Murine cell surface glycoproteins. Purification of the polymorphic Pgp-1 antigen and analysis of its expression on macrophages and other myeloid cells

We previously reported the initial characterization of a polymorphic major cell surface glycoprotein of about 80,000 daltons from mouse embryo 3T3 cells. This glycoprotein has now been purified 1800-fold to apparent homogeneity by monoclonal antibody affinity chromatography. The purified molecule retained the total antigenic activity of the cell, as determined by antibody binding assays. The quantity of the glycoprotein, 0.06% of the total protein of the crude cell extract, confirmed its presence as a major constituent of the cell plasma membrane. The monoclonal antibody was also used to detect related antigens in cells and tissues of C57BL/6J mice. The antigen was present in high concentration in macrophages and subpopulations of bone marrow and blood polymorphonuclear cells. Much lower concentrations of antigen were detected in spleen cells, thymocytes, and extracts of solid tissues. The apparent Mr of the target antigen of myeloid cells was 92,000. This molecule was a major surface constituent of myeloid cells with 10(6) antibody binding sites per cell containing 10% of total 125I incorporated by the lactoperoxidase procedure. The macrophage glycoprotein labeled on the cell surface with 125I was highly sensitive to trypsin, yielding an antigenically active soluble glycopolypeptide of about 65,000 daltons, that contained all of the incorporated 125I. A similar 65,000-dalton glycopeptide was released from 3T3 cells by trypsin cleavage. These data indicate that a major cell surface constituent of mouse myeloid cells is a 92,000-dalton glycoprotein closely related to the 80,000-dalton glycoprotein of mouse embryo 3T3 cells.     

Platelet-collagen adhesion: inhibition by a monoclonal antibody that binds glycoprotein IIb

To identify platelet surface structures involved in adhesion to collagen, the effect of 16 murine antiplatelet membrane hybridoma antibodies were tested in a defined, in vitro assay. Four of these antibodies inhibited platelet-collagen adhesion and reacted with a polypeptide with Mr approximately 125,000, as determined by immunoblots after gel electrophoresis under reducing conditions. Through detailed studies with one of these antibodies, the monoclonal antibody PMI-1, the relevant antigen was identified as platelet glycoprotein IIb alpha, based upon (a) co-migration with this glycoprotein in two-dimensional gel electrophoresis and (b) co-purification by immunoaffinity chromatography with a protein with apparent Mr identical to that of glycoprotein III, under conditions in which glycoproteins IIb and III form a complex. Univalent antibody fragments prepared from monoclonal antibody PMI-1 inhibited greater than 80% of platelet-collagen adhesion, and inhibition was completely blocked by the immunopurified antigen. These results indicate that glycoprotein IIb participates in some aspect of platelet-collagen adhesion. In contrast, the purified antigen only partially neutralized a polyclonal antiserum that blocked platelet-collagen adhesion, to a maximum of approximately 25%, at saturating antigen concentrations. Thus, by these immunological criteria, glycoprotein IIb is not the only molecule involved in this process.     

Expression of platelet glycoprotein Ib alpha in HEL cells

We have previously shown that platelet glycoprotein Ib is expressed in a minority of cells of the human leukemic cell line HEL (Tabilio, A., Rosa, J. P., Testa, U., Kieffer, N., Nurden, A. T., Del Canizo, M. C., Breton-Gorius, J., and Vainchenker, W. (1984) EMBO J. 3, 453-459). In this report, we have selected a stable HEL subclone with increased expression of glycoprotein (GP) Ib as assessed by 6 different monoclonal antibodies in order to investigate the biochemical characteristics of this glycoprotein. A single polypeptide chain of apparent Mr = 60,000 was precipitated under reducing and nonreducing conditions by a specific polyclonal anti-platelet glycocalicin antibody and two anti-GPIb alpha monoclonal antibodies (AN51 and AP1), both from surface-labeled and metabolically labeled HEL cells. We were unable to demonstrate the presence of a polypeptide corresponding to the beta subunit of GPIb or GPIX which is closely associated with GPIb. Competitive immunoprecipitation performed in the presence of an excess amount of cold platelet glycocalicin completely displaced the Mr = 60,000 polypeptide. Synthesis of N-linked oligosaccharide chains on this Mr = 60,000 polypeptide was inhibited by the antibiotic tunicamycin, and a shift of the apparent Mr from 60,000 to 48,000 was observed. O-Linked oligosaccharide chains identical to platelet GPIb hexasaccharides were deficient or incomplete since no peanut agglutinin binding to the Mr = 60,000 polypeptide was observed after neuraminidase treatment of HEL cells. Thus, our results provide evidence that the Mr = 60,000 polypeptide expressed on the surface membrane of HEL cells is closely related to platelet GPIb and corresponds to an incompletely or abnormally O-glycosylated GPIb alpha subunit.     

Lysosome-associated membrane proteins: characterization of LAMP-1 of macrophage P388 and mouse embryo 3T3 cultured cells

Lysosome-associated membrane protein (LAMP)-1, a major glycoprotein of mouse embryo 3T3 cells and specifically associated with the lysosomal membrane, has been identified in P388 macrophage cells and compared with the homologous glycoprotein of NIH 3T3 cells. Immunofluorescence microscopy with anit-LAMP-1 monoclonal antibodies shows that the antigen was distributed throughout P388 cells including the ruffled edges or pseudopodia, identical to the pattern of acridine orange accumulation. LAMP-1 was purified from P388 cells by affinity chromatography with 1D4B monoclonal antibody, yielding a homogeneous glycoprotein comprising 0.1% of the total detergent-extracted cell protein. The apparent mass of P388 LAMP-1 was 130,000 to 150,000 compared to the 3T3 glycoprotein of 105,000 to 115,000. Analysis of tryptic peptides indicated that the two purified glycoproteins were highly homologous. Protein synthesis was analyzed in a variety of cell lines by pulse-chase labeling with [35S]methionine; in every case, LAMP-1 was synthesized as a precursor of apparent Mr 92,000, and then converted to heterogeneous mature forms differing in average Mr from 110,000 to 140,000. The basis for these apparent differences in mass was examined by studies of the biosynthesis and oligosaccharide composition of the glycoprotein. Core polypeptides of 45,000 Da were obtained from both HaNIH and P388 cells by treating immunoprecipitates of [35S]methionine pulse-labeled molecules with endoglycosidase H. Cells treated with monensin contained heterogeneous molecules of 80,000 to 85,000 Da. Isoelectric heterogeneity of mature LAMP-1 was markedly reduced by treatment with neuraminidase whereas there was little effect on the apparent molecular weight of the molecules or the differences between the various cell lines. beta-D-Xyloside inhibition of glycosaminoglycan synthesis had little effect on the apparent mass of LAMP-1.     

Purification and characterization of the human brain insulin receptor

The insulin receptor from human brain cortex was purified by a combination monoclonal antibody affinity column and a wheat germ agglutinin column. This purified receptor preparation exhibited major protein bands of apparent Mr = 135,000 and 95,000, molecular weights comparable to those for the alpha and beta subunits of the purified human placental and rat liver receptors. A minor protein band of apparent Mr = 120,000 was also observed in the brain receptor preparation. Crosslinking of 125I-insulin to all three receptor preparations was found to preferentially label a protein of apparent Mr = 135,000. In contrast, cross-linking of 125I-labeled insulin-like growth factor I to the brain preparation preferentially labeled the protein of apparent Mr = 120,000. The purified brain insulin receptor was found to be identical with the placental insulin receptor in the amount of neuraminidase-sensitive sialic acid and reaction with three monoclonal antibodies to the beta subunit of the placental receptor. In contrast, a monoclonal antibody to the insulin binding site recognized the placental receptor approximately 300 times better than the brain receptor. These results indicate that the brain insulin receptor differs from the receptor in other tissues and suggests that this difference is not simply due to the amount of sialic acid on the receptor.     

Transforming growth factor beta 1 treatment of AKR-2B cells is coupled through a pertussis-toxin-sensitive G-protein(s).

Spermidine synthase was purified to apparent homogeneity from human spleens (8700-fold) by affinity chromatography. The native enzyme was composed of two subunits of identical Mr (35,000) and showed an apparent Mr of 62,000 in pore-gradient gel electrophoresis. Its pI was 5.1, Spermine synthase was purified to apparent homogeneity from placenta (5300-fold) and from kidney (4600-fold). The native enzyme was composed of two subunits of identical Mr (45,000) and showed an apparent Mr of 78,000 in pore-gradient gel electrophoresis. In isoelectric focusing it revealed two bands, with pI values of 4.9 and 5.0. Both synthases were present in all human tissues studied, but revealed a clear tissue-specific pattern. Mouse antisera against spermidine synthase revealed only one band, of Mr 35,000, in all purified enzyme preparations and in crude human tissue extracts in immunoblotting. Antisera against spermine synthase showed an immunoreactive band corresponding to the Mr of the subunit of spermine synthase. These antisera did not indicate any cross-reactivity in immunoblotting. Thus spermine synthase and spermidine synthase do not share homologous antigenic sites and are totally different proteins.     

Isolation and characterization of a human T lymphocyte-associated glycoprotein (gp40)

The biosynthetic and structural characteristics of the human thymocyte/T cell antigen defined by the monoclonal antibody WT1 have been studied. WT1 identified a monomeric cell surface glycoprotein of Mr = 40,000 ( gp40 ). Cross-absorption experiments and two-dimensional gel analyses indicate that WT1 and another monoclonal antibody, 3A1, react with the same structure. This glycoprotein was asymmetrically inserted into the rough endoplasmic reticulum as a transmembrane structure. At this stage, the polypeptide chain possessed two N-linked, "high-mannose" type glycans; these were subsequently processed into endo-H-insensitive, complex oligosaccharides during intracellular transport to the cell surface. Inhibition of N-linked glycosylation with tunicamycin failed to block the processing of the nonglycosylated Mr = 29,000 polypeptide to a glycoprotein of Mr = 33,000. Cleavage of the mature Mr = 40,000 form with endo-F yielded a similar Mr = 33,000 product. The kinetics of synthesis of the Mr = 33,000 intermediate in conjunction with gal-NAc oligosaccharidase digestion indicated the presence of O-linked glycans in the mature cell surface WT1 antigen. The fully processed cell surface form of the polypeptide also contains covalently associated fatty acid, and was labeled by 32P phosphate, the predominantly labeled phosphoamino acid being phosphoserine. We also demonstrate biochemically that the reactivity of WT1 with cells from a few patients with acute myeloid leukemia reflects genuine expression of the gp40 structure on myeloid cells.     

Monoclonal antibody to a diagnostic Mr 24,000 antigen of Gnathostoma spinigerum.

Crude water extract (CA) was prepared from the advanced third-stage larvae of Gnathostoma spinigerum collected from livers of naturally infected eels. The extract was partially purified by chromatofocussing column chromatography and the fraction which contained specific antigen of G. spinigerum which was an Mr 24,000 glycoprotein was used to immunize five Balb/c mice for preparing immune splenocytes. Spleen cells were collected from one mouse which showed high serum titre by indirect enzyme-linked immunosorbent assay and contained specific antibody to the Mr 24,000 antigen as checked by Western blot analysis. The spleen cells were fused with myeloma Sp2/0 cells at a ratio of 10 spleen cells per one myeloma cell using polyethylene glycol 3350 as a fusogen. Thirteen out of 174 growing polyclones (7.5%) produced antibodies to the partially purified CA fraction. Among them, two polyclones produced antibody directed to the Mr 24,000 protein. These two polyclones were subjected to monocloning by limiting dilution and a monoclone GN6/24 which produced monoclonal antibody to the specific Mr 24,000 protein of G. spinigerum was obtained.     

Production of a monoclonal antibody directed against rat liver glutathione-insulin transhydrogenase

A hybridoma cell line secreting monoclonal antibody specific for glutathione-insulin transhydrogenase has been produced by fusing mouse myeloma cells with spleen cells from mice immunized to purified rat liver glutathione-insulin transhydrogenase. The secreted antibody isotypes were found to be: Ig gamma 1 heavy chains and kappa light chains. This monoclonal antibody has been used to screen glutathione-insulin transhydrogenase in various rat tissue extracts (liver, fat, heart, testis, spleen, lung and kidney) following separation on NaDodSO4/urea polyacrylamide disc-gel electrophoresis and electrophoretic transfer to nitrocellulose. Screening with the monoclonal antibody showed the presence of one immunoreactive protein band equal in molecular weight to that of purified rat liver GIT (Mr 53,000) in extracts of all tissues studied and a second immunoreactive protein band of lower molecular weight (Mr 49,000) in spleen and lung tissue extracts. Separation of these two proteins by HPLC using a TSK-DEAE column demonstrated that both proteins exhibit insulin degrading activity. These data indicate that GIT may occur in multiple forms in some tissues.     

The hyaluronate receptor is identical to a glycoprotein of Mr 85,000 (gp85) as shown by a monoclonal antibody that interferes with binding activity

The cell surface receptor for hyaluronate is an integral membrane glycoprotein of Mr 85,000 (Underhill, C. B., Thurn, A. L., and Lacy, B. E. (1985) J. Biol. Chem. 260, 8128-8133), which appears to be associated with actin filaments. This protein is similar in many respects to another protein, termed gp85, which was originally identified by Tarone, G., Ferracini, R., Galeto, G., and Comoglio, P. (1984) J. Cell Biol. 99, 512-519), using a monoclonal antibody designated as K-3. The gp85 is also a membrane glycoprotein of Mr 85,000 which is associated with the cytoskeleton. Indeed, immunohistological staining has shown that it is distributed in patches along stress fibers of spread baby hamster kidney (BHK) cells. In the present study, we have used the K-3 monoclonal antibody to determine whether gp85 is identical to the hyaluronate receptor. Initial studies showed that the K-3 antibody reacted with material at Mr 85,000 on immunoblots of a purified preparation of the hyaluronate receptor. In addition, the K-3 antibody specifically blocked the binding of [3H]hyaluronate to detergent extracts of the receptor from both BHK and polyoma virus transformed baby hamster kidney (PY-BHK) cells, as well as to intact PY-BHK cells. These results indicate that the K-3 antibody is directed against the hyaluronate receptor, which therefore must be identical to gp85. The K-3 antibody was then used to determine the relative number of hyaluronate receptors associated with parent (BHK) and transformed (PY-BHK) cells. Using an enzyme-linked assay, we found that parent cells had a substantially greater number of receptors than their transformed counterparts. These results were consistent with those obtained when detergent extracts of cells were directly assayed for [3H]hyaluronate binding activity.     

A spectrin-like protein in retinal rod outer segments

Biochemical and immunochemical studies indicate that rod outer segments (ROS) of bovine photoreceptor cells contain a Mr 240,000 polypeptide related to the alpha-subunit of red blood cell (RBC) spectrin. With the use of sodium dodecyl sulfate gel electrophoresis in conjunction with the immunoblotting technique, monoclonal antibody 4B2 was found to bind to a Mr 240,000 polypeptide in ROS that is distinct from the prominent Mr 220,000 concanavalin A binding glycoprotein. The Mr 240,000 polypeptide is highly susceptible to degradation by endogenous proteases. It does not appear to be an integral membrane protein but is tightly membrane associated since it can be partially extracted from ROS membranes with urea in the absence of detergent. The 4B2 antibody cross-reacted with RBC ghosts and bovine brain microsomal membranes. Radioimmune assays and immunoblotting analysis of purified bovine RBC spectrin further revealed that the 4B2 antibody predominantly labeled the alpha-chain of RBC spectrin having an apparent molecular weight of 240,000. Polyclonal anti-spectrin antibody that bound to both the alpha- and beta-chain of RBC spectrin predominantly labeled a Mr 240,000 polypeptide of ROS membranes. Two faintly labeled bands in the molecular weight range of 210,000-220,000 were also observed. These components may represent variants of the beta-chain of spectrin that are weakly cross-reacting or present in smaller quantities than the alpha-chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum angiotensin-converting enzyme. Isolation and relationship to the pulmonary enzyme.

Angiotensin-converting enzyme from rabbit serum was purified almost 60,000-fold to apparent homogeneity by a procedure exploiting its affinity for antibodies prepared against the enzyme from lung. The pure serum and pulmonary enzymes exhibited identical behavior during gel filtration, sucrose gradient centrifugation, and disc gel electrophoresis in the reduced, denatured state. Their catalytic properties with hippurylhistidylleucine, angiotensin I, and bradykinin as substrates were similar and their reactivity with antilung enzyme antibody was indistinguishable as examined by immunodiffusion, inhibition dose-response curves, and radioimmunoassay. Their content of fucose, mannose, galactose, and N-acetylglucosamine was also comparable; however, N-acetylneuraminic acid was much more abundant in the serum glycoprotein. This difference may reflect selective removal of sialic acid-deficient enzyme molecules from the circulation by the hepatic lectin which has been postulated to initiate the catabolic phase for plasma glycoproteins (Ashwell, G., and Morell, A.G. (1974) Adv. Enzymol. Relat. Areas Mol. Biol. 41, 91-128).     

One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes

Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fy(a)/Fy(b) blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy protein expressed in K562 cells has three N-linked oligosaccharide chains, which are mainly of complex-type. Here we report a one-step purification method of Duffy protein from human erythrocytes. DARC was extracted from erythrocyte membranes in the presence of 1% n-dodecyl-β-D-maltoside (DDM) and 0.05% cholesteryl hemisuccinate (CHS) and purified by affinity chromatography using immobilized anti-Fy6 2C3 mouse monoclonal antibody. Duffy glycoprotein was eluted from the column with synthetic DFEDVWN peptide containing epitope for 2C3 monoclonal antibody. In this single-step immunoaffinity purification method we obtained highly purified DARC, which migrates in SDS-polyacrylamide gel as a major diffuse band corresponding to a molecular mass of 40-47?kDa. In ELISA purified Duffy glycoprotein binds anti-Duffy antibodies recognizing epitopes located on distinct regions of the molecule. Results of circular dichroism measurement indicate that purified DARC has a high content of α-helical secondary structure typical for chemokine receptors. Analysis of DARC glycans performed by means of lectin blotting and glycosidase digestion suggests that native Duffy N-glycans are mostly triantennary complex-type, terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core.     

Purification of an 86-70 kDa nuclear DNA-associated protein complex

In the course of studies on nucleolar antigens, monoclonal antibodies were developed, one of which recognized an 86 kDa antigen as shown by analysis of nuclear extracts from HeLa or Namalwa cells. Immunofluorescence studies on HeLa cells showed a nucleoplasmic and phase-dependent nucleolar localization of the monoclonal antibody was decreased after digestion with DNAase I but not with RNAase A. For purification, the antigen was released from nuclei by digestion with DNAase I and then purified by chromatography on DEAE cellulose, phosphocellulose and antibody-Sepharose affinity chromatography. Interestingly, the immunoaffinity purified product contained two polypeptide chains; the immunoreactive polypeptide had an Mr of 86 000 and a pI of 6.0. The complex also contained a 70 kDa, pI 6.5 nonantigenic polypeptide in a 1:1 ratio. The overall purification of the complex was 5700-fold. Both polypeptides contained approx. 15 mol% glutamic acid and the 70 kDa polypeptide contained approx. 15 mol% serine.     

Molecular cloning and characterization of chick sialoprotein associated with cones and rods, a developmentally regulated glycoprotein of interphotoreceptor matrix

MY-174 is an IgM class monoclonal antibody originally established against chick PG-M/versican. The antibody specifically stains the photoreceptor layer, where we recently reported an absence of PG-M/versican. In this study, we re-characterized the antibody and identified the molecule that reacts to MY-174 at the photoreceptor layer. Immunohistochemistry localized the antigen to the matrix surrounding photoreceptors. A variety of glycosidase digestions showed that the antigen is the 150-kDa glycoprotein that has sialylated N- and O-linked glycoconjugates having a molecular mass of more than 30-kDa. The peptide sequences obtained from purified MY-174 antigen showed we had sequenced a full-length cDNA with an open reading frame of 2787 base pairs, encoding a polypeptide of 928 amino acids, with 56 and 54% identities to human and mouse sialoprotein associated with cones and rods (SPACRs), respectively, and with the structural features observed in SPACRs. The specific sialylated O-glycoconjugates here are involved in the epitope structure for MY-174. SPACR first appeared by embryonic days 15-16, and expression increased with developmental age, paralleling the adhesion between neural retina and retinal pigment epithelium. Thus, we concluded that the MY-174 antigen at the photoreceptor layer, a developmentally regulated glycoprotein, is identical to chick SPACR and may be involved in a novel system mediating adhesion between neural retina and retinal pigment epithelium.     

Radioimmunoassay of a glycoprotein associated with malignancy

A glycoprotein associated with malignancy was purified from the 0.6M perchloric acid-soluble fraction of serum obtained from cancer patients. The purified glycoprotein contained sialic acid, which was responsible for binding to wheat-germ agglutinin-Sepharose. Gel electrophoresis showed one band with an apparent Mr of 50 000-55 000, and the isoelectric point was 4.4 +/- 0.1. The glycoprotein could be distinguished from carcinoembryonic antigen and alpha-fetoprotein. Iodination of this material with chloramine-T permitted development of a radioimmunoassay.     

Purification and characterization of a protein antigen (p94) from human brain and its identification in other species.

A protein antigen was chromatographically purified from human brain by its immunoaffinity to 44E3 monoclonal IgG and its chemical nature was investigated. The yield of antigen was estimated at 71%, and a 3160-fold purification was achieved relative to the homogenate. The antigen preparation from brain showed a very high degree of purity when analysed by SDS/polyacrylamide-gel electrophoresis and was composed of a single polypeptide of Mr 94,000. Amino-sugar and neutral-sugar analyses indicated that the protein was not glycosylated. The amino acid composition of the purified protein from brain was compared with that of the analogous protein purified from an acute-lymphoblastic-leukaemic cell line, HOON. The compositions were very similar, suggesting that the two proteins were closely related. Both purified proteins were equivalent in their ability to inhibit the reactivity of monoclonal antibodies 44E3 and 44H4 with leukaemic cells. These two antibodies appear to recognize spatially related, if not identical, epitopes on the same molecule. The antibodies were shown to cross-react with a polypeptide of Mr 94,000 in homogenates of human, bovine and guinea-pig brain white matter. Indirect immunoperoxidase staining of human grey- and white-matter acetone-fixed tissue sections incubated with either antibody indicated that the antigen was present on neuronal and glial cells; the staining was seen as clusters in the cytoplasm, starting at the plasma membrane, but leaving the nucleus unstained. The concentration of the protein in human brain was shown to be similar throughout postnatal development and aging.     

Immunochemical evidence for a transmembrane orientation of both the (Na+, K+)-ATPase subunits

Antibodies were raised against the large catalytic subunit (apparent Mr 96000) and the glycoprotein (apparent Mr 60000) of the sodium- and potassium-dependent adenosine triphosphatase [(Na+, K+)-ATPase] from Bufo marinus. The specificity of each antiserum was assessed by two-dimensional immunoelectrophoresis using toad kidney microsomes or the purified holoenzyme as a source of antigen and by indirect immunoprecipitation of detergent-solubilized (Na+, K+)-ATPase subunits from radioiodinated or biosynthetically labeled kidney holoenzyme, microsomes, or postnuclear supernatant. The anticatalytic subunit serum reacted exclusively with a 96000-dalton protein. The antiserum to the glycoprotein was rendered specific to this subunit by absorption with purified catalytic subunit. The two antisera were agglutinating and lytic in the presence of complement when toad erythrocytes were used as targets, indicating that antigenic determinants of both subunits were exposed on the cell surface. The specific reactivities with surface-exposed antigenic determinants of both subunits could be absorbed with toad red blood cells. Such absorbed antisera still reacted with detergent-treated or untreated kidney microsomes, revealing the presence of cytoplasmic and/or intramembranous antigenic sites. Our immunochemical data demonstrate that the glycoprotein subunit of (Na+, K+)-ATPase spans the lipid bilayer and confirm the transmembrane orientation of the catalytic subunit postulated from functional studies.     

Purification and protein sequence analysis of rat liver prolactin receptor

Prolactin receptors were purified from rat liver membranes by single-step immunoaffinity chromatography using a specific monoclonal antibody to the rat liver prolactin receptor. Scatchard analysis of 125I-human growth hormone binding to the purified receptor revealed two classes of specific binding sites with Ka = 18.5 x 10(9) and 1.2 x 10(9) M-1. Considering that both classes of binding sites are responsible for high affinity prolactin binding, the partially purified receptor preparation had a binding activity of 1.69 nmol/mg protein, representing 1000-fold purification over microsomal receptors with a recovery of 52%. From three separate purifications, 6 mg of partially purified prolactin receptor were obtained with a purity of approximately 4 to 6.5%. Thus, the use of monoclonal antibody for affinity chromatography resulted in a large improvement of prolactin receptor purification compared to previous hormone affinity chromatography (300-fold purification, 15% recovery). The purified receptor was run on preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a homogeneous preparation of prolactin receptor was obtained by electroelution from gel slices corresponding to Mr 38,000-43,000. Immunoblot analysis using a radiolabeled monoclonal antibody revealed two separate but closely located bands of Mr 42,000 and 40,000 in microsomal, partially purified, and electroeluted preparations. The homogeneous receptor protein was extensively digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone trypsin, and 10 internal amino acid sequences of the rat liver prolactin receptor were determined by gas-phase sequence analysis. Oligonucleotide probes were prepared against two of these internal sequences, and a prolactin receptor cDNA was isolated from a rat liver library using one of these probes (Boutin, J. M., Jolicoeur, C., Okamura, H., Gagnon, J., Edery, M., Shirota, M., Banville, D., Dusanter-Fourt, I., Djiane, J., and Kelly, P. A. (1988) Cell 53, 69-77). The amino acid sequence deduced from the cDNA reveals three potential sites of N-linked glycosylation, two of which were confirmed during protein sequencing. The prolactin receptor was characterized by affinity labeling with 125I-human growth hormone. Cross-linking of microsomes revealed a single band for the hormone-receptor complex with Mr 62,000. On the other hand, cross-linking of Triton X-100-solubilized or partially purified receptor with labeled hormone resulted in the appearance of two bands with Mr 62,000 and 102,000, suggesting the existence of a subunit structure of the prolactin receptor, or alternatively, the existence of two types of prolactin receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of a Candida albicans germ tube specific antigen

Abstract In a previous work, Marot-Leblond et al. identified a Candida albicans germ tube-specific antigen by the use of a monoclonal antibody (mAb 3D9.3). In the present report, we used a two-step procedure to obtain a purified preparation of this antigen from a Zymolyase extract of Candida albicans germ tubes. The extract was first fractionated by gel filtration chromatography. The immunoreactive fractions were pooled, and the 3D9.3 antigen was further purified by hydrophobic interaction chromatography using a Phenyl-superose column. Analysis by SDS-PAGE, immunoblotting and Concanavalin A staining, revealed a single, polydisperse band ranging from 110 to 170 kDa. The antigen was purified 126-fold by protein content and 16.4-fold by carbohydrate content. Recovery of the antigen was 6.8% following the two-step purification.