 0.25 M sodium carbonate; extraction with EDTA solutions; sonication; repeated washing in buffers of low ionic strength according to the method of Abrams (28); aqueous pyridine extraction by the method of Blumenfeld (29); and enzyme digestion with trypsin and chymotrypsin. None of the abovementioned procedures have proved successful for solubilizing the Neurospora plasma membrane ATPase in an active form.

on the trypsin inactivation. These results CONCLUSION may indicate conformational changes in As mentioned in the introduction, rethe ATPase molecule upon binding and/or hydrolysis of ATP, which is of interest in cent studies with intact cells (l-3) and isolight of the role of this enzyme as an elec- lated plasma membrane vesicles (4) have demonstrated that the Neurospora plasma trogenic pump. In order to determine the orientation of membrane ATPase is an electrogenic the trypsin-sensitive site(s) of the ATPase pump. ATP hydrolysis catalyzed by the Neurosporu plasma membrane ATPase in the plasma membrane, an experiment similar in design to that described above leads to the generation of a transmemfor the localization of the organic mercu- brane electrical potential in uiuo and in rial sensitive site(s) was carried out. Intact vitro. As part of an overall plan to elucicells were treated with trypsin (0.1 mg/ml) date the molecular mechanism by which in the presence or absence of NaN, (1O--2M) the Neurospora plasma membrane ATPand the plasma membranes were subse- ase generates a membrane potential, the quently isolated and assayed for ATPase present studies were carried out to characactivity. Plasma membranes were also iso- terize the biochemical properties of this lated from untreated cells for comparison. enzyme in its membrane bound state. The plasma membranes isolated from Hopefully, knowledge of the properties of this enzyme as it exists in the plasma trypsin-treated cells (with or without

Neurosporu

crassa

PLASMA

membrane will facilitate progress toward isolation and purification of the enzyme in an active form and reconstitution of its physiological activity in a defined system. ACKNOWLEDGMENT The author would like to acknowledge the technical assistance of Ms. Randa Grisham and Mrs. Esperanza Alvarez. REFERENCES 1. SLAYMAN, C. L., Lu, C. Y.-H., AND SHANE, L. (1970) Nature (London) 226, 274-276. 2. SLAYMAN, C. L., LONG, W. S., AND Lu, C. Y.-H. (1973) J. Memb. Biol. 14, 305-338. 3. SLAYMAN, C. L., AND GRADMANN, D. (1975) Biophys.J. 15, 968-971. 4. SCARBOROUGH, G. A. (1976) Proc. Nat. Acud. Sci. USA 73, 1485-1488. 5. HENDRICKSON, H. S., AND BALLOU, C. E. (1964) J. Biol. Chem. 239, 1369-1373. 6. MICHELL, R. H., HAWTHORNE, J. N., COLEMAN, R., AND KARNOVSKY, M. L. (1970) Biochim. Biophys. Acta 210, 86-91. 7. SCARBOROUGH, G. A. (1975) J. Biol. Chem. 250, 1106-1111. 8. STANTON, M. G. (1968)Anal. Biochem. 22,27-34. 9. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951)J. Biol. Chem. 193, 265-275. 10. PATTERSON, M. S., AND GREENE, R. C. (1965) Anal. Chem. 37, 854-857. 11. MORRISON, J. F. (1968)Anal. Biochem. 24, 106111. 12. DAHL, J. L., AND HOKIN, L. E. (1974)Ann. Reu. Biochem. 43, 327-356. 13. SCARBOROUGH, G. A. (1971) B&hem. Biophys.

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Res. Commun. 43, 968-975. 14. SLAYMAN, C. L., AND SLAYMAN, C. W. (1974) Proc. Nat. Acad. Sci. USA 71, 1935-1939. 15. HAROLD, F. M., BAARDA, J. R., BARON, C., AND ABRAMS, A. (1969) J. Biol. Chem. 244, 22612268. 16. BULOS, B., AND RACKER, E. (196815. Biol. Chem. 243,3891-3900. 17. VAN GRONINGEN, H. E. M., AND SLATER, E. C. (1963) Biochim. Biophys. Actu 73, 530-532. 18. HASSELBACH, W. (1974) in The Enzymes (Boyer, P. D., ed.), Vol. 10, pp. 431-467, Academic Press, New York/London. 19. SKOU, J. C., AND HILBERG, C. (1965) Biochim. Biophys. Acta 110, 359-369. 20. SKOU, J. C. (1974) Biochim. Biophys. Actu 339, 234-245. 21. PATZELT-WENCZLER, R., PAULS, H., ERDMANN, E., AND SCHONER, W. (1975)Eur. J. Biochem. 53, 301-311. 22. FAHN, S., HURLEY, M. R., KOVAL, G. J., AND ALBERS, R. W. (1966) J. Biol. Chem. 241,18901895. 23. PANET, R., AND SELINGER, Z. (1970) Eur. J. Biothem .14,440-444. 24. VANSTEVENINCK, J., WEED, R. I., AND ROTHSTEIN, A. (1965) J. Gen. Physiol. 48, 617-632. 25. GIOTTA, G. J. (1975) J. Biol. Chem. 250, 51595164. 26. STEWART, P. S., AND MACLENNAN, D. H. (1974) J. Biol. Chem. 249, 985-993. 27. HOKIN, L. E., DAHL, J. L., DEUPREE, J. D., DIXON, J. F., HACKNEY, J. F., AND PERDUE, J. F. (1973) J. Biol. Chem. 248, 2593-2605. 28. ABRAMS, A. (1965) J. Biol. Chem. 240, 36753681. 29. BLUMENFELD, 0. 0. (1968) Biochem. Biophys. Res. Commun. 30, 200-205.

Properties of Neurospora crassa plasma membrane ATPase.

ARCHIVES OF BIOCHEMISTRY Properties AND BIOPHYSICS of the Neurospora 180, 384-393 (1977) crassa Plasma Membrane ATPasel GENE A. SCARBOROU...
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