Wheat Germ agglutinin-Sepharose Research Articles

Adenosine triphosphate regulates the activity of guinea pig Cav1.2 channel by direct binding to the channel in a dose-dependent manner

The present study is to investigate the mechanism by which ATP regulates Cav1.2 channel activity. Ventricular tissue was obtained from adult guinea pig hearts using collagenase. Ca(2+) channel activity was monitored using the patch-clamp technique. Proteins were purified using wheat germ agglutinin-Sepharose, and the concentration was determined using the Coomassie brilliant blue technique. ATP binding to the Cav1.2 channel was examined using the photoaffinity method. EDA-ATP-biotin maintains Ca(2+) channel activity in inside-out membrane patches. ATP directly bound to the Cav1.2 channel in a dose-dependent manner, and at least two molecules of ATP bound to one molecule of the Cav1.2 channel. Low levels of calmodulin (CaM) increased ATP binding to the Cav1.2 channel, but higher levels of CaM decreased ATP binding to the Cav1.2 channel. In addition, Ca(2+) was another regulator for ATP binding to the Cav1.2 channel. Furthermore, ATP bound to GST-fusion peptides of NH2-terminal region (amino acids 6-140) and proximal COOH-terminal region (amino acids 1,509-1,789) of the main subunit (α1C) of the Cav1.2 channel. Our data suggest that ATP might regulate Cav1.2 channel activity by directly binding to the Cav1.2 channel in a dose-dependent manner. In addition, the ATP-binding effect to the Cav1.2 channel was both CaM- and Ca(2+) dependent.

Ligand Stimulation Reduces Platelet-derived Growth Factor β-Receptor Susceptibility to Tyrosine Dephosphorylation

Ligand binding to the platelet-derived growth factor (PDGF) beta-receptor leads to increased receptor tyrosine phosphorylation as a consequence of dimerization-induced activation of the intrinsic receptor tyrosine kinase activity. In this study we asked whether ligand-stimulated PDGF beta-receptor tyrosine phosphorylation, to some extent, also involved reduced susceptibility to tyrosine dephosphorylation. To investigate this possibility we compared the sensitivity of ligand-stimulated and non-stimulated forms of tyrosine-phosphorylated PDGF beta-receptors to dephosphorylation using various preparations containing protein-tyrosine phosphatase activity. Ligand-stimulated or unstimulated tyrosine-phosphorylated receptors were obtained after incubation of cells with pervanadate only or pervanadate, together with PDGF-BB, respectively. Dephosphorylation of receptors immobilized on wheat germ agglutinin-Sepharose, as well as of receptors in intact cell membranes, was investigated under conditions when rephosphorylation did not occur. As compared with unstimulated receptors the ligand-stimulated PDGF beta-receptors showed about 10-fold reduced sensitivity to dephosphorylation by cell membranes, a recombinant form of the catalytic domain of density-enhanced phosphatase-1, or recombinant protein-tyrosine phosphatase 1B. We conclude that ligand-stimulated forms of the PDGF beta-receptor display a reduced susceptibility to dephosphorylation. Our findings suggest a novel mechanism whereby ligand stimulation of PDGF beta-receptor, and possibly other tyrosine kinase receptors, leads to a net increase in receptor tyrosine phosphorylation.

Vascular Endothelial Growth Factor (VEGF) Receptor II-derived Peptides Inhibit VEGF

Vascular endothelial growth factor (VEGF) directly stimulates endothelial cell proliferation and migration via tyrosine kinase receptors of the split kinase domain family. It mediates vascular growth and angiogenesis in the embryo but also in the adult in a variety of physiological and pathological conditions. The potential binding site of VEGF with its receptor was identified using cellulose-bound overlapping peptides of the extracytosolic part of the human vascular endothelial growth factor receptor II (VEGFR II). Thus, a peptide originating from the third globular domain of the VEGFR II comprising residues 247RTELNVGIDFNWEYP261 was revealed as contiguous sequence stretch, which bound 125I-VEGF165. A systematic replacement with L-amino acids within the peptide representing the putative VEGF-binding site on VEGFR II indicates Asp255 as the hydrophilic key residue for binding. The dimerized peptide (RTELNVGIDFNWEYPAS)2K inhibits VEGF165 binding with an IC50 of 0.5 microM on extracellular VEGFR II fragments and 30 microM on human umbilical vein cells. VEGF165-stimulated autophosphorylation of VEGFR II as well as proliferation and migration of microvascular endothelial cells was inhibited by the monomeric peptide RTELNVGIDFNWEYPASK at a half-maximal concentration of 3-10, 0.1, and 0.1 microM, respectively. We conclude that transduction of the VEGF165 signal can be interrupted with a peptide derived from the third Ig-like domain of VEGFR II by blockade of VEGF165 binding to its receptor.

Purification and Characterization of a Polyisoprenyl Phosphate Phosphatase from Pig Brain: POSSIBLE DUAL SPECIFICITY

Microsomal fractions from pig and calf brain catalyze the enzymatic dephosphorylation of endogenous and exogenous dolichyl monophosphate (Dol-P) (Sumbilla, C. A., and Waechter, C. J. (1985) Methods Enzymol. 111, 471-482). The Dol-P phosphatase (EC 3.1.3.51) has been solubilized by extracting pig brain microsomes with the nonionic detergent Nonidet P-40 and purified approximately 1,107-fold by a combination of anion exchange chromatography, polyethylene glycol fractionation, dye-ligand chromatography, and wheat germ agglutinin affinity chromatography. Treatment of the enzyme with neuraminidase prevented binding to wheat germ agglutinin-Sepharose, indicating the presence of one or more N-acetylneuraminyl residues per molecule of enzyme. When the highly purified polyisoprenyl phosphate phosphatase was analyzed by SDS-polyacrylamide gel electrophoresis, a major 33-kDa polypeptide was observed. Enzymatic dephosphorylation of Dol-P by the purified phosphatase was 1) optimal at pH 7; 2) potently inhibited by F-, orthovanadate, and Zn2+ > Co2+ > Mn2+ but unaffected by Mg2+; 3) exhibited an approximate Km for C95-Dol-P of 45 microM; and 4) was sensitive to N-ethylmaleimide, phenylglyoxal, and diethylpyrocarbonate. The pig brain phosphatase did not dephosphorylate glucose 6-phosphate, mannose 6-phosphate, 5'-AMP, or p-nitrophenylphosphate, but it dephosphorylated dioleoyl-phosphatidic acid at initial rates similar to those determined for Dol-P. Based on the virtually identical sensitivity of Dol-P and phosphatidic acid dephosphorylation by the highly purified enzyme to N-ethylmaleimide, F-, phenylglyoxal, and diethylpyrocarbonate, both substrates appear to be hydrolyzed by a single enzyme with an apparent dual specificity. This is the first report of the purification of a neutral Dol-P phosphatase from mammalian tissues. Although the enzyme is Mg2+-independent and capable of dephosphorylating Dol-P and PA, several enzymological properties distinguish this lipid phosphomonoesterase from PAP2.

1] Purification, hydrodynamic properties, and glycosylation analysis of glycine transporters

Publisher Summary This chapter describes a biochemical procedure to obtain a highly purified glycine transport activity with GLYT2-like properties from pig brain stem. The main advantage of this procedure is to provide the transporter in its native state, what readily permits the study of its biochemical, structural, and functional properties. Glycine acts as an inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates, mainly in the spinal cord and the brain stem. An additional role of glycine is the potentiation of glutamate excitatory action on postsynaptic N-methyl-D-aspartate (NMDA) receptors. The reuptake of glycine into presynaptic nerve terminals or neighboring glial cells provides one way of clearing the neurotransmitter from the extracellular space and constitutes an efficient mechanism by which its postsynaptic action can be terminated. This process is carried out by a transport system that actively accumulates glycine and that is energized by the electrochemical gradient of sodium. The purification of glycine transporter involves the solubilization of the transporter protein from plasma membrane vesicles followed by three chromatographic steps on phenyl-Sepharose, wheat germ agglutinin-Sepharose and hydroxylapatite columns, and a final sucrose density gradient fractionation.

Tissue specific differences in insulin receptor characteristics in the mature bovine animal

Insulin is believed to play a key role in the partitioning of nutrients towards fat and protein deposition. The biological effects of insulin are a function of plasma insulin concentration, insulin-receptor concentration and the affinity of the receptor. The number and affinity of insulin receptors on adipocytes differs between genetically lean and obese pigs (Camara and Mourot, 1996) while receptor number, but not affinity, was shown to differ in a variety of bovine muscles (Boge et al., 1995). The objective of the current work was to determine if insulin receptor affinity and number vary between the principal target tissues (i.e. liver, muscle and adipose tissue) in the bovine animal.Five Charolais-cross steers were offered grass silage ad libitum from 18 months of age until slaughter, at 701± 22.9 kg, at a commercial abattoir. Samples of liver [L], of two skeletal muscles [Ml, M3] and of subcutaneous [S], omental [0]and renal [R] fats were taken as soon as possible after slaughter (typically <30min for L, M3, S and O and <45min for Mland R), wrapped and snap frozen in liquid nitrogen prior to storage at -70 °. Insulin receptors were partially purified by solubilising tissues in 1% Triton X-100 for 24 hours at 4 °C followed by centrifugation (100000 x g, lh, 4°) and affinity chromatography of the supernatants on 0.5 ml wheatgerm agglutinin Sepharose 6MB columns. Bound receptors were eluted from the column with 0.3M N-acetyl-D-glucosamine. Receptor binding was assessed using a tracer amount of A-14 125I-insulin (∼35 pM) and increasing concentrations of unlabelled insulin (0-10 μM) in a total volume of 150 μl of pH 7.4 buffer as described by Magri et al (1990).

A Subpopulation of Estrogen Receptors Are Modified by O-Linked N-Acetylglucosamine

Estrogen receptors (ER) are ligand-inducible transcription factors regulated by Ser(Thr)-O-phosphorylation. Many transcription factors and eukaryotic RNA polymerase II itself are also dynamically modified by Ser(Thr)-O-linked N-acetylglucosamine moieties (O-GlcNAc). Here we report that subpopulations of murine, bovine, and human estrogen receptors are modified by O-GlcNAc. O-GlcNAc moieties were detected on insect cell-expressed, mouse ER (mER) by probing with bovine milk galactosyltransferase, followed by structural analysis. Wheat germ agglutinin-Sepharose affinity chromatography also readily detected terminal GlcNAc residues on subpopulations of ER purified from calf uterus, from human breast cancer cells (MCF-7), or from mER produced by in vitro translation. These data suggest that greater than 10% of these populations of estrogen receptors bear O-GlcNAc. Site mapping of insect cell expressed mER localized one major site of O-GlcNAc addition to Thr-575, within a PEST region of the carboxyl-terminal F domain. Based upon their relative resistance to both hexosaminidase and to in vitro galactosylation, O-GlcNAc moieties appear to be largely buried on native mER. This dynamic saccharide modification, like phosphorylation, may play a role in modulating the dimerization, stability, or transactivation functions of estrogen receptors.

Characterization of p92, karyopherin beta, co-purified with N-acetylglucosamine-bearing nucleoporins from rat liver nuclear envelopes.

A 92k protein (p92) was purified from the wheat germ agglutinin-Sepharose (WGA-Sepharose) bound fraction of a rat liver nuclear envelope salt-extract by DEAE-5PW and hydroxyapatite HPLCs. Partial amino acid sequence analysis of p92 revealed that it is karyopherin beta, which was found recently in the cytosolic fraction. It was shown using anti-p92 antiserum that the protein is present in the nuclear envelope and cytosolic fractions, in almost the same amounts, but not in other subcellular fractions of rat liver. p92 bound to N-acetylglucosamine bearing nucleoporins (GNPs) on WGA-Sepharose, but not directly to WGA. The amount of p92 found in the rat liver nuclear envelope fraction corresponded to about 10% of the nuclear pore complex in mass, and to as much as 140 mol of p92 per mol of nuclear pore complex. Hydrodynamic analysis of the purified p92 suggested that the molecule is present as a monomer and that it is a rod-shaped molecule. The interaction of p92 and GNPs seemed to be hydrophobic and ionic. Based on these results, the participation of nuclear envelope p92 in protein nuclear transport is discussed.

Biochemical Characterization of Liver Microsomal, Golgi, Lysosomal, and Serum β-Glucuronidases in Dibuty1 Phosphate-Treated Rats1

Organophosphate compounds are known to cause the selective release of rat liver microsomal beta-glucuronidase into plasma. To investigate the alterations of molecular forms and oligosaccharide moieties of liver beta-glucuronidase in organophosphate compound-administered rats, beta-glucuronidase was isolated from microsomal, Golgi, lysosomal, and serum fractions. In SDS-polyacrylamide gel electrophoresis, a single polypeptide band was observed on gels in Golgi and serum beta-glucuronidases. This result indicated that Golgi and serum beta-glucuronidases of treated rats did not undergo post-translational proteolytic processing, in contrast to those in control rat livers. Biochemical characterization of the isolated beta-glucuronidases by employing lectin affinity chromatography revealed that interaction of serum and Golgi enzymes with Ricinus communis agglutinin- and wheat germ agglutinin-Sepharose was fairly strong, and that microsomal and lysosomal enzymes were poorly retained on those columns. These results suggested that the serum and Golgi beta-glucuronidases are sialoglycoproteins. A clearance study also showed that infused serum beta-glucuronidase was slowly cleared from plasma with a half-life of about 60 min, but the asialo-serum enzyme was rapidly cleared with a half-life of about 5 min. These results imply that microsomal beta-glucuronidase undergoes extensive modification of the oligosaccharide moieties by terminal glycosyltransferases at the trans Golgi when it is destined for secretion into serum in response to treatment with an organophosphate compound.

Purification and Characterization of Human Serum N-Acetylglucosamine-1-phosphodiester α-N-Acetylglucosaminidase

Many lysosomal enzymes are recognized and selected by a unique marker in the form of mannose 6-phosphate groups which are present exclusively on their N-linked oligosaccharides. Two enzymes act sequentially to catalyze the addition of mannose g-phosphate groups to the proteins: N-acetylglucosamine phosphotransferase (GlcNAc phosphotransferase) and N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (phosphodiester α-GlcNAcase). We report here the purification and partial characterization of phosphodiester α-GlcNAcase from human serum. The enzyme was purified over 600,000-fold by utilizing ammonium sulfate precipitation, fractionation on wheat germ agglutinin-Sepharose, Fe3+-chelating Sepharose, and Cu2+-chelating Sepharose, and renaturation from gel slices after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein observed after renaturation and subsequent SDS-PAGE and silver staining had an apparent molecular mass of 118 kDa, which is slightly smaller than bovine liver phosphodiester α-GlcNAcase (Mullis et al. (1994) J. Biol. Chem. 269, 1718-1726). Serum enzyme activity does not require Triton X-100 and is not stimulated by its addition. These results suggest that the enzyme found in serum represents a form secreted after proteolysis in the Golgi of the membrane-bound enzyme. The serum enzyme hydrolyzed UDP-GlcNAc to UDP and GlcNAc and hydrolyzed GlcNAc-P-ManαMe into αMeMan-P and GlcNAc. The enzyme had no hydrolyzing activity toward UDP-GalNAc, UDP-Glc, [6-3H] GlcNAcβ1-3Galβ1-4Glc, p-nitrophenyl-α-N-acetylglucosaminide, p-nitrophenyl-β-N-acetylglucosaminide, p-nitrophenyl-α-N-galactopyranoside, or p-nitrophenyl-β-N-galactopyranoside. The enzyme activity was strongly inhibited by UDP-GlcNAc and GlcNAc-1-phosphate, had a pH optimum between pH 6.0 and 7.0, and was inhibited by FeCl3, FeSO4, and CuSO4. The Km values for UDP-GlcNAc and GlcNAc-P-ManαMe were 0.94 and 0.45 mM, respectively. Over 77% of enzyme activity remained after incubation for 10 min at 70°C, demonstrating an unusual thermostability of the serum enzyme.

Identification of a receptor candidate for the carboxyl-terminal cell binding domain of thrombospondins.

The carboxyl-terminal cell binding domain (CBD) of thrombospondin-1 (TS1) contains two cell attachment peptides, 4N1s (RFYVVMWK) and 7N3 (FIRVVMY-EGKK), which share the sequence VVM. These peptides, and more soluble derivatives have been radiolabeled with 125I and used in conjunction with a variety of membrane impermeant cross-linking reagents to identify and characterize receptor candidates on several cell types. All of the VVM containing peptides tested with five different cross-linking reagents specifically labeled a 52-kDa protein, which was also affinity labeled by the recombinant TS1 CBD. After cross-linking peptide to K562 cells to block the 52-kDa protein, both cell adhesion to and affinity labeling by VVM peptides were inhibited in a concentration-dependent manner. Peptide labeling, like cell adhesion, was partially inhibited by heparin and stimulated by EDTA. The 52-kDa protein did not appear to contain sulfated glycan chains and was trypsin sensitive. It was recovered in a membrane fraction and was readily solubilized with Triton X-100 and X-114. Upon phase separation of the Triton X-114, the 52-kDa protein partitioned into the hydrophobic detergent phase. The detergent-solubilized receptor candidate bound selectively to wheat germ agglutinin-Sepharose, and after cell surface labeling with a membrane impermeant biotinylating reagent, bound to streptavidin-Sepharose. Furthermore, fluorescent beads covalently derivatized with peptide specifically decorated intact K562 cells. Thus the properties of the 52-kDa protein are consistent with those of a receptor for the CBD of TS1 and other TS isoforms.

Clathrin assembly protein AP-3 is phosphorylated and glycosylated on the 50-kDa structural domain.

AP-3 (AP180) in rat sympathetic neurons maintained in culture was analyzed by pulse-chase labeling with [35S]methionine to look for post-translational modifications. At early times, two lower molecular weight precursors of the mature species were detected. By 10 min, all of the AP-3 was found in the mature form which is stable for at least 9 h. We show here that at least one of these processing events is due to the addition of O-linked N-acetylglucosamine (GlcNAc) which is present on the mature form of the protein. Wheat germ agglutinin, a GlcNAc-specific probe, bound to AP-3 and the binding was blocked by excess GlcNAc but not by excess mannose. Purified AP-3, and AP-3 in coated vesicles derived from bovine brain, served as substrates for beta-D-galactosyltransferase which is specific for terminal GlcNAc residues. Analysis of the disaccharide released by beta-elimination indicated that single GlcNAc residues are attached to AP-3 through an O-glycosidic linkage to threonine or serine residues. In vivo 32P-labeled AP-3, the result of serine phosphorylation (Keen, J. H., and Black, M.M. (1986) J. Cell Biol. 102, 1325-1333), bound to wheat germ agglutinin-Sepharose indicating that phosphorylation and glycosylation can occur simultaneously on the same molecule. Both modifications have been mapped to the central 50-kDa structural domain that is responsible for the anomalous migration of AP-3. Consistent with localization to the nonclathrin binding domain, the O-GlcNAc modification does not play a discernible role in the interaction of AP-3 with clathrin.

Biosynthesis of the inositol trisphosphate receptor in WB rat liver epithelial cells.

We have studied the biosynthesis and turnover of the inositol 1,4,5-trisphosphate receptor (IP3R) in WB cells, a rat liver epithelial cell line. In detergent extracts of 35S-labeled WB cells, an affinity-purified antibody directed at the C terminus of the IP3R immunoprecipitated two labeled polypeptides, a major band at 233 kDa and a minor band at 222 kDa. The major band was shown to correspond to the IP3R by immunoblotting. The minor band was not a proteolytic clip of the IP3R because, unlike the IP3R, the 222-kDa band was not a glycoprotein or a substrate for protein kinase A and was not recognized by three different IP3R antibodies on immunoblots. The identity and function of this co-immunoprecipitating protein is unknown. The IP3R in WB cells was 5 kDa smaller than the rat cerebellar IP3R. Significant changes in the molecular weight of the IP3R were not observed in pulse-chase experiments, indicating that extensive proteolytic or carbohydrate processing events do not occur during biosynthesis of the receptor. From the analysis of such experiments in confluent WB cells, it was determined that the half-life of the IP3R protein was 11 h. Chromatography of the 35S-labeled extracts on Sephacryl S-400 columns revealed rapid oligomerization of newly synthesized protein with > 95% of newly synthesized protein forming tetramers during a short (10 min) pulse labeling period. However, significant amounts of mature IP3R were found as incompletely oligomerized forms. Mature IP3R was bound by concanavalin A-Sepharose beads, and binding was greatly reduced by endoglycosidase H treatment of the receptor. Endoglycosidase H sensitivity, absence of binding to wheat germ agglutinin-Sepharose, and insensitivity of biosynthesis or oligomerization to brefeldin A suggest that processing of the WB IP3R does not involve transit through the Golgi apparatus.

Purification of a 24 kD parasitism‐specific hemolymph protein from pharate pupae of the caribbean fruit fly, Anastrepha suspensa

We report purification of a 24 kD parasitism-specific protein (24 kD PSP) from pharate pupal hemolymph of the Caribbean fruit fly, Anastrepha suspensa, after parasitization by the braconid wasp, Diachasmimorpha (= Biosteres) longicaudata. We previously utilized isoelectric focusing (IEF) and two-dimensional (2-D) gel electrophoresis to demonstrate that the 24 kD PSP consists of two variants with pl 6.7 (more abundant) and pl 6.3. Purification of the more abundant 24 kD PSP variant was accomplished by Concanavalin A (Con A) sepharose B affinity chromatography followed by DEAE column chromatography. A second protocol, utilizing wheat germ agglutinin (WGA) sepharose 6MB affinity chromatography between the ConA and DEAE chromatographic steps, resulted in the purification of a partially deglycosylated form of the 24 kD PSP which retained its immunoreactivity with anti-PSP serum but which exhibited a greater relative migration in sodium dodecyl sulfate (SDS)-PAGE than the pl 6.7 24 kD PSP variant. For structural studies both 24 kD PSP variants were purified from whole hemolymph by flat bed IEF followed by SDS-PAGE. Peptide cleavage profiles in 1-D SDS-PAGE after treatment with BNPS-skatole, CNBr, and endproteinases Lys-C and Asp-N were identical for both 24 kD PSP variants. Primary N-terminus sequences of at least the first 20 amino acid residues of both variants were identical. A secondary sequence of five amino acids residues was detected in both variants at Thr, the seventh amino acid residue from the N-terminus of the primary sequence. These data indicate that both 24 kD PSPs are glycoforms of a branched, apparently homogeneous polypeptide.

Partial characterization of the rH1 sodium channel protein from rat heart using subtype-specific antibodies.

Three subtype-specific antisera were generated against peptides corresponding to portions of the amino terminus, interdomain 1-2, and carboxy terminus of the rH1 sodium channel primary sequence to confirm the expression of this protein in the adult rat heart and to determine selected biochemical properties of this protein that might contribute to its subtype-specific characteristics. All three antisera identify a 240-kD band on Western blots of partially purified cardiac membrane proteins and by immunoprecipitation of iodinated partially purified membrane proteins. Unlike other characterized mammalian sodium channels, no beta subunit is detected in association with the rH1 alpha subunit. The rH1 alpha subunit is a complex sialoglycoprotein as evidenced by its interaction with wheat germ agglutinin-Sepharose and by reduction in its apparent molecular weight after treatment with neuraminidase; deglycosylation with N-glycanase confirms that the rH1 protein contains significantly less carbohydrate than other sodium channel proteins characterized to date (5% versus 25% to 30%). Consistent with electrophysiological studies indicating a role of phosphorylation in channel regulation, the rH1 alpha subunit can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase A. The possible functional significance of these findings is discussed.

Affinity labeling of the mu opioid receptor in bovine striatal membranes with [3H]-14 beta-(bromoacetamido)-7,8-dihydromorphine.

[3H]-14 beta-(Bromoacetamido)-7,8-dihydromorphine ([3H]H2BAM) was synthesized and tested for its ability to selectively label mu opioid receptors in bovine striatal membranes. Incubating membranes with N-tosyl-L-phenylalanine chloromethyl ketone and dithiothreitol before the addition of [3H]H2BAM reduced nonspecific [3H]H2BAM binding so that [3H]H2BAM binding to opioid receptors was up to 70% of the total [3H]H2BAM binding and was dependent on [3H]H2BAM concentration, incubation time, and pH of the reaction. At pH 7.5, [3H]H2BAM bound selectively to the mu opioid receptor, but mainly noncovalently. After the initial binding of [3H]H2BAM to the receptor, membranes were washed and then incubated at 37 degrees C in 50 mM Tris-HCl, pH 8.5, for 3 h, a time that resulted in greater than 80% of the [3H]H2BAM associated with the receptor becoming covalently bound to the opioid receptor. The mu-selective peptide [D-Ala2,(Me)Phe4,Gly(ol)5]enkephalin inhibited [3H]H2BAM labeling of membranes, while delta- or kappa-selective compounds were ineffective. Both NaCl and the nonhydrolyzable guanine nucleotide analog guanylyl 5'-imidodiphosphate reduced the incorporation of [3H]H2BAM into membranes. When [3H]H2BAM-labeled striatal membranes were separated under reducing conditions on a sodium dodecyl sulfate-polyacrylamide gel, two proteins with molecular weights of 54,000 and 44,000 were specifically labeled. The 54-kDa protein was present in a greater amount than the 44-kDa protein. Both proteins bound to wheat germ agglutinin-Sepharose and concanavalin A-Sepharose, suggesting that both proteins contain multiple carbohydrate moieties. Despite the inclusion of protease inhibitors, the 44-kDa protein may be a proteolytic fragment of the 54-kDa protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial purification and characterization of the human placental serotonin transporter

The human placental serotonin transporter was solubilized from purified brush border membranes using digitonin as the solubilizing agent. The solubilizate was subjected to wheat germ agglutinin-Sepharose 6B column chromatography, Centricon-100 ultrafiltration and Sepharose 6B gel filtration to yield a partially purified preparation of the serotonin transporter. Specific binding of the high affinity ligand paroxetine was used to monitor the transporter during the solubilization and the purification steps. The enrichment of paroxetine binding in the final preparation was 51-fold compared to the intact brush border membranes, taking into account the inactivation that occurred during purification. The partially purified transporter exhibited paroxetine binding characteristics which were similar to those of the transporter in intact membranes. The transporter in the partially purified preparation bound paroxetine with a high affinity (dissociation constant, 0.21 nM). The binding was inhibitable by serotonin but not by other monoamines, dopamine and norepinephrine, nor by the serotonin precursor 5-hydroxytryptophan. The antidepressants, imipramine, fluoxetine and desipramine inhibited the binding with a rank order of potency of imipramine = fluoxetine > desipramine. The approximate molecular weight of the transporter was assessed by molecular sieve chromatography on Sepharose 6B and was found to be 300,000. When reconstituted into proteoliposomes, the partially purified transporter was able to catalyse NaCl-dependent serotonin transport in these proteoliposomes. The results of this study show that the human placental serotonin transporter can be solubilized, partially purified and reconstituted in a transport-competent form and, in addition, provide some insight into the protein nature of the transporter.

Purification and characterization of a nuclear pore glycoprotein complex containing p62.

It is known that nucleoporins, a family of glycoproteins with N-acetylglucosamine that are found in nuclear pore complexes, are essential for nuclear import and export. A major component of the family, p62, was purified from a salt extract of rat liver nuclear envelopes by wheat germ agglutinin-Sepharose affinity chromatography and DEAE-anion exchange HPLC. p62 was purified as a complex with two glycoproteins of 60 and 54 kDa. The presence of the complex was confirmed by gel filtration, glycerol density gradient centrifugation, and cross-linking experiments. The molecular ratio of the 62-, 60-, and 54-kDa components of the complex was estimated to be 1: 1.1 +/- 0.2: 1.7 +/- 0.3 from the intensity of Coomassie Blue staining of SDS-PAGE gels. The complex was stable against 1 M NaCl, 1% Triton X-100, and 2 M urea. The Stokes' radius and sedimentation coefficient of the complex are 8.0 nm and 6.7 S. The molecular mass and frictional ratio of the complex were estimated to be about 231 kDa and 2.0, respectively. p62 and p54 were acidic and neutral proteins, respectively, exhibiting charge heterogeneities, and p60 was assumed to be a basic protein. p60 tended to undergo proteolytic degradation to a 47-kDa fragment.

The lymphoma transmembrane glycoprotein GP85 (CD44) is a novel guanine nucleotide-binding protein which regulates GP85 (CD44)-ankyrin interaction.

In this study, we have used photoaffinity labeling by [32P]azido-GTP as well as [32P]ADP-ribosylation by pertussis toxin (PT) and cholera toxin (CT) to identify GTP-binding proteins associated with mouse T-lymphoma plasma membranes. Our results indicate that GP85 (CD44) can be photoaffinity labeled by [32P] azido-GTP and [32P]ADP-ribosylated by both PT and CT. Using purified GP85 (CD44) obtained by Triton X-100 extraction, wheat germ agglutinin-Sepharose, and anti-GP85 (CD44) antibody affinity chromatographies, we have further characterized GP85 (CD44) as a GTP-binding protein. GP85 (CD44) is found to bind guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) in a time- and dose-dependent manner with a dissociation constant of 0.83 nM. Importantly, GP85 (CD44) appears to display a GTPase activity which hydrolyzes [gamma-32P]GTP at a rate of 0.011 mol of Pi released/mol of GP85 (CD44)/min. This GTPase activity can be readily inhibited by PT- or CT-mediated ribosylation of GP85 (CD44). Most interestingly, GTP binding significantly enhances the interaction of purified GP85 (CD44) with ankyrin, whereas ADP-ribosylation of GP85 (CD44) by PT or CT inhibits the GTP-induced increase in ankyrin binding to GP85 (CD44). In addition to GP85 (CD44) being the first reported transmembrane GTP-binding protein, these results suggest that GTP plays an important role in promoting the interaction between GP85 (CD44) and its underlying membrane cytoskeleton through ankyrin.

Identification of molecular aggregates containing glycoproteins III, J, K (carboxypeptidase H), and H (Kex2-related proteases) in the soluble and membrane fractions of adrenal medullary chromaffin granules.

An investigation of the molecular properties of glycoprotein III has shown this to be a major component of molecular aggregates present in the membrane and soluble fractions of secretory vesicles from bovine adrenal medulla. These aggregates also contain components identified as glycoproteins H, J, and K which are molecular forms of Kex2-related proteases (glycoprotein H) and carboxypeptidase H (glycoprotein components J and K) and which have functions concerned with the processing of prohormones. A number of experiments indicated that these glycoproteins were associated. These components were coimmunoprecipitated from the soluble and membrane fractions of chromaffin granules. Purification of soluble glycoprotein III using wheat germ agglutinin-Sepharose resulted in the recovery of similar proportions of glycoproteins H, J, and K and gel filtration of the eluted material in combination with immunoprecipitation revealed the presence of heteroaggregates containing all of the glycoproteins. Similar results were obtained following octylglucoside solubilization of chromaffin granule membranes. Glycoprotein components III, H, J, and K were also found to have identical distributions following fractionation of chromaffin granule membranes with Triton X-114. It was concluded that the aggregates seen in the soluble fraction reflect an association of these components in the chromaffin granule membrane. This raises the possibility that these interactions are important for the targetting of these glycoproteins to secretory granules.