A study of the dicyclohexylcarbodiimide-binding component of the mitochondrial ATPase complex from beef heart

1. The binding of [14C]-dicyclohexylcarbodiimide to membrane proteins of beef heart mitochondria has been investigated using dodecylsulphate/polyacrylamide gel electrophoresis. Upon incubation of submitochondrial particles with low concentrations of dicyclohexylcarbodiimide (5 nmol/mg protein) radioactivity was incorporated into three components with apparent molecular weights of 30000, 18000 and less than 6500. Only the two smaller components were found to be extracted into chloroform/methanol. The same two components were labelled when the isolated ATPase complex or a reconstituted F0F1 system was incubated with low concentrations of dicyclohexylcarbodiimide. High concentrations of dicyclohexylcarbodiimide (20-100 nmol/mg protein) resulted in binding to several mitochondrial proteins. 2. The maximal amount of dicyclohexylcarbodiimide which can bind to submitochondrial particles, the isolated ATPase complex, and the reconstituted F0F1 system was found to exceed the amount required for maximal inhibition of the ATPase activity by several-fold. The distribution of the bound [14C]dicyclohexylcarbodiimide between the different dicyclohexylcarbodiimide-binding components was investigated as a function of dicyclohexylcarbodiimide concentration. The smallest and largest components revealed a high affinity for dicyclohexylcarbodiimide-binding which paralleled the inhibition of ATPase activity. The intermediate component had a markedly lower affinity for dicyclohexylcarbodiimide-binding. 3. The larger dicyclohexylcarbodiimide-binding component of the isolated ATPase complex can be converted into the smaller component by treatment of the ATPase complex with performic acid. Partial conversion can also be achieved by extraction of the band from the dodecylsulphate-polyacrylamide gel after electrophoresis, followed by re-electrophoresis. The observations suggest that the larger component may be an oligomer of the smaller one. 4. Using concentrations of oligomycin and dicyclohexylcarbodiimide which were equal to or greater than those required for maximal inhibition of the ATPase activity, oligomycin was found to diminish the binding of [14C]dicyclohexylcarbodiimide to both dicyclohexylcarbodiimide-binding components of the isolated ATPase complex.     

The sodium ion translocating adenosinetriphosphatase of Propionigenium modestum pumps protons at low sodium ion concentrations

The purified ATPase (F1F0) of Propionigenium modestum has its pH optimum at pH 7.0 or at pH 6.0 in the presence or absence of 5 mM NaCl, respectively. The activation by 5 mM NaCl was 12-fold at pH 7.0, 3.5-fold at pH 6.0, and 1.5-fold at pH 5.0. In addition to its function as a primary Na+ pump, the ATPase was capable of pumping protons. This activity was demonstrated with reconstituted proteoliposomes by the ATP-dependent quenching of the fluorescence of 9-amino-6-chloro-2-methoxyacridine. No delta pH was formed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone or by blocking the ATPase with dicyclohexylcarbodiimide. In the presence of valinomycin and K+, the delta pH increased, in accord with the operation of an electrogenic proton pump. The proton pump was only operative at low Na+ concentrations (less than 1 mM), and its activity increased as the Na+ concentration decreased. Parallel to the decrease of H+ pumping, the velocity of the Na+ transport increased about 6-fold from 0.1 to 4 mM NaCl, indicating a switch from H+ to Na+ pumping, as the Na+ concentration increases. Due to proton leaks in the proteoliposomal membranes, fluorescence quenching was released after blocking the ATPase with dicyclohexylcarbodiimide, by trapping residual ATP with glucose and hexokinase, or by the Na+-induced conversion of the proton pump onto a Na+ pump. Amiloride, an inhibitor of various Na+-coupled transport systems, was without effect on the kinetics of Na+ transport by the P. modestum ATPase.     

Characterization of the ATP synthase of Propionigenium modestum as a primary sodium pump

The ATP synthase (F1F0) of Propionigenium modestum has been purified to a specific ATPase activity of 5.5 units/mg of protein, which is about 6 times higher than that of the bacterial membranes. Analysis by SDS gel electrophoresis indicated that in addition to the five subunits of the F1 ATPase, subunits of Mr 26,000 (a), 23,000 (b), and 7500 (c) have been purified. The ATPase activity of F1F0 was specifically activated about 10-fold by Na+ions. The enzyme was strongly inhibited by dicyclohexylcarbodiimide, venturicidin, tributyltin chloride, and azide. After incubation with [14C]dicyclohexylcarbodiimide, about 3-4 mol of the inhibitor was bound per 500,000 g of the enzyme. The radioactive label was specifically bound to submit c. These subunits form stable aggregates which resist dissociation by SDS at 100 degrees C. The monomer is formed upon heating with SDS to 121 degrees C or by extraction of the membranes with chloroform/methanol. The ATP synthase was incorporated into liposomes by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes catalyzed the transport of Na+ions upon ATP hydrolysis. The transport was completely abolished by dicyclohexylcarbodiimide. Whereas monensin prevented the accumulation of Na+ions, the uptake rate was stimulated 4-5-fold in the presence of valinomycin or carbonyl cyanide m=chlorophenylhydrazone. These results indicate an electrogenic Na+ transport and also that it is a primary event and not accomplished by a H+-translocating ATP synthase in combination with a Na+/H+ antiporter.     

Characterization of the Na+-stimulated ATPase of Propionigenium modestum as an enzyme of the F1F0 type

The ATP-hydrolyzing activity of Propionigenium modestum was extracted from the membranes with Triton X-100 or by incubation with EDTA at low ionic strength. The ATPase in the Triton extract was highly sensitive to dicyclohexylcarbodiimide but not to vanadate. These properties are characteristic for enzymes of the F1 F0 type. The ATPase was specifically activated by Na+ ions yielding a 15-fold increase in catalytic activity at 5 mM Na+ concentration. The additional presence of 1% Triton X-100 caused a further 1.5-fold activation. In the absence of Na+ Triton stimulated the ATPase about 13-fold. The Triton-stimulated ATPase was further activated about 1.5-2-fold by Na+ addition. The ATPase extracted by the low-ionic-strength treatment was purified to homogeneity by fractionation with poly(ethylene glycol) and gel chromatography. The enzyme had the characteristic F1-ATPase subunit structure with Mr values of 58,000 (alpha), 56,000 (beta), 37,600 (gamma), 22,700 (delta), and 14,000 (epsilon). The F1-ATPase was not stimulated by Na+ ions. The membrane-bound ATPase was reconstituted from the purified F1 part and F1-depleted membranes, thus further indicating an F1 F0 structure for the ATPase of P. modestum. Upon reconstitution the ATPase recovered its stimulation by Na+ ions, suggesting that the binding site for Na+ is localized on the membrane-bound F0 part of the enzyme complex.     

Proteins of bacterial membranes. Purification of soluble ATPase from Acholeplasma laidlawii

A purified preparation of ATPase (factor F1) from the Acholeplasma laidlawii was obtained. The purification procedure included extraction of the enzyme complex from the isolated membranes by ultrasonication, chromatography on DEAE-cellulose and gel filtration on Sepharose 6B. The specific activity of the ATPase was increased 30-fold as compared to the original activity. The Km value for ATP hydrolysis was 7,4 . 10(-4) M. ADP competitively inhibited the enzyme (Ki = 2,0 . 10(-4) M). Ouabain (2,5 . 10(-4) M) and dicyclohexylcarbodiimide (1,0 . 10(-4) M) did not inhibit the ATPase activity. The enzyme was activated by Mg2+, but was inhibited by a combination of Na+ and K+. The enzyme is cold-labile, but can be stabilized by storage in buffer solutions, containing methanol, glycerol or lecithin.     

Quercetin interaction with the chloroplast ATPase complex

1. Quercetin, a flavonoid which acts as an energy transfer inhibitor in photophosphorylation is shown to inhibit the P-ATP exchange activity of membrane-bound CF1 and the ATPase activity of isolated CF1. Quercetin, affects also the proton uptake in chloroplasts in a manner similar to that of dicyclohexylcarbodiimide. 2. The light-dependent proton uptake in EDTA-treated chloroplasts is stimulated by quercetin. In untreated chloroplasts quercetin has a dual effect: it enhances at pH above 7.5 while at lower pH values it decreases the extent of H+ uptake. Similar effects were obtained with dicyclohexylcarbodiimide. 3. Like quercetin, dicyclohexylcarbodiimide was also found to inhibit the ATPase activity of isolated CF1. 4. Quercetin inhibits uncoupled electron transport induced by either EDTA-treatment of chloroplasts or by addition of uncouplers. Quercetin restores H+ uptake in both types of uncoupled chloroplasts. 5. The mode of action of quercetin and dicyclohexylcarbodiimide in photophosphorylation is discussed, and interaction with both CF1 and F0 is suggested.     

Extraction of mitochondrial protein-lipid complexes into organic solvents: an approach to study the interaction between the ATPase and the mitochondrial ATPase-inhibitor protein

Protein-lipid complexes were transferred directly from mitochondria and submitochondrial particles into hexane and ether. The protein-lipid residue left after solvent removal from these extracts was used to form liposomes which display low-temperature-resistant ATPase activity. Centrifugation experiments indicate that the ATPase activity is associated to the vesicles. Most of the F1-ATPases appear to be accessible to the external water phase of the liposomes. The ATPase activity of these particles was insensitive to dicyclohexylcarbodiimide and oligomycin. Incubation of these vesicles at room temperature activated (4--10-fold) the ATPase through a process that is partially sensitive to phenylmethylsulfonyl fluoride. The results with purified ATPase-inhibitor protein and (F1--ATPase)-inhibitor complex indicate that the activation process in the liposomes is due to the abolition of the inhibitory action of the inhibitor protein bound to a large fraction of the extracted ATPases. Liposomes prepared from hexane extracts obtained from submitochondrial particles having different levels of ATPase activity displayed an activation ratio which correlated with the number of ATPases that are inhibited by the inhibitor protein in the submitochondrial particles. The extraction of mitochondrial ATPase and its incorporation into liposomes followed by activity measurements may be used to judge the number of ATPases that in a given preparation contain the inhibitor protein in its inhibiting site.     

The ATP synthetase of Escherichia coli K12: purification of the enzyme and reconstitution of energy-transducing activities.

The ATP synthetase of Escherichia coli K12 was purified by a simple procedure. The dicyclohexylcarbodiimide-sensitive ATPase activity was enriched 21-fold. The ATP synthetase preparation contained the eight polypeptides (alpha, beta, gamma, a,delta, b,espilon, c) of the enzyme and a residual contamination (4% of the total protein) as shown by dodecylsulfate/polyacrylamide electrophoresis. The polypeptide c was specifically labelled with [14C]dicyclohexylcarbodiimide. Energy-transducing activities were reconstituted from soybean phospholipids and the purified enzyme. The proteoliposomes exhibited a significantly higher ATP-32Pi exchange activity and a higher proton-translocating activity as compared to the untreated membranes.     

Physiological and genetic modifications of the expression of the yeast mitochondrial adenosine triphosphatase inhibitor.

1. The oligomycin-sensitive ATPase activity of submitochondrial particles of the glycerol-grown "petite-negative" yeast: Schizosaccharomyces pombe is markedly stimulated by incubation at 40 degrees C and by trypsin activations are treatment. Both increased in Triton-X 100 extracts of the submitochondrial particles. 2. A trypsin-sensitive inhibitory factor of mitochondrial ATPase with properties similar to that of beef heart has been extracted and purified from glycerol-grown and glucose-grown S. pombe wild type, from the nuclear pleiotropic respiratory-deficient mutant S. pombe M126 and from Saccharomyces cerevisiae. 3. ATPase activation by heat is more pronounced in submitochondrial particles isolated from glycerol-grown than from glucose-grown S. pombe. An activation of lower extent is observed in rat liver mitochondrial particles but is barely detectable in the "petite-positive" yeast: S. cerevisiae. No activation but inhibition by heat is observed in the pleitotropic respiratory-deficient nuclear mutant S. pombe M126. 4. The inhibition of S. pombe ATPase activity by low concentrations of dicyclohexylcarbodiimide dissapears at inhibitor concentrations above 25 muM. In Triton-extract of submitochondrial particles net stimulation of ATPase activity is observed at 100 muM dicyclohexylcarbodiimide. The pattern of stimulation of ATPase activity by dicyclohexylcarbodiimide in different genetic and physiological conditions parallels that produced by heat and trypsin. A similar mode of action is therefore proposed for the three agents: dissociation or inactivation of an ATPase inhibitory factor. 5. We conclude that "petite-positive" and "petite-negative" yeasts contain an ATPase inhibitor factor with properties similar to those of the bovine mitochondrial ATPase inhibitor. The expression of the ATPase inhibitor, measured by ATPase activation by heat, trypsin or high concentrations of dicyclohexylcarbodiimide, is sensitive to alterations of the hydrophobic membrane environment and dependent on both physiological state and genetic conditions of the yeast cells.     

Physiological and genetic modifications of the expression of the yeast mitochondrial adenosine triphosphatase inhibitor

1. The oligomycin-sensitive ATPase activity of submitochondrial particles of the glycerol-grown “petite-negative” yeast: Schizosaccharomyces pombe is markedly stimulated by incubation at 40°C and by trypsin activations are treatment. Both increased in Triton-X 100 extracts of the submitochondrial particles.

2. A trypsin-sensitive inhibitory factor of mitochondrial ATPase with properties similar to that of beef heart has been extracted and purified from glycerolgrown and glucose-grown S. pombe wild type, from the nuclear pleiotropic respiratory-deficient mutant S. pombe M126 and from Saccharomyces cerevisiae.

3. ATPase activation by heat is more pronounced in submitochondrial particles isolated from glycerol-grown than from glucose-grown S. pombe. An activation of lower extent is observed in rat liver mitochondrial particles but is barely detectable in the “petite-positive” yeast: S. cerevisiae. No activation but inhibition by heat is observed in the pleitotropic respiratory-deficient nuclear mutant S. pombe M126.

4. The inhibition of S. pombe ATPase activity by low concentrations of dicyclohexylcarbodiimide dissapears at inhibitor concentrations above 25 μM. In Triton-extract of submitochondrial particles net stimulation of ATPase activity is observed at 100 μM dicyclohexylcarbodiimide. The pattern of stimulation of ATPase activity by dicyclohexylcarbodiimide in different genetic and physiological conditions parallels that produced by heat and trypsin. A similar mode of action is therefore proposed for the three agents: dissociation or inactivation of an ATPase inhibitory factor.

5. We conclude that “petite-positive” and “petite-negative” yeasts contain an ATPase inhibitor factor with properties similar to those of the bovine mitochondrial ATPase inhibitor. The expression of the ATPase inhibitor, measured by ATPase activation by heat, trypsin or high concentrations of dicyclohexylcarbodiimide, is sensitive to alterations of the hydrophobic membrane environment and dependent on both physiological state and genetic conditions of the yeast cells.     

Purification of N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes

An N-ethylmaleimide-sensitive ATPase was purified 100-fold from chromaffin granule membranes. The purification procedure included solubilization with polyoxyethylene 9 lauryl ether, chromatography on hydroxylapatite and DEAE-cellulose columns, and glycerol gradient centrifugations. Inclusion of phosphatidylserine and a mixture of protease inhibitors during the purification procedure was necessary to maintain the activity of the preparation. The purified preparation contained four major polypeptides with molecular masses of about 115, 72, 57, and 39 kDa, which were copurified with the ATPase activity. The 115-kDa subunit binds [14C]dicyclohexylcarbodiimide and the subunits of 115 and 39 kDa bind [14C]N-ethylmaleimide. The ATP-dependent proton uptake activity of chromaffin granule membranes is inhibited 50% with about 20 microM N-ethylmaleimide, while over 5 mM concentrations of the inhibitor were required to block the ATPase activity of the membranes. The ATPase activity of the purified enzyme was inhibited via two different affinities: a high affinity site with a Ki in the microM range and a low affinity site in the mM range, each contributing to about 50% inhibition of the enzyme. It is concluded that the proton-ATPase of chromaffin granule membranes contains at least four subunits with the 115-kDa polypeptide being the main subunit having the active site for the ATPase activity of the enzyme.     

Characterization and solubilization of the membrane-bound ATPase of Mycoplasma gallisepticum.

The membrane-bound ATPase of Mycoplasma gallisepticum selectively hydrolyzed purine nucleoside triphosphates and dATP. ADP, although not a substrate, inhibited ATP hydrolysis. The enzyme exhibited a pH optimum of 7.0 to 7.5 and an obligatory requirement for divalent cations. Dicyclohexylcarbodiimide at a concentration of 1 mM inhibited 95% of the ATPase activity at 37 degrees C, with 50% inhibition occurring at 22 microM dicyclohexylcarbodiimide. Sodium or potassium (or both) failed to stimulate activity by greater than 37%. Azide (2.6 mM), diethylstilbestrol (100 micrograms/ml), p-chloromercuribenzoate (1 mM), and vanadate (50 microM) inhibited 50, 91, 89, and 60%, respectively. The ATPase activity could not be removed from the membrane without detergent solubilization. Although most detergents inactivated the enzyme, the dipolar ionic detergent N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (0.1%) solubilized approximately 70% of the enzyme with only a minor loss in activity. The extraction led to a twofold increase in specific activity and retention of inhibition by dicyclohexylcarbodiimide and ADP. Glycerol greatly increased the stability of the solubilized enzyme. The properties of the membrane-bound ATPase are not consistent with any known ATPase. We postulate that the ATPase functions as an electrogenic proton pump.     

The plasma membrane ATPase of Dictyostelium discoideum

During centrifugation of Dictyostelium membranes on sucrose and metrizamide gradients, an ATPase activity resistant to azide and molybdate but sensitive to diethylstilbestrol was found to copurify with the plasma membrane markers alkaline phosphatase and ¹²⁵I in cells surface-labelled by lactoperoxidase catalyzed iodination. This ATPase was enriched 50-fold in purified plasma membranes and could be separated from the mitochondrial ATPase on metrizamide gradients. The plasma membrane ATPase is very specific for ATP as substrate and Mg²⁺ as essential cofactor. Its pH optimum is 6.5 and it is inhibited by dicyclohexylcarbodiimide, diethylstilbestrol, vanadate, mercurials and Cu²⁺, but not by ouabain, molybdate, azide or oligomycin. It was not specifically affected by either monovalent cations or anions. These results suggest that the plasma membranes of Dictyostelium contain an ATPase similar to the proton-pumping ATPases recently identified in fungal and plant plasma membranes (Serrano, R. (1984) Curr. Top. Cell. Regul. 23, 87–126).     

Purification and properties of a vanadate- and N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes

A vanadate- and N-ethylmaleimide-sensitive ATPase was purified about 500-fold from chromaffin granule membranes. The purified preparation contained a single major polypeptide with an apparent molecular mass of about 115 kDa, which was copurified with the ATPase activity. Immunological studies revealed that this polypeptide has no relation to subunit I (115 kDa) of the H+-ATPase from chromaffin granules. The ATPase activity of the enzyme is inhibited about 50% by 100 microM N-ethylmaleimide or 5 microM vanadate. The enzyme is not sensitive to dicyclohexylcarbodiimide, ouabain, SCH28080, and omeprazole, which distinguishes it from Na+/K+-ATPase and the gastric K+/H+-ATPase. ATP and 2-deoxy ATP are equally effective substrates for the enzyme. However, the enzyme exhibited only 10% activity with GTP as a substrate. UV illumination of the purified enzyme in the presence of [alpha-32P]ATP exclusively labeled the 115 kDa protein. This labeling was increased by Mg2+ and strongly inhibited by Ca2+ ions. Similarly, the ATPase activity was dependent on Mg2+ and inhibited by the presence of Ca2+ ions. The ATPase activity of the enzyme was largely insensitive to monovalent anions and cations, except for F-, which inhibited the vanadate-sensitive ATPase. Incubation of the enzyme in the presence of [14C]N-ethylmaleimide labeled the 115-kDa polypeptide, and this labeling could be prevented by the addition of ATP during the incubation. A reciprocal experiment showed that preincubation with N-ethylmaleimide inhibited the labeling of the 115-kDa polypeptide by [alpha-32P]ATP by UV illumination. This suggests a close proximity between the ATP-binding site and an essential sulfhydryl group. A possible connection between the isolated ATPase and organelle movement is discussed.     

Improved purification for thermophilic F1F0 ATP synthase using n-dodecyl beta-D-maltoside

Here we report a fast, simple purification for thermophilic F1F0 ATP synthase (TF1F0) that utilizes a cocktail of stabilizing reagents and the detergent n-dodecyl beta-D-maltoside to yield enzyme with an ATPase activity of 41 micromol/min/mg, 2.5-fold higher than that previously reported. ATPase activity was 80% inhibited by the F0-reactive reagent dicyclohexylcarbodiimide, indicating that F1-F0 interactions were largely intact. To measure ATP-driven proton pumping activity, purified TF1F0 was incorporated into liposomes, and the ATP-induced change in internal pH was measured using the fluorescent probe pyranine. In the presence of valinomycin, a maximum ATP-driven deltapH of 0.8 units was obtained. To measure ATP synthesis activity, TF1F0 was incorporated into liposomes with the light-dependent proton pump bacteriorhodopsin. Proteoliposomes were illuminated to generate an electrochemical gradient, after which ADP and inorganic phosphate were added to initiate ATP synthesis. A steady state ATP synthesis activity of 490 nmol/min/mg was achieved after an initial approximately 30-min lag phase.     

Characterization of a vacuolar proton ATPase in Dictyostelium discoideum

Of the total ATPase activity in homogenates of the ameba, Dictyostelium discoideum, approximately one-third was inhibited at pH 7 by 25 microM 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Upon isopycnic sucrose density gradient centrifugation, the bulk of the NBD-CI-sensitive ATPase activity was recovered in a major membrane fraction with a broad peak at 1.16 g/ml, well-resolved from markers for plasma membranes, mitochondria, lysosomes and contractile vacuoles. The gradient peak had a specific activity of 0.5 mumol/min per mg protein. The activity was half-inhibited by 1 microM silicotungstate, 2 microM diisothiocyanatostilbene disulfonate (DIDS), 2.5 microM dicyclohexylcarbodiimide (DCCD), 4 microM NBD-CI and 20 microM N-ethylmaleimide (NEM) but was resistant to conventional inhibitors of mitochondrial and plasma membrane ATPase. That this ATPase activity constituted a proton pump was shown by the MgATP-dependent uptake and quenching of Acridine orange fluorescence by partially purified vacuoles. The Acridine orange uptake was specifically blocked by the aforementioned inhibitors. The generation of proton electrochemical gradients was suggested by the stimulation of enzyme activity by protonophores (fatty acids) and cation exchangers (nigericin). Uncoupling stimulated the ATPase activity as much as 20-fold, revealing an unusually high impermeability of the membranes to protons. ATPase activity was also stimulated by halide ions, apparently through a parallel conductance pathway. Under a variety of sensitive test conditions, the reverse enzyme reaction (i.e., incorporation of 32Pi into ATP) was not detected. We conclude that this major H+-ATPase serves to acidify the abundant prelysosomal vacuoles found in D. discoideum (Padh et al. (1989) J. Cell Biol. 108, 865-874). The finding of a vacuolar H+-ATPase in a protist suggests the ubiquity of this enzyme among the eukaryotic kingdoms.     

Characterization of the Golgi Complex Cleared of Proteins in Transit and Examination of Calcium Uptake Activities

To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were >99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that >90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200- to 400-fold with and 400- to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.     

The properties of arginine transport in vacuolar membrane vesicles of Neurospora crassa

We have measured the uptake of arginine into vacuolar membrane vesicles from Neurospora crassa. Arginine transport was found to be dependent on ATP hydrolysis, Mg2+, time, and vesicle protein with transported arginine remaining unmodified after entry into the vesicles. The Mg2+ concentration required for optimal arginine transport varied with the ATP concentration so that maximal transport occurred when the MgATP2- concentration was at a maximum and the concentrations of free ATP and Mg2+ were at a minimum. Arginine transport exhibited Michaelis-Menten kinetics when the arginine concentration was varied (Km = 0.4 mM). In contrast, arginine transport did not follow Michaelis-Menten kinetics when the MgATP2-concentration was varied (S0.5 = 0.12 mM). There was no inhibition of arginine transport when glutamine, ornithine, or lysine were included in the assay mixture. In contrast, arginine transport was inhibited 43% when D-arginine was present at a concentration 16-fold higher than that of L-arginine. Measurements of the internal vesicle volume established that arginine is concentrated 14-fold relative to the external concentration. Arginine transport was inhibited by dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl-hydrazone, and potassium nitrate (an inhibitor of vacuolar ATPase activity). Inhibitors of the plasma membrane or mitochondrial ATPase such as sodium vanadate or sodium azide did not affect arginine transport activity. In addition, arginine transport had a nucleoside triphosphate specificity similar to that of the vacuolar ATPase. These results suggest that arginine transport is dependent on vacuolar ATPase activity and an intact proton channel and proton gradient.     

Plasma membrane proton-ATPase of a turtle bladder epithelial cell line

Urinary acidification by the turtle bladder is mediated by a proton ATPase located in the apical membrane. The present study describes a proton ATPase in the plasma membrane of a cell line of turtle bladder epithelial cells. In the presence of ouabain to inhibit Na+,K+-ATPase and in the absence of Ca2+ to inhibit Ca2+-ATPase, we measured ATPase activity of the plasma membranes of the cultured cells. This ATPase was resistant to oligomycin but sensitive to dicyclohexylcarbodiimide, N-ethylmaleimide, and vanadate. In the presence of ATP, the ATPase was capable of acidification as assessed by quenching of acridine orange. Acidification could not be elicited by other nucleotides (GTP, UTP). Acidification was inhibited by dicyclohexylcarbodiimide, N-ethylmaleimide, and vanadate but was not affected by replacement of Na+ by K+. The acidification response was dependent on the presence of chloride, abolished in the presence of gluconate, and inhibited partially by nitrate. Experiments utilizing the voltage-sensitive dye 3,3'-dipropylthiodicarbocyanine iodide showed that the proton ATPase was electrogenic and capable of responding to a favorable electric gradient. In summary, the turtle bladder epithelial cell line has a plasma membrane proton ATPase which is similar to the proton ATPase of turtle bladder epithelium and thus should allow purification and characterization of this enzyme.     

Interaction between ubiquinone and ATPase in mitochondrial membranes

The extraction of ubiquinone from mitochondrial membranes produces alterations of ATPase activity including a reversible loss of oligomycin sensitivity which is restored by long-chain Q-homologs. Short-chain ubiquinones like Q₃ produce a loss of oligomycin and dicyclohexylcarbodiimide (DCCD) sensitivity in submitochondrial particles. The effect shows uncompetitive or noncompetitive kinetics with respect to oligomycin or DCCD respectively. Long-chain ubiquinones have a competitive effect with Q₃, thus restoring oligomycin sensitivity; they behave, however, in about the same way as Q₃ in lowering the DCCD sensitivity in submitochondrial particles. On the basis of these observations we suggest that ubiquinone may be a physiological modulator of ATPase activity in the mitochondrial membrane.Abbreviations used: BHM, beef heart mitochondria; DCCD, dicyclohexylcarbodiimide; ETP, electron transfer particles (submitochondrial particles); Q, ubiquinone.