Characterization of the ATP transporter in the reconstituted rough endoplasmic reticulum proteoliposomes

Adenosine triphosphate (ATP) transporter from rat liver rough endoplasmic reticulum (RER) was solubilized and reconstituted into phosphatidylcholine liposomes. The RER proteoliposomes, resulting from optimizing some reconstitution parameters, had an apparent K(m) value of 1.5 microM and a V(max) of 286 pmol min(-1) (mg protein)(-1) and showed higher affinity for ATP and a lower V(max) value than intact RER (K(m) of 6.5 microM and V(max) of 1 nmol). ATP transport was time- and temperature-dependent, inhibited by 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid, which is known as an inhibitor of anion transporters including ATP transporter, but was not affected by atractyloside, a specific inhibitor of mitochondrial ADP/ATP carrier. The internal and external effects of various nucleotides on the ATP transport were examined. ATP transport was cis-inhibited strongly by ADP and weakly by AMP. ADP-preloaded RER proteoliposomes showed a specific increase of ATP transport activity while AMP-preloaded RER proteoliposomes did not show the enhanced overshoot peak in the ATP uptake plot. These results demonstrate the ADP/ATP antiport mechanism of ATP transport in rat liver RER.     

Selective transport of adenosine into porcine coronary smooth muscle

Adenosine (ADO), an endogenous regulator of coronary vascular tone, enhances vasorelaxation in the presence of nucleoside transport inhibitors such as dipyridamole. We tested the hypothesis that coronary smooth muscle (CSM) contains a high-affinity transporter for ADO. ADO-mediated relaxation of isolated large and small porcine coronary artery rings was enhanced 12-fold and 3.4-fold, respectively, by the transport inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI). Enhanced relaxation was independent of endothelium and was selective for ADO over synthetic analogs. Uptake of [(3)H]ADO into freshly dissociated CSM cells or endothelium-denuded rings was linear and concentration dependent. Kinetic analysis yielded a maximum uptake (V(max)) of 67 +/- 7.0 pmol. mg protein(-1). min(-1) and a Michaelis constant (K(m)) of 10. 5 +/- 5.8 microM in isolated cells and a V(max) of 5.1 +/- 0.5 pmol. min(-1). mg wet wt(-1) and a K(m) of 17.6 +/- 2.6 microM in intact rings. NBTI inhibited transport into small arteries (IC(50) = 42 nM) and cells. Analyses of extracellular space and diffusion kinetics using [(3)H]sucrose indicate the V(max) and K(m) for ADO transport are sufficient to clear a significant amount of extracellular adenosine. These data indicate CSM possess a high-affinity nucleoside transporter and that the activity of this transporter is sufficient to modulate ADO sensitivity of large and small coronary arteries.     

Purification and partial characterization of membrane-associated type II (cGMP-activatable) cyclic nucleotide phosphodiesterase from rabbit brain

Membrane-associated, Type II (cGMP-activatable) cyclic nucleotide phosphodiesterase (PDE) from rabbit brain, representing 75% of the total homogenate Type II PDE activity, was purified to apparent homogeneity. The enzyme was released from 13,000 x g particulate fractions by limited proteolysis with trypsin and fractionated using DE-52 anion-exchange, cGMP-Sepharose affinity and hydroxylapatite chromatographies. The enzyme showed 105 kDa subunits by SDS-PAGE and had a Stokes radius of 62.70 A as determined by gel filtration chromatography. Hydrolysis of cAMP or cGMP showed positive cooperativity, with cAMP kinetic behavior linearized in the presence of 2 microM cGMP. Substrate concentrations required for half maximum velocity were 28 microM for cAMP and 16 microM for cGMP. Maximum velocities were approx. 160 mumol/min per mg for both nucleotides. The apparent Kact for cGMP stimulation of cAMP hydrolysis at 5 microM substrate was 0.35 microM and maximal stimulation (3-5-fold) was achieved with 2 microM cGMP. Cyclic nucleotide hydrolysis was not enhanced by calcium/calmodulin. The purified enzyme can be labeled by cAMP-dependent protein kinase as demonstrated by the incorporation of 32P from [gamma-32P]ATP into the 105 kDa enzyme subunit. Initial experiments showed that phosphorylation of the enzyme did not significantly alter enzyme activity measured at 5 microM [3H]cAMP in the absence or presence of 2 microM cGMP or at 40 microM [3H]cGMP. Monoclonal antibodies produced against Type II PDE immunoprecipitate enzyme activity, 105 kDa protein and 32P-labeled enzyme. The 105 kDa protein was also photoaffinity labeled with [32P]cGMP. The purified Type II PDE described here is physicochemically very similar to the isozyme purified from the cytosolic fraction of several bovine tissues with the exception that it is predominantly a particulate enzyme. This difference may reflect an important regulatory mechanism governing the metabolism of cyclic nucleotides in the central nervous system.     

ATP-dependent transport of bile salts by rat multidrug resistance-associated protein 3 (Mrp3)

We have previously shown that cloned rat multidrug resistance-associated protein 3 (Mrp3) has the ability to transport organic anions such as 17beta-estradiol 17-beta-D-glucuronide (E(2)17betaG) and has a different substrate specificity from MRP1 and MRP2 in that glutathione conjugates are poor substrates for Mrp3 (Hirohashi, T., Suzuki, H., and Sugiyama, Y. (1999) J. Biol. Chem. 274, 15181-15185). In the present study, the involvement of Mrp3 in the transport of endogenous bile salts was investigated using membrane vesicles from LLC-PK1 cells transfected with rat Mrp3 cDNA. The ATP-dependent uptake of [(3)H]taurocholate (TC), [(14)C]glycocholate (GC), [(3)H]taurochenodeoxycholate-3-sulfate (TCDC-S), and [(3)H]taurolithocholate-3-sulfate (TLC-S) was markedly stimulated by Mrp3 transfection in LLC-PK1 cells. The extent of Mrp3-mediated transport of bile salts was in the order, TLC-S > TCDC-S > TC > GC. The K(m) and V(max) values for the uptake of TC and TLC-S were K(m) = 15.9 +/- 4.9 microM and V(max) = 50.1 +/- 9.3 pmol/min/mg of protein and K(m) = 3.06 +/- 0.57 microM and V(max) = 161.9 +/- 21.7 pmol/min/mg of protein, respectively. At 55 nM [(3)H]E(2)17betaG and 1.2 microM [(3)H]TC, the apparent K(m) values for ATP were 1.36 and 0.66 mM, respectively. TC, GC, and TCDC-S inhibited the transport of [(3)H]E(2)17betaG and [(3)H]TC to the same extent with an apparent IC(50) of approximately 10 microM. TLC-S inhibited the uptake of [(3)H]E(2)17betaG and [(3)H]TC most potently (IC(50) of approximately 1 microM) among the bile salts examined, whereas cholate weakly inhibited the uptake (IC(50) approximately 75 microM). Although TC and GC are transported by bile salt export pump/sister of P-glycoprotein, but not by MRP2, and TCDC-S and TLC-S are transported by MRP2, but not by bile salt export pump/sister of P-glycoprotein, it was found that Mrp3 accepts all these bile salts as substrates. This information, together with the finding that MRP3 is extensively expressed on the basolateral membrane of human cholangiocytes, suggests that MRP3/Mrp3 plays a significant role in the cholehepatic circulation of bile salts.     

Large-scale purification,dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK(2)) of Salmonella typhimurium

The maltose ATP-binding cassette (ABC) transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK(2)) and a periplasmic substrate binding protein. To further elucidate protein-protein interactions between the subunits, we have studied the dissociation and reassembly of the MalFGK(2) complex at the level of purified components in proteoliposomes. First, we optimized the yield in purified complex protein by taking advantage of a newly constructed expression plasmid that carries the malK, malF and malG genes in tandem orientation. Incorporated in proteoliposomes, the complex exhibited maltose binding protein/maltose-dependent ATPase activity with a V(max) of 1.25 micromol P(i)/min/mg and a K(m) of 0.1 mM. ATPase activity was sensitive to vanadate and enzyme IIA(Glc), a component of the enterobacterial glucose transport system. The proteoliposomes displayed maltose transport activity with an initial rate of 61 nmol/min/mg. Treatment of proteoliposomes with 6.6 M urea resulted in the release of medium-exposed MalK subunits concomitant with the complete loss of ATPase activity. By adding increasing amounts of purified MalK to urea-treated proteoliposomes, about 50% of vanadate-sensitive ATPase activity relative to the control could be recovered. Furthermore, the phenotype of MalKQ140K that exhibits ATPase activity in solution but not when associated with MalFG was confirmed by reassembly with MalK-depleted proteoliposomes.     

Diacylglycerol acyltransferase in maturing sunflower seeds

Developing sunflower seeds exhibit a high diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) activity. The distribution of the enzyme has been studied in subcellular fractions prepared by differential centrifugation of seed homogenate. Its activity was characterized using [1-(14)C]oleoyl-CoA and diolein dispersed in Tween 20. Some properties of the microsomal fraction of DAGAT were investigated. Hyperbolic kinetics were observed, the apparent K(m) was 60 microM and the specific activity of the reaction 15 pmol/min/mg of protein. Addition of BSA (0.1%) stimulated oleate incorporation, which was not dependent on the presence of exogenous diacylglycerol. Detergents which might solubilize DAGAT, Triton X-100 and CHAPS, were tested for enzyme inhibition, and CHAPS was found to be the least denaturing.     

Protein kinase activities in neoplastic squamous epithelia and normal epithelia from the upper aero-digestive tract

In the present study the activities of three different protein kinase were determined in squamous cell carcinoma from the upper aero-digestive tract, and compared with the activities in normal oral mucosa. The protein kinases investigated are: a) cAMP-dependent protein kinase; b) cGMP-dependent protein kinase, and c) casein kinase II. The basal protein kinase activity, when histone IIa was used as substrate, was about 3-fold higher in tumors, as compared to normal mucosa, in the soluble fraction (32.0 +/- 4.2 and 10.9 +/- 2.4 pmol 32P/mg prot. X min, respectively). In the particulate fraction the basal protein kinase activity was about 9 times higher in tumors as compared to normal mucosa (19.4 +/- 5.2 and 2.1 +/- 0.3 pmol 32P/mg prot X min, respectively). The protein kinase activity in the presence of cyclic nucleotide (cAMP/cGMP) minus the basal protein kinase activity was taken as the cAMP- and the cGMP-dependent protein kinase activity, respectively. Maximal protein kinase activity was obtained in the presence of 0.5 microM of cyclic nucleotide both in squamous cell carcinoma and normal mucosa. In the cytosolic fraction the cAMP-dependent protein kinase activity was 33.9 +/- 13.0 pmol 32P/mg prot. X min in tumors, and 28.2 +/- 5.8 pmol 32P/mg prot. X min in normal tissue, after stimulation with 0.5 microM cAMP. The cGMP-dependent protein kinase activity was 5-10% of the cAMP-dependent protein kinase activity, and no concentration-dependent stimulation with cGMP was seen. The cGMP-dependent protein kinase activity in the presence of 0.5 microM cGMP was 2.4 +/- 1.3 and 1.8 +/- 0.6 pmol 32P/mg prot. X min in tumors and normal mucosa, respectively. Casein kinase II activity was determined only in the cytosolic fraction and was found to be 3-fold higher in tumors as compared to normal mucosa (31.8 +/- 5.2 and 8.6 +/- 3.5 pmol 32P/mg prot X min, respectively). This study shows a general increase in histone phosphorylation and casein kinase activity in neoplastic squamous epithelia compared to normal epithelia. No evidence for an increase in cyclic nucleotide dependent protein kinase activities in neoplastic squamous epithelia was found. This study thus supports the idea that phosphorylation/dephosphorylation reactions may play an important role in the control of cell growth, differentiation and proliferation.     

Kinetic studies on mitochondrial F(1)-ATPase from crayfish (Orconectes virilis) gills

The substrate kinetics and the role of free Mg(2+) and free ATP were studied in membrane-bound F(1)-ATPase from crayfish (Orconectes virilis) gills. It was shown that the MgATP complex was the true substrate for the ATPase activity with a K(m) value of 0.327 mM. In the absence of bicarbonate, the maximum azide-sensitive activities in the presence and absence (<18 microM) of free ATP were 0.878 and 0.520 micromol P(i)/mg protein/min, respectively, while the maximum bicarbonate-stimulated activity in absence of free ATP was 1.486 micromol P(i)/mg protein/min. Free ATP was a competitive inhibitor (K(i)=0.77 mM) and free Mg(2+) was a mixed inhibitor (K(i)=0.81 mM, K(i)'=5.89 mM). However, free ATP also acted as an activator. Lineweaver-Burk plots for MgATP hydrolysis at high free Mg(2+) concentrations exhibited an apparent negative cooperativity, which was not the case for high free ATP levels. These results suggest that, although free ATP inhibited the enzyme by binding to catalytic sites, it stimulated ATPase activity by binding to non-catalytic sites and promoted the dissociation of inhibitory MgADP from the catalytic site.     

ATPase activity of purified and reconstituted multidrug resistance protein MRP1 from drug-selected H69AR cells.

The ATP-binding cassette transporter protein, multidrug resistance protein MRP1, was purified from doxorubicin-selected H69AR lung tumor cells which express high levels of this protein. A purification procedure comprised of a differential two-step solubilization of MRP1 from plasma membranes with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate followed by immunoaffinity chromatography using the MRP1-specific monoclonal antibody QCRL-1 was developed. Approximately 300 microgram of MRP1 was obtained from 6 mg of plasma membranes at 80-90% purity, as indicated by silver staining of protein gels. After reconstitution of purified MRP1 into proteoliposomes, kinetic analyses indicated that its K(m) for ATP hydrolysis was 104+/-22 microM with maximal activity of 5-10 nmol min(-1) mg(-1) MRP1. MRP1 ATPase activity was further characterized with various inhibitors and exhibited an inhibition profile that distinguishes it from P-glycoprotein and other ATPases. The ATPase activity of reconstituted MRP1 was stimulated by the conjugated organic anion substrates leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) with 50% maximal stimulation achieved at concentrations of 150 nM and 1.6 microM, respectively. MRP1 ATPase was also stimulated by glutathione disulfide but not by reduced glutathione or unconjugated chemotherapeutic agents. This purification and reconstitution procedure is the first to be described in which the ATPase activity of the reconstituted MRP1 retains kinetic characteristics with respect to ATP-dependence and substrate stimulation that are very similar to those deduced from transport studies using MRP1-enriched plasma membrane vesicles.     

The effect of Mg2+, nucleotides and ATPase inhibitors on the uptake of [3H]-cGMP to inside-out vesicles from human erythrocytes.

An ATP-dependent transport system is responsible for the cellular extrusion of cGMP. The objective of the present study was to determine the effect of Mg2+, ATP and other nucleotides (2'-dATP, GTP and ADP), exogenous ATPase modulators (such as metavanadate, ouabain, EGTA, NEM, bafilomycin A1 and oligomycin A) on the cGMP transport. The uptake of [3H]-cGMP (1 microM) at 37 degrees C was studied in inside-out vesicles from human erythrocytes. Magnesium caused a maximal activation between 5 and 10 mM and the optimal ATP concentration was 1.25 mM with K50-values of 0.3-0.5 mM. Among other nucleotides tested, 2'-dATP (K50 of 0.7 mM) was nearly as effective as ATP, whereas cGMP accumulated slowly in the presence of GTP. ADP and metavanadate (P-type ATPase inhibitor) showed to be competitive inhibitors with Ki values of 0.15 mM and 10 microns, respectively. NEM (a sulphydryl agent) reduced the ATP-dependent uptake in a concentration-dependent manner with a Ki value of 10 microM. Ouabain (Na+/K(+)-ATPase inhibitor) had no effect. Bafilomycin A1 (V-type ATPase inhibitor) and oligomycin (F-type ATPase inhibitor) were the most potent inhibitors with Ki values of 0.7 and 1.8 microM, respectively. The present study suggests that the cellular cGMP extrusion is energized by an ATPase with a unique inhibitor profile, which clearly differentiates it from the other major classes of membrane-bound ATPases.     

Purification and functional reconstitution of the rat organic cation transporter OCT1

The rat organic cation transporter rOCT1 with six histidine residues added to the C-terminus was expressed in Sf9 insect cells, and expression of organic cation transport was demonstrated. To purify rOCT1 protein, Sf9 cells were lysed with 1% (w/v) CHAPS [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate], centrifuged, and subjected to sequential affinity chromatography using lentil-lectin Sepharose and nickel(II)-charged nitrilotriacetic acid-agarose. This procedure yielded approximately 70 microg of purified rOCT1 protein from 10 standard culture plates. Using a freeze-thaw procedure, purified rOCT1 was reconstituted into proteoliposomes formed from phosphatidylcholine, phosphatidylserine, and cholesterol. Proteoliposomes exhibited uptake of [3H]-1-Methyl-4-phenylpyridinium ([3H]MPP) that was inhibited by quinine and stimulated by an inside-negative membrane potential. MPP uptake was saturable with an apparent K(m) of 30 +/- 17 microM. MPP uptake (0.1 microM) was inhibited by tetraethylammonium, tetrabutylammonium, and tetrapentylammonium with IC50 values of 197 +/- 11, 19 +/- 1, and 1.8 +/- 0.03 microM, respectively. With membrane potential clamped to 0 mV using valinomycin in the presence of 100 mM potassium on both sides of the membrane, uptake of 0.1 microM MPP was trans stimulated 3-fold by 2.5 mM intracellular choline, and efflux of 0.1 microM MPP was trans stimulated 4-fold by 9.5 mM extracellular choline. The data show that rOCT1 is capable and sufficient to mediate transport of organic cations. The observed trans stimulation under voltage-clamp conditions shows that rOCT1 operates as a transporter rather than a channel. Purification and reconstitution of functional active rOCT1 protein is an important step toward the biophysical characterization and crystallization.     

Physiological characterization of 45Ca2+ and 65Zn2+ transport by lobster hepatopancreatic endoplasmic reticulum

The crustacean hepatopancreas is an epithelial-lined, multifunctional organ that, among other activities, regulates the flow of calcium into and out of the animal's body throughout the life cycle. Transepithelial calcium flow across this epithelial cell layer occurs by the combination of calcium channels and cation exchangers at the apical pole of the cell and by an ATP-dependent, calcium ATPase in conjunction with a calcium channel and an Na+/Ca2+ antiporter in the basolateral cell region. The roles of intracellular organelles such as mitochondria, lysosomes, and endoplasmic reticulum (ER) in transepithelial calcium transport or in transient calcium sequestration are unclear, but may be involved in transferring cytosolic calcium from one cell pole to the other. The ER membrane has a complement of ATP-dependent calcium ATPases (SERCA) and calcium channels that regulate the uptake and possible transfer of calcium through this organelle during periods of intense calcium fluxes across the epithelium as a whole. This investigation characterized the mechanisms of calcium transport by lobster hepatopancreatic ER vesicles and the effects of drugs and heavy metals on them. Kinetic constants for 45Ca2+ influx under control conditions were K(n) (m)=10.38+/-1.01 microM, J(max)=14.75+/-1.27 pmol/mg protein x sec, and n=2.53+/-0.46. The Hill coefficient for 45Ca2+ influx under control conditions, approximating 2, suggests that approximately two calcium ions were transported for each transport cycle in the absence of ATP or the inhibitors. Addition of 1 mM ATP to the incubation medium significantly (P<0.01) elevated the rate of 45Ca2+ influx at all calcium activities used and retained the sigmoidal nature of the transport relationship. The kinetic constants for 45Ca2+ influx in the presence of 1 mM ATP were K(n) (m)=12.76+/-0.91 microM, J(max)=25.46+/-1.45 pmol/mg protein x sec, and n=1.95+/-0.15. Kinetic analyses of ER 65Zn2+ influx resulted in a sigmoidal relationship between transport rate and zinc activity under control conditions (K(n) (m)=38.63+/-0.52 microM, J(max)=19.35+/-0.17 pmol/mg protein x sec, n=1.81+/-0.03). The Addition of 1 mM ATP enhanced 65Zn2+ influx at each zinc activity, but maintained the overall sigmoidal nature of the kinetic relationship. The kinetic constants for zinc influx in the presence of 1 mM ATP were K(n) (m)=34.59+/-2.31 microM, J(max)=26.09+/-1.17 pmol/mg protein x sec, and n=1.96+/-0.17. Both sigmoidal and ATP-dependent calcium and zinc influxes by ER vesicles were reduced in the presence of thapsigargin and vanadate. This investigation found that lobster hepatopancreatic ER exhibited a thapsigargin- and vanadate-inhibited, SERCA-like, calcium ATPase. This transporter displayed cooperative calcium transport kinetics (Hill coefficient, n approximately 2.0) and was inhibited by the heavy metals zinc and copper, suggesting that the metals may reduce the binding and transport of calcium when they are present in the cytosol.     

Endogenous phosphorylation of sarcoplasmic reticulum fragments of rabbit fast skeletal muscles

The purified membrane fragments of sarcoplasmic reticulum (SR) of rabbit fast skeletal muscles were found to incorporate 32P from[gamma-32P]ATP in endogenous membrane substrates and in histone H1. The existence of membrane-bound protein kinase of SR was demonstrated by steady state binding of [3H]-cAMP to the SR membranes. The constant of [3H]cAMP binding to the membranes is 2.5 +/- 0.003 x 10(6) M-1, the number of binding sites is 6.1 +/- 0.8 pmol per 1 mg of protein. The endogenous phosphorylation of SR components was inhibited by cAMP and cGMP at concentrations of 10(-7)-10(-6) and depended on Mg2+ and Ca2+. The thermostable protein inhibitor of cAMP-dependent protein kinase inhibited the endogenous phosphorylation of SR membranes by 30-40%. The protein phosphoproduct of SR membranes revealed the properties of a phosphoester. The membrane-bound protein kinase was active towards the exogenous substrate--histone H1. Phosphorylation in the presence of histones was independent of cyclic nucleotides, Mg2+ and Ca2+. Fractionation of 32P-labelled solubilized membranes in polyacrylamide gel in the presence of Na-SDS showed that the radioactivity is bound to protein zones with molecular weights of 95 000 and 6000.     

Functional expression of the human breast cancer resistance protein in Pichia pastoris

We report functional expression of BCRP in Pichia pastoris in which BCRP was produced as a 62 kDa underglycosylated protein. BCRP expression level in P. pastoris was comparable to that in HEK cells. The basal BCRP ATPase activity in the yeast membranes was approximately 40-80 nmol Pi/min/mg protein, which can be modulated by known BCRP substrates and inhibitors. Photolabeling of BCRP with 8-azido[alpha-32P]ATP was dependent preferentially on the presence of Co2+ than Mg2+ and could be inhibited by a molar excess of ATP. Vanadate-induced trapping of 8-azido[alpha-32P]ADP by BCRP was much more significant in the presence of Co2+ than that with Mg2+. The Km and Vmax values of BCRP for [3H]E1S transport were 3.6+/-0.3 microM and 55.2+/-1.6 pmol/min/mg protein, respectively. This efficient and cost-effective expression system should facilitate large scale production and purification of BCRP for further structural and functional analyses.     

The effect of lipid environment and retinoids on the ATPase activity of ABCR, the photoreceptor ABC transporter responsible for Stargardt macular dystrophy

ABCR is a photoreceptor-specific ATP-binding cassette transporter that has been linked to various retinal diseases, including Stargardt macular dystrophy, and implicated in retinal transport across rod outer segment (ROS) membranes. We have examined the ATPase and GTPase activity of detergent-solubilized and reconstituted ABCR. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-solubilized ABCR had ATPase and GTPase activity (K(m) approximately 75 micrometer V(max) approximately 200 nmol/min/mg) that was stimulated 1.5-2-fold by all-trans-retinal and dependent on phospholipid and dithiothreitol. The K(m) for ATP decreased to approximately 25 micrometer after reconstitution, whereas the V(max) was strongly dependent on the lipid used for reconstitution. ABCR reconstituted in ROS phospholipid had a V(max) for basal and retinal activated ATPase activity that was 4-6 times higher than for ABCR in soybean or brain phospholipid. This enhanced activity was mainly due to the high phosphatidylethanolamine (PE) content of ROS membranes. PE was also required for retinoid-stimulated ATPase activity. ATPase activity of ABCR was stimulated by the addition of N-retinylidene-PE but not the reduced derivative, retinyl-PE. ABCR expressed in COS-1 cells also exhibited retinal-stimulated ATPase activity similar to that of the native protein. These results support the view that ABCR is an active retinoid transporter, the nucleotidase activity of which is strongly influenced by its lipid environment.     

Taurine transporter is expressed in vascular smooth muscle cells

Summary. The regulation of vascular smooth muscle cells (VSMCs) function by taurine has been a subject of increasing interest and investigation, and taurine is taken up into cells through a specific transporter system, the taurine transporter (TAUT). In the present study, we examined the expression of TAUT in VSMCs and the kinetic parameters of the uptake process of TAUT in VSMCs. RT-PCR and western blot demonstrated that the mRNA and protein of TAUT was expressed in VSMCs in vitro. Immunohistochemistry using antibody for TAUT revealed the expression of this protein in rat thoracic aorta. The maximal [³H]taurine uptake rate in VSMCs was 37.75 ± 3.13 pmol/min per mg of protein, with a K _m value of 5.42 ± 0.81 μM. Thus, VSMCs are able to express a functional taurine transporter. The regulation and detailed function of taurine and TAUT in VSMCs remain unclear, but our findings suggest a functional role for them in VSMCs metabolism.     

Functional characterization and ATP-induced dimerization of the isolated ABC-domain of the haemolysin B transporter

Nucleotide-binding domains (NBD) are highly conserved constituents of ATP-binding cassette (ABC) transporters. Members of this family couple ATP hydrolysis to the transfer of various molecules across cell membranes. The NBD of the HlyB transporter, HlyB-NBD, was characterized with respect to its uncoupled ATPase activity, oligomeric state, and stability in solution. Experimental data showed that both the nature and pH of an assay buffer influenced the level of protein activity. Comparative analysis of protein stability and ATPase activity in various buffers suggests an inverse relationship between the two. The highest ATPase activity was detected in HEPES, pH 7.0. A kinetic analysis of the ATPase activity in this buffer revealed an enzyme concentration dependence and ATP-induced protein oligomerization. Assuming that the dimer is the active form of enzyme, at least half of the purified HlyB-NBD was estimated to be a dimer at 1.2 microM under the most optimal conditions for ATP hydrolysis. This is about 2 orders of magnitude lower than reported for other canonical ABC-ATPases. The maximum reaction velocity of 0.6 micromol/mg x min at 22 degrees C and the apparent kinetic constant K(app)(0.5) of 0.26 mM for ATP were determined for the dimerized HlyB-NBD. Gel filtration experiments with the wild-type protein and HlyB-NBD mutated in a key catalytic residue, H662A, provided further evidence for ATP-induced protein dimerization. ATPase activity experiments with protein mixtures composed of wild-type and the ATPase-deficient H662A mutant demonstrated that one intact NBD within a dimer is sufficient for ATP hydrolysis. This single site turnover might suggest a sequential mechanism of ATP hydrolysis in the intact HlyB transporter.     

Reconstitution in liposomes of the functionally active human OCTN1 (SLC22A4) transporter overexpressed in Escherichia coli

The hOCTN1 (human organic cation transporter 1) overexpressed in Escherichia coli and purified by Ni-chelating chromatography has been reconstituted in liposomes by detergent removal with a batch-wise procedure. The reconstitution was optimized with respect to the protein concentration, the detergent/phospholipid ratio and the time of incubation with Amberlite XAD-4 resin. Time-dependent [(14)C]tetraethylammonium, [(3)H]carnitine or [(3)H]ergothioneine uptake was measured in proteoliposomes with activities ratios of 8:1.3:1 respectively. Optimal activity was found at pH 8.0. The transport depended on intraliposomal ATP. [(14)C]tetraethylammonium transport was inhibited by several compounds. The most effective were acetyl-choline and γ-butyrobetaine, followed by acetylcarnitine and tetramethylammonium. Reagents such as pyridoxal 5-phosphate, MTSES [sodium (2-sulfonatoethyl) methanethiosulfonate] and mercurials strongly inhibited the transport. From kinetic analysis of tetraethylammonium transport a K(m) of 0.77 mM was calculated. Acetylcholine and γ-butyrobetaine behaved as competitive inhibitors of TEA (tetraethylammonium) transport with K(i) values of 0.44 and 0.63 mM respectively.     

Concentration of components of the adenylate system in heavy and light fractions of the sarcoplasmic reticulum of skeletal muscles and sensitivity of these fractions to the effects of imidazole-containing compounds and caffeine]

Fragments of sarcoplasmic reticulum from rabbit sceletal muscles sedimented within the range from 2000 g to 8000 g (heavy fraction) and 8000 g to 40000 g (light fraction) and washed with 0.6 M KCl, were practically free of adenylatecyclase activity. Phosphodiesterase cAMP was not found in the light fraction, while its activity in the heavy fraction was 500 pmol of cAMP/min per mg of protein. Both fractions contain bound cAMP (1-2 pmol/mg of protein) and specific sites of cAMP binding, the binding constant being approximately 10(6)M-1. The number of binding sites is 60 pmol/mg of protein for the heavy and 30 pmol/mg of protein for the light fractions. The level of phosphodiesterase activity in the heavy fraction correlates with its sensitivity to imidazole, anserine and caffeine. Imidazole and anserine increase in 1.5-1.8 times the value of Ca2+/ATP in the heavy fraction and produce no effect on Ca2+ transport by the light fraction. Caffeine decreases almost twice the Ca2+/ATP value in the heavy fraction and has practically no effect on Ca2+ absorption by enzymes of the light reticulum fraction. Imidazole and anserine activate membrane-bound phosphodiesterase, while caffeine inhibits it. It is suggested that structural rearrangements of membrane-bound phosphodiesterase under the effect of caffeine, imidazole and anserine are responsible for changes in the efficiency of Ca2+ transport by fragments of the heavy reticulum fractions.     

Solubilization of Peripheral-Type Benzodiazepine Binding Sites from Cat Cerebral Cortex

The present study demonstrates for the first time the solubilization of peripheral-type benzodiazepine binding sites (PBS) from cat cerebral cortex. Of all detergents tested [digitonin, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), Tween 20, deoxycholate, and Triton X-100] in the presence of NaCl, the best solubilization (15% of initial activity) was obtained using 0.5% of the zwitterionic detergent CHAPS plus 2 M NaCl. Specific binding of [3H]PK 11195 to membrane-bound and solubilized PBS was saturable, yielding equilibrium dissociation constants (KD) of 1.3 +/- 0.2 and 1.9 +/- 0.3 nM, respectively, and maximal numbers of binding sites of 1,435 +/- 150 and 980 +/- 126 fmol/mg protein, respectively. The KD value of PK 11195 binding to solubilized PBS obtained from experimental kinetic analysis was 0.95 +/- 0.09 nM. The relative potencies of various compounds (PK 11195, Ro 5-4864, diazepam, flunitrazepam, clonazepam, methyl-beta-carboline-3-carboxylate, and Ro 15-1788) in displacing [3H]PK 11195 specific binding from membrane-bound and solubilized PBS were similar. Most of the solubilized binding activity was destroyed by heating at 60 degrees C for 30 min or by treatment with 2 M guanidinium chloride, which indicates the presence of a protein-binding site in the solubilized preparation. Over 85% of the solubilized binding activity was retained after 1 week at 4 degrees C, which will enable future application of purification procedures without major concern for stability of the material.