Putative Plasma Membrane Research Articles

Differential localization of syntaxin isoforms in polarized Madin-Darby canine kidney cells.

Syntaxins, integral membrane proteins that are part of the ubiquitous membrane fusion machinery, are thought to act as target membrane receptors during the process of vesicle docking and fusion. Several isoforms of the syntaxin family have been previously identified in mammalian cells, some of which are localized to the plasma membrane. We investigated the subcellular localization of these putative plasma membrane syntaxins in polarized epithelial cells, which are characterized by the presence of distinct apical and basolateral plasma membrane domains. Syntaxins 2, 3, and 4 were found to be endogenously present in Madin-Darby canine kidney cells. The localization of syntaxins 1A, 1B, 2, 3, and 4 in stably transfected Madin-Darby canine kidney cell lines was studied with confocal immunofluorescence microscopy. Each syntaxin isoform was found to have a unique pattern of localization. Syntaxins 1A and 1B were present only in intracellular structures, with little or no apparent plasma membrane staining. In contrast, syntaxin 2 was found on both the apical and basolateral surface, whereas the plasma membrane localization of syntaxins 3 and 4 were restricted to the apical or basolateral domains, respectively. Syntaxins are therefore the first known components of the plasma membrane fusion machinery that are differentially localized in polarized cells, suggesting that they may play a central role in targeting specificity.

Evidence for the role of cell protein phosphorylation in human cytomegalovirus/host cell fusion.

The mechanism by which human cytomegalovirus (HCMV) enters cells is unknown. We sought evidence that protein phosphorylation plays a role in HCMV infection in two ways: (1) by determining whether the degree of phosphorylation of a constitutively phosphorylated 92.5 kDa putative cell membrane receptor for HCMV gH is changed following exposure to HCMV or monoclonal anti-idiotype antibodies (MAb2) that antigenically mimic HCMV gH, and (2) by studying the effects of protein kinase inhibition on receptor phosphorylation and HCMV adsorption or fusion. Phosphorylation of the 92.5 kDa cell membrane protein was specifically increased within 10 min of incubation with HCMV or MAb2 that had been crosslinked by goat anti-mouse antibodies. In addition, fusion of viral envelope with the cell membrane was inhibited by certain protein kinase inhibitors which also inhibited receptor phosphorylation, while the adsorption of [3H]HCMV to human embryonic lung cells was not affected. Tyrosine kinase inhibitors inhibited virus/cell fusion to a greater extent than protein kinase C (PKC) inhibitors, and an inhibitor which primarily affects cAMP and cGMP kinases had little effect. In addition, fusion was stimulated by preincubating cells with agents that stimulated receptor phosphorylation including a phorbol ester, tyrosine phosphatase inhibitor and serine/threonine phosphatase inhibitor. These data indicate that increased phosphorylation of a 92.5 kDa putative cell membrane protein receptor for gH is an early event in response to HCMV, and that cell protein phosphorylation by tyrosine kinase(s) and PKC may facilitate HCMV/cell membrane fusion.

Cold-shock regulation of the Arabidopsis TCH genes and the effects of modulating intracellular calcium levels.

The Arabidopsis TCH genes, which encode calmodulin-related proteins and a xyloglucan endotransglycosylase, are shown to be up-regulated in expression following cold shock. We investigated a possible role of fluctuations in intracellular calcium ion concentrations ([Ca2+]) in the cold-shock-induced TCH gene expression. Transgenic plants harboring the apoaequorin gene were generated to monitor [Ca2+]) and to test the necessity of cold-induced [Ca2+] increases for TCH expression. Cold-shock-induced [Ca2+] increases can be blocked by La3+ and Gd3+, putative plasma membrane Ca2+ channel blockers, and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, an extracellular Ca2+ chelator. Cold-shock-induced expression of the TCH genes is inhibited by levels of La3+, Gd3+, and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, that have been shown to block [Ca2+] increases. These data support the hypotheses that (a) intracellular [Ca2+] increases following cold shock require extracellular Ca2+ and may derive from a Ca2+ influx mediated by plasmalemma Ca2+ channels, and (b) cold up-regulation of expression of at least a subset of the TCH genes requires an intracellular [Ca2+] increase. The inhibitors are also shown to have stimulus-independent effects on gene expression, providing strong evidence that these commonly used chemicals have more complex effects than generally reported.

Roots of Iron-Efficient Maize also Absorb Phytosiderophore-Chelated Zinc.

To investigate the recognition of Zn-phytosiderophores by the putative Fe-phytosiderophore transporter in maize (Zea mays L.) roots, short-term uptake of 65Zn-labeled phytosiderophores was compared in the Fe-efficient maize cultivar Alice and the maize mutant ys1 carrying a defect in Fe-phytosiderophore uptake. In ys1, uptake and translocation rates of Zn from Zn-phytosiderophores were one-half of those in Alice, but no genotypical difference was found in Zn uptake and translocation from other Zn-binding forms. In ys1 and in tendency also in Alice, Zn uptake decreased with increasing stability constant of the chelate in the order: ZnSO4 [greater than or equal to] Zn-desferrioxamine > Zn-phytosiderophores > Zn-EDTA. Adding a 500-fold excess of free phytosiderophores over Zn to the uptake solution depressed Zn uptake in ys1 almost completely. In uptake studies with double-labeled 65Zn-14C-phytosiderophores, ys1 absorbed the phytosiderophore at similar rates when supplied as a Zn-chelate or the free ligand. By contrast, in Alice 14C-phytosiderophore uptake from the Zn-chelate was 2.8-fold higher than from the free ligand, suggesting that Alice absorbed the complete Zn-phytosiderophore complex via the putative plasma membrane transporter for Fe-phytosiderophores. We propose two pathways for the uptake of Zn from Zn-phytosiderophores in grasses, one via the transport of the free Zn cation and the other via the uptake of nondissociated Zn-phytosiderophores.

TRAINING-INDUCED INCREASE IN FABPPM IS ASSOCIATED WITH ELEVATIONS IN PALMITATE UPTAKE AND OXIDATION BY CONTRACTING MUSCLE 456

Endurance training has been shown to be associated with an increased contribution of lipids to oxidative metabolism in exercising muscles. To evaluate a mechanism for this change, we measured the uptake and oxidation of palmitate in isolated rat hindquarters as well as the content of fatty acid-binding proteins in the plasma membranes (FABPPM) of red and white muscles from 16 trained (T) and 18 untrained (UT) rats. Hindquarters were perfused with 6 mM glucose, 1800 μM albumin-bound palmitate and albumin-bound [1-14C]palmitate at rest and during electrical stimulation (100 ms trains at 100 Hz, 20 trains/min) for 25 min (ES). FABPPM content was 1.5 to 3-fold higher in red than in white muscles and was increased by 56% after endurance training in red muscles. A positive correlation (r=0.78, p<0.05) was found to exist between SDH activity and FABPPM content in red muscles. Palmitate uptake increased by 60-65% from rest to ES in both T and UT and was 30-35% higher in T than UT both at rest (29.4±2.7 vs 39.6±2.5 nmol·min-1·g-1 for UT and T, respectively) and during ES (48.7±4.2 vs 63.5±5.1 nmol·min-1·g-1 for UT and T, respectively). Resting palmitate oxidation was not affected by training and averaged 1.7±0.3 nmol·min-1·g-1. During ES, palmitate oxidation was higher in T than UT (12.1±1.7 vs 7.3±0.5 nmol·min-1·g-1, respectively). It is concluded that endurance training increases plasma FFA uptake in resting and contracting perfused muscle and plasma FFA oxidation in contracting perfused muscle. Furthermore, endurance training increases FABPPM content in red muscles, suggesting that an increased number of transporters may be available in the trained muscle. The data are consistent with the notion that the putative plasma membrane carrier of FFA may be implicated in the regulation of plasma FFA metabolism in skeletal muscle.

Molecular Cloning and Expression of Receptor Peptides That Block Human Cytomegalovirus/Cell Fusion

Clones expressing a partial human cytomegalovirus putative fusion receptor were selected by binding specifically to monoclonal anti-idiotypic antibodies that mimic glycoprotein H. cDNA was isolated from 2 of the clones (131 and 611) and fused in frame with the glutathione S-transferase gene in a pGEX-4T-1 vector. Two purified peptides (FR131 and FR611) were produced: both were shown to bind specifically to the monoclonal anti-idiotypic antibodies and inhibit virus/cell fusion and viral plaque formation in a specific and dose-dependent manner. This is the first demonstration of cloned peptides encoding a putative cell membrane receptor that are able to block cytomegalovirus infection.

Tissue-specific expression of the gene for a putative plasma membrane H(+)-ATPase in a seagrass.

A cDNA clone corresponding to the gene (ZHA1) for a putative plasma membrane H(+)-ATPase of a seagrass (Zostera marina L.) was isolated and sequenced. Comparison of the amino acid predicted sequence from the nucleotide sequence of ZHA1 with those encoded by known genes for plasma membrane H(+)-ATPases from other plants indicated that ZHA1 is most similar to the gene (PMA4) for a plasma membrane H(+)-ATPase in a tobacco (84.4%). Northern hybridization indicated that ZHA1 was strongly expressed in mature leaves, which are exposed to seawater and have the ability of tolerate salinity; ZHA1 was weakly expressed in immature leaves, which are protected from seawater by tightly enveloping sheaths and are sensitive to salinity. In mature leaves, in situ hybridization revealed that ZHA1 was expressed specifically in epidermal cells, the plasma membranes of which were highly invaginated and morphologically similar to those of typical transfer cells. Therefore, the differentiation of the transfer cell-like structures, accompanied by the high-level expression of ZHA1, in the epidermal cells of mature leaves in particular may be important for the excretion of salt by these cells.

Regulation of cation transport in Saccharomyces cerevisiae by the salt tolerance gene HAL3.

Dynamic regulation of ion transport is essential for homeostasis as cells confront changes in their environment. The gene HAL3 encodes a novel component of this regulatory circuit in the yeast Saccharomyces cerevisiae. Overexpression of HAL3 improves growth of wild-type cells exposed to toxic concentrations of sodium and lithium and suppresses the salt sensitivity conferred by mutation of the calcium-dependent protein phosphatase calcineurin. Null mutants of HAL3 display salt sensitivity. The sequence of HAL3 gives little clue to its function. However, alterations in intracellular cation concentrations associated with changes in HAL3 expression suggest that HAL3 activity may directly increase cytoplasmic K+ and decrease Na+ and Li+. Cation efflux in S. cerevisiae is mediated by the P-type ATPase encoded by the ENA1/PMR24 gene, a putative plasma membrane Na+ pump whose expression is salt induced. Acting in concert with calcineurin, HAL3 is necessary for full activation of ENA1 expression. This functional complementarity is also reflected in the participation of both proteins in recovery from alpha-factor-induced growth arrest. Recently, HAL3 was isolated as a gene (named SIS2) which when overexpressed partially relieves loss of transcription of G1 cyclins in mutants lacking the protein phosphatase Sit4p. Therefore, HAL3 influences cell cycle control and ion homeostasis, acting in parallel to the protein phosphatases Sit4p and calcineurin.

Increases in [Ca2+]i mediated by the 92.5-kDa putative cell membrane receptor for HCMV gp86

We determined that changes in intracellular Ca2+ concentration ([Ca2+]i) occur in human fibroblasts within the first hour of human cytomegalovirus (HCMV) infection when viral adsorption and fusion take place, and we investigated whether such changes also occur in response to monoclonal anti-idiotype antibodies (MAb2) that mimic HCMV gp86 and bind to a 92.5-kDa putative cell membrane receptor for gp86. Digitized image analysis of fura 2-loaded human embryonic lung fibroblasts indicated specific transient increases in [Ca2+]i beginning in some cells within the first 5 min of incubation with cross-linked MAb2 (70-750 nM, P < 0.01), which were similar in timing and intracellular distribution to those induced by HCMV. A primary source of Ca2+ appeared to be intracellular Ca2+ stores, since prior depletion of these stores with 30 nM thapsigargin inhibited the response (91.7 +/- 8.6%, P < 0.01); influx of Ca2+ from the extracellular medium was apparently necessary to maintain the intracellular Ca2+ stores. Transient increases in inositol trisphosphate (IP3) occurred in response to MAb2 (up to 3,329 +/- 84%, P < 0.001) or HCMV (92.8 +/- 19%, P < 0.01) during this same time period. These data suggest that the 92.5-kDa receptor for HCMV gp86 mediates an increase in IP3 and subsequent release of Ca2+ from intracellular stores.

Purification of plasma membranes from leaves of conifer and deciduous tree species by phase partitioning and free‐flow electrophoresis

Purified plasma membrane fractions were obtained from leaves of Picea abies L., Pinus sylvestris L., Fagus sylvatica L. and Quercus robur L., whereas plasma membranes from Pinus halepensis Mill, proved to be more difficult to obtain, perhaps due to the higher content of volatile substances in this plant species. Plasma membranes were purified by both phase partitioning and free‐flow electrophoresis from microsomal fractions and identified on the basis of biochemical and in some cases morphological and cytochemical markers. Electron micrographs revealed that membrane vesicles from Pinus sylvestris exhibited a very clear dark‐light‐dark pattern and measurements of membrane thickness showed that it ranged from 6 to 10 nm. Most membranes were 8 nm thick and stained with phosphotungstic acid at low pH, both typical characteristics of the plasma membrane. Enzymatic identification of plasma membranes consisted in the determination of the vanadate‐sensitive ATPase (EC 3.6.1.3) activity. The specific activity in the upper phase (U2) fraction was 10–25 times higher than those in the lower phase and microsomal fractions, depending on plant species. 1,3‐β‐glucan synthase II (EC 2.4.1.3), another putative plasma membrane marker, was not detected in the plasma membrane‐enriched fractions of conifer needles and showed a very low specific activity in membranes of deciduous trees. Contamination by membranes of other origin was determined by analysis of membrane markers: cytochrome c oxidase (EC 1.9.3.1) for mitochondria, inosine diphosphatase (EC 3.6.1.6) for Golgi apparatus, cytochrome c reductase (EC 1.6.2.4) for endoplasmic reticulum, and pyrophosphatase (EC 3.6.1.1) for tonoplasts. The main, but relatively low contamination, was due to tonoplasts, as determined by the activity of pyrophosphatase. Plasma membrane characteristics were quite different depending on the season during which needles were taken. Membrane preparations of better quality were more easily obtained from samples taken during winter.

Cellular pathway of photosynthate transport in coats of developing seed ofVicia fabaL. andPhaseolus vulgarisL. II. Principal cellular site(s) of efflux

The cells responsible for the photosynthate efflux from coats of developing seed of Vicia faba L. and Phaseolus vulgaris L. were elucidated using known properties of the efflux mechanism. Sensitivity of sucrose efflux to NEM and high potassium concentrations was retained by seed-coat halves of Phaseolus following pectinase removal of the branch parenchyma cell layer. In contrast, removal of the thin-walled parenchyma transfer cell layer from Vicia seed-coat halves abolished this sensitivity. The membrane-impermeant thiol-binding fluorochrome, qBBr, selectively stained the surface of the thin-walled parenchyma transfer cells. This phenomenon was inhibited by the slowly permeable sulphydryl agent, PCMBS, indicating that the plasma membranes of these cells are enriched in sulphydryl groups characteristic of membrane porter proteins. On the basis that carrier-mediated sucrose efflux from seed coats appears to be proton coupled, the putative plasma membrane H + -ATPase was used as a marker for the cells responsible for carrier-mediated photosynthate efflux. When seed-coat halves were exposed briefly at pH 8.5 to the weak acid fluorochrome, SRG, the ground parenchyma and thin-walled parenchyma transfer cell layers selectively accumulated the dye. The apparent low pH environment in the walls of these cells that renders SRG membrane permeant appeared to be maintained by a VAN-sensitive proton pump. The findings with SRG were corroborated by the cytochemical localization of plasma membrane ATPase activity to the ground parenchyma and thin-walled parenchyma transfer cells using precipitation of cerium phosphate. Together, our observations provide qualified support for the conclusion that carriermediated photosynthate efflux from coats of Phaseolus and Vicia seed is primarily restricted to the ground parenchyma and thin-walled parenchyma transfer cell layers, respectively

High-affinity Ca 2+,Mg 2+-ATPase in plasma membrane-rich preparations from olfactory epithelium of Atlantic salmon

High-affinity Ca 2+,Mg 2+-ATPase was identified in a plasma membrane-rich fraction of olfactory epithelium from Atlantic salmon ( Salmo salar). The enzyme required both Ca 2+ and Mg 2+ for activation. The apparent K m for Ca 2+ was 9.5 nM and V max was 0.85 μmol P i/mg of protein per min. Stimulation by Ca 2+ was optimal at 5–100 μM MgCl 2. Bovine brain calmodulin had no effect on Ca 2+,Mg 2+-ATPase, even after multiple washes of the membrane preparation with EDTA or EGTA. Endogenous calmodulin was somewhat resistant to removal and could be detected with immunobloting after multiple washes of the membrane preparation with EDTA or EGTA. This endogenous calmodulin may regulate Ca 2+,Mg 2+-ATPase activity because the activity was inhibited by calmidazolium. Vanadate inhibited Ca 2+,Mg 2-ATPase activity and thapsigargin, a specific inhibitor for Ca 2+,Mg 2+-ATPase of endoplasmic reticulum, had no effect on the enzyme activity. High affinity Ca 2+,Mg 2+-ATPase exists in both ciliary and nonciliary membranes with a similar K m for Ca 2+. Ca 2+,Mg 2+-ATPase activity is greater in cilia preparations than in membranes from the deciliated olfactory epithelium. As a putative plasma membrane Ca 2+ pump, this high-affinity Ca 2+,Mg 2+-ATPase may play an important role in the regulation of intracellular Ca 2+ in olfactory epithelia. In particular, the ciliary membrane may play a prominent role in the removal of Ca 2+ from ciliated olfactory receptor cells after odorant stimulation.

A slowly ADP-ribosylated pertussis-toxin-sensitive GTP-binding regulatory protein is required for vasopressin-stimulated Ca2+ inflow in hepatocytes.

The roles of heterotrimeric GTP-binding regulatory proteins (G-proteins) and inositol polyphosphates in the mechanism by which vasopressin stimulates Ca2+ inflow in hepatocytes were investigated by using single cells loaded with fura2 by microinjection. Vasopressin-stimulated Ca2+ inflow was mimicked by microinjection of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) or guanosine 5'-[beta gamma-imido]triphosphate to the cells, but not adenosine 5'-[gamma-thio]triphosphate (ATP[S]) or guanosine 5'-[beta-thio]diphosphate (GDP[S]). Extracellular Gd3+ (5 microM) inhibited both vasopressin- and GTP[S]-stimulated Ca2+ inflow. GDP[S], but not GMP, administered to hepatocytes by microinjection, completely inhibited vasopressin-stimulated Ca2+ inflow and partially inhibited vasopressin-induced release of Ca2+ from intracellular stores. The microinjection of pertussis toxin had no effect either on the release of Ca2+ from intracellular stores or on Ca2+ inflow induced by vasopressin, but completely inhibited changes in these processes induced by epidermal growth factor (EGF). Hepatocytes isolated from rats treated with pertussis toxin for 24 h exhibited no vasopressin- or GTP[S]-stimulated Ca2+ inflow, whereas the vasopressin-stimulated release of Ca2+ from intracellular stores was similar to that observed for control cells. Heparin or ATP[S] inhibited, or delayed the onset of, both vasopressin-induced release of Ca2+ from intracellular stores and vasopressin-stimulated Ca2+ inflow. Vasopressin-induced oscillations in intracellular [Ca2+] were observed in some heparin-treated cells. It is concluded that the stimulation by vasopressin of Ca2+ inflow to hepatocytes requires inositol 1,4,5-trisphosphate (InsP3) and, by implication, the pertussis-toxin-insensitive G-protein required for the activation of phospholipase C beta [Taylor, Chae, Rhee and Exton (1991) Nature (London) 350, 516-518], and another G-protein which is slowly ADP-ribosylated by pertussis toxin and acts between InsP3 and the putative plasma-membrane Ca2+ channel. EGF-stimulated Ca2+ inflow involves at least one G-protein which is rapidly ADP-ribosylated and is most likely required for InsP3 formation.

Voltage-dependent Ca2+ influx into right-side-out plasma membrane vesicles isolated from wheat roots: characterization of a putative Ca2+ channel.

We report on the identification of a voltage-dependent Ca2+ transport system that mediates Ca2+ influx across the plasma membrane (PM) of wheat (Triticum aestivum) root cells. The experimental approach involved the imposition of transmembrane electrical potentials (via K+ diffusion potentials) in populations of purified, right-side-out PM vesicles isolated from wheat roots. Using 45Ca2+ to quantify Ca2+ influx into the PM vesicles, the voltage-dependent characteristics of Ca2+ transport were found to be similar to those exhibited by L-type voltage-gated Ca2+ channels in animal cells. The putative PM Ca2+ channel opened upon depolarization of the membrane potential, and Ca2+ flux increased to a maximum upon further depolarization and then decreased back to zero upon further successive depolarizations. This channel was found to be selective for Ca2+ over Mg2+, Sr2+, K+, and Na+; was blocked by very low concentrations of La3+; was unaffected by high concentrations of the K+ channel blocker tetraethylammonium; and exhibited Michaelis-Menten-type transport kinetics. Based on these transport properties, we argue that this transport system is a PM Ca2+ channel. We suggest that the use of radiotracer flux analysis of voltage-clamped PM vesicles derived from plant roots is a straightforward approach for the characterization of certain voltage-gated ion channels functioning in cellular membranes of higher plant cells.

Multiphasic control of proteolysis by leucine and alanine in the isolated rat hepatocyte.

Autophagically mediated proteolysis in the perfused rat liver is under complex multiphasic control by a small group of amino acids dominated by leucine. Because there have been no prior reports of such regulation in the isolated hepatocyte, our goal was to determine whether it is a manifestation of interactions between diverse cells in the intact liver or, alternatively, the expression of a unique control mechanism within a single population of cells. Hepatocytes were isolated from livers of ad libitum-fed rats and incubated with cycloheximide at low density (approximately 10(6) cells/ml) for the determination of valine release. As in perfusion experiments with synchronously fed rats, proteolytic responses to leucine in cells from fed rats were mediated through two inhibitory mechanisms that alternated randomly on a day-to-day basis. The first (L) represented a typical multiphasic dose-response with low- and high-concentration inhibition separated by a sharp zonal loss of inhibition that could be abolished by alanine. The second (H) mediated inhibition only at high concentrations. It disappeared after 24 h of starvation, leaving L as the prevailing mode. The findings indicate that both macroautophagy and the multiphasic mechanism for regulating it coexist in a single population of hepatocytes, making the cells suitable for studies aimed at defining the putative plasma membrane site of leucine recognition.

A putative cytoprotective receptor in the kidney: Relation to the neuronal strychinine-sensitive glycine receptor

The neutral amino acid glycine has been demonstrated to prevent cell death in numerous cell types exposed to a variety of toxic insults. Recently, the central nervous system (CNS) glycine antagonist strychine was demonstrated to bind specifically to the plasma membrane of renal proximal tubules (RPT) and mimic glycine cytoprotection. Further, it has been demonstrated in RPT that glycine and strychnine block chloride influx in the late stages of cell injury. The aim of this study was to determine if the RPT cytoprotective site is related to neuronal glycine receptors. Only antagonists tot he CNS strychnine-sensitive glycine receptor (strychnine, brucine), and not antagonists to the glycine modulatory site of the NMDA receptor (DCQX, 7-CKA, HA-966) or the GABA A receptor (bicuculline methiodide, picrotoxin), prevented mitochondrial inhibitor (antimycin A)-induced RPT cell death. Using immunoblot analysis, proteins corresponding to the 58kDa β-subunit of the strychnine-sensitive glycine receptor and the associated protein gephyrin were identified in rabbit kidney cortical membrane fractions and RPT. No protein corresponding to the 48 kDa α-subunit was identified. Thus, glycine and strychnine may exert their cytoprotective effects via a putative plasma membrane receptor that is related to the strychnine-sensitive glycine receptor found in the CNS.

True transport: one or more sodium-dependent bile acid transporters?

Sodium-dependent bile acid transport is a well-established function of the sinusoidal segment of the hepatocyte plasma membrane. Evidence has been provided previously by the authors for the existence of a putative sinusoidal plasma membrane sodium-dependent bile acid transporter with a mass of 49 kD. This protein has been partially characterized with a monoclonal antibody and by reconstitution in proteoliposomes. Further characterization is provided in the paper under discussion. The transporter was isolated from plasma membranes and from endoplasmic reticulum by immunoprecipitation with a monoclonal antibody. The protein was separated from antibody by means of HPLC and SDS-PAGE with electroelution. Amino acid analysis and NH2-terminal amino acid sequencing were performed. For both preparations, it was shown that the NH2-terminal amino acid sequence was identical, and the amino acid composition was similar to results obtained for the microsomal enzyme epoxide hydrolase. Purified mEH was immunoprecipitated with the monoclonal antibody which precipitated the plasma membrane protein. Isoelectric focusing established that the isoelectric points for epoxide hydrolase and the proteins isolated from the endoplasmic reticulum and plasma membranes were identical. The peptide maps prepared by subjecting the three proteins to enzymatic fragmentation with subtilisin were essentially identical. Because of the known contamination of plasma membrane fractions with endoplasmic reticulum and because of the high specific activity of epoxide hydrolase in endoplasmic reticulum, special attention was devoted to the question of the localization of the enzyme/transporter in plasma membrane. It was possible to show that virtually all of the immunoprecipitated protein obtained from intact cells derived from plasma membrane and that only a small fraction could be explained as having originated in endoplasmic reticulum. Finally, a hydrophobic plot of the amino acid sequence of epoxide hydrolase suggested that the protein contained four potential transmembrane domains. It could be concluded that a/the 49-kD sinusoidal plasma membrane sodium-dependent bile acid transporter protein was closely similar or identical to epoxide hydrolase. In view of the relatively small mass of the protein and its limited number of potential transmembrane domains, it appeared probable that the physiological membrane transporter consists of an assembly of 49-kD monomers or an assembly of heterologous proteins including the 49-kD protein.

Cholesterol kinetics in subjects with bile fistula. Positive relationship between size of the bile acid precursor pool and bile acid synthetic rate.

Our aim was to identify and quantitate cholesterol pools and transport pathways in blood and liver. By studying bile fistula subjects, using several types of isotopic preparations, simultaneous labeling of separate cholesterol pools and sampling all components of blood and bile at frequent intervals, we developed a comprehensive multicompartmental model for cholesterol within the rapidly miscible pool. Data in six components (bile acids, esterified cholesterol in whole plasma, and free cholesterol in blood cells, bile, alpha lipoproteins, and beta lipoproteins) were modeled simultaneously with the SAAM program. The analysis revealed extensive exchange of free cholesterol between HDL and liver, blood cells, and other tissues. There was net free cholesterol transport from HDL to the liver in most subjects. The major organ that removed esterified cholesterol from blood was the liver. A large portion (4,211 mumol) of total hepatic cholesterol comprised a pool that turned over rapidly (t1/2 of 72 min) by exchanging mainly with plasma HDL and was the major source of bile acids and biliary cholesterol. Only 6% of hepatic newly synthesized cholesterol was used directly for bile acid synthesis: the analysis showed that 94% of newly synthesized cholesterol was partitioned into the large hepatic pool (putative plasma membrane free cholesterol) which exchanged rapidly with plasma lipoproteins. Bile acid synthetic rate correlated directly with the size of the large hepatic pool. In conclusion, hepatic and blood cholesterol pools and transports have been quantitated. HDL plays a central role in free cholesterol exchange/transport between all tissues and plasma. In humans, the metabolically active pool comprises a large portion of total hepatic cholesterol that, in part, regulates bile acid synthesis.

Identification and characterization of a second plasma membrane H+-ATPase gene subfamily in Nicotiana plumbaginifolia

A cDNA clone was isolated for a fourth pma gene encoding a putative plasma membrane H(+)-ATPase of Nicotiana plumbaginifolia. The sequence of the predicted 952 residue PMA4 polypeptide was compared with those of other known plant PMAs, revealing a higher identity with the Arabidopsis thaliana proteins (86-89%) than with the other three N. plumbaginifolia PMA proteins (80-82%). This supports the view that there are two pma subfamilies which probably arose from a gene duplication predating the separation of the Dilleniidae and Asteridae plant subclasses. Measured pma4 transcript levels indicate that pma4 is similarly expressed in root, stem, leaf, and flower tissues, contrary to the pmal-3 subfamily whose members displayed differential expression according to the organ.

Amino acid substitutions in the sixth transmembrane domain of P-glycoprotein alter multidrug resistance.

Eukaryotic cells can display resistance to a wide range of natural-product chemotheraputic agents by the expression of P-glycoprotein (pgp), a putative plasma membrane transporter that is thought to mediate the efflux of these agents from cells. We have identified, in cells selected for multidrug resistance with actinomycin D, a mutant form of pgp that contains two amino acid substitutions within the putative sixth transmembrane domain. In transfection experiments, this altered pgp confers a cross-resistance phenotype that is altered significantly from that conferred by the normal protein, displaying maximal resistance to actinomycin D. These results strongly implicate the sixth transmembrane domain in the mechanism of pgp drug recognition and efflux. Moreover, they indicate a close functional homology between pgp and the cystic fibrosis transmembrane regulator in which the sixth transmembrane domain has also been shown to influence substrate specificity.