Plasma Membrane Marker Enzyme Research Articles

ISOLATING NA+K+ATPASE IN BOAR (Sus scrofa) SPERMATOZOA MEMBRANES

Na+K+ATPase exists in the membranes of bull spermatozoa, concentrated in the head plasma membrane. In sperm, Na+K+ATPase actively transports ions contributing to the intracellular pH change in capacitation, and has very recently been implicated as a direct signalling molecule leading to tyrosine phosphorylation in capacitating bull sperm. The characteristics of Na+K+ATPase in boar sperm are unknown, and were the focus of this study. To determine the existence of Na+K+ATPase, and any preferential location of it within boar sperm, Western blot analyses were performed on whole sperm and purified boar sperm head plasma membrane (HPM). The HPM was isolated (n=8 ejaculates) by nitrogen cavitation and differential centrifugation. Its purity was confirmed by its significant enrichment in two plasma membrane marker enzymes (alkaline phosphatase 224 ± 18%, Mean ± SE; acid phosphatase 248 ± 65% vs activity in homogenised whole sperm); and by the significant diminution of activity of marker enzymes associated with the acrosome (acetylglucosaminidase 41.9 ± 8%) and mitochondria (succinate dehydrogenase; 3.21 ± 1%). Equal protein amounts from whole sperm (WS), HPM and sperm body membranes (SB; whole sperm minus HPM) from 3 ejaculates were analyzed by Western blotting for the alpha (isoforms 1 and 3), and beta (isoforms 1,2 and 3) subunits of Na+K+ATPase. Alpha 1 and 3, and beta 1 were less prevalent in HPM than in WS or SB, while alpha 2 and beta 3 were equally prominent in all fractions, based on densitometry of equal protein concentrations. Monoclonal antibodies for each subunit bound at the kD of the rat brain positive control, and several additional bands for each subunit; these are being investigated as multimers, glycosylated residues, and/or breakdown products. Immunofluorescence of the same monoclonal antibodies then delineated specific subunit locations on the sperm. Fresh sperm (n=4 ejaculates) on poly-LLysine slides at 4°C were permeabilized with Methanol or left intact, and incubated at 37°C with antibodies for each enzyme subunit, and compared to binding that occurred when all steps were at room temperature. The α3 subunit bound post-equatorially regardless of temperature; all other isoforms bound only at room temperature, indicating a high degree of temperature sensitivity. The antibodies to all beta subunits bound over the anterior acrosomes of permeabilised cells only, suggesting the antigens are on internal surface, while the alpha 1 bound to the anterior acrosome of intact cells, indicating an extracellular antigen. These results indicate that Na+K+ATPase exists in specific regions of boar spermatozoa membranes, and its configuration is temperature-sensitive. Funding from NSERC, OMAFRA, Bioniche and Semex Canada gratefully acknowledged. (poster)

Estrogen and growth factor receptor interactions in human breast and non-small cell lung cancer cells

Extranuclear estrogen receptors may mediate rapid effects of estradiol that communicate with nuclear receptors and contribute to proliferation of human cancers bearing these signaling proteins. To assess these growth-promoting pathways, we undertook controlled homogenization and fractionation of NIH-H23 non-small cell lung cancer cells. As many breast tumors, NIH-H23 cells express estrogen receptors (ER), with the bulk of specific estradiol binding in nuclear fractions. However, as in breast cells, a significant portion of specific, high-affinity estradiol-17β binding-sites are also enriched in plasma membranes of lung tumor cells. These estrogen binding-sites co-purify with plasma membrane-marker enzymes and are not significantly contaminated by cytosol or nuclei. On further purification of membrane caveolae from lung tumor cells, proteins recognized by monoclonal antibodies to nuclear ER-α and to ER-β were identified in close association with EGF receptor in caveolae. In parallel studies, ER-α and ER-β are also detected in nuclear and extranuclear sites in archival human breast and lung tumor samples and are noted to occur in clusters at the cell membrane by using confocal microscopy to visualize fluorescent-labeled monoclonal antibodies to ER-α. Data on site-directed mutagenesis of cysteine-447 in ER-α suggest that association of ER forms with membrane sites may depend on acylation of cysteine by palmitate. Estrogen-induced growth of MCF-7 breast cancer and NIH-H23 lung cancer cells in vitro correlated closely with acute hormonal activation of mitogen-activated protein kinase signaling and was significantly reduced by treatment with Faslodex, a pure anti-estrogen. Further, combination of Faslodex with selected growth factor receptor inhibitors elicited a more pronounced inhibiton of tumor cell growth. Thus, extranuclear forms of ER play a role in promoting downstream signaling for hormone-mediated proliferation and survival of breast, as well as lung, cancers and offer a new target for anti-tumor therapy.

Membrane-associated binding sites for estrogen contribute to growth regulation of human breast cancer cells.

Membrane-associated binding sites for estrogen may mediate rapid effects of estradiol-17beta that contribute to proliferation of human breast cancers. After controlled homogenization and fractionation of MCF-7 breast cancer cells, the bulk of specific estradiol binding is found in nuclear fractions. However, a significant portion of specific, high-affinity estradiol-17beta binding-sites are also enriched in plasma membranes. These estradiol binding-sites co-purify with 5'-nucleotidase, a plasma membrane-marker enzyme, and are free from major contamination by cytosol or nuclei. Electrophoresis of membrane fractions allowed detection of a primary 67-kDa protein and a secondary 46-kDa protein recognized by estradiol-17beta and by a monoclonal antibody directed to the ligand-binding domain of the nuclear form of estrogen receptor. Estrogen-induced growth of MCF-7 breast cancer cells in vitro was blocked by treatment with the antibody to estrogen receptor and correlated closely with acute hormonal activation of mitogen-activated protein kinase and Akt kinase signaling. Estrogen-promoted growth of human breast cancer xenografts in nude mice was also significantly reduced by treatment in vivo with the estrogen receptor antibody. Thus, membrane-associated forms of estrogen receptor may play a role in promoting intracellular signaling for hormone-mediated proliferation and survival of breast cancers and offer a new target for antitumor therapy.

Subcellular localization of cyclic ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities in porcine airway smooth muscle

Recent studies have provided evidence for a role of cyclic ADP-ribose (cADPR) in the regulation of intracellular calcium in smooth muscles of the intestine, blood vessels and airways. We investigated the presence and subcellular localization of ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of β-NAD + to cADPR, and cADPR hydrolase, the enzyme that degrades cADPR to ADPR, in tracheal smooth muscle (TSM). Sucrose density fractionation of TSM crude membranes provided evidence that ADP-ribosyl cyclase and cADPR hydrolase activities were associated with a fraction enriched in 5′-nucleotidase activity, a plasma membrane marker enzyme, but not in a fraction enriched in either sarcoplasmic endoplasmic reticulum calcium ATPase or ryanodine receptor channels, both sarcoplasmic reticulum markers. The ADP-ribosyl cyclase and cADPR hydrolase activities comigrated at a molecular weight of approximately 40 kDa on SDS–PAGE. This comigration was confirmed by gel filtration chromatography. Investigation of kinetics yielded K m values of 30.4±1.5 and 695.3±171.2 μM and V max values of 330.4±90 and 102.8±17.1 nmol/mg/h for ADP-ribosyl cyclase and cADPR hydrolase, respectively. These results suggest a possible role for cADPR as an endogenous modulator of [Ca 2+] i in porcine TSM cells.

Characterization of plasma membrane fraction from filamentous fungus Rhizopus nigricans

In the filamentous fungus Rhizopus nigricans a steroid hydroxylating multienzyme system is inducible by progesterone and by several other steroids. The biological signal carried by progesterone might be mediated by receptors, located either in the plasma membrane or inside the cell. To elucidate the first possibility, plasma membrane fraction was examined for the presence of progesterone receptors. The isolation of plasma membrane from fungal homogenate containing different other membranes is difficult because of the rigid cell wall. Three different membrane fractions were prepared by differential centrifugation of the fungal homogenate and characterized by plasma membrane and mitochondrial membrane marker enzymes, H+-ATPase and mit-ATPase, respectively. The same fractions were examined for the presence of specific progesterone-binding molecules. Two of these fractions comprising the highest level of plasma membrane enzyme activity contained also the highest level of specific progesterone-binding compounds: 27,6 fmol/mg protein and 18,8 fmol/mg protein. The correlation between plasma membrane marker enzyme activity and the amount of progesterone-binding proteins in plasma membrane fraction of Rhizopus nigricans might indicate the involvement of these molecules in the induction process.

Characterization of plasma membrane fraction from filamentous fungus Rhizopus nigricans.

In the filamentous fungus Rhizopus nigricans a steroid hydroxylating multienzyme system is inducible by progesterone and by several other steroids. The biological signal carried by progesterone might be mediated by receptors, located either in the plasma membrane or inside the cell. To elucidate the first possibility, plasma membrane fraction was examined for the presence of progesterone receptors. The isolation of plasma membrane from fungal homogenate containing different other membranes is difficult because of the rigid cell wall. Three different membrane fractions were prepared by differential centrifugation of the fungal homogenate and characterized by plasma membrane and mitochondrial membrane marker enzymes, H+-ATPase and mit-ATPase, respectively. The same fractions were examined for the presence of specific progesterone-binding molecules. Two of these fractions comprising the highest level of plasma membrane enzyme activity contained also the highest level of specific progesterone-binding compounds: 27,6 fmol/mg protein and 18,8 fmol/mg protein. The correlation between plasma membrane marker enzyme activity and the amount of progesterone-binding proteins in plasma membrane fraction of Rhizopus nigricans might indicate the involvement of these molecules in the induction process.

The subcellular localization of phospholipase D activities in rat Leydig cells

Rat Leydig cells contain a phospholipase D (PLD), which can be activated by vasopressin and phorbol ester. In order to clarify which Leydig cell organelles that express PLD activity, the subcellular localization of two differently regulated PLD activities was investigated by subcellular fractionation on a 40% (v/v) self-generating Percoll gradient. PLD activities in broken cells were estimated using radiolabeled didecanoylphosphatidylcholine as a substrate. Initial experiments revealed the presence of an oleate Mg 2+-activated PLD and a phosphatidylinositol 4,5-bisphosphate-activated PLD (PIP 2-PLD) in the microsomal fraction of Leydig cells. The latter activity could be further stimulated by recombinant nonmyristoylated ADP ribosylating factor 1 (ARF1) plus GTPγS. The peak of oleate Mg 2+-PLD activity colocalized with the plasma membrane marker, whereas the highest specific activity of the PIP 2-PLD activity was found in fractions with a slightly lower density than those containing the plasma membrane and trans-Golgi marker enzymes. In order to localize phorbol ester-stimulated PLD activity in intact Leydig cells, the cells were prelabeled with [ 14C]-palmitate and then stimulated for 15 min with 100 nM 4-β-phorbol-12-myristate-13-acetate (PMA) in the presence of ethanol or butanol. The PLD product [ 14C]-phosphatidylethanol, expressed as the percentage of total labeled phospholipids in the fraction, was slightly increased in all Percoll fractions and showed a prominent peak in the fractions containing plasma membrane, trans-Golgi, and fractions of slightly lower density. The PMA-induced formation of [ 14C]-phosphatidylbutanol could be inhibited dose-dependently with brefeldin A suggesting that the activation of PLD by the phorbol ester was mediated by ARF.

Lipid and fatty acid composition of different fractions from rat urinary transitional epithelium.

The phospholipid composition of rat urinary transitional epithelium (TE) and the fatty acid composition of microsomal, mitochondrial, cytosolic, and plasma membrane (PM) subcellular fractions were investigated. PM marker enzymes and electron microscopy analysis were used to characterize the PM fraction, which showed a distinctive lipid composition compared to the general profile of PM from different sources. The levels of cholesterol and sphingomyelin were not enriched in the PM fraction; on the other hand, the increased amounts of glycosphingolipids and phosphatidylserine, and the decreased level of phosphatidylcholine followed the general features of a PM profile. This differential PM lipid composition may reflect the unique morphology of this mammal TE, consisting of concave plaques with an asymmetrical membrane unit. The distribution of the double bond across the PM indicated a higher unsaturation of the inner relative to the outer part of the PM hemileaflet. In addition, the presence of 20:3n-9 nonessential fatty acid in a normal TE may represent a characteristic fatty acid metabolism of this epithelium.

Subcellular distribution of the R-subunits of cAMP-dependent protein kinase in LS-174T human colon carcinoma cells.

The analysis of the particulate preparations of LS-174T human colon carcinoma cells in confluent stage of growth revealed different distribution for regulatory subunits (R) of cAMP-dependent protein kinase (PKA) in the subcellular compartments. The LS-174T cell lysates were subjected to differential and discontinuous sucrose density gradient centrifugation. The obtained fractions were assayed for marker enzymes and photoaffinity labeled with 8-N3[32P]cAMP. The whole lysates and cytoplasmic fraction exhibited the presence of both RI alpha and RII alpha--subunits of PKA. The fractions exhibiting high activity of the marker enzymes for plasma membranes, Golgi apparatus and mitochondria contained mainly RII alpha. In the fractions of lysosomes and microsomes RI alpha and RII alpha were found in nearly equal amounts.

Specific Non-Genomic, Membrane-Localized Binding Sites for Progesterone in the Bovine Corpus Luteum1

Fractionation of bovine corpus luteum (CL) homogenates on continuous sucrose density gradients with and without preincubation with 3H-progesterone demonstrated high levels of tracer binding and high content of endogenous progesterone associated with particulate membrane fractions. Analysis of gradient fractions for a range of luteal plasma membrane and intracellular organelle marker enzyme activities indicated that endogenous progesterone content and 3H-progesterone-binding activity were associated with fractions enriched in luteal plasma membrane markers. This was confirmed by pretreatment of homogenates with the saponin, digitonin, prior to fractionation. Digitonin perturbed the buoyant density of luteal surface membrane markers and 3H-progesterone binding to a similar extent, but did not perturb the buoyant densities of other intracellular markers to the same degree. Interestingly, digitonin pretreatment also increased the proportion of progesterone tracer that entered the gradients. We consistently failed to demonstrate significant binding of 3H-progesterone to membrane fractions incubated with progesterone tracer in vitro. However, when digitonin was included in the in vitro binding assay, we observed a dramatic, dose-dependent stimulation of 3H-progesterone binding by digitonin. Other radiolabeled steroids tested (3H-cortisol, 3H-testosterone) bound poorly in the presence or absence of digitonin. 3H-Progesterone binding in the presence of optimal digitonin concentrations increased linearly with increasing luteal membrane concentration; was dependent on the pH, duration, and temperature of incubation; and low levels of progesterone (68 nM) competed for tracer binding. A range of other steroids tested (androgens, estrogens, corticosteroids, steroid precursors) competed at higher concentrations (10- to 100-fold) or did not compete at all for 3H-progesterone binding. There was no correlation between the hydrophobicity of various steroids and their ability to compete for binding. Moreover, a number of agonists and antagonists specific for the genomic progesterone receptor, agonists of peripheral benzodiazepine receptors, and inhibitors of a range of steroidogenic enzymes did not compete for 3H-progesterone binding. Western blots confirmed that detergent-solubilized progesterone-binding sites could be resolved from cytochrome P450 side-chain cleavage and 3beta-hydroxysteroid dehydrogenase. Moreover, extraction of bound steroid from the binding site and HPLC demonstrated identity to progesterone, suggesting that no metabolism of the progesterone tracer had occurred during incubation. Progesterone binding to membranes of large luteal cells was higher compared with binding to small luteal cells, and levels were similar in membranes prepared from CL at all stages of the luteal phase. We suggest that bovine luteal progesterone-binding sites may play a role either in sequestration of newly synthesized progesterone or in the mediation of autocrine and/or paracrine actions of progesterone in the CL.

The hemopoietic growth factor, interleukin-3, promotes glucose transport by increasing the specific activity and maintaining the affinity for glucose of plasma membrane glucose transporters.

Most mammalian cells rely on an external supply of glucose for survival, proliferation, and function. Glucose enters cells through specific transporter molecules at the plasma membrane by a facilitative process that does not expend energy. Regulation of glucose transport into cells is thought to occur largely through transporter expression at the cell surface, but the extent to which the intrinsic properties of glucose transporters are regulated is at present controversial. Using a bone marrow-derived cell line that responds to the hemopoietic growth factor, interleukin-3 (IL-3), we investigated IL-3 regulation of glucose transport. IL-3 significantly increased 2-deoxyglucose (2-DOG) uptake within 1 h (26 +/- 8.0%, n = 11) with a maximum 73% increase after 6 h. Withdrawal of IL-3 resulted in decreased uptake within 1 h and this continued to decline to 43% of initial uptake by 16 h. To determine whether these changes in 2-DOG uptake were associated with corresponding changes in glucose transporter expression, subtype-specific antisera against Glut-1 and Glut-3 were used. Little change in membrane expression of these transporters was observed prior to 16 h. Fractionation of cell membranes on Nycodenz gradients showed that the majority of each transporter subtype was associated with the plasma membrane (63-93%) and that transporter distribution did not change markedly in response to addition or withdrawal of IL-3. These results demonstrate that IL-3 regulates glucose uptake by modulating the intrinsic transporting ability of glucose transporters. Decreased transporter affinity for 2-DOG and 3-O-methylglucose was observed following IL-3 withdrawal. Similar affinity changes were observed with 2-DOG following exposure of IL-3-stimulated cells to the protein kinase inhibitors, genistein and staurosporine. In contrast, the tyrosine phosphatase inhibitor, vanadate, acted like IL-3 to increase transporter affinity for glucose. Together these results demonstrate that IL-3 acts to maintain the intrinsic transport properties of glucose transporters without markedly affecting their expression or translocation.

Sodium dependent ion transporters in trout gills

Biochemical procedures developed to isolate plasma membranes from the branchial epithelium of rainbow trout (Oncorhynchus mykiss) yield membrane fractions that are specifically enriched in the plasma membrane marker enzyme Na+/K+-ATPase. As the bulk of the branchial Na+/K+-ATPase is assumed to be confined to the mitochondria-rich chloride cells, such membrane preparations must contain the essence of the enzymatic machinery of the chloride cells. Basal Na+ activity in branchial (chloride) cells is around 10 millimolar and, accordingly, we find a Km for Na+ of the Na+/K+-ATPase of 13 millimolar, indicating that the enzyme may be regulated by changes in cytosolic sodium. The Na+-gradient across the serosal plasma membrane created by this pump provides energy for 3Na+/Ca2+-exchange and bumetanide-sensitive Na+/K+/2Cl--cotransport. Here we further postulate the presence of a Na+/Cl--cotransporter, indicated by thiazide-sensitive, bumetanide-insensitive transport of Na+ and Cl-; this cotransporter activity awaits the characterization of its kinetics. The Na+/Ca2+-exchanger has kinetic characteristics compatible with a regulatory role of cytosolic Na+ in the activity of this carrier. Both Na+/Ca2+-exchange and Ca2+-ATPase activity may contribute to transport of Ca2+, the former having lower affinity for calcium but a higher capacity than the latter carrier. The Na+/K+/2Cl--cotransporter has kinetics that favor a regulatory role for plasma K+ in the activity of this carrier. Seawater adaptation leads to increased activity of cotransporter molecules in the plasma membrane fractions (the activity increases relative to that of the Na+/K+-ATPase) and this may reflect a function in Cl--extrusion performed by the chloride cells in a seawater environment. A function for the cotransporter in the gills of freshwater fish may be the regulation of cell volume.

A survey of gtp-binding proteins and other potential key regulators of exocytotic secretion in eosinophils : Apparent absence of rab3 and vesicle fusion protein homologues

We set out to identify potential key regulators of exocytotic fusion in the eosinophil, in the knowledge that granule exocytosis can be stimulated in these cells by intracellular application of nonhydrolyzable analogues of guanosine triphosphate, with Ca2+ acting as a modulator of guanine nucleotide-dependent secretion. To screen for GTP-binding proteins, guinea pig eosinophils were purified from peritoneal washings and subjected to western blotting analysis using specific immune sera raised against recombinant proteins or consensus peptide sequences within proteins of interest. We found a number of heterotrimeric G proteins (G alpha i3, G alpha o, G alpha q11, G alpha s and G beta subunits) and members of the small GTP-binding proteins expressed in eosinophils. Two subtypes of G-protein alpha subunits (G alpha i1 and G alpha z) could not be detected. Separation of subcellular organelles from homogenized eosinophils by density gradient centrifugation revealed that all of the detected GTP-binding proteins were mainly expressed in fractions containing peak plasma membrane and Golgi marker enzyme activities, while G beta subunits were also detected in secretory granule fractions. However, isoforms of Rab3, a putative GTP-binding regulator of exocytotic fusion, were undetectable in eosinophils. Neither, with the exception of syntaxin-3, could we detect any of the proteins belonging to the proposed synaptic vesicle fusion complex (SNAP-25; synaptobrevin (VAMP) and its non-neuronal homologue, cellubrevin; synaptophysin; synaptotagmin). The results from this study, based on western blotting, suggest that eosinophils express a different class of exocytotic fusion complex proteins from those found in neuronal tissues, although a number of potential candidates fulfilling the role of GE were identified in this important inflammatory cell.

HeLa plasma membranes bind the antitumor sulfonylurea LY181984 with high affinity

Homogenates, total particulate and plasma membranes of cultured HeLa S cells bound the tritiated antitumor sulfonylurea [ 3H]LY181984 with high affinity ( K d of 20 to 50 nM). Highest affinity binding ( K d of 25 nM) was to purified plasma membrane. The number of binding sites, estimated to represent 30 to 35 pmol/mg protein, would represent a low abundance constituent representing about 1 1000 of the total plasma membrane proteins. When corrected for mitochondrial uptake, binding recoveries of about 80% were achieved. Of the recovered specific radioactivity bound, approximately 90% was associated with the total particulate fraction. Of this, nuclei- and plasma membrane-free total membranes bound little or no [ 3HILY181984 with high affinity. The high-affinity binding was restricted primarily to the plasma membranes. All fractions exhibited varying degrees of lower affinity binding indicative of a heterogeneous array of components capable of binding [ 3H]LY181984 at high concentrations of LY181984. Enrichment of 5-fold over total homogenates of high-affinity binding compared favorably to a 6.7-fold enrichment of the plasma membrane marker enzyme 5′-nucleotidase determined in parallel. We conclude that plasma membranes of HeLa cells contain high-affinity binding sites of low abundance for the antitumor sulfonylurea LY181984 and that the high-affinity sites are associated predominantly with the plasma membrane.

LLC-PK1 epithelia as a model for in vitro assessment of proximal tubular nephrotoxicity.

LLC-PK1 cells, an established epithelial cell line derived from pig kidney, were used as a model system for assessment of nephrotoxic side effects of three cephalosporin antibiotics: cephaloridine, ceftazidime, and cefotaxime. Toxic effects of these xenobiotics were monitored on confluent monolayers by light and electron microscopy and by the release of cellular marker enzyme activities into the culture medium. In addition, LLC-PK1 cells were grown on microporous supports, and cephalosporin-induced alteration of epithelial functional integrity was monitored by a novel electrophysiologic approach. For this purpose, an Ussing chamberlike experimental setup was used. The dose-dependent effects on transepithelial ionic permselectivity were monitored under conditions in which defined fractions of the apical culture medium NaCl contents were replaced iso-osmotically by mannitol. This method of determining the functional intactness of the epithelial barrier by measuring dilution potentials was found to be far more sensitive than monitoring cell injury by means of morphology or measurement of enzyme release. As expected from animal experimental data, a dose-dependent disruption of monolayer integrity was detected with all three methodologies applied. Cephaloridine was found the most toxic compound followed by ceftazidime, where a 3-fold, and cefotaxime, where a 10-fold dose of that of cephaloridine was needed to produce cell injury. Measurement of transepithelial dilution potentials was more sensitive as compared to the release of the apical plasma membrane marker enzyme activities alkaline phosphatase and gamma-glutamyltranspeptidase, the cytosolic lactate dehydrogenase, or the mitochondrial glutamate dehydrogenase. The data were compared to the effects of the aminoglycoside antibiotic gentamicin, which at least with respect to its effects on LLC-PK1 morphology and enzyme release, but not transepithelial electrical properties, was already investigated.

Separation of K+-and Cl−-selective ion channels from rye roots on a continuous sucrose density gradient

Microsomal membranes from rye (Secale cereale L.) roots were separated by isopycnic sucrose density gradient centrifugation. The ion channels present in gradient fractions were assayed by reconstitution into planar 1-palmitoyl-2-oleoyl phosphatidylethanolamine bilayers (PLB) and the distributions of ion channel activities were compared with membrane markerenzyme activities. A number of ion channel activities were observed and could be distinguished on the combined bases of their conductance, selectivity, kinetics and pharmacology. A voltage-dependent maxi (498 pS) cation-channel, a voltage-dependent 199-pS cationchannel, 48-pS and 18-pS K + channels, and a 148-pS Cl − channel (all unitary conductances determined in asymmetrical cis: trans 325: 100 mM KCl) colocalized with the plasma membrane marker-enzyme, vanadatesensitive ATPase. A weakly K + -selective (108 pS) channel, a 1249-pS cation-channel and a 98-pS K + channel colocalized with the tonoplast markerenzyme, nitrate-sensitive ATPase. A 706-pS K + channel colocalized with the expected distribution of intact plastids and a 38-pS Cl − channel colocalized with either plastid or ER membranes. The membrane location of several other channels including a hypervoltage-sensitive maxi (497 pS) cation-channel, a 270-pS K + channel, an 8-pS K + channel and a 4-pS K + channel was equivocal, but they were tentatively assigned to the Golgi. Thus, the plasma membrane and tonoplast origin of ion channels previously characterized following the incorporation of plasma membrane prepared by aqueous-polymer two-phase partitioning or tonoplast derived from isolated vacuoles into PLB was confirmed and the ion channel complement of previously unassayed membranes was defined. This demonstrates the usefulness of PLB in identifying and characterizing ion channels from plant cell membranes, in particular, those of membranes which are inaccessible to patch-clamp electrodes

Isolation and partial purification of plasma membrane from porcine oocytes.

Egg plasma membrane (EPM) was isolated in comparatively large amounts from porcine slaughterhouse ovaries. Ovaries were minced, and the oocyte containing fluid was filtered to retrieve zona pellucidae-intact oocytes. The oocytes were homogenized and filtered again to remove zona pellucidae. The egg filtrate was subjected to differential centrifugation to remove membrane bound organelles and the remaining plasma membrane containing material was pelleted by ultracentrifugation. Plasma membranes were further separated from cellular material by sucrose density gradient centrifugation and were collected from portions of the gradient that correspond to the densities of plasma membrane. The purity of isolated plasma membranes was assessed by membrane marker enzyme analysis and transmission electron microscopy. Activities of the plasma membrane marker enzymes 5' nucleotidase and alkaline phosphatase increased from nondetectable levels in the egg filtrate to relatively high levels in the plasma membrane preparation. Marker enzymes for mitochondrial and lysosomal membranes fell from detectable levels in the egg filtrate to levels that were at the lower limits of the assays to detect in the final preparation. Evidence provided by binding of biotin-labeled EPM to capacitated sperm suggests that the isolated EPM retains its biological activity. The procedure presented here represents a novel method of isolating porcine egg plasma membranes for further study involving sperm-egg interaction.

Isolation of sinusoidal and canalicular liver plasma membranes: Effects of frozen storage of human material

Sinudoisal and canalicular plasma membranes were prepared from rat and fresh or frozen human liver and characterized using plasma membrane marker enzymes. The distributions of these enzymes were used as a means of comparison between the plasma membrane fractions and homogenates from the rat and fresh and frozen human livers. Compared with their homogenate, all the sinusoidal plasma membrane preparations were poorly enriched with Na +-K +-ATPase (2.6–8.4-fold), a sinusoidal marker enzyme. Activities of Na +-K +-ATPase and Mg 2+-ATPase were much greater in rat membranes whereas leucine aminopeptidase and alkaline phosphatase activities were much greater in the human membranes. Consideration of the extent of enrichment showed that Mg 2+-ATPase and alkaline phosphatase are better markers in rat canalicular plasma membranes and leucine aminopeptidase in human membranes from both fresh and frozen tissue. Intracellular organelle marker enzymes were not enriched in the plasma membranes, thus indicating no significant contamination. Importantly, the relative enrichments of the marker enzymes showed no difference between fresh and frozen human liver. Absolute activities of the enzymes were decreased, however (except alkaline phosphatase), after frozen storage. This work indicates that human liver tissue may be frozen and stored in liquid nitrogen before plasma membranes are isolated with no effect on the distribution of marker enzymes in the membranes.

Role of Plasma Membrane Ganglioside Sialidase of Human Neuroblastoma Cells in Growth Control and Differentiation

In cultured human neuroblastoma cells (SK-N-MC), a plasma membrane-bound besides a lysosomal ganglioside GM3 sialidase was detected. Both activities can be distinguished by the specific activation with detergents, as well as differential inhibition by Cu ++. Plasma membrane and lysosomal sialidase specific activities showed strikingly different behaviour during the growth phase of neuroblastoma cells. Thus, the plasma membrane sialidase increased about 15-fold and mirrored cell growth; it differed from the kinetics of ornithine decarboxylase, an early marker of cell proliferation. The lysosomal sialidase, on the other hand, exhibited constant specific activities during growth of the cells, as did lysosomal and plasma membrane marker enzymes. When the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid was included in the culture medium, a profound change in proliferation kinetics was observed, indicating a release from density-dependent control of cell division. Additionally, the inhibitor abolished the increase of the biochemical differentiation marker acetylcholinesterase. The results suggest an important role of the ganglioside sialidase of the plasma membrane in the processes of proliferation control and differentiation in this neuronal cell system.

Solubilization of Functional Plasma Membrane-Localized Hepta-[beta]-Glucoside Elicitor-Binding Proteins from Soybean

Total membranes prepared from roots of soybean (Glycine max L.) seedlings have previously been shown to contain proteinaceous binding site(s) for a hepta-beta-glucoside elicitor of phytoalexin accumulation. The hepta-beta-glucoside elicitor-binding proteins have now been shown to co-migrate with a plasma membrane marker enzyme (vanadate-sensitive H(+)-ATPase) on linear sucrose density gradients. With the use of detergents, the elicitor-binding proteins have been solubilized in functional form from soybean root membranes. The nonionic detergents n-dodecylsucrose, n-dodecylmaltoside, and Triton X-114, at concentrations of 5 to 10 mg/mL, each solubilizes between 50 and 60% of the elicitor-binding activity in a single extraction of the membranes. A zwitterionic detergent, N-dodecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (ZW 3-12), also solubilizes about 40% of the total binding activity at detergent concentrations between 1 and 2 mg/mL, but the total binding activity recovered is only approximately 50% of that recovered with the nonionic detergents. The elicitor-binding proteins solubilized with either n-dodecylsucrose or ZW 3-12 retain the high affinity for radiolabeled hepta-beta-glucoside elicitor (apparent dissociation constant [Kd] = 1.8 nM and 1.4 nM, respectively) that was observed with the membrane-localized binding proteins (apparent Kd = 1 nM). Competitive ligand-binding experiments with several structurally related synthetic oligoglucosides demonstrate that the solubilized binding proteins retain specificity for elicitor-active oligosaccharides, irrespective of the detergent used for solubilization. Moreover, the binding affinities of the oligoglucosides for the solubilized binding proteins correlate well with their abilities to induce phytoalexin accumulation in soybean cotyledon tissue. Gel-permeation chromatography of n-dodecylsucrose-solubilized elicitor-binding proteins demonstrate that the bulk of the elicitor-binding activity is associated with large detergent-protein micelles (relative molecular weight > 400,000). Our results suggest that n-dodecylsucrose is a suitable detergent for solubilizing elicitor-binding proteins from soybean root membranes with minimal losses of binding activity. More importantly, we demonstrate that solubilization does not significantly after the binding properties of the proteins for elicitor-active oligoglucosides.