Neurochern. Int. Vol. 11, No. 4, pp. 407-415, 1987 0197-0186/87 $3.00 +0.00 Printed in Great Britain. All rights reserved © 1987 Pergamon Journals Ltd

DRUG MODIFICATION OF PROTEIN THIOPHOS- PHORYLATION IN PERMEABILIZED CHROMAFFIN CELLS

J. C. BROOKS* and M. BROOKS Marquette University School of Dentistry, Milwaukee, WI 53233, U.S.A.

(Received 4 May 1987; accepted 5 June 1987)

Al~tract--Treatment of permeabilized chromaffin cells with low concentrations of the ATP analog adenosine-5'-O-(3-thiotriphosphate)[35S] results in 35S incorporation into a small number of cellular proteins. Of these proteins, a 47 kilodalton protein is most heavily thiophosphorylated. Permcabilized cells were treated with various drugs known to influence cell functions, more specifically chromatlin granule function, to determine the kinase responsible for thiophosphorylation of the 47 kilodalton protein and if its thiophosphorylation is associated with a specific cell function.

Several drugs which influence the activity of cell kinases were examined for their effect on secretion and thiophosphorylation of the 47 kilodalton protein. There was no qualitative effect of cAMP, cGMP or trifluoperazine on thiophosphorylation of the protein. Both cyclic nucleotides slightly enhanced secretion, while trifluoperazine enhanced only unstimulated catecholamine release. Phorbol 12-myristate 13-acetate had no effect on secretion or 35S incorporation into cell proteins. Only the free calcium concentration of the medium influenced thiophosphorylation of the 47 kilodalton protein, with increased calcium producing increased thiophosphorylation.

Drugs affecting chromaffin vesicle functions were used to assess the relationship between specific functions and thiophosphorylation of the protein. Inhibition of nucleotide translocation with atractyloside or 4,4'diisothiocyanatostilbene-2,2'disulfonic acid or inhibition of the proton translocating ATPase by N-ethylmaleimide inhibited thiophosphorylation of the 47 kilodalton protein, with little effect on secretion. Treatment with rotenone markedly enhanced secretion and thiophosphorylation of the protein. Calcium ionophores had no effect on thiophosphorylation of the protein. Dichloroacetic acid, which inhibits phosphorylation of mitochondrial pyruvate dehydrogenase, had no effect on secretion and a variable effect on thiophosphorylation of the 47 kilodalton protein. The data suggest that thio- phosphorylation of the protein may be associated with nucleotide translocation across the vesicle membrane.

We have used the saponin-permeabilized chromaffin cell as a model system for studying the role o f protein phosphorylat ion in secretion. Use of permeabilized cells allows direct access of exogenous nucleotides and nucleotide analogs to the cellular secretory appa- ratus. Such cells have been treated with the A T P analog adenosine-5'-O-(3-thiotriphosphate) (ATP? S) to lock phosphorylat ion dependent reactions in the thiophosphorylated state. Various aspects of the secretory process can then be evaluated for a re- lationship to the phosphorylat ion reaction. Thio- phosphorylation o f chromaffin cells with ATPyS

*Address correspondence to: Dr Jack C. Brooks, De- partment of Basic Sciences, Marquette University School of Dentistry, 604 North 16th Street, Milwaukee, WI 53233, U.S.A.

N.C.I. 11/4~D

irreversibly inhibits catecholamine secretion (Brooks et al., 1984).

If permeabilized cells are treated with trace levels of [~-asS]ATP, label is incorporated into a small number of cellular proteins (Brooks and Brooks, 1985). Predominant among these is a 47ki lodal ton (kDa) protein. A causal relationship between the secretory inhibition and thiophosphorylat ion has not been established. An effort has been made here to determine the kinase responsible for thio- phosphorylat ion of the 47 kDa protein and if thio- phospborylat ion is related to chromatfin cell secre- tion. Since this would presumably involve chromaffin vesicles, we have examined a variety of drugs which are known to influence chromaffin vesicle functions. Several of the drugs influence thiophosphorylation of the 47 kDa protein but have little effect on cate-

407

408 J .C. BROOKS and M. BROOKS

cholamine secretion. The data suggest that thio- phosphorylat ion of this protein is influenced by fac- tors which affect the translocation of ATP across the

vesicle membrane.

EXPERIMENTAL PROCEDURES

Materials All reagents were analytical grade. Culture media and

antibiotics were purchased from GIBCO (Grand Island, N.Y.). Cytosine arabinoside, 5-fluorodeoxyuridine, ethyleneglycolbis-(amino-ethyl ether) N,N'-tetraacetic acid (EGTA), cyclic nucleotides, 4-phorbol 12-myristate 13-acetate (TPA), epinephrine and norepinephrine bit- artrates, MgATP, atractyloside, rotenone, N,N'-dichloro- hexylcarbodiimide (DCCD), 4-acetamido-4'isothiocyanato- stilbene-2,2'-disulfonic acid (SITS) 4,4'diisothiocyanato- stilbene-2,2'disulfonic acid (DIDS), and palmitoyl- Coenzyme A were obtained from Sigma Chemical Co. (St. Louis, Mo.). Piperazine-N,N'-bis (2-ethanesulfonic acid) (PIPES), glutamic acid and N-ethylmaleimide (NEM) were obtained from Aldrich Chemical Co. (Milwaukee, Wis). Saponin and culture plates were obtained from Fisher Scientific (Itaska, I11). b,-3~S]ATP, specific activity I136 Ci/mmol, was purchased from New England Nuclear, Inc. (Boston, Mass.). Fluoro-Hance was purchased from Research Products International (Mt. Prospect, ILL). The calcium ionophores A 23187 and X 537A were gifts from Eli Lilly Company (Indianapolis, Ind.). and Hoffmann-La Roche (Nutly, N.J.), respectively.

Analytical methods Total catecholamines secreted to the medium by the cells

were determined fluorometricaUy (Brooks and Treml, 1983). The catecholamine content of individual samples was deter- mined from a standard curve relating relative fluorescence to known concentrations of a 1:1 mixture of norepinephrine and epinephrine. Data are expressed as ng catecholamine/2 × 105 ceils. Statistical analysis was per- formed by one-way ANOVA on a VAX 11/780 computer at a significance level of P < 0.01.

For electrophoresis experiments, the monolayers were dissolved directly in the sample buffer used for sodium dodecyl sulfate electrophoresis (SDS-PAGE) in 10% gels (Brooks et al., 1985). Proteins containing 35 S-thiophosphate were detected by fluorography on Kodak X-Omat AR film after treatment of the stained gels with Fluoro-Hance. In some instances, radiolabelled bands identified by fluorography were cut from dried gels, solubilized for 2 h at 50°C in 0.5 ml of a 9:1 (v/v) mixture of NCS and water and counted in 7 ml of OCS (Amersham Corp., Arlington Heights, Ill.). Similar pieces of gel from non-radiolabelled regions were used for background determinations. The molecular weight of the separated proteins was determined from a curve relating log molecular weight to migration distance for protein standards of known molecular weight.

General experimental protocol Bovine chromatfin ceils were prepared and maintained in

primary culture as described previously (Brooks and Treml, 1983). Isolated cells were suspended at 106 cells/ml in culture medium and plated at 200,000 cells/well in 96 well culture plates (Falcon, 3070). The medium was changed at intervals of 4 days and the cells were used between 7 10 days in

culture. Cytosine arabinoside (10 -5 M) was present in the medium to inhibit fibroblast growth (Keningsberg and Trifaro, 1980). Cell monolayers to be used for experiments were washed with 0.2 ml/well of Lockes medium for three 5 min time periods and permeabilized by a brief exposure to 0.01% saponin in potassium glutamate (KG) medium, pH 6.66 (Brooks and Treml, 1983). The composition of the medium was (mM): potassium glutamate, 140; EGTA, 0.4; PIPES, 20; MgATP, 2; MgCI, 2; and glucose, 5. This was followed by three rapid washes with KG medium free of saponin and ATP. All experimental treatments were carried out in KG medium free of saponin and supplemented with ATP, [y-35S]ATP, calcium or drugs as required by the experiment. The catecholamine content of permeabilized cells was about 2,600ng/2 x 105 cells. In all instances, control cells received medium without calcium and stimu- lated cells medium plus 0.5 mM added calcium. Free cal- cium was calculated to be 100 ~M in the absence of added ATP and 30/~M in the presence of 2 mM MgATP (Brooks and Treml, 1983). When required, drugs were administered in dimethyl sulfoxide (DMSO) to a final concentration of 0.5%. Concentrations of DMSO up to 1% had no effect on catecholamine secretion or thiophosphorylation of the cells.

RESULTS

Cyclic nucleotides or drugs known to influence the activity of kinases were chosen to correspond to the major categories of kinases described by Nestler and Greengard (1984): a. cAMP-dependent protein

kinase; b. cGMP-dependen t protein kinase; c. calcium/calmodulin-dependent protein kinases; and d. protein kinase-C. The cyclic nucleotides, c A M P and c G M P enhanced both unstimulated and calcium- stimulated secretion (Table 1). TFP, an inhibitor of

Table I. Effect of drugs which influence kinases on catecholamine secretion by saponin-permeabilized chromaffin cells

Catecholamine secretion (ng/2 × 105 cells)

Treatment Control Stimulated*

No drug 79.5 + 3.0t 249.0 + 8.0 cAMP 122.0 _+ 7.5 314.9 _+ 11.92; cGMP 129.3 _+ 5.1 299.9 + 5.8:~ TFP I 17.5 + 4.2 223.3 + 9.4

No drug 81.8 _+ 1.1 209.0 + 7.7 TPA 95.8 _ 5.2 216.0 _+ 8.7

*All stimulated groups are significantly different from their re- spective control groups at P < 0.01.

tAll drug control groups, except that for TPA, are significantly different from the group "control" at p < 0.01.

~Signiflcantly different from the group "stimulated, no drug" at P < 0.01.

Permeabilized cells were treated for 20 min at 37'C with 0. I ml/well of KG medium containing 10.uCi/ml of D,-35SlATP and the indicated drugs at 10/IM concentration. The media were recovered for the determination of secreted catecholamines. The experiment with TPA was done separately from the other drugs. For this experiment, TPA was presented to the cells in DMSO to a final concentration of 0.5%; cells not treated with TPA received DMSO to the same final concentration. Each table is the mean + SEM for 5 replicates.

Drugs and thiophosphorylation 409

47' kDa - -

D rugs

cAMP -I- -I-

cGMP + +

T F P -I- +

Co z+ + -I- -I- -t-

Fig. 1. Fluorogram of SDS-PAGE showing the effect of cyclic nucleotides and TFP on thio- phosphorylation of the 47 kDa protein. Permeabilized cells were treated for 20 rain with 0.1 ml/well of KG medium containing 10 # Ci/ml of b' - 35 S]ATP and the drugs at 10 # M concentration. The" +" beneath

the lanes on the gel indicates the presence of a component in the medium.

calcium/calmodulin-dependent phosphorylation in a ~ariety of cellular systems (for reviews, see Public- over, 1985, Browning et al., 1985), increased un- stimulated secretion but had no effect on stimulated secretion. TPA had no effect on secretion (Table 1).

Neither the cyclic nucleotides nor TFP had any qualitative influence upon thiophosphorylation of the 47 kDa protein (Fig. 1), although some other proteins were affected. The only factor which affected thio- phospborylation of the 47 kDa protein was the cal- cium concentration of the medium. The ~5S incorpo- ration increased with calcium concentration from 0 to 0.4raM and then remained constant with calcium concentrations up to 1.0 mM (Fig. 2).

Drugs were selected for established effects on known chromaflin vesicle functions. In general, these included drugs known to inhibit the proton trans- locating ATPase, nucleotide transport or calcium transport across the chromaffin vesicle membrane. The effect of drugs which influence the proton trans-

locating ATPase is shown in Fig. 3. N-ethyl- maleimide (NEM) inhibits the proton translocating ATPase of the chromaffin vesicle, and secondarily, nucleotide, catecholamine and calcium translocation across the chromaffin vesicle membrane (Kostron et al., 1977; Carmichael et al., 1980; Aberer et al., 1978). NEM inhibited thiophosphorylation of the 47 kDa protein. It also increased unstimulated catecholamine secretion, but had no effect on calcium-stimulated secretion. The effect of the drugs tested on secretion is shown in Table 2. DCCD, another inhibitor of the ATPase (Cidon and Nelson, 1983; Burger et al., 1984) had no effect on thiophosphorylation of the protein (Fig. 3) or secretion (not shown). Inhibition of the chromaffin vesicle nucleotide translocation system by 0.2mM atractyloside (Kostron et aL, 1977) and 10ttM DIDS (Weber et al., 1983)inhibited thio- phosphorylation of the protein (Figs 4 and 3, re- spectively). The less potent nucleotide translocase inhibitor SITS (Capasso and Hirschberg, 1984) had

410 J.C. BROOKS and M. BROOKS

47 kDa --

CaLcium :

Added (p.M) 0 0.2 0.4 0.6 0.8 1.0

Free (nM) 0 1.0 19.6 200 400 600

Fig. 2. Fluorogram from SDS-PAGE of permeabilized cells exposed to various concentrations of calcium. Cells were exposed for 20 min at 37°C to 0.1 ml/well of KG medium containing I0 #Ci/ml of [~,:SS]ATP and various calcium concentrations. The total medium calcium concentration and calculated free calcium

are indicated below the lanes on the gel.

no effect on thiophosphorylation of the 47 kDa pro- tein at 10 #M concentration (Fig. 3). Atractyloside inhibited thiophosphorylation at concentrations as low as 20#M (data not shown). Atractyloside slightly increased unstimulated secretion, but had no effect on calcium-stimulated secretion (Table 2). An- other inhibitor of nucleotide translocation, palmitoyl Coenzyme A (Woldegiorgis et al., 1982), had no effect on secretion or protein thiophosphorylation (Fig. 4). Inhibition of calcium transport into the chromaflin vesicle by rotenone (Niedermaier and Burger, 1981) markedly increased thiophosphorylation of the pro- tein and increased both unstimulated and calcium- stimulated secretion (Fig. 4). In contrast to the effect of rotenone, the calcium ionophores A23187 and X-537A had no effect on thiophosphorylation of the protein (not shown). A23187 increased unstimulated secretion, while X 537A caused a large, probably non-physiological release of catecholamines from both unstimulated and calcium-stimulated cells (Table 2).

There is a 40-43 kDa phosphoprotein in mito- chondria, the alpha subunit of pyruvate dehy- drogenase, that is rapidly phosphorylated upon stim-

ulation of brain tissue (Browning et al., 1981). Because its molecular weight is similar to that of the 47 kDa thiophosphoprotein, it seemed possible that the two proteins might be the same. Phosphorylation of the mitochondrial protein is 90% inhibited by 1 mM dichloroacetic acid (DCA) (Yang and Smith, 1983). This concentration of DCA had no effect on catecholamine secretion (Table 2). The drug had a variable effect on thiophosphorylation of the 47 kDa protein, ranging from 0 to 40% inhibition, as deter- mined by counting bands cut from solubilized gels. Another inhibitor, fluphenazine (Dunkley and Robinson, 1983) inhibited calcium-stimulated secre- tion (Table 2) and nearly doubled thio- phosphorylation of the 47 kDa protein (not shown).

DISCUSSION

We have used drugs affecting chromaflin vesicle functions in an effort to relate changes in the thio- phosphorylation state of the 47 kDa protein to mod- ulation of these functions. In this way, it should help identify the functional system containing the protein.

Drugs and thiophosphorylation 411

4 " / k D a - ~ ~

Drugs - _ ~

NEM d- +

S I T S + +

D IDS + +

DCCD

Ca 2+ + + + +

+

+

Fig. 3. Fluorogram of SDS-PAGE demonstrating the effect of drugs which influence the proton translocating ATPase on thiophosphorylation of the 47 kDa protein. Permeabilized cells were exposed for 20 min at 37°C to 0.1 ml/well of KG medium containing 10 t~ Ci/ml of [~,-35 S]ATP and the indicated drugs at 10/~M concentration. Drugs were freshly prepared in DMSO and added to a final DMSO concentration of 0.5%; cells not receiving drugs received only DMSO. The " + " beneath each lane indicates the presence of a component in the medium. The fluorogram is slightly overexposed to demonstrate the lighter bands.

It is difficult to distinguish with certainty between chromaffin vesicle and mitochondrial functions with any of the drugs used, since none are totally specific in their actions. We have been concerned that the 47kDa protein could be the 40-43kDa phos- phorylated subunit of pyruvate dehydrogenase, which has been identified in chromaffin vesicle (Burgoyne and Giesow, 1982) and synaptic mem- brane preparations (Magilen et al., 1981; Dunkley and Robinson, 1983). This protein is rapidly phos- phorylated in stimulated brain tissue (Browning et al., 1981). Our overall data indicate that the proteins are different. Phosphorylation of the mitochondrial protein is inhibited by calcium, rotenone, DCA, fluphenazine, and TFP, and is increased by cAMP (Browning et al., 1981; Burgoyne and Giesow, 1982; Sieghart, 1981; Yang and Smith, 1983). All of these agents, except DCA (variable effect) and cAMP (no

effect), stimulated thiophosphorylation of the 47 kDa protein. Also, atractyloside dramatically increases phosphorylation of the mitochondrial protein (Sieghart, 1983) while it abolishes thiophosphoryla- tion of the 47 kDa protein. While phosphorylation and thiophosphorylation of the two protein are being compared, it appears that the 47 kDa protein is different from the alpha subunit of mitochondrial pyruvate dehydrogenase.

We could find no evidence that thio- phosphorylation of the 47 kDa protein was mediated by any of the well Characterized kinases. Incubation of permeabilized cells with various concentrations of cAMP, cGMP, trifluoperazine, and phorbol ester showed no changes in the extent of thio- phosphorylation of the protein; thiophosphorylation depended only on the calcium concentration of the medium. The lack of clear evidence for a specific

412 J.C. BROOKS and M. BROOKS

Table 2. Effect of drug treatments on catecholamine secretion by permeabilized ceils

Catecholamine Secretion (ng/2 × l0 s cells)

Drug Control Stimulated*

N-Ethylmaleimide ( 1 mM) None 138.7 ± 4.9 394.9 +± 1.3 N-Ethylmaleimide 212.3 + 11.9t 357.4 ± 13,4

Atractyloside (0.2mM) None 81.8 + 1.1 209.0 ___ 7.7 Atractyloside 104.8 _+ 5.6t 190.8 ± 2,3

SITS (IOltM); DIDS (lOpM) None 95.8 _+ 6.4 220,7 ± 14.0 SITS 113.5 + 5.7 203.9 _+ 14.8 DIDS 99.0 _+ 8.9 244.0 + 12.0

Palmitoyl coenzyme A ( lO,uM) None 100.7 ± 3.9 182.5 ± 7.7 Palmitoyl Coenzyme A 94,0 _+ 4.9 191.0 ± 21.0

Rotenone (IO#M) None 96.5 + 4.8 163,6 ± 9.7 Rotenone 159.5 + 2.9t 417.3 + 17.5~

Calcium ionophores : A 23187 ( 31~g /ml); X 537/1 (31tg/ml)

None 128.3 + 6.9 252.3 + 10,8 A 23187 212.3 + 8.3t 298.9 ± 14,0 X 537A 1498,7 ±41 .4 t 1320.1 + 32.8:~

Dichloroacetic acid ( 1 raM); Fluphenazine (0.2raM)

None 99.0 _+ 3.2 184,3 _+ 3,7 Dichloroacetic acid 96.2 _+ 2.9 187.6 ± 9,0 Fluphenazine 101,7 + 8,0 110.0 + 6.8~

*All stimulated groups were significantly different from their respective control groups at P < 0.01, except for X 537A and fluphenazine.

tSignificantly different from the respective control group "None" at P < 0.01. :~Significantly different from the respective stimulated group at P < 0.01.

Permeabilized ceils were incubated for 20rain at 37"C with 0.1 ml/well of KG medium containing the drugs at the concentrations indicated in the table. For stimulated cells, the medium contained 0.5 mM added CaCI 2. Solutions of NEM, DCA and fluphenazine were prepared in K G medium; controls received only medium. All other drugs were freshly prepared in DMSO and added to a final DMSO concentration of 0.5%. The groups not receiving drugs received an equivalent concentration of DMSO. This concentration of DMSO had no effect on either catecholamine secretion or thiophosphorylation. At the end of the incubation period, the medium was recovered for catecholamine determination.

kinase involvement in thiophosphorylation of the protein, except for a possible undefined calcium- dependent kinase, suggested that the protein might be thiophosphorylated by a non-kinase mediated mech- anism. Other data indicate that the protein might be part of an ATPase enzyme, such as the proton translocating ATPase, or the nucleotide translocator known to be present in the chromaffin vesicle mem- brane (Kostron et al., 1977) and cholinergic synaptic vesicle (Stadler and Fenwick, 1983). Perhaps a phos- phointermediate not normally evident is now locked in the thiophosphorylated state. Several drugs were selected to evaluate the~ possibilities. The drugs were chosen for known effects on specific functions. How- ever, none were completely specific in their actions and may have unrecognized effects on other func- tions. Inhibition of the proton translocating ATPase with NEM inhibited thiophosphorylation of the pro- tein and slightly increased unstimulated cate- cholamine secretion. This result implicates an essen-

tial sulfhydryl group in thiophosphorylation of the protein, although it does not necessarily have to be on the 47 kDa protein. Atractyloside and palmitoyl Coenzyme A inhibit the mitochondrial nucleotide translocator and have been used to isolate the protein (Woldegiorgis et al., 1982). In chromaffin cells, atractyloside inhibits the chromaffin vesicle nucleo- tide translocator (Kostron et al., 1977), ATP- stimulated calcium transport into chromaffin vesicles (Burger, 1984) and transport of adenosine T-phosphate 5'-phosphosulfate (PAPS) into the Golgi apparatus (Lee et al., 1984). The PAPS carrier protein and the mitochondrial translocator protein are about 34 kDa in size, considerably smaller than the 47 kDa thiophosphoprotein. The cholinergic syn- aptic vesicle protein (Stadler and Fenwick, 1983) is also smaller, about 30 kDa molecular weight. There is a discrepancy concerning the molecular weight of the chromaffin vesicle nucleotide translocator. Taugner and Wunderlich (1981) have reported a

Drugs and thiophosphorylation 413

4 7 k O a - ~ ~ ~

P a t m i t o y L Co. A -I- -I-

A t roc t yLos ide -I- -l-

Ro tenone -I- -I-

Ca z+ -I- + -I- +

Fig. 4. Effect of drugs which influence vesicular nucteotide and calcium transl0cation on thio- phosphorylation of the 47 kDa protein. The cells were exposed for 20 rain at 37°C to KG medium containing 10/,Ci/ml of D,-35S]ATP and drugs at the following concentrations: Palmitoyl Coenzyme A, 1/~M; Atraetyloside, 0.2 mM; and, Rotenone, 10/~M. The drugs were freshly prepared and administered in DMSO. All groups received DMSO at a final concentration of 0.5%. After treatment, the monolayers

were subjected to SDS-PAGE and fluorography.

molecular weight of 47 kDa based upon gel filtration measurements. Winkler et al. (1986), using an anti- body against the mitochondrial nucleotide trans- locator, have shown binding of the antibody to a 70 kDa component of the granule membrane. The discrepant molecular weights does not support the 47 kDa protein as the nucleotide translocator. How- ever, it does not eliminate the protein as a possible subunit of such a translocator. The 47 kDa protein is probably membraneous since it is not extracted into aqueous media or organic solvents from permeabilized cells and sediments with chromattin granules prepared from the cells (unpublished data). Therefore, the protein appears to at least be in a physical location where it could be a translocator or have its phosphorylation state controlled by such a

translocator. The chromaffin vesicle nucleotide trans- locator is dependent upon sulfhydryl groups for phosphoryl group transferase activity (Taugner and Wunderlich, 1981). There is no evidence that it exists in a phosphointermediate form. Another known transporter of the chromaffin granule, the cate- cholamine transporter, is 45 kDa (Gabizon and Schuldiner, 1985); however, its activity is inhibited by DCCD and is unaffected by atractyloside.

Most of the data can be explained as a result of inhibition of a translocation system or decreased ATP (or ATPv S) availability to a compartment such as the chromafiin vesicle. The simplest explanation of the data is that the 47 kDa protein might be a phosphointermediate of an ATPase or translocase that is evident only because it is locked in the

414 J .C. BROOKS and M. BROOKS

thiophosphorylated state during thiophosphoryl transferase activity. Alternatively, thiophos- phorylation of the 47 kDa protein could depend upon its compartmentalization within the cell. It could be unavailable as a substrate when ATP~ S is prevented from reaching the compartment due to blockade of an ATP translocator with atractyloside. N E M in- hibits ATP uptake into vesicles, as does atractyloside. Direct inhibition of the proton transtocating ATPase by N E M could secondarily inhibit ATP utilization, and thus ATP translocation, to produce the same effect. Since calcium uptake into vesicles is also dependent upon ATP translocation, the result of N E M and atractyloside treatments could be loss of a calcium uptake essential to thiophosphorylation of the 47 kDa protein. However, neither the enhanced thiophosphorylation due to rotenone nor the lack of effect of calcium ionophores supports this inter- pretation.

The failure of atractyloside or N E M to inhibit catecholamine secretion, while inhibiting thio- phosphorylation of the 47 kDa protein, does not appear to support a direct role for the 47 kDa protein in control of the secretory response. The present results suggest that the protein may be involved in nucleotide translocation across the chromaffin vesicle membrane or be a substrate in a phosphorylation reaction directly dependent upon such translocation.

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