The sodium channel from rat brain. Separation and characterization of subunits

Procedures are described for separation of the alpha, beta 1, and beta 2 subunits of the voltage-sensitive sodium channel from rat brain by gel filtration in sodium dodecyl sulfate (SDS) before and after reduction of intersubunit disulfide bonds or by preparative SDS-gel electrophoresis. Partial proteolytic maps of the SDS-denatured subunits indicate that they are nonidentical polypeptides. They are all heavily glycosylated and contain complex carbohydrate chains that bind wheat germ agglutinin. The apparent molecular weights of the separated subunits were estimated by gradient SDS-gel electrophoresis, by Ferguson analysis of migration in SDS gels of fixed acrylamide concentration, or by gel filtration in SDS or guanidine hydrochloride. For the alpha subunit, SDS-gel electrophoresis under various conditions gives an average Mr of 260,000. Gel filtration methods give anomalously low values. Removal of carbohydrate by sequential treatment with neuraminidase and endoglycosidase F results in a sharp protein band with apparent Mr = 220,000, suggesting that 15% of the mass of the native alpha subunit is carbohydrate. Electrophoretic and gel filtration methods yield consistent molecular weight estimates for the beta subunits. The average values are: beta 1, Mr = 36,000, and beta 2, Mr = 33,000. Deglycosylation by treatment with endoglycosidase F, trifluoromethanesulfonic acid, or HF yields sharp protein bands with apparent Mr = 23,000 and 21,000 for the beta 1 and beta 2 subunits, respectively, suggesting that 36% of the mass of the native beta 1 and beta 2 subunits is carbohydrate.     

Multimeric structure of the tumor necrosis factor receptor of HeLa cells

The tumor necrosis factor (TNF) receptor of HeLa cells was solubilized in Triton X-100 and characterized by gel filtration, affinity labeling, and ligand blotting studies. Receptors solubilized with Triton X-100 eluted in gel filtration as a major peak of Mr = 330,000 and retained high affinity binding (KD = 0.25 nM). Affinity labeling of soluble receptor/125I-TNF complexes using the reversible, bifunctional bis[2-(succinimidooxycarbonyl-oxy)ethyl] sulfone resulted in the formation of cross-linked species of Mr = 310,000, 150,000-175,000, 95,000, and 75,000. The formation of these complexes was competitively inhibited by unlabeled TNF. Partial reversal of cross-linking in these complexes and their analysis by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved 125I-TNF dimers cleaved from the 95,000 band and 125I-TNF monomer cleaved from the 75,000 band, providing evidence for a Mr approximately 60,000 subunit. In addition, the 95,000 and 75,000 bands were resolved as components of larger complexes (Mr = 150,000-175,000), which presumably contain two receptor subunits. The Mr 95,000 and 75,000 bands were also released from the Mr 310,000 complex by reduction with dithiothreitol, suggesting a role for disulfide bond stabilization. To investigate the association of the putative receptor subunits, Triton X-100 extracts from HeLa membranes were fractionated by SDS-PAGE without reduction and transferred electrophoretically to nylon membranes for TNF binding assays. Only two bands of Mr = 60,000 and 70,000 specifically bound TNF, and higher Mr binding activity was not observed. These results indicate that TNF receptors in HeLa cells are high molecular weight complexes containing Mr = 60,000 and 70,000 subunits each capable of binding TNF and that the complexes are primarily stabilized by non-covalent, hydrophobic interactions.     

Isolation and characterization of a third proteoglycan (PG-Lt) from chick embryo cartilage which contains disulfide-bonded collagenous polypeptide

Chick embryo epiphyseal cartilage has been shown to contain three different proteoglycan species (PG-H, PG-Lb, and PG-Lt). This report is concerned with the purification and characterization of the third proteoglycan, PG-Lt. The proteoglycan can be separated from the other two by virtue of its low buoyant density in a CsCl density gradient and further purified by consecutive ion exchange and gel chromatography. The final preparation is composed of PG-Lt monomer and PG-Lt oligomer. The amino acid composition of PG-Lt is quite different from that of PG-H and PG-Lb and rather resembles that of collagens with respect to high content of glycine and high degrees of hydroxylation of proline and lysine. PG-Lt monomer is composed of disulfide-bonded subunits of Mr congruent to 120,000 and 190,000 as demonstrated by its gel electrophoretic behavior after reduction with 2-mercaptoethanol. The latter, but not the former, contains dermatan sulfate chains with glucuronic acid/iduronic acid residues and yields a protein-enriched core molecule of Mr congruent to 100,000 after digestion with chondroitinase ABC. Both of the protein subunits are completely digestible with bacterial collagenase. Immunofluorescence microscopic examination of cartilage tissues, using an antibody against PG-Lt, shows that this proteoglycan exists in both the cartilage matrix and perichondrial noncartilagenous region. When chondrocytes are plated onto tissue culture dishes, the antibody stains strands found on the cell surfaces and in the intercellular space of substrate-attached cell layers, suggesting that PG-Lt mediates cell-to-cell and cell-to-substrate contacts.     

Arylphorin from Manduca sexta: carbohydrate structure and immunological studies

The major hemolymph protein in the last larval stage of Manduca sexta is a hexameric glycoprotein, arylphorin (Mr = 450,000). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified arylphorin reveals the presence of two subunits, A1 and A2. Both subunits are glycosylated and have apparent Mr = 77,000 and 72,000, respectively. Pronase digestion of arylphorin yielded a single major glycopeptide. 250 MHz NMR spectroscopy of arylphorin glycopeptide revealed a Man9GlcNAc2 oligosaccharide structure similar to that observed in mammalian glycoproteins. Endoglycosidase-H treatment of arylphorin was employed to remove covalently linked carbohydrate residues. The carbohydrate removal lowered the apparent Mr of subunits A1 and A2 to 72,000 and 69,000, respectively, indicating that the difference in arylphorin subunit size is not due to levels of glycosylation. Immunoblotting experiments with anti-arylphorin antiserum and Bombyx mori storage proteins indicated cross reactivity with the corresponding arylphorin of this insect. Preparation of subunit A2 monospecific antibodies, followed by immunoblotting of arylphorin showed a close immunological relationship between subunits A1 and A2.     

Isolation and biochemical characterization of the subunits of the rabbit sperm acrosome stabilizing factor

The rabbit Acrosome Stabilizing Factor (ASF) is a glycoprotein synthesized in the corpus epididymis that demonstrates the ability to reversibly decapacitate sperm. Separation of the molecule into its individual subunits (92,000 Da and 38,000 Da) was accomplished via electroelution from polyacrylamide gels or via gel filtration on a Sephadex G-200 column in the presence of 0.1% sodium dodecyl sulfate. Column separation of the subunits revealed an entity of low molecular mass (500 daltons) associated with the ASF molecule. Amino acid compositional analysis of the subunits revealed the lack of cysteine and high glycine in the small subunit (38,000 Da) and high proline and glycine in the large subunit (92,000 Da). Lysine and aspartic acid were identified as the N-terminal amino acids for the large and small subunits, respectively. Identification of a 20 amino acid N-terminal sequence was accomplished for both of the subunits. Carbohydrate compositional analysis demonstrated that the small subunit contained N-asparagine-linked high mannose sugar chains while the large subunit contained N-asparagine-linked complex sugar chains. Endoglycosidase-H and N-Glycanase treatment of ASF indicated that the small subunit appears to contain four high mannose chains and the large subunit contains three complex chains.     

Characterization of the trimeric, self-recognizing Geodia cydonium lectin I

A D-galactose-specific lectin I was extracted from the sponge Geodia cydonium and purified by affinity chromatography. The molecular weight of lectin I as determined by high-pressure liquid gel chromatography, was found to be 36500 +/- 1300. Disc gel electrophoresis in the presence and in the absence of sodium dodecyl sulfate showed that lectin I is a trimer composed of three different subunits (Mr: 13800, 13000 and 12200); two of the three subunits are linked by one disulfide bond. Isoelectric focusing gave a pI of 5.6 for the native molecule and a pI of 4.4 and of 7.4 for the subunits. The three subunits carry carbohydrate side chains, composed of D-galactose (94%) and of arabinose (5%). Based on experiments with lectins, the terminal D-galactose residues are bound by beta 1 leads to 6 and/or beta 1 leads to 4 glycosidic linkages. The Geodia lectin I contains, besides two carbohydrate recognition sites, at least one receptor site for a second lectin I molecule.     

The active form of tumor necrosis factor is a trimer

Natural human and recombinant human and murine tumor necrosis factors (TNF) were fractionated by gel filtration chromatography on Sephadex G-75. The active form of TNF was identified by its inhibitory activity in receptor binding assays with HeLa cells and was eluted as a protein of Mr approximately 55,000. Radioiodinated human and murine TNF were fractionated by gel filtration into a major peak of Mr approximately 55,000, corresponding to a trimer, and a minor peak of Mr approximately 17,000, corresponding to a monomer. Binding assays showed that the timer was at least 8-fold more active than the monomer. The human TNF partially dissociated into monomers upon addition of the nonionic detergent Triton X-100. Isolated monomers showed low binding affinity (KD = 70 nM) and reduced cytotoxicity, whereas trimers showed high binding affinity (KD = 90 pM) and cytotoxicity. When 125I-TNF was bound to cells, no release of monomer was detectable, suggesting that the trimer could directly bind to cellular receptors without dissociating into subunits. Further evidence for such binding was obtained by cross-linking 125I-TNF trimers with bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone. These trimers were bound to HeLa cells, could be dissociated from cellular receptors, and elicited a cytotoxic response. These results show that trimers, whether native or cross-linked, bind to receptors and are the biologically active form of TNF.     

Isolation and characterization of the receptor on human neutrophils that mediates cellular adherence

The receptor on human neutrophils (polymorphonuclear leukocytes or PMN) that mediates cellular adherence has been purified from the peripheral blood PMN obtained from an individual with chronic myelogenous leukemia (CML). This receptor consists of two noncovalently associated subunits, designated alpha M (Mac-1 alpha, CD11b) (Mr = 170,000) and beta (Mac-1 beta, CDw18) (Mr = 100,000), respectively, which are identical on normal and CML PMN. The subunits were purified by monoclonal antibody 60.1-Sepharose (anti-alpha M) affinity chromatography and separated in 5-nmol quantities by high pressure liquid chromatography on a TSK-4000 gel filtration column. Subunits were characterized by amino acid composition, NH2-terminal amino acid sequence, and carbohydrate content. The NH2-terminal sequence of the human PMN alpha M subunit contains regions of homology with the human platelet glycoprotein IIb alpha. We conclude that nanomole amounts of individual alpha M and beta subunits of the receptor on human PMN that mediates cellular adherence can be isolated and separated using CML PMN.     

Molecular structure and immunochemical properties of highly purified hemagglutinin from Clostridium botulinum type A

A procedure for isolation of highly purified hemagglutinin from a toxic complex of culture filtrates of Cl. botulinum type A is described. This procedure includes precipitation with (NH4)2SO4, chromatography on Sephadex G-100, G-200 and DEAE-cellulose, specific adsorption on human erythrocytes and affinity chromatography. Using polyacrylamide gel electrophoresis, it was shown that hemagglutinin is a heteropolymeric protein consisting of a monomer (Mr 53 000) and a trimer (Mr 160 000). The monomer is made up of two subunits with Mr 13 000 and one subunit with Mr 27 000 covalently linked by SS-crosslinks. The number and nature of the SS-crosslinks and SH-groups in the protein molecule were determined and a hypothetical structural model of hemagglutinin was proposed. Using immunochemical analysis, it was shown that some (but not all) serological properties of the highly purified protein from Cl. botulinum type A and of its partially purified counterpart are similar to those of hemagglutinin from Cl. botulinum type B.     

Photoaffinity cross-linked A1 adenosine receptor-binding subunits. Homologous glycoprotein expression by different tissues

Mammalian A1 adenosine receptor-binding peptides can be visualized by covalently labeling them with the photoaffinity cross-linking ligand N6-2-(4-amino-3-[125I] iodophenyl)ethyladenosine followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography. The proteins comprising the A1 adenosine receptor-binding subunit of rat brain and fat migrate with Mr 38,000. In this study, the glycoproteins representing the radiolabeled A1 adenosine receptor-binding subunit expressed in each of these tissues (brain and fat) were compared through the use of peptide mapping and exo- and endoglycosidase treatments. Peptide mapping studies with several enzymes demonstrate that the protein component of the radiolabeled A1 adenosine receptor-binding subunit is conserved between different tissues. Both labeled receptor peptides demonstrate a sensitivity to neuraminidase as evidenced by increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the receptors contain complex-type carbohydrate chains. Insensitivity to alpha-mannosidase suggests a lack of high mannose-type carbohydrate chains. Deglycosylation of the labeled receptor-binding subunits with endoglycosidase F results in a single labeled polypeptide of Mr 32,000 for both systems. These data suggest that the A1 adenosine receptor-binding subunits expressed in the rat brain and fat are similar glycoproteins as evidenced by similar overall molecular weights, identical peptide maps, and equivalent responses to endo- and exoglycosidase treatment.     

Quantitation of the rabbit sperm acrosome stabilizing factor utilizing a sensitive immunoradiometric assay

Acrosome Stabilizing Factor (ASF) has been previously demonstrated to reversibly decapacitate sperm, presumably through preventing the acrosome reaction. ASF is a 259,000 Mr glycoprotein composed of two dissimilar subunits and is synthesized by the corpus epididymis. This report takes advantage of monoclonal antibodies directed toward different antigenic determinants of the ASF macromolecule to develop an immunoradiometric assay. Because the immunoradiometric assay sandwiches the native antigen between two antibodies (one iodinated), this type of assay circumvented the denaturation of ASF caused by iodination. Conditions established for maximal sensitivity with reasonable efficiency were determined to be a 12-24-h equilibrium incubation of ASF with the first bound antibody followed by a carefully timed 3-h incubation with the iodinated second antibody. These conditions provide an assay for ASF that is sensitive to 200 pg/ml and covers a concentration range of more than 2.5 orders of magnitude. The ejaculates from nine male rabbits were evaluated semiweekly over a two-month period for ASF concentration, volume, and sperm number. The average ASF concentration was 265 micrograms/ml of ejaculate and while relatively consistent for any one buck, varied widely between bucks. This variation may relate to the previously reported differences of capacitation of sperm from different bucks.     

Comparison between the monomeric and binary-complex forms of procarboxypeptidase A from whole pig pancreas.

Two different forms of procarboxypeptidase A (I and II) were obtained from pig pancreas extracts. The Mr values, the pattern found on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, and the sedimentation coefficients indicate that form I is a binary complex formed by two different subunits, whereas form II is a monomer. The carboxypeptidase A-precursor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx.sor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx.sor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx. 12500) and, in the case of the binary complex, the activation with trypsin follows a rather complex pattern, suggesting that the accompanying subunit of form I might play a modulating role in the activation process. Although the appearance of enzymic activity is rather slow, a protein with an Mr equivalent to that of active carboxypeptidase A is found very early in the activation process. Both zymogens are glycoproteins (so far no carbohydrate has been reported in any procarboxypeptidase A) and both contain two strongly bound Zn2+ ions/molecule. Other chemical and physical properties were also determined.     

Electrophoretically monodisperse cytochrome c oxidases

A discontinuous gradient polyacrylamide gel electrophoresis under nondenaturing conditions has been used to demonstrate monodispersity of procaryotic and eucaryotic cytochrome c oxidase preparations. Alkaline treated bovine enzyme which contains nine subunits as analysed by subsequent discontinuous SDS-polyacrylamide gel electrophoresis is a monodisperse dimer in 0.1% Triton X-100 and a monomer in 0.1% dodecyl maltoside. The Mr-values corrected for bound detergent are 286,000 in Triton X-100 and 152,000 in dodecyl maltoside respectively. The two-subunit bacterial cytochrome c oxidase of Paracoccus denitrificans is proved to be a monomer with a corrected Mr of 76,000 in both nonionic detergents Triton X-100 and dodecyl maltoside.     

Isolation and partial amino acid sequence of three subunit species of porcine spleen ferritin: evidence of multiple H subunits

A partial amino acid sequence for three different subunits of the iron storage protein, ferritin, has been determined. Ferritin (Mr approximately 480,000) was isolated from porcine spleen and dissociated into its component subunits (Mr approximately 20,000). The subunits, in turn, were separated into three fractions by reversed-phase HPLC. The fractions appeared to be of equal size by sedimentation velocity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and size-exclusion chromatography in 6 M guanidinium chloride. All three fractions were shown to be monomeric and to have no covalently attached carbohydrate (J. F. Collawn et al. (1984) Arch. Biochem. Biophys. 233, 260-266). Determination of the amino acid sequence of the C-terminal 70-80 residues from each of the fractions demonstrated three different sequences. Comparison with human liver H and L subunit sequences indicates that two of the porcine ferritin subunits are H-type subunits and one is an L-type subunit. Application of the Chou-Fasman algorithm on the three partial sequences suggests that these respective regions from each of the three subunits would probably adopt the same conformation.     

Subunit composition of oligomeric human von Willebrand factor

The oligomerization of human endothelial cell-synthesized von Willebrand factor (vWf) has been studied by gel chromatography in columns of Sephacryl S-500 and by discontinuous agarose gel electrophoresis. A quantitative recovery of high Mr vWf oligomers has been obtained after binding to a monoclonal anti-vWf-Sepharose adduct. This reagent has been used to analyze gel filtration chromatographic elution profiles of [35S]methionine-labeled culture medium and cell lysate. It was determined that high Mr oligomers are present in endothelial cell lysates as well as in the medium overlying these cells and are composed of Mr 225,000 subunits. When vWf oligomers were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of a reducing agent, the Mr 240,000 subunit (provWf) was not observed to oligomerize beyond the dimer stage to a significant degree. Therefore, vWf oligomerization appears to be facilitated by conversion of provWf subunits to mature vWf subunits, most likely by proteolytic removal of sequences unique to the intracellular precursor.     

Purification and characterization of an estrogen-inducible membrane glycoprotein. Evidence that it is a transferrin receptor

A number of N-linked membrane glycoproteins are induced during chick oviduct differentiation. We have purified a major estrogen-inducible glycoprotein (Mr = 91,000) to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of partial NH2-terminal sequence data with membrane glycoproteins having similar Mr showed a limited homology with human and murine transferrin receptors. We observed that oviduct membranes contain estrogen-inducible transferrin receptor activity (Kd = 2-8 x 10(-8) M). Analytical purification of the putative receptor on an ovotransferrin-Affi-Gel affinity column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis reveals a protein of Mr, 180,000, which contains two disulfide-linked subunits of Mr 91,000. The receptor reacts very strongly with antibodies prepared against the 91-kDa glycoprotein on Western blots. Western blot analysis confirms that the 91-kDa glycoprotein is induced by estrogen. The protein has 2% total carbohydrate with Man, GlcNAc, Gal, GalNAc, and NeuAc in a molar ratio of 6:4:2:1:1. The protein contains at least one O-linked moiety. Analysis of the O-linked moiety by glycosidase digestions and gel filtration indicates there are sialo tetra- and trisaccharides and a neutral disaccharide(s). Labeled N-linked glycopeptides were prepared by pronase digestion, beta-elimination, and 3H-acetylation. The N-linked oligosaccharides include high mannose and complex neutral nonbisected biantennary types in an approximate ratio of 3:1 as determined by serial lectin affinity chromatography.     

The microheterogeneity of androgen-binding protein in rat serum and epididymis is due to differences in glycosylation of their subunits

The microheterogeneity of androgen-binding protein (ABP) from rat serum and epididymis was examined by subjecting purified native or deglycosylated preparations to analysis by one- or two-dimensional polyacrylamide gel electrophoresis (PAGE) followed by electrophoretic transfer to nitrocellulose and immunochemical localization. Analysis of native ABP by one-dimensional sodium dodecyl sulfate-PAGE confirmed earlier observations that it is composed of subunits and that the subunits of serum ABP had higher apparent molecular weights than those of epididymal ABP. Treatment with neuraminidase, N-glycanase, or O-glycanase, alone or in combination, resulted in decreases in the apparent molecular weight of the subunits. These analyses indicated that terminal sialic acid residues and Asn-linked oligosaccharides were present on both subunits of ABP from the two sources. The fact that the greatest reduction in the Mr of the heavy subunit occurred following treatment with all three enzymes provides evidence that O-linked sugars are present on it. While enzyme treatment did not result in the appearance of a single subunit, chemical deglycosylation did (Mr 39,600). The carbohydrate composition of the heavy and light subunits of intact serum and epididymal ABP was 22 and 9% and 19 and 8%, respectively. Analysis by two-dimensional PAGE indicated that both subunits of the ABPs were composed of isoelectric variants. Although ABP from the two sources had several variants in common, differences were also observed. Treatment of the ABPs with the enzymes resulted in a shift of the pI values to a more basic pH range, indicating that carbohydrate removal also removed charged moieties. The most dramatic shift in the pI values of the isoforms occurred when O-glycanase was present in the enzyme mixture, providing further evidence for the presence of O-linked oligosaccharides on ABP. Isoelectric variants were present even after chemical deglycosylation of ABP.     

Comparative cross-linking activities of lactose-specific plant and animal lectins and a natural lactose-binding immunoglobulin G fraction from human serum with asialofetuin

Plant and animal lectins bind and cross-link certain multiantennaryoligosaccharides, glycopeptides, and glycoproteins. This canlead to the formation of homogeneous cross-linked complexes,which may differ in their stoichiometry depending on the natureof the sugar receptor involved. As a precisely defined ligand,we have employed bovine asialofetuin (ASF), a glycoprotein thatpossesses three asparagine-linked triantennary complex carbohydratechains with terminal LacNAc residues. In the present study,we have compared the carbohydrate cross-linking properties oftwo Lac-specific plant lectins, an animal lectin and a naturallyoccurring Lac-binding polyclonal iminunoglobulin G subfractionfrom human serum with the ligand. Quantitative precipitationstudies of the Lac-specific plant lectins, Viscum album agglutininand Ricinus communis agglutinin, and the Lac-specific 16 kDadimenc galectin from chicken liver demonstrate that these lectinsform specific, stoichiometric cross-linked complexes with ASF.At low concentrations of ASF, 1:9 ASF/lectin (monomer) complexesformed with both plant lectins and the chicken lectin. Withincreasing concentrations of ASF, 1:3 ASF/lectin (monomer) complexesformed with the lectins irrespective of their source or size.The naturally occurring polyclonal antibodies, however, revealeda different cross-linking behavior. They show the formationof 1:3 ASF/antibody (per Fab moiety) cross-linked complexesat all concentrations of ASF. These studies demonstrate thatLac-specific plant and animal lectins as well as the Lac-bindingimmunoglobulin subfraction form specific stoichiometric cross-linkedcomplexes with ASF. These results are discussed in terms ofthe structure-function properties of multivalent lectins andantibodies. asialofetuin Lac-specific lectins immunoglobulin subfraction     

Deglycosylation and proteolysis of photolabeled D2 dopamine receptors of the porcine anterior pituitary

Dopamine D2 receptor binding subunits of the porcine anterior pituitary were visualized by autoradiography following photoaffinity labeling with [125I]N-azidophenethylspiperone and sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The ligand binding subunit comprising the pituitary D2 dopamine receptor migrated as two distinct bands of apparent Mr approximately equal to 150,000 and 118,000, substantially higher than neuronal D2 receptor subunits from porcine or canine brain. The glycoprotein nature of pituitary D2 receptor binding subunits was investigated by the use of exo- and endo-glycosidase treatments and peptide mapping experiments. Photoaffinity labeled polypeptides of the anterior pituitary were susceptible to both neuraminidase and alpha-mannosidase digestion as indexed by their increased electrophoretic mobility on sodium dodecyl-sulfate polyacrylamide gels, and suggests the presence of both complex type and terminal mannose carbohydrate residues. Moreover, the additive effects of sequential treatment with these enzymes suggests that both types of carbohydrate chains are present on each receptor peptide. N-linked deglycosylation of pituitary D2 photolabeled receptors with glycopeptidase-F produced a further increase in the mobility of the labeled protein to apparent Mr approximately equal to 44,000, similar to that of deglycosylated D2 binding subunits of porcine and canine brain. Peptide mapping experiments following limited proteolysis with Staphylococcus aureus V8 proteinase and papain demonstrated that deglycosylated D2 dopamine receptors (Mr = 44,000), in different tissues and species, were homologous. Taken together, these data suggest that despite the differences in the overall molecular weight and tissue specific glycosylation pattern of pituitary D2 dopamine receptors, the primary structure of mammalian D2 receptors appears to be conserved.     

Biosynthesis and glycosylation of the insulin receptor. Evidence for a single polypeptide precursor of the two major subunits

The biosynthesis and carbohydrate processing of the insulin receptor were studied in cultured human lymphocytes by means of metabolic and cell surface labeling, immunoprecipitation with anti-receptor autoantibodies, and analysis on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions. In addition to the two major subunits of Mr = 135,000 and Mr = 95,000, two higher molecular weight bands were detected of Mr = 210,000 and Mr = 190,000. The Mr = 210,000 band and the two major subunits were labeled by [3H]mannose, [3H]glucosamine, [3H]galactose, and [3H]fucose, and were bound by immobilized lentil, wheat germ, and ricin I lectins. On the other hand, the Mr = 190,000 band was labeled only by [3H]mannose and [3H]glucosamine and was bound only by lentil lectin. All four components could be labeled with [35S] methionine; however, in contrast with the other three polypeptides, the Mr = 190,000 band was not labeled by cell surface iodination with lactoperoxidase, suggesting that it is not exposed at the outer surface of the plasma membrane. Pulse-chase studies with [3H]mannose showed that the Mr = 190,000 was the earliest labeled component of the receptor; radioactivity in this band reached a maximum 1 h after the pulse, clearly preceded the appearance of the other components, and had a very brief half-life (t1/2 = 2.5 h). The Mr = 210,000, Mr = 135,000, and Mr = 95,000 bands were next in appearance and reached a maximum 6 h in the chase period. Monensin, an ionophore which interferes with maturation of some proteins, blocked both the disappearance of the Mr = 190,000 protein and the appearance of the Mr = 135,000 and Mr = 95,000 subunits. The mannose incorporated in the Mr = 190,000 component was fully sensitive to treatment with endoglycosidase H while that in the Mr = 210,000 band and the two major subunits was only partially sensitive. Tryptic fingerprints of the 125I-labeled Mr = 210,000 band suggested that this component contains peptides of both the Mr = 135,000 and Mr = 95,000 subunits. In conclusion, the Mr = 190,000 component appears to represent the high mannose precursor form of the insulin receptor that undergoes carbohydrate processing and proteolytic cleavage to generate the two major subunits. In addition, the Mr = 210,000 band is probably the fully glycosylated form of the precursor that escapes cleavage and is expressed in the plasma membrane.