Detergent-solubilized Membranes Research Articles

Developmental regulation of protein kinase-A and -C activities in the baboon fetal adrenal.

We have previously demonstrated that the estrogen-regulated change in transuteroplacental metabolism of cortisol (F) and cortisone (E) from preferential reduction (E to F) at midgestation to oxidation (F to E) near term results in activation of the hypothalamic-pituitary-adrenal axis of the baboon and the ontogenesis of rate-limiting steroidogenic enzymes, culminating in de novo F secretion. It is well established that transcription of messages activated by peptide-mediated binding to membrane receptors can occur via cAMP-dependent protein kinase-A (PKA) and/or phospholipid/calcium-dependent protein kinase-C (PKC). The present study was designed to determine whether basal levels of PKA and PKC in the fetal adrenal are developmentally regulated during baboon gestation and, thus, could provide the mechanism(s) by which activation of the fetal adrenal near term is mediated. Fetal adrenal glands were obtained on day 100 (n = 8) and day 165 (n = 6) of gestation (term = day 184) from untreated baboons and on day 100 after treatment of the mother with estradiol benzoate, injected sc between days 70-100 to increase estrogen production. PKA activity (picomoles of 32P incorporated into kemptide per min/mg protein) was determined by incubation of adrenal cytosol (12,000 x g; 0.3-30 micrograms protein) in reaction mixtures containing 0.25 mM ATP, 1 x 10(6) dpm [lambda-32P]ATP, and 3 micrograms kemptide in the presence or absence of 0.02 mM cAMP. PKC activity (picomoles of 32P incorporated into histone IIIS per min/mg protein) was determined in cytosol (105,000 x g) and detergent-solubilized membrane fractions after incubation with 0.02 mM ATP, 50 micrograms histone IIIS, and 1 x 10(6) dpm [lambda-32P]ATP in the presence or absence of calcium and phospholipids. Mean (+/- SE) maternal serum estradiol concentrations (nanograms per ml) were 3-fold greater (P < 0.05) at term (1.9 +/- 0.3) than at midgestation and increased (P < 0.05) after treatment with estradiol. PKA activity was greater at term (3965 +/- 546) than at midgestation (2130 +/- 240) and increased (P < 0.05) 2-fold after treatment with estrogen (3525 +/- 416) at midgestation. PKC activity was always 3- to 4-fold lower than that of PKA and was similar in the cytosol and membrane fractions of the cell. In contrast to PKA, cytosolic PKC activity was similar at mid (265 +/- 98)-and late (353 +/- 99) gestation and was not altered by treatment with estradiol (223 +/- 27).(ABSTRACT TRUNCATED AT 400 WORDS)

Differential Glycosylation of the 5A11/HT7 Antigen by Neural Retina and Epithelial Tissues in the Chicken

The 5A11/HT7 antigen, a member of the immunoglobulin supergene family, has been implicated in heterotypic cell-cell interactions during retina development. Immunopurified 5A11 antigen isolated from Nonidet P-40-solubilized retina membranes had two components as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 45.5-kDa doublet and a 69-kDa polypeptide. Immunoreactive bands of 46-50 kDa were recognized following SDS-PAGE of detergent-solubilized membrane proteins from liver, kidney, and erythrocytes. Treatment with N-glycosidase F (EC 3.2.2.18) converted the 45.5-50-kDa immunoreactive polypeptides from all tissues to 32 kDa, indicating that the observed differences in molecular mass were due to differences in glycosylation. N-Glycosidase F treatment also converted the 69-kDa form from retina to 46 kDa, indicating a different polypeptide core than the 32-kDa species. Treatment with endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96) resulted in modest increases in electrophoretic mobility due to hydrolysis of high mannose or hybrid oligosaccharides and lack of hydrolysis of complex oligosaccharides resistant to endo-beta-N-acetylglucosaminidase H digestion. Immunoreactivity was retained after deglycosylation. Much of the difference in molecular weight could be attributed to variations in sialylation. The higher molecular mass species of the 45.5-kDa doublet from retina and the polypeptides from other tissues were susceptible to neuraminidase (EC 3.2.1.18) and O-glycosidase (endo-alpha-N-acetylgalactosaminidase; EC 3.2.1.97) digestion. Labeling with elderberry bark lectin (specific for alpha 2,6-linked sialic acid) was confined to the higher molecular mass species of the 45.5-kDa doublet and was considerably greater in antigen derived from epithelia rather than neural retina. In paraffin sections of chick retina, elderberry bark lectin staining was confined to the retinal pigmented epithelium, photoreceptor cells, and bipolar cells with no staining of the Müller cells, which bear the bulk of the 5A11 antigen. These results indicate tissue-specific posttranslational modifications, particularly differences in sialylation of antigen-bearing polypeptides.

Membrane topology and biogenesis of eukaryotic signal peptidase.

The signal peptidase complex (SPC) is a hetero-oligomeric membrane protein containing subunits of 12, 18, 21, 22/23, and 25 kDa. The 18- and 21-kDa subunits are mammalian homologs of SEC11 protein, which is necessary for signal peptide processing and cell viability in the yeast Saccharomyces cerevisiae. The functional and/or structural contributions of the 12-, 22/23-, and 25-kDa subunits to SPC activity have not yet been elucidated. To explore the structure of SPC subunits and their relationships to signal peptide processing and protein translocation, we have examined their endoplasmic reticulum (ER) membrane topology and biogenesis. Signal peptidase activity and SPC subunits are resistant to protease treatment in intact and detergent-solubilized membranes. Heat-denatured SPC subunits and SPC subunits translated in vitro are, however, protease sensitive, suggesting that the assembly of the oligomeric complex confers protease resistance. To define the membrane topology of SPC subunits, both wild-type subunits and subunit fusion proteins containing additional sites for N-linked glycosylation were assembled into microsomal membranes in vitro. Despite the presence of multiple hydrophobic domains, each subunit is anchored to the ER membrane by a single amino-terminal transmembrane domain in an Ncytoplasmic Cexoplasmic (type II) orientation. This topology places the bulk of the protein mass in the ER lumen and positions a putative serine-containing active site domain in SPC 18 and 21 at the same relative distance from the membrane as the analogous region in Escherichia coli leader peptidase. These studies have also revealed that, in spite of the temporal and perhaps physical association of the SPC with the process of protein translocation, SPC subunits integrate into the ER membrane by a signal recognition particle-dependent pathway and, hence, rely on the existence of a preformed translocation apparatus for their own membrane assembly.

A novel 95 kilodalton membrane polypeptide associated with lung cancer drug resistance

A novel 95 kilodalton (kDa) membrane protein has been previously shown to be induced in breast cancer cells made resistant in vitro to adriamycin. We have used antisera raised against the 95 kDa protein to examine a panel of early passage small cell lung cancer cell lines from patients with untreated extensive stage disease. Two lines, demonstrating marked de novo resistance to etoposide, showed expression of the 95 kDa protein by Western blotting. The protein was enriched in detergent-solubilized membrane preparations, and detected by silver staining and immune blotting. The 95 kDa protein is frequently detected in small cell lung cancer cell lines obtained from patients relapsed after chemotherapy, suggesting that it may be selected for by chemotherapy in vivo.

Actions of a monoclonal antibody Tor 23 on rat brain presynaptic cholinergic processes.

Tor 23 is a monoclonal antibody, generated against cholinergic terminals of the Torpedo californica, that has been found to bind to the extracellular surface of cholinergic neurons in a variety of tissues. This study shows that Tor 23 inhibits: 1) high affinity [3H]hemicholinium-3 binding to detergent-solubilized membranes prepared from rat neocortices; 2) high affinity [3H]choline uptake in rat neocortical and striatal P2 preparations; and 3) [3H]acetylcholine synthesis in isolated nerve terminals. Tor 23 does not appear to affect low affinity [3H]choline uptake or [3H]acetylcholine release. These results are consistent with the hypothesis that Tor 23 may bind to nerve terminal high affinity choline transporters in the rat brain.

Multiple carbohydrate moieties on the Na+/H+ exchanger.

Affinity-purified antibodies against the C-terminal region of the Na+/H+ exchanger (NHE-1) were used to analyse the carbohydrate moiety of the protein. The Na+/H+ exchanger in human placental brush-border membranes has an apparent molecular mass of 105 kDa. Incubation of intact or detergent-solubilized membranes with glycopeptidase F removed the carbohydrate moiety and increased the apparent mobility of the exchanger. Digestion with endoglycosidase-F caused a similar change in mobility, but endoglycosidase-H had no effect, suggesting that the placental Na+/H+ exchanger is a glycoprotein of the biantennary complex type. Removal of the carbohydrate moiety with glycopeptidase F had no effect on the ability of the protein to promote the exchange of Na+ for H+, and had no detectable effect on the sensitivity of the exchanger to trypsin. Limited digestion with glycopeptidase F and neuraminidase indicated the presence of two intermediate forms between the fully glycosylated and the deglycosylated protein. This suggests the presence of at least two, and possibly three, N-linked carbohydrate moieties.

Analysis of the streptococcal hyaluronic acid synthase complex using the photoaffinity probe 5-azido-UDP-glucuronic acid.

The mucopolysaccharide, hyaluronic acid, is an important component of both mammals and pathogenic streptococci. This high molecular weight polymer is synthesized by a membrane-associated, multisubunit hyaluronate synthase which utilizes UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. Using the photoaffinity probe, [beta-32P]5-azido-UDP-glucuronic acid, three streptococcal membrane proteins (42, 33, and 27 kDa) specifically photoincorporated this probe. Labeling of these proteins was enhanced in the presence of UDP-N-acetylglucosamine, whereas UDP-galactose or UDP-glucose had no effect on incorporation. UDP-glucuronic acid inhibited the labeling of the three proteins in a dose-dependent manner. Detergent-solubilized membrane proteins from transposon-inactivated hyaluronic acid capsule mutants no longer incorporated the probe. This was also the case when membranes from stationary phase organisms were tested. Finally, glucuronic acid no longer was incorporated into high molecular weight hyaluronic acid with either the mutant or stationary phase preparations. Further biochemical analysis will be required to demonstrate the exact role each of the proteins play in hyaluronic acid biosynthesis.

Efficient precipitation and accurate quantitation of detergent-solubilized membrane proteins

The protein assay method of I. Polacheck and E. Cabib ( Anal. Biochem. 117, 311–314, 1981 ) has been modified to provide a general method for quantitating protein samples in the presence of detergents. Dilute detergent-solubilized membrane proteins, by using ribonucleic acid as a carrier, have been efficiently precipitated here by trichloroacetic acid (TCA) in the presence of sodium dodecyl sulfate (SDS). Washing the pellets once with TCA solution has removed most of the reagents present in the original sample. The washed sample could then be quantitated by the Lowry method ( O. J. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265–275, 1951 ). This procedure could be used to assay protein solutions of a concentration as low as 5 μg/ml in the presence of the following reagents: Triton X-100, Triton X-114, Tween 20, N-octylglucoside, deoxycholate, cholate, Thesit, octanoyl- N-methylglucamide, isotridecylpoly (ethyleneglycoether) n , Nonidet P-40, glucose, methyl- d-glucopyranoside, methyl- d-mannopyranoside, N-acetyl-glucosamine, Mn 2+, Ca 2+, Mg 2+, and many buffer reagents. Proteins solubilized from porcine brain myelin sheath and synaptic plasma membranes were quantitated by amino acid analysis and by the TCA SDS precipitation method described here. The resultant protein concentrations were almost identical. The results have suggested this TCA SDS precipitation method to be useful for quantitating dilute protein samples containing high concentrations of detergents and other reagents commonly employed in studying membrane proteins.

Specific antibody to the thyrotropin receptor identifies multiple receptor forms in membranes of cells transfected with wild-type receptor complementary deoxyribonucleic acid: characterization of their relevance to receptor synthesis, processing, structure, and function.

An antibody to a peptide of the TSH receptor, residues 352-366 which are not present in gonadotropin receptors, specifically identifies three major forms of the receptor on Western blots of detergent-solubilized membrane preparations from Cos-7 cells transfected with full-length rat and human TSH receptor cDNA: 230, 180, and 95-100 kilodaltons (kDa), based on simultaneously run protein standards. The 95- to 100-kDa protein is absent in cells transfected with a mutant receptor with no signal peptide and is sensitive to endoglycosidase-F. Its size is consistent with the sum of amino acids predicted from its cDNA sequence (84 kDa after subtracting the signal peptide) plus its carbohydrate content (14 kDa estimated from glycosylation mutants). It alone is absent in two deletion mutants that have lost TSH binding and activity after transfection: M1 missing residues 37-121 and M2 missing residues 110-307. It, thus, appears to be the processed glycosylated functional receptor on the cell surface. The 230-kDa protein is a nonprocessed form of the receptor, as evidenced by its insensitivity to endoglycosidase-F and its continued presence in cells transfected with a mutant receptor with no signal peptide. It is the primary form identified in rat FRTL-5 thyroid cells that have a functioning TSH receptor; it is not present in rat FRT thyroid cells with no functioning TSH receptor or receptor RNA. It appears, therefore, to be a early synthetic form of the functional TSH receptor. The 180-kDa protein is endoglycosidase-F sensitive and appears to be a processed intermediate between the 230-kDa early synthetic form and the 95- to 100-kDa functional receptor, rather than a dimer of the latter. Thus, with decreases in size appropriate to a receptor monomer, it remains present in membranes from the M1 and M2 deletion mutants that contain the 230-kDa protein but are missing the 95- to 100-kDa receptor form in association with lost TSH binding and activity after transfection. Minor receptor forms (54 kDa in rat receptor transfectants, 54 and 48 kDa in human receptor transfectants) appear to be degraded forms of the processed and glycosylated 95- to 100-kDa receptor. The presence or absence of reducing agents in the detergent solubilization mixture does not change the pattern or amount of the receptor forms recognized by the antibody, including the 54-kDa form; however, boiling does.(ABSTRACT TRUNCATED AT 400 WORDS)

Reconstituted membranes of tumour cells (proteoliposomes) induce specific protection to murine lymphoma cells.

Antigens presented on cell membranes or on liposomes are usually more immunogenic than antigens in soluble form, this being one of the reasons for the weak immunogenicity of extracted tumour-associated transplantation antigens (TATA). The main objective of this study is to solubilize TATA from tumour cells and to present them on a membrane-like structure to the immune system. Crude tumour cell membranes of SL2 lymphosarcoma cells (a spontaneously arising, weakly immunogenic tumour) were solubilized with octylglucoside or sodium deoxycholate, and reconstituted membranes (proteoliposomes) were prepared by detergent removal. Mice immunized s.c. with reconstituted membranes were protected against an i.p. challenge with tumour cells. Although octylglucoside solubilized only 41% of the membrane proteins, the reconstituted membranes were as immunoprotective as crude membranes. (Glyco)proteins were probably the major membrane components in the reconstituted membranes that induce immunoprotection, as mice immunized with preparations constituted of (glyco)lipids from SL2 cells could not reject SL2 cells. If Freund's complete adjuvant was used with the first immunization injection, no potentiation of the elicited immune responses was observed. Besides the membrane TATA, SL2 cells contained an apparently non-membrane-bound TATA, which was found in the cytoplasm. It is concluded that detergent solubilization of membranes and subsequent preparation of reconstituted membranes can be used to obtain membrane tumour-associated antigens that retain activity for induction of protective tumour immunity. The major advantage of this method is that membrane proteins are solubilized and are subsequently presented on a membrane-like structure that resembles the tumour cell membrane. On theoretical and practical grounds it provides a promising alternative for whole-cell vaccines.

Kinetic analyses of the binding of leukemia inhibitory factor to receptor on cells and membranes and in detergent solution.

The equilibrium and kinetic properties of leukemia inhibitory factor (LIF) binding to a range of cell types have been compared. When binding was examined at 4 degrees C, the majority of cells were found to express a single class of high affinity LIF receptor (KD = 20-100 pM; ka = 2-8 x 10(8) min-1 M-1; kd = 0.0004-0.0011 min-1). In contrast, certain activated macrophage populations expressed apparently independent classes of high and low affinity LIF receptor. The low affinity receptors differed from the high affinity receptors in terms of the dissociation rate of the receptor-ligand complex (KD = 1-2 nM; ka = 3-7 x 10(8) min-1 M-1; kd = 0.30-0.67 min-1). At 37 degrees C, the interaction of LIF with its high affinity receptor was more complicated, since occupied LIF receptors were internalized more rapidly than unoccupied receptors, internalized LIF was hydrolyzed and released from the cell, and new receptors were synthesized and expressed on the cell surface. Interestingly, when membranes were prepared from cells that expressed only high affinity receptors, both high and low affinity receptors were detected, while after detergent solubilization of membranes only low affinity receptors were apparent. These results are discussed in terms of a structural model for the LIF receptor in which interaction of a low affinity binding subunit and a second nonbinding subunit is required for the generation of the high affinity receptor.

Time-resolved fluorescence of erythrocyte plasma membrane ca 2+-atpase in different functional states

The fluorescence decay of the plasma membrane calmodulin-activated Ca 2+-ATPase from the erythrocyte was measured for the first time. The availability of a novel procedure for on-line blank subtraction in frequency-domain lifetime data acquisition (G.G. Reinhart, B. Feddersen, D. Jameson and E. Gratton, Biophys. J. 57 (1990) 189a) permitted the elimination of background interference from detergent-solubilized purified plasma membrane ATPase samples. The fluorescence decay of the erythrocyte Ca 2+-ATPase was measured in the absence of Ca 2+, or in the presence of Ca 2+ or Ca 2+ plus calmodulin. In the three different experimental conditions the fluorescence decay was very heterogeneous and could be best described by Lorentzian distributions of lifetime values. In the absence of Ca 2+ the decay was described by a broad lifetime distribution centered at 4.4 ns with a width of 3.2 ns, indicating heterogeneity of tryptophan microenvironments in the ATPase. Calcium ion binding promoted an 11% increase in the center and a 27% decrease in the width of the distribution. By contrast, addition of calmodulin in the presence of Ca 2+ caused a 15% decrease in the center of the distribution, revealing structural difference between calmodulin-activated and Ca 2+-activated states of the ATPase. These results indicate the usefulness of on-line blank subtraction in frequency-domain lifetime measurements to investigate conformational changes in detergent-solubilized membrane protein samples.

Surface-bound biomembranes incorporating receptors: electrochemical and structural characterization

A generic method is described for forming surface-bound structures that incorporate protein receptors in a membrane-like environment. Silane reagents (octadecyltrichlorosilane and dimethyloctadecylchlorosilane) were used to produce primed substrates bearing full and partial monolayers, respectively. Biomembranes were formed by dialysis of detergent-solubilized membranes in the presence of two different alkylsilanized substrates: Si/SiO 2 electrodes and glass microspheres. Electrochemical analysis of the capacitance was used to determine apparent thickness and degree of surface coverage at each stage in the deposition process. Elemental analysis on glass beads gave the hydrocarbon incorporation. Glass bead substrates were also examined by Fourier transform infrared spectroscopy to evaluate the alkylsilanized substrate before and after dialysis. Both vertebrate rhodopsin and the nicotinic acetylcholine receptor could be incorporated into structures with composition and dimensions similar to natural bilayer membranes. The techniques reported here are applicable for coupling membrane receptors to a variety of transducing substrates used in biosensors.

31P-NMR study of pig intestinal brush-border membrane structure: effect of zinc and cadmium ions

31P-NMR experiments on intact pig small intestine brush-border membrane vesicles (BBMV) and detergent-solubilized membranes gave direct insights into the organization of the phospholipids (PL) and their interaction with zinc and cadmium ions. Various endogenous PL were identified from well resolved BBM micelle spectra. These experiments revealed a strong interaction of Zn2+ and Cd2+ with the negatively charged phosphatidylinositol and phosphatidylserine. In BBM micelles, a progressive time-dependent PL degradation occurred in the absence of ions and indicated the presence of active phospholipases. The presence of zinc inhibited the degradation process whereas cadmium had the opposite influence. 31P spectra of BBMV were carefully characterized. Neither zinc nor cadmium affected the PL bilayer structural organization. A degradation of PL, monitored by the increase of the inorganic phosphate (Pi) signal, also occurred in vesicles but to a lesser extent than in micelles. A 2/3 internal, 1/3 external PL asymmetry was observed in the absence and presence of ions.

Differential response of Apis mellifera acetylcholinesterase towards pirimicarb

The kinetic analysis of Apis mellifera acetylcholinesterase inhibition by the carbamate pirimicarb showed that native and detergent-solubilized membrane enzyme exhibited slightly different carbamylation kinetics. The acetylcholinesterase form sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) was carbamylated more rapidly (kapp = 36.4 X 10(-3) min-1) than the PI-PLC-resistant counterpart (kapp = 10.13 X 10(-3) min-1) which had a behavior close to that of the soluble tryptic enzyme (kapp = 11.89 X 10(-3) min-1). A difference in acetylcholinesterase sensitivity towards pirimicarb was also observed between foraging and emerging bees. These results show that the molecular structure, the mode of preparation and the source of acetylcholinesterase from the bee head should be taken into account in accurate toxicological studies.

A transferrin-binding protein of Trypanosoma brucei is encoded by one of the genes in the variant surface glycoprotein gene expression site.

A transferrin-binding protein (TFBP) with an apparent molecular weight of 42 kd was purified from detergent-soluble membrane proteins of bloodstream forms of Trypanosoma brucei. The protein is not expressed in the insect-borne stage of the parasite's life-cycle. Purified TFBP can be converted from an amphiphilic to a hydrophilic form by cleavage with T.brucei glycosylphosphatidylinositol (GPI)-specific phospholipase C, demonstrating that the C-terminus is modified by a GPI-membrane anchor. The TFBP is encoded by an expression-site-associated gene [ESAG 6 in the nomenclature of Pays et al. (1989) Cell, 57, 835-845] which is under the control of the promoter transcribing the expressed variant surface glycoprotein gene. The possible function of TFBP as a receptor for the uptake of transferrin in bloodstream forms is discussed.

Angiotensin-converting enzyme in the rabbit stomach wall

Abstract To examine the potential role of angiotensinconverting enzyme as a physiological regulator of neuropeptide activity in the alimentary tract, we purified and partially characterized this enzyme from the crude membrane fraction of rabbit gastric muscles. A single-step purification from detergent-solubilized gastric membranes by affinity chromatography, using lisinopril-Sepharose, yielded an electrophoretically homogeneous peptidase composed of 180-kilodalton polypeptide. The purified enzyme represented approximately 2% of [Leu 5 ]enkephalin-degrading activity of the original membrane preparation, and 275-fold purification was achieved. The gastric angiotensin-converting enzyme hydrolyzed synthetic substrates specific to the lung enzyme. It hydrolyzed angiotensin I and several other bioactive peptides, by removing their carboxyl-terminal dipeptides. The activity was completely inhibited with 10 −6 mol/L captopril. The hydrolysis of enkephalin was enhanced twofold by addition of 0.3 mol/L NaCl to the assay buffer. These properties were comparable with those reported for the rabbit lung enzyme. Therefore, it was concluded that rabbit gastric muscle tissue contains membrane-bound angiotensinconverting enzyme. The results suggest a role for this enzyme as a local inactivator of bioactive peptides in the stomach wall.

Identification of a putative Na(+)-H+ exchanger regulatory cofactor in rabbit renal BBM

Previous in vitro studies with detergent-solubilized rabbit renal brush-border membrane (BBM) proteins have suggested that adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA)-mediated inhibition of the Na(+)-H+ exchanger requires the presence of 42-kDa cofactor that is distinct from the exchanger itself. We sought to determine whether there was a protein in native rabbit renal BBM vesicles that has characteristics similar to that of the 42-kDa cofactor. Incubation of native BBM vesicle proteins with a hypotonic phosphorylation solution containing purified catalytic subunit of PKA resulted in phosphorylation of a number of BBM proteins, including a protein with an apparent molecular weight that was similar but not identical to that of the 42-kDa cofactor obtained from anion-exchange column chromatography of n-octyl glucoside-extracted BBM proteins. The identity between the BBM vesicle protein and the 42-kDa cofactor was established by phosphopeptide maps and radioiodinated peptide maps. These results indicate that native BBM vesicles contain a number of proteins that are phosphorylated by PKA when the PKA and ATP are present inside the vesicle space. One of these proteins appears to be identical to the 42-kDa protein that, as previously suggested by in vitro studies, acts as a regulatory cofactor mediating the inhibitory effect of PKA on the renal Na(+)-H+ exchanger.

Covalent binding of detergent-solubilized membrane glycoproteins to ‘chemobond’ plates for ELISA

An ELISA method is presented which is based on covalent binding of detergent-solubilized membrane proteins to surface-modified polystyrene plates (Chemobond plates). These plates carried 0.52–0.65 nmol of aldehyde groups per well (150 μl) and allowed coupling of protein by Schiff base formation either at high pH and subsequent reduction with NaBH 4 or by trapping reduced imines at pH 6–6.8 with cyanoborohydride. They bound 15 times the amount of normal plates. Sodium chloride (0.5 M) increased binding 2–3-fold. Binding was essentially resistant to elution by 1% sodium dodecyl sulfate. Reduction of uncoated plates with NaBH 4 eliminated the high extent of binding. ELISA tests on Chemobond plates with a rabbit anti-band 3 antibody gave a ten-fold higher signal than plates to which band 3 protein was merely adsorbed. The use of an antigen-enzyme conjugate to detect bound antibody allowed to perform antibody binding and detection of bound antibody simultaneously in the presence of 0.05% Triton X-100. A competitive, one step ELISA system allowed determination of rabbit anti-band 3 antibodies in diluted serum with a sensitivity range of 0.02–0.4 μg/ml.

A Mr 80K hepatocyte surface protein(s) interacts with basement membrane components

We have identified a M mr 80K cell surface protein(s) from adult rat hepatocytes that binds basement membrane components, including collagen IV, heparan sulfate proteoglycan, and laminin. Freshly isolated hepatocytes were cell surface-labeled with 125I using the lactoperoxidase-catalyzed method, and detergent-solubilized membrane proteins were chromatographed on affinity columns prepared with purified basement membrane components. A M r 80K protein was eluted with 0.15–1 M NaCl from a collagen IV column. Two proteins ( M r 80K and 38K) were eluted from a heparan sulfate proteoglycan column. The larger protein was also eluted from a column made with heparan sulfate side chains. Several proteins ( M r 80K, 67K, 45K, and 32K) bound to an affinity chromatography column made with the laminin A chain-derived synthetic peptide PA22-2, which is active for promoting cell attachment. When fractions eluted from these columns were analyzed by two-dimensional gel electrophoresis, the M r 80K proteins showed similar patterns with a p I ranging from 8 to 9. The M r 80K protein(s) may have an important role in the interaction of hepatocytes with basement membrane.