Characterization of native and reconstituted hydrogen ion pumping adenosinetriphosphatase of chromaffin granules

The ATP-dependent H+ pump from adrenal chromaffin granules is, like the platelet-dense granule H+ pump, essentially insensitive to the mitochondrial ATPase inhibitors sodium azide, efrapeptin, and oligomycin and also insensitive to vanadate and ouabain, agents that inhibit the Na+,K+-ATPase. The chromaffin granule H+ pump is, however, sensitive to low concentrations of NEM (N-ethylmaleimide) and Nbd-Cl (7-chloro-4-nitro-2,1,3-benzoxadiazole). These transport ATPases may thus belong to a new class of ATP-dependent ion pumps distinct from F1F0-and phosphoenzyme-type ATPases. Comparisons of ATP hydrolysis with ATP-dependent serotonin transport suggest that approximately 80% of the ATPase activity in purified chromaffin granule membranes is coupled to H+ pumping. Most of the remaining ATPase activity is due to contaminating mitochondrial ATPase and Na+,K+-ATPase. When extracted with cholate and octyl glucoside, the H+ pump is solubilized in a monodisperse form that retains NEM-sensitive ATPase activity. When reconstituted into proteoliposomes with crude brain phospholipid, the extracted enzyme recovers ATP-dependent H+ pumping, which shows the same inhibitor sensitivity and nucleotide dependence as the native pump. These data demonstrate that the predominant ATP hydrolase of chromaffin granule membrane is also responsible for ATP-driven amine transport and granule acidification in both native and reconstituted membranes.     

Ca2+ and proton transport in chromaffin granule membranes: a proton NMR study

High-resolution proton NMR spectroscopy has been used to monitor the internal pH of chromaffin granule ghosts during Ca2+ influx through the membrane. For this purpose, ghosts were prepared by lysing and resealing chromaffin granules in a medium containing the disodium-ethylenediaminetetraacetic acid complex (Na2.EDTA). Uncomplexed EDTA and Ca.EDTA give rise to distinct sets of methylene peaks in the proton NMR spectrum. Free EDTA titrates with a pK near 6.6 in deuterated media; the chemical shifts that accompany titration have been used to monitor intravesicular pH changes which occur inside chromaffin granule ghosts as a result of ATPase activity and deprotonation of EDTA during Ca2+ influx and complex formation. ATPase activity results in an NMR-detectable proton gradient which is dissipated by nigericin. Experiments monitoring Ca2+ uptake showed that protons which are liberated inside ghosts as a result of Ca.EDTA complex formation are not extruded from the ghosts via a process coupled to Ca2+ entry. This suggests that the Ca2+ transport system of the chromaffin granule membrane occurs without concurrent proton antiport and is not directly coupled energetically to the transmembrane pH gradient.     

Biochemical and immunological evidence for a calcium pump in chromaffin granules

ATP stimulated the accumulation of 45Ca2+ by chromaffin granule ghosts which contained 5 mM oxalate to trap transported calcium within the lumen. Inasmuch as the ATP-dependent 45Ca2+ transport was resistant to 25 mM ammonium acetate as well as the proton ionophore, carbonylcyanide-m-chlorophenylhydrazone, the chromaffin granule proton translocating ATPase does not provide the energy for this process. Instead, we suggest that chromaffin granules contain a calcium translocating ATPase which catalyzes the 45Ca2+ uptake directly. The observation that chromaffin granules bind to a monoclonal antibody raised against a calcium pump from bovine brain supports this hypothesis.     

Relative lack of ATP-driven H+ translocase activity in isolated parotid secretory granules

The possible presence of ATP-driven H+ translocase activity in isolated rat parotid secretory granules has been examined by several approaches. First the transmembrane pH difference measured by either [14C] methylamine or [3H]acetate distribution is not substantially affected by ATP in the presence of membrane-permeating anions. Second, despite a low intrinsic H+ permeability of parotid granule membranes, only a small variably detectable inside-positive transmembrane potential is observed (by altered distribution of radioactive ions) when ATP is added in the absence of permeant anions. Third, ATP-induced lysis of parotid granules is minor and appears to be independent of ATP hydrolysis. Finally, ATP-hydrolase activity of the parotid granule fraction is not stimulated by an H+ ionophore, nor is it susceptible to inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole at a concentration which decreases the measured ATPase of purified chromaffin granule membranes by more than 80%. These findings suggest that this exocrine secretory granule type, which is characterized by storage of a heterogeneous mixture of secretory proteins, exhibits H+ pump activity which is at most a small fraction of that observed in biogenic amine storage granules of neural and endocrine tissues.     

ATP-dependent uptake of 5-hydroxytryptamine by secretory granules isolated from thyroid parafollicular cells.

The current study was done to test the hypotheses that parafollicular granules contain a vacuolar ATPase (V-ATPase) similar to that found in chromaffin granules, that the transport of H+ into granules mediated by this enzyme drives the granular uptake of 5-hydroxytryptamine (5-HT, serotonin), and that secretagogues stimulate both the acidification of parafollicular granules and their ability to take up 5-HT by opening an anion channel in the granular membrane. Our studies indicate that parafollicular granules contain a V-ATPase that is antigenically similar to that of the V-ATPase of adrenal chromaffin granules; however, the parafollicular granular membrane differs from that of chromaffin granules in permeability to Cl- and K+. The membranes of granules derived from resting parafollicular cells appear to be relatively impermeable to Cl- but permeable to K+. Parafollicular granules (and ghosts derived from them) manifest ATP-dependent transmembrane transport of 5-HT. This transport is more dependent on the pH difference (delta pH) than on the membrane potential component of the proton electrochemical gradient across the granular membrane. Transport of 5-HT is thus inhibited more by exposure of parafollicular granules to agents, such as nigericin, that collapse delta pH than by those, such as valinomycin, that decrease transmembrane difference in potential. ATP-dependent uptake of 5-HT by granules isolated from secretagogue-stimulated parafollicular cells is greater than that into granules isolated from unstimulated cells. Since secretagogues open a Cl- channel in parafollicular granule membranes, which enhances acidification of the granules, the facilitation of 5-HT uptake by secretagogues is probably due to an increase in delta pH.     

Inhibition of N-ethylmaleimide of the MgATP-driven proton pump of the chromaffin granules

The thiol reagent N-ethylmaleimide (NEM) completely inhibits the proton pump activity of the H+-ATPase in chromaffin granule 'ghosts' at concentrations which only partly (approximately 20%) inhibit the Mg2+-dependent ATP hydrolysis. Half-maximal inhibition was obtained at approximately 13 microM NEM as compared to 18 microM for the classical proton channel inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), and the apparent stoichiometry of the inhibitors at complete inhibition was NEM : DCCD congruent to 1 : 2. HIgh concentrations of NEM (greater than 100 microM) induce a dissipation of the transmembrane potential generated by MgATP. These findings establish NEM as a valuable proton channel inhibitor in chromaffin granules and explain the rather complex effect of NEM previously reported for catecholamine accumulation in this organelle.     

Participation of a transmembrane proton gradient in 5-hydroxytryptamine transport by platelet dense granules and dense-granule ghosts.

Dense granules, the storage organelles for 5-hydroxytryptamine in blood platelets, have been isolated from porcine platelets and are shown to transport 5-hydroxytryptamine in response to a transmembrane proton gradient (delta pH). Transport in the absence of delta pH is minimal, and it is shown that a rapid increase in transport takes place as delta pH increases. Direct measurements with [14C]methylamine show a delta pH of 1.1 units (acid inside) for intact granules. Osmotically active ghosts of dense granules from which 95% of the endogenous 5-hydroxytryptamine content has been released have also been prepared. Ghosts swell in the presence of ATP and Mg2+, and this swelling is shown to be due to the entry of protons via a process linked to ATP hydrolysis. Proton entry is also apparently linked to anion penetration in ghosts. Steady-state 5-hydroxytryptamine transport in ghosts is stimulated approx. 3-fold on the addition of ATP to the incubation medium, and the stimulation of 5-hydroxytryptamine transport in ghosts correlates with the formation of a transmembrane delta pH. Ghosts generate a delta pH of 1.1-1.3 pH units (acid inside) in the presence of 5 mM-ATP/2.5 mM-MgSO4. delta pH is generated within 3 min at 37 degrees C and is dissipated by the ionophore nigericin and by NH4Cl. It is shown that an Mg2+-stimulated ATPase activity is present on the ghost membrane, and inhibition of the ATPase leads to a corresponding decrease in 5-hydroxytryptamine transport. The results presented support the idea that 5-hydroxytryptamine transport into platelet dense granules is dependent on the presence of a transmembrane delta pH and, together with previous findings by others, suggest a generalized mechanism for biogenic amine transport into subcellular storage organelles.     

Biological amine transport in chromaffin ghosts. Coupling to the transmembrane proton and potential gradients.

The effect of the transmembrane proton gradient (delta pH) and potential gradient (delta psi) upon the rate and extent of amine accumulation was investigated in chromaffin ghosts. The chromaffin ghosts were formed by hypo-osmotic lysis of isolated bovine chromaffin granules and extensive dialysis in order to remove intragranular binding components and dissipate the endogenous electrochemical gradients. Upon ATP addition to suspensions of chromaffin ghosts, a transmembrane proton gradient alone, a transmembrane gradient alone, or both, could be established, depending upon the compositions of the media in which the ghosts were formed and resuspended. When chloride was present in the medium, addition of ATP resulted in the generation of a transmembrane proton gradient, acidic inside of 1 pH unit (measured by [14C]methylamine distribution), and no transmembrane potential (measured by [14C]-thiocyanate distribution). When ATP was added to chromaffin ghosts suspended in a medium in which chloride was substituted by isethionate, a transmembrane potential, inside positive, of 45 mV and no transmembrane proton gradient, was measured. In each medium, the addition of agents known to affect proton or potential gradients, respectively, exerted a predictable mechanism of action. Accumulation of [14C]epinephrine or [14C]5-hydroxytryptamine was over 1 order of magnitude greater in the presence of the transmembrane proton gradient or the transmembrane potential than in the absence of any gradient and, moreover, was related to the magnitude of the proton or potential gradient in a dose-dependent manner. When ghosts were added to a medium containing chloride and isethionate, both a delta pH and delta psi could be generated upon addition of ATP. In this preparation, the maximal rate of amine accumulation was observed. The results indicate that amine accumulation into chromaffin ghosts can occur in the presence of either a transmembrane proton gradient, or a transmembrane potential gradient, and that the maximal rate of accumulation may exist when both components of the protonmotive force are present.     

Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 microM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 microM reserpine, 30 microM NH4+, or 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules with digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 microM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-beta-hydroxylase. The studies demonstrate that 20 microM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.     

Effects of the sulfhydryl reagentN-ethylmaleimide on reserpine binding to the catecholamine transporter in chromaffin granule membranes

1. Catecholamines are transported into chromaffin granules via a carrier-mediated, active-transport process which is inhibited by micromolar concentrations of the sulfhydryl reagent, N-ethylmaleimide (NEM). Reserpine is a very potent, competitive inhibitor of the catecholamine transporter and can be used to investigate the characteristics of the catecholamine transporter. 2. The purpose of this study was to determine whether [3H]reserpine binding to the catecholamine transporter present in chromaffin granule membranes isolated from bovine adrenal glands was also inhibited by NEM and, if so, whether this was a direct or an indirect effect of NEM on the catecholamine transporter. 3. Both [3H]norepinephrine transport into and [3H]reserpine binding to the chromaffin granule ghosts isolated from bovine adrenal glands are inhibited by NEM, with IC50 values of 0.63 +/- 0.02 and 2.8 +/- 0.66 microM, respectively. 4. Mg and ATP protected both the [3H]norepinephrine transport into the ghosts and the [3H]reserpine binding to the transporter from inhibition by NEM, shifting the IC50 values to 260 +/- 43 and 120 +/- 29 microM, respectively. 5. NEM inhibition of the catecholamine transport and reserpine binding appears to be due to an action on the proton translocator associated with the Mg ATPase enzyme rather than a direct action on the catecholamine transporter since (a) the concentration of NEM required to inhibit formation of a membrane potential is similar to that required to inhibit [3H]norepinephrine transport into and [3H]reserpine binding to the ghosts and (b) Mg and ATP protected the proton translocation and [3H]norepinephrine transport into the ghosts, and [3H]reserpine binding to the ghosts, from inhibition by NEM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of the H+-ATPase in bovine adrenal chromaffin granule ghosts by diethylstilbestrol. Evidence for a tight coupling between ATP hydrolysis and proton translocation

Diethylstilbestrol (DES) was found to inhibit reversibly the hydrolysis of MgATP (80% at 100 microM) and proton pump activity (I50 approximately equal to 15 microM, complete at 100 microM) in chromaffin granule ghosts. The parallel inhibition suggests a tight kinetic coupling between the two activities. The Mg2+-ATPase activity, but not proton pumping, was partially restored by N,N'-dicyclohexylcarbodiimide, indicating that the two inhibitors in combination cause a partial uncoupling. The non-competitive type of inhibition shows that the action of DES is distal to the site of ATP binding and hydrolysis. Although unspecific, the interaction of DES with the chromaffin granule membrane seems primarily to affect the H+-ATPase.     

The assignment of the Ca2+-ATPase activity of chromaffin granules to the proton translocating ATPase

CaATP is shown to function as a substrate for the proton translocating ATPase of chromaffin granule ghosts at concentrations which are comparable to that of MgATP. Using the initial rate of the proton pump activity as the measure (delta pH/delta t), an apparent Km-value of 139 +/- 8 microM was estimated for CaATP and 59 +/- 3 microM for MgATP. The maximal rate was markedly higher with MgATP than with CaATP, partly due to an inhibition of the hydrolytic activity at the higher concentrations of CaATP. The proton pump activity with CaATP was inhibited by N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide at concentrations similar to that found for MgATP. No inhibition was observed with sodium vanadate in the concentration range 0-15 microM. Calmodulin and trifluoperazine had no effect on the overall ATPase activity with CaATP. These findings establish this activity as an intrinsic property of the chromaffin granules, i.e., linked to the H+-ATPase. No evidence was obtained for the presence of a Ca2+-translocating ATPase [Ca2+ + Mg2+)-ATPase) in the chromaffin granules.     

Accessibility of phospholipids in the chromaffin granule membrane.

1. The accessibility of phospholipids in the membrane of the adrenomedullary storage vesicles (chromaffin granules) has been studied. 2. The reaction of 2,4,6-trinitrobenzenesulphonic acid with both intact granules and their ghosts, results in the labelling of 70% of the phosphatidylethanolamine. 3. The action of phospholipase A2 (from bee venom), phospholipase C (from Bacillus cereus) and sphingomyelinase C (from Staphylococcus aureus) on granules and their ghosts was followed as a function of time. No significant difference was observed between the intact granules and their ghosts. 4. In the intact granules the various treatments led to varying amounts of lysis although again no evidence was obtained that such lysis in any way increased the amount of accessible phospholipid. 5. Highly purified granule preparations were also compared with the so-called "large granule" fraction and no significant differences were detected. 6. Approx. 67% of phosphatidylethanolamine + phosphatidic acid, 50% of phosphatidylserine + phosphatidylinositol, 65% of phosphatidylcholine and 20% of sphingomyelin is accessible to enzymatic degradation. In total, approx. 50% of all the phospholipids reacted. 7. It is also shown that, unlike in enzymatic treatment, all the phosphatidylcholine can be exchanged in the presence of a phospholipid exchange protein (prepared from beef liver). 8. It is concluded that transmembrane movement of phosphatidylcholine is slow in isolated membranes of chromaffin granules. The presence of the exchange protein, however, in conjunction with membrane proteins and specific phospholipid arrangements may catalyse this transmembrane movement.     

Localization of the phlorizin site on Na, K-ATPase in red cell membranes.

Phlorizin at 2 X 10(-4) M inhibited Na+ and Rb+-activated ATPase activities in human red cell membranes by 43%. It inhibited the 86Rb uptake activity of erythrocytes by only 15%. 86Rb uptake into resealed ghosts was inhibited strongly when phlorizin and ATP were preloaded in the ghosts before resealing. Na,K-ATPase activity in the resealed ghosts was also inhibited in the presence of phlorizin inside but not outside the ghosts. These findings suggested that the phlorizin site is located inside the cell.     

Existence of an adenosine 5'-triphosphate dependent proton translocase in bovine neurosecretory granule membrane

The addition of ATP to bovine neurohypophysial secretory granules suspended in isotonic sucrose medium induces a positive polarization, delta psi, of their interior without affecting their internal pH. In KCl-containing media, ATP failed to generate large delta psi but induced a pH gradient (delta pH; interior acidic). These observations are consistent with the existence in the neurosecretory granule membrane of an ATP-dependent inward electrogenic H+ translocase (H+ pump), capable in KCl-containing media of acidifying the granule matrix by H+-Cl- cotransport. The delta psi and delta pH generated by the H+ pump, defined as the ATP-induced changes sensitive to the H+ ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), were blocked by N,N'-dicyclohexylcarbodiimide, an inhibitor of all H+ pumps, and were insensitive to oligomycin, a mitochondrial ATPase inhibitor. In sucrose medium, measurements were complicated by a Donnan equilibrium reflecting the presence in the granule of peptide hormones and neurophysins which resulted in a CCCP-resistant resting delta pH. In KCl-containing media, the Donnan equilibrium was destroyed since the membrane is permeable to cations, but under these conditions a CCCP-resistant K+-diffusion potential was observed. The ATP-induced delta psi was also monitored by the extrinsic fluorescent probe bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol. The hypothesis of a granule H+ pump is further supported by the presence of an oligomycin-resistant ATPase in the preparation and the ultrastructural localization of such an activity on the granule membrane. The H+ pump has been found in both newly formed and aged neurosecretory granules. Its possible physiological function is discussed with reference to that of chromaffin granules, with which it has many similarities.     

Proton-translocating Mg2+-dependent ATPase activity in insulin-secretory granules

Insulin-secretory granules isolated from a pancreatic islet-cell tumour by centrifugation on Percoll density gradients exhibited a membrane-associated Mg(2+)-dependent ATPase activity. In granule suspensions incubated in iso-osmotic media, activity was increased 2-3-fold by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K(2)SO(4) or by the addition of a detergent. Permeant anions also increased Mg(2+)-dependent ATPase activity under iso-osmotic conditions when combined with K(+) and nigericin, or NH(4) (+). It was deduced that a major component of the activity was coupled to the translocation of protons into the granule interior. The granule membrane appeared poorly permeable to H(+), K(+), NH(4) (+) and SO(4) (2-) but permeable, in increasing order, to phosphate or acetate, Cl(-), I(-) and SCN(-). Like the proton-translocating ATPase of mammalian mitochondria the granule enzyme when membrane-bound was inhibited by up to 85% by tributyltin or NN'-dicyclohexylcarbodi-imide and was solubilized in a tributyltin-insensitive form after extraction with dichloromethane. It was clearly not a mitochondrial contaminant as evidence by the distribution of marker proteins on density gradients. Unlike mitochondrial activity it was insensitive to oligomycin, efrapeptin, atractyloside, azide and oxyanions. Its properties, however, were indistinguishable from those of the proton-translocating ATPase found in the chromaffin granules of the adrenal medulla. Moreover, insulin granules and chromaffin granules exhibited similar levels of activity. This indicated that in spite of the differences in their internal composition, granules from tissues involved in polypeptide and amine hormone secretion possess catalytic components in common. Only a minor role for the ATPase in amine transport in insulin granules was apparent. Rather, its presence here may relate to the process of secretory vesicle morphogenesis or to the exocytotic mechanism.     

The gastric [H,K]ATPase:H+/ATP stoichiometry

An H+/ATP ratio of 2 for H+ transport was determined from initial rate measurements at pH 6.1 in a purified gastric microsomal fraction containing the [H,K]ATPase. This ratio was independent of external KCl, though the apparent K0.5 for ATP was increased from 10.78 +/- 0.51 (n = 3) to 64.6 +/- 11.9 (n = 3) microM ATP and from 5.13 +/- 0.64 (n = 3) to 65.2 +/- 0.64 (n = 3) microM ATP for H+ transport and the K+-stimulated ATPase, respectively, as K+external was increased from 12 to 150 mM. The H+/ATP ratio was also relatively independent of ATP concentration. Maximum initial rates obtained in KCl-equilibrated vesicles were independent of added valinomycin, though net H+ transport was increased 29.3 +/- 1.03% (n = 6) by the addition of ionophore. Maximum net H+ transport in this vesicle preparation was 185 +/- 2.1 (n = 14) nmol mg-1 of protein. Initial rate measurements of ATPase represent a burst of K+-dependent activity of approximately 10-15 s duration. The H+/ATP stoichiometry was calculated based on the K+-stimulated component of hydrolysis. Under most conditions, the Mg2+-dependent component of hydrolysis was less than 10% of the (Mg2+ + K+) component of hydrolysis.     

Spectrophotometric measurements of transmembrane potential and pH gradients in chromaffin granules

The electrical potential (delta psi) and proton gradient (alpha pH) across the membranes of isolated bovine chromaffin granules and ghosts were simultaneously and quantitatively measured by using the membrane- permeable dyes 3,3'dipropyl-2,2'thiadicarbocyanine (diS-C3-(5)) to measure delta psi and 9-aminoacridine or atebrin to measure delta pH. Increases or decreases in the delta psi across the granular membrane could be monitored by fluorescence or transmittance changes of diS-C3- (5). Calibration of the delta psi was achieved by utilization of the endogenous K+ and H+ gradients, and valinomycin or carbonyl cyanide-p- trifluoromethoxyphenylhydrazone (FCCP), respectively, with the optical response of diS-C3-(5) varying linearly with the Nernst potential for H+ and K+ over the range -60 to +90 mV. The addition of chromaffin granules to a medium including 9-aminoacridine or atebrin resulted in a rapid quenching of the dye fluorescence, which could be reversed by agents known to cause collapse of pH gradients. From the magnitude of the quenching and the intragranular water space, it was possible to calculate the magnitude of the alpha pH across the chromaffin granule membrane. The time-course of the potential-dependent transmittance response of diS-C3-(5) and the delta pH-dependent fluorescence of the acridine dyes were studied simultaneously and quantitatively by using intact and ghost granules under a wide variety of experimental conditions. These results suggest that membrane-permeable dyes provide an accurate method for the kinetic measurement of delta pH and delta psi in an amine containing subcellular organelle.     

The hydrogen ion-pumping adenosine triphosphatase of platelet dense granule membrane. Differences from F1F0- and phosphoenzyme-type ATPases

Using a coupled transport assay which detects only those ATPase molecules functionally inserted into the platelet dense granule membrane, we have characterized the inhibitor sensitivity, substrate specificity, and divalent cation requirements of the granule H+ pump. Under identical assay conditions, the granule ATPase was insensitive to concentrations of NaN3, oligomycin, and efrapeptin which almost completely inhibit ATP hydrolysis by mitochondrial membranes. The granule ATPase was inhibited by dicyclohexylcarbodiimide but only at concentrations much higher than those needed to maximally inhibit mitochondrial ATPase. Vanadate (VO3-) ion and ouabain also failed to inhibit granule ATPase activity at concentrations which maximally inhibited purified Na+,K+-ATPase. Two alkylating agents, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and N-ethylmaleimide both completely inhibited H+ pumping by the granule ATPase under conditions where ATP hydrolysis by mitochondrial membranes or Na+,K+-ATPase was hardly affected. These results suggest that the H+-pumping ATPase of platelet granule membrane may belong to a class of ion-translocating ATPases distinct from both the phosphoenzyme-type ATPases present in plasma membrane and the F1F0-ATPases of energy-transducing membranes.     

Localization of lysophosphatidylcholine in bovine chromaffin granules.

One of the unique features of the chromaffin granule membrane is the presence of about 17 mol% lysophosphatidylcholine. Lysophosphatidylcholine isolated from the granules could be degraded by approx. 94% by lysophospholipase. This result is consistent with chemical analyses data showing that about 9% of this lysophospholipid is 1'-alkenyl glycerophosphocholine. The localization of the acylglycerophosphocholine in the chromaffin granule membrane was studied by using pure bovine liver lysophospholipases. In intact granules only about 10% of the total lysophosphatidylcholine was directly available for enzymic hydrolysis. In contrast, when granule membranes (ghosts) were treated with lysophospholipases approx. 60% of the lysophosphatidylcholine was deacylated. These values did not increase after pre-treatment of intact granules or ghosts with trypsin. Added 1-[1-14C]palmitoyl-sn-glycero-3-phosphocholine did not mix with the endogenous lysophosphatidylcholine pool(s) and remained completely accessible to added lysophospholipases.