ATP-driven calcium transport in membrane vesicles of Streptococcus sanguis.

Calcium transport was investigated in membrane vesicles prepared from the oral bacterium Streptococcus sanguis. Procedures were devised for the preparation of membrane vesicles capable of accumulating 45Ca2+. Uptake was ATP dependent and did not require a proton motive force. Calcium transport in these vesicles was compared with 45Ca2+ accumulation in membrane vesicles from Streptococcus faecalis and Escherichia coli. The data support the existence of an ATP-driven calcium pump in S. sanguis similar to that in S. faecalis. This pump, which catalyzes uptake into membrane vesicles, would be responsible for extrusion of calcium from intact cells.     

The high affinity (Ca2+-Mg2+)-ATPase in liver plasma membranes is a Ca2+ pump. Reconstitution of the purified enzyme into phospholipid vesicles

The purified (Ca2+-Mg2+)-ATPase from rat liver plasma membranes (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215) was incorporated into soybean phospholipid vesicles, together with its activator. In the presence of millimolar concentrations of Mg2+, the reconstituted proteoliposomes displayed a rapid, saturable, ATP-dependent Ca2+ uptake. Half-maximal Ca2+ uptake activity was observed at 13 +/- 3 nM free Ca2+, and the apparent Km for ATP was 16 +/- 6 microM. Ca2+ accumulated into proteoliposomes (2.8 +/- 0.2 nmol of Ca2+/mg of protein/90 s) was totally released upon addition of the Ca2+ ionophore A-23187. Ca2+ uptake into vesicles reconstituted with enzyme alone was stimulated 2-2.5-fold by the (Ca2+-Mg2+)-ATPase activator, added exogenously. The (Ca2+-Mg2+)-ATPase activity of the reconstituted vesicles, measured using the same assay conditions as for ATP-dependent Ca2+ uptake activity (e.g. in the presence of millimolar concentrations of Mg2+), was maximally activated by 20 nM free Ca2+, half-maximal activation occurring at 13 nM free Ca2+. The stoichiometry of Ca2+ transport versus ATP hydrolysis approximated 0.3. These results provide a direct demonstration that the high affinity (Ca2+-Mg2+)-ATPase identified in liver plasma membranes is responsible for Ca2+ transport.     

Role of the Plasma Membrane H+-ATPase in K+ Transport

The role of the plant plasma membrane H+-ATPase in K+ uptake was examined using red beet (Beta vulgaris L.) plasma membrane vesicles and a partially purified preparation of the red beet plasma membrane H+-ATPase reconstituted in proteoliposomes and planar bilayers. For plasma membrane vesicles, ATP-dependent K+ efflux was only partially inhibited by 100 [mu]M vanadate or 10 [mu]M carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. However, full inhibition of ATP-dependent K+ efflux by these reagents occurred when the red beet plasma membrane H+-ATPase was partially purified and reconstituted in proteoliposomes. When reconstituted in a planar bilayer membrane, the current/voltage relationship for the plasma membrane H+-ATPase showed little effect of K+ gradients imposed across the bilayer membrane. When taken together, the results of this study demonstrate that the plant plasma membrane H+-ATPase does not mediate direct K+ transport chemically linked to ATP hydrolysis. Rather, this enzyme provides a driving force for cellular K+ uptake by secondary mechanisms, such as K+ channels or H+/K+ symporters. Although the presence of a small, protonophore-insensitive component of ATP-dependent K+ transport in a plasma membrane fraction might be mediated by an ATP-activated K+ channel, the possibility of direct K+ transport by other ATPases (i.e. K+-ATPases) associated with either the plasma membrane or other cellular membranes cannot be ruled out.     

Novel ATP-dependent calcium transport component from rat liver plasma membranes. The transporter and the previously reported (Ca2+-Mg2+)-ATPase are different proteins

An ATP-dependent calcium transport component from rat liver plasma membranes was solubilized by cholate and reconstituted into egg lecithin vesicles by a cholate dialysis procedure. The uptake of Ca2+ into the reconstituted vesicles was ATP-dependent and the trapped Ca2+ could be released by A23187. Nucleotides, including ADP, UTP, GTP, CTP, GDP, AMP, and adenyl-5'-yl beta, gamma-imidophosphate, and p-nitrophenylphosphate did not substitute for ATP. The concentration of ATP required for half-maximal stimulation of Ca2+ uptake into the reconstituted vesicles was 6.2 microM. Magnesium was required for calcium uptake. Inhibitors of mitochondrial calcium-sequestering activities, i.e. oligomycin, sodium azide, ruthenium red, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and valinomycin did not affect the uptake of Ca2+ into the vesicles. In addition, strophanthidin and p-chloromercuribenzoate did not affect the transport. Calcium transport, however, was inhibited by vanadate in a concentration-dependent fashion with a K0.5 of 10 microM. A calcium-stimulated, vanadate-inhibitable phosphoprotein was demonstrated in the reconstituted vesicles with an apparent molecular weight of 118,000 +/- 1,300. These properties of Ca2+ transport by vesicles reconstituted from liver plasma membranes suggest that this ATP-dependent Ca2+ transport component is different from the high affinity (Ca2+-Mg2+)-ATPase found in the same membrane preparation (Lotersztajn, S., Hanoune, J. and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215; Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020). When the entire reconstituted vesicle population was treated with ATP and 45Ca in a buffer containing oxalate, the vesicles with Ca2+ transport activity could be separated from other vesicles by centrifugation in a density gradient and the ATP-dependent Ca2+ transport component was purified approximately 9-fold. This indicates that transport-specific fractionation may be used to isolate the ATP-dependent Ca2+ transport component from liver plasma membrane.     

F0F1-ATPase from Vibrio alginolyticus. Subunit composition and proton pumping activity.

An F0F1-ATPase was isolated from the membranes of the marine bacterium Vibrio alginolyticus. Homology between the subunits of the F0-complexes from E. coli and V. alginolyticus was found using antibodies against subunits a, b and c of the E. coli F0F1-ATPase. The F0F1-complex from V. alginolyticus was reconstituted into proteoliposomes, which were competent in ATP-dependent proton uptake. This process was inhibited by triphenyltin, DCCD, and venturicidin. Na+ did not affect proton translocation.     

The blocking effect of aliphatic hydrocarbons on Ca2+ leakage channels formed by aggregates of Ca2+-ATPase of the sarcoplasmic reticulum

The effects of aliphatic hydrocarbons within the liposomes on the Ca2+ transport function of isolated sarcoplasmic reticulum (SR) membranes of rabbit skeletal muscle, vesiculate preparation of Ca2+ dependent ATPase and proteoliposomes reconstituted from Ca2+-ATPase and egg phosphatidylcholine, were studied. It was shown that liposomes prepared from dipalmitoyl phosphatidylcholine containing aliphatic hydrocarbons increase 2 to 3 times Ca2+ accumulation by Ca2+-dependent ATPase from rabbit skeletal muscle SR. Ca2+ transport by SR vesicles increases in the presence of hydrocarbons by 15--20%. The activating effect of hydrocarbons on Ca2+ transport by proteoliposomes depends on the lipid/protein ratio. The proteoliposomes with a high lipid/protein ratio are practically insensitive to the effects of hydrocarbons. It was suggested that activation of Ca2+ transport by hydrocarbons is due to blocking of Ca2+ leakage channels formed during the aggregation of Ca2+-ATPase molecules. Treatment of membranes by formaldehyde results in the oligomerization of Ca2+-ATPase and decreases 2--4-fold the ATP-dependent accumulation of Ca2+. Subsequent addition of decane restores Ca2+ transport practically completely.     

Effect of gradients of monovalent cations on active transport of Ca2+ in the sarcoplasmic reticulum and proteoliposomes

Artificially generated K+ gradient from the sarcoplasmic reticulum vesicles enhances the ATP-dependent Ca2+ transport. The effect is not specific for K+, and is observed when K+ is replaced by Na+ or choline. Dissipation of the K+, Na+, choline gradient does not influence the ATP-dependent Ca2+ transport in proteoliposomes from asolectin and purified Ca2+-ATPase. The K gradient in the presence of valinomycin stimulates the ATP-dependent Ca2+ transport in proteoliposomes.     

Reconstitution of the sodium-calcium exchanger from cardiac sarcolemmal vesicles

The Na+-Ca2+ exchanger was extracted from cardiac sarcolemmal vesicles and reconstituted into phospholipid vesicles by a cholate-dialysis method. Reconstitution was attempted with different phospholipids. Phosphatidylcholine alone was ineffective, whereas phosphatidylcholine and phosphatidylethanolamine (1:1, w/w) showed high activity, but a significant Ca2+ uptake in the absence of Na+ gradient. Optimal reconstitution was obtained with a mixture of phosphatidylcholine and phosphatidylserine (9:1, mol/mol). The reconstituted proteoliposomes showed an ouabain-sensitive (Na+ + K+)-ATPase activity and a Na+-Ca2+ exchange with a specific activity comparable to that of the original vesicles. The specificity toward Na+ was also recovered. A partial purification of the exchanger was obtained by the method of transport-specificity fractionation ( Goldin , S.M. and Rhoden , V. (1978) J. Biol. Chem. 253, 2575-2583). When proteoliposomes were reconstituted with sodium oxalate inside and incubated with calcium in the presence of an outwardly directed Na+ gradient, the vesicles containing the Na+-Ca2+ exchanger specifically accumulated calcium which precipitated inside as calcium oxalate. The resulting increase in density allowed separation of the proteoliposomes containing the Na+-Ca2+ exchanger from the rest of the vesicles on a sucrose density gradient.     

Membrane ATPase of Vibrio alginolyticus. Ion transport activity and homology with F0F1-ATPase from E. coli]

F0F1-ATPase has been isolated from the marine alkali-resistant bacterium Vibrio alginolyticus. The enzyme subunits cross-reacted with antibodies against subunits alpha, beta, gamma, epsilon, and b of E. coli ATPase. The purified ATPase was reconstituted into liposomes effecting an ATP-dependent uptake of H+. Proton transport was inhibited by the ATPase blockers DCCD, triphenyltin, and venturicidin. Na+ ions had no effect on ATP-dependent proton transport. No ATP-dependent transport of Na+ was detected in proteoliposomes.     

Bacterial anion exchange. Use of osmolytes during solubilization and reconstitution of phosphate-linked antiport from Streptococcus lactis

Membranes of Streptococcus lactis were solubilized with 1.1% octyl-beta-D-glucopyranoside in the presence of 0.37% acetone/ether-washed phospholipid from several sources. After adding excess Escherichia coli phospholipid as bath-sonicated liposomes, phosphate:sugar phosphate antiport was reconstituted in proteoliposomes by a 25-fold dilution in 0.1 M KPi (pH 7). Assays of 32Pi:Pi exchange showed that antiport was subject to an inactivation which varied in severity according to the lipid present at solubilization. Recovery of Pi-linked exchange was improved by the presence of 10-20% glycerol or other osmolyte during extraction. The osmolytes tested in this regard have included polyols (glycerol, erythritol, xylitol, sorbitol), sugars (glucose, trehalose), and two amino acids (glycine, proline). Each gave 10--20-fold increased recoveries of 32Pi:Pi antiport compared to controls using only detergent and lipid; these precautions were not required for the efficient reconstitution of F0F1-ATPase. Antiport in the artificial system was studied most carefully when glycerol was the stabilizing additive. For that case, the Kt values for Pi or 2-deoxyglucose 6-phosphate transport (275 and 25 microM, respectively) were the same as in native membranes. Maximal rates of Pi and 2-deoxyglucose 6-phosphate transport (200 and 42 nmol/min/mg of protein, respectively) and the turnover number for Pi exchange (25--50/s) suggested that antiporters were recovered without loss of activity. We conclude that the quantitative aspects of bacterial anion exchange are amenable to study in an artificial system, and that the use of osmolytes as general stabilants can be a valuable adjunct to current techniques for reconstitution of integral membrane transport proteins.     

Regulation of arginine-ornithine exchange and the arginine deiminase pathway in Streptococcus lactis.

Streptococcus lactis metabolizes arginine by the arginine deiminase (ADI) pathway. Resting cells of S. lactis grown in the presence of galactose and arginine maintain a high intracellular ornithine pool in the absence of arginine and other exogenous energy sources. Addition of arginine results in a rapid release of ornithine concomitant with the uptake of arginine. Subsequent arginine metabolism results intracellularly in high citrulline and low ornithine pools. Arginine-ornithine exchange was shown to occur in a 1-to-1 ratio and to be independent of a proton motive force. The driving force for arginine uptake in intact cells is supplied by the ornithine and arginine concentration gradients formed during arginine metabolism. These results confirm studies of arginine and ornithine transport in membrane vesicles of S. lactis (A. J. M. Driessen, B. Poolman, R. Kiewiet, and W. N. Konings, Proc. Natl. Acad. Sci. USA, 84:6093-6097). The activity of the ADI pathway appears to be affected by the internal concentration of (adenine) nucleotides. Conditions which lower ATP consumption (dicyclohexylcarbodiimide, high pH) decrease the ADI pathway activity, whereas uncouplers and ionophores which stimulate ATP consumption increase the activity. The arginine-ornithine exchange activity matches the ADI pathway most probably by adjusting the intracellular levels of ornithine and arginine. Regulation of the ADI pathway and the arginine-ornithine exchanger at the level of enzyme synthesis is exerted by glucose (repressor, antagonized by cyclic AMP) and arginine (inducer). An arginine/ornithine antiport was also found in Streptococcus faecalis DS5, Streptococcus sanguis 12, and Streptococcus milleri RH1 type 2.     

Ca2+-transporting properties of myometrial plasma cell membrane Ca2+, Mg2+-ATPase reconstituted in liposomes

Purified myometrium cells plasma membrane Ca2+, Mg(2+)-ATPase was reconstitute in liposomes in functionally active state by the method of cholate dialysis: it showed ATP-hydrolase activity increased by 0.8 microM A23187 average 4 times and it showed Mg2+, ATP-dependent Ca(2+)-transporting activity. Reconstituted system transported Ca2+ at an initial rate of 114.4 +/- 16.3 nmol.min-1.mg-1 with the stoichiometry Ca2+: ATP = 1: (3.2-3.7). Calmodulin increased by 30% the initial rate of Ca(2+)-accumulation by the proteoliposomes with reconstituted Ca2+, Mg(2+)-ATPase; 0.1 mM orthovanadate decreased by 80% Ca(2+)-accumulation by this system. Ca2+, Mg(2+)-ATPase reconstituted in liposomes is just Ca(2+)-transporting ATPase of the plasma membrane. Obtained enzyme preparate can be utilised for study of the properties of this important energy-dependent Ca(2+)-transporting system of smooth muscle cell.     

Ca~(2+)通过膜脂影响肌质网Ca~(2+)-ATP酶的活性与Ca~(2+)转运功能

重建在大豆磷脂脂质体上的兔骨骼肌肌质网Ca~(2+)—ATP酶在ATP驱动下可将溶液中的Ca~(2+)转运到脂酶体内部;外加EGTA则可除去脂酶体外部的Ca~(2+),由此可得到四种含Ca~(2+)状态不同的脂酶体:(1)内、外都无Ca~(2+);(2)仅外部有Ca~(2+);(3)内、外都有Ca~(2+);(4),仅内部有Ca~(2+).用DPH和AS系列萤光探针对这四种含Ca~+状态不同的脂酶体的膜脂流动性进行了测定,结果表明:脂酶体外部加入Ca~(2+),脂双层外表面的流动性降低.当Ca~(2+)进入脂酶体内部后,内表面膜脂的流动性也降低,而且外层膜脂流动性进一步降低.脂酶体内、外的Ca~(2+)含量不同时,Ca~(2+)—ATP酶功能状态也不同.转运到脂酶体内部的ca~(2+)积累到一定浓度后,通过Ca~(2+)泵向内转运的Ca~(2+)及Ca~(2+)—ATP酶活力都受到了抑制.转运进行到第四分钟时的酶活只有第一分钟的9%.但在相同的实验条件下,失去了完整的膜结构的纯化的Ca~(2+)—ATP酶蛋白没有被抑制.这提示完整的膜结构是这种抑制作用所必需的,而且膜两侧Ca~(2+)浓度的梯差可通过影响膜脂来调节Ca~(2+)—ATP酶的功能.     

The vanadate-sensitive ATPase of Streptococcus faecalis pumps potassium in a reconstituted system

The vanadate-sensitive ATPase of Streptococcus faecalis, purified to homogeneity, was reconstituted into soybean phospholipid vesicles in a functional state. Freeze-fracture electron micrographs revealed a relatively uniform population of unilamellar liposomes of 50-100 nm in diameter, with particles protruding from both fracture faces. Transport studies with 42K+ and with a K+-selective electrode showed that the ATP-ase catalyzes electrogenic potassium extrusion in proteoliposomes. The following parameters for potassium transport in the reconstituted system were determined: K+/ATP stoichiometry = 1, Km for potassium = 1.4 mM, Vmax = 0.1 mumol/min/mg. The ATPase could be activated by an electrical membrane potential, vesicle interior positive. This ATPase thus appears to function as a potential regulated, ATP-driven pump that serves in electrogenic potassium accumulation by the bacterial cell.     

Solubilization and functional reconstitution of the DCCD-sensitive Na+/H(+)-antiporter from Halobacterium halobium

Na+/H+ exchange activity was solubilized from Halobacterium halobium with octyl-beta-D-glucoside (OG) and was reconstituted into the bacterio-rhodopsin incorporated liposomes (BR-liposomes) by the detergent-dialysis method. Light illumination stimulated uphill 22Na+ uptake into the reconstituted conjugate proteoliposomes. The 22Na+ uptake was FCCP-sensitive and was dependent on the amounts of OG-extract applied. On the other hand, the proteoliposomes reconstituted with the membrane fraction pretreated with N,N'-dicyclohexylcarbodiimide (DCCD) did not exhibit the light-dependent 22Na+ uptake, thus, DCCD-sensitive. When the reconstituted proteoliposome was incubated with [14C]DCCD, radio-labels appeared slightly on 50K but mainly on 11K-Dalton component, which are the same components labeled in the intact membrane vesicles. It is concluded that halobacterial DCCD-sensitive Na+/H(+)-antiporter was solubilized and reconstituted in the conjugate BR-liposomes with preserved functional unit.     

Modulation of coated vesicle chloride channel activity and acidification by reversible protein kinase A-dependent phosphorylation.

We have previously shown that activity of a Cl- channel is required for acidification of clathrin-coated vesicles by the coated vesicle (H+)-ATPase (Arai, H., Pink, S. and Forgac, M. (1989) Biochemistry 28, 3075-3082). We demonstrate that activity of the coated vesicle Cl- channel is modulated by phosphorylation. Cl- conductance was measured in a reconstituted preparation of coated vesicle membrane proteins using the Cl(-)-sensitive fluorescence probe, 6-methoxy-N-(3-sulfopropyl)quinolinium. Treatment of coated vesicle membranes with alkaline phosphatase resulted in a 25 +/- 5% decrease in Cl- channel activity. A parallel decrease in ATP-dependent acidification of coated vesicles was also observed. The decrease in Cl- conductance and ATP-dependent acidification was reversed by treatment with protein kinase A and MgATP; the alkaline phosphatase inhibitor, sodium orthovanadate, blocked the inhibition of acidification. These results indicate that Cl- conductance in coated vesicles is modulated by a protein kinase A-dependent phosphorylation and that this modulation in turn affects ATP-dependent acidification.     

Molecular and genetic characterization of lactose-metabolic genes of Streptococcus cremoris.

Lac+ plasmid DNA from Streptococcus cremoris H2 was subcloned with an Escherichia coli vector on a 3.5-kilobase-pair PstI-AvaI fragment. Genetic analysis of the cloned DNA was possible because linear Lac+ DNA fragments were productive in the S. sanguis transformation system. Complementation of S. sanguis Lac-mutants showed that the 3.5-kilobase-pair fragment included the structural gene for 6-phospho-beta-D-galactosidase and either enzyme II-lac or factor III-lac of the lactose-specific phosphoenolpyruvate-dependent phosphotransferase system. Expression of the S. cremoris-like 40,000-dalton 6-phospho-beta-D-galactosidase in S. sanguis Lac+ transformants, rather than the 52,000-dalton wild-type S. sanguis enzyme, demonstrated the occurrence of gene replacement and not gene repair. The evidence supports chromosomal integration as the mechanism by which S. sanguis Lac- recipients are converted to a Lac+ phenotype after transformation with Lac+ DNA. Southern blot data suggest that the Lac+ DNA does not reside on a transposon, but that integration always occurs within a specific HincII fragment of the recipient chromosome. Hybridization experiments demonstrate homology between the S. cremoris Lac+ DNA and cellular DNA from Lac+ strains of Streptococcus lactis, S. mutans, S. faecalis, and S. sanguis.     

The purified and functionally reconstituted multidrug transporter LmrA of Lactococcus lactis mediates the transbilayer movement of specific fluorescent phospholipids

Lactococcus lactis possesses an ATP-binding cassette transporter, LmrA, which is a homolog of the mammalian multidrug resistance (MDR) P-glycoprotein, and is able to transport a broad range of structurally unrelated amphiphilic drugs. A histidine tag was introduced at the N-terminus of LmrA to facilitate purification by nickel affinity chromatography. The histidine-tagged protein was overexpressed in L. lactis using a novel protein expression system for cytotoxic proteins based on the tightly regulated, nisin-inducible nisA promoter. This system allowed us to get functional overexpression of LmrA up to a level of 30% of total membrane protein. For reconstitution, LmrA was solubilized with dodecylmaltoside, purified by nickel-chelate affinity chromatography, and reconstituted in dodecylmaltoside-destabilized, preformed liposomes prepared from L. lactis phospholipids. The detergent was removed by adsorption onto polystyrene beads. The LmrA protein was reconstituted in a functional form, and mediated the ATP-dependent transport of the fluorescent substrate Hoechst-33342 into the proteoliposomes. Interestingly, reconstituted LmrA also catalyzed the ATP-dependent transport of fluorescent phosphatidylethanolamine, but not of fluorescent phosphatidylcholine. These data demonstrate that LmrA activity is independent of accessory proteins and support the notion that LmrA may be involved in the transport of specific lipids or lipid-linked precursors in L. lactis.     

Purification of a putative K+-ATPase from Streptococcus faecalis

We have purified a novel membrane ATPase from Streptococcus faecalis by the following procedure: extraction of membranes with Triton X-100 followed by fractionation of the extract by successive DEAE-cellulose chromatography, hydroxylapatite chromatography and Cm-Sepharose chromatography. The overall yield was 5%. The purified ATPase appears to consist of a single polypeptide component of Mr = 78,000. The Triton-solubilized purified enzyme has a specific activity of approximately 50 mumol of ATP hydrolyzed per min per mg, is dependent on phospholipids for activity, and is strongly inhibited by vanadate (I50 = 3 microM). Maximal ATPase activity is displayed at pH 7.3. Mg2+-ATP, for which the enzyme has a Km of 60 microM, is the best substrate. The ATPase forms an acylphosphate intermediate that can also be detected in native membranes as the major acylphosphate component. The purified ATPase, when reconstituted into soybean phospholipid vesicles, exhibits coupling, e.g. the ATPase activity can be stimulated at least 8-fold by valinomycin in the presence of potassium. Based on these observations we conclude that the enzyme we have purified is an ion-motive ATPase, most likely a K+-ATPase.