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PubMed1985

Characterization of wild type and mutant glucocorticoid receptors from rat hepatoma and mouse lymphoma cells.

Using a combination of immunological blotting techniques and hormone affinity labeling, we have characterized the glucocorticoid receptors present in wild type and mutant rat hepatoma (HTC) and mouse thymoma (S49 and WEHI7) cells. Mutant HTC and WEHI7 cells of the receptorless phenotype, which contain greatly reduced amounts of glucocorticoid hormone binding activity, show parallel decreases in immunoreactive material using a monoclonal antibody raised against the rat liver glucocorticoid receptor. This indicates that these receptorless mutant cells harbor defects in either the production or accumulation of receptor protein. Quantitation of immunoreactivity and hormone binding activity present in wild type and mutant S49 cells indicates that these cells contain significantly more immunoreactive material than hormone binding activity. We conclude that S49 cells produce, in addition to their well characterized wild type or mutant receptors, a mutant receptor from a second allele which is of wild type size, is immunologically reactive, but is unable to bind hormone. The S49 mutant cell line nti (nuclear transfer increase) contains a glucocorticoid receptor which has a molecular weight of 40,000, while the wild type receptor has a molecular weight of 94,000. Affinity labeling of glucocorticoid receptors in nti cells with [3H]dexamethasone mesylate indicates that nti cells do not contain wild type sized precursor molecules which bind hormone, nor do they contain immunoreactive fragments of a molecular mass smaller than 94 kDa. It is proposed that the 40-kDa nti receptor is produced as a truncated protein most likely resulting from a nonsense mutation or from a truncated messenger RNA.

Affinity Labels↗
PubMed1993

Role of the conserved Lys-X-Gly-Gly sequence at the ADP-glucose-binding site in Escherichia coli glycogen synthase.

Although bacterial and mammalian glycogen synthases differ in the primary structure and specificity for glucosyl donor, lysyl residues identified at their substrate-binding sites by affinity labeling are present in a conserved tetrapeptide sequence, Lys-X-Gly-Gly, where X is a residue not conserved (Tagaya, M., Nakano, K., and Fukui, T. (1985) J. Biol. Chem. 260, 6670-6676; Furukawa, K., Tagaya, M., Inouye, M., Preiss, J., and Fukui, T. (1990) J. Biol. Chem. 265, 2086-2090). To elucidate the functional role of this conserved sequence, Lys-15, Gly-17, and Gly-18 in Escherichia coli glycogen synthase have been replaced by other amino acid residues via site-directed mutagenesis. Kinetic analyses of the Lys-15 mutant enzymes showed that the epsilon-amino group of Lys-15 is mainly involved in binding of the phosphate moiety adjacent to the glycosidic linkage in the substrate ADP-glucose, presumably through an ionic interaction. The mutant enzyme in which Ala was substituted for Gly-17 had a catalytic rate constant 3 orders of magnitude smaller than that of the wild-type enzyme with a slightly increased Michaelis constant for ADP-glucose, whereas the Gly-18-->Ala mutant showed a rate constant only 3.2-fold smaller. In addition, mutations of Gly-17 and Gly-18 resulted in marked changes in the reactivity of Lys-15 with affinity labeling reagents. These results suggest that the 2 glycyl residues in the conserved Lys-X-Gly-Gly sequence, in particular the one closer to the ADP-glucose-binding lysyl residue, participate in catalysis by assisting conformational change(s) of the active site or stabilizing the transition state.

Adenosine Diphosphate Glucose↗
PubMed1977

Interaction of substrates and inhibitors with the homoserine dehydrogenase of kinase-inactivated aspartokinase I.

The aspartokinase activity of the aspartokinase-homoserine dehydrogenase complex of Escherichia coli was affinity labeled with substrates ATP, aspartate, and feedback inhibitor threonine. Exchange-inert ternary adducts of Co(III)-aspartokinase and either ATP, aspartate or threonine were formed by oxidation of corresponding Co(II) ternary complexes with H2O2. The ternary enzyme-Co(III)-threonine adduct (I) had 3.8 threonine binding sites per tetramer, one-half that of the native enzyme. The binding of threonine to I was still cooperative as determined by equilibrium dialysis (nH = 2.2) or by studying inhibition of residual dehydrogenase activity (nH = 2.7). Threonine still protected the SH groups of I against 5,5'-dithiobis(2-nitrobenzoate) (DTNB) reaction but the number of SH groups reacting with thiol reagents (DTNB) was reduced by 1-2 per subunit in the absence of threonine. This suggests either that Co(III) is bound to the enzyme via sulfhydryl groups or that 1-2SH groups are buried or rendered inaccessible in I. The binding of threonine to sites not blocked by the affinity labeling produced changes in the circular dichroism of the complex comparable to changes produced by threonine binding to native enzyme and also protected against proteolytic digestion. The major conformational changes produced by threonine are thus ascribable to binding at this one class of regulatory sites. The interactions of kinase substrates with various aspartokinase-Co(III) complexes containing ATP, aspartate, or threonine and a threonine-insensitive homoserine dehydrogenase produced by mild proteolysis were studied. The inhibition of homoserine dehydrogenase by kinase substrates is not due to binding of these inhibitors at the kinase active site but was shown to be due to binding to sites within the dehydrogenase domain of the enzyme. L-alpha-Aminobutyrate, a presumed threonine analogue, also inhibits the dehydrogenase by binding at the same or similar sites in the dehydrogenase domain and not at threonine regulatory site.

Adenosine Diphosphate↗
PubMed1986

Photolabeling of protein components in the pactamycin binding site of rat liver ribosomes.

The antitumoral and antibacterial drug pactamycin can be radioactively labeled by iodination without loss of biological activity. Using the labeled pactamycin, the ribosomal binding site of the drug on rat liver ribosomes has been studied by affinity labeling techniques taking advantage of the photoreactive acetophenone group present in the molecule. When 40 S ribosomal subunits are labeled, one major spot of radioactivity is found associated to protein S25. In addition, weaker spots related to proteins S14/15, S10, S17 and S7 can also be detected in the autoradiogram of the two-dimensional gel slab. Since pactamycin inhibits protein synthesis initiation, the proteins forming its binding site must be related to some step of this process. By comparison with results from pactamycin affinity labeling of Escherichia coli ribosomes (Tejedor, F., Amils, R. and Ballesta, J.P.G. (1985) Biochemistry 24, 3667-3672) these proteins could lie in the mRNA and initiation factors binding region of the rat liver ribosome.

Animals↗
PubMed1990

Cysteinyl proteinases and their selective inactivation.

The affinity-labeling of cysteinyl proteinases may now be carried out with a number of peptide-derived reagents with selectivity, particularly for reactions carried out in vitro. These reagents have been described with emphasis on their selectivity for cysteine proteinases and lack of action on serine proteinases, the most likely source of side reactions among proteinases. Perhaps a crucial feature of this selectivity is an enzyme-promoted activation due to initial formation of a hemiketal, which may destabilize the reagent. Prominent among the reagent types that have this class selectivity are the peptidyl diazomethyl ketones, the acyloxymethyl ketones, the peptidylmethyl sulfonium salts, and peptidyl oxides analogous to E-64. The need for specific inhibitors capable of inactivating the target enzyme in intact cells and animals is inevitably pushing the biochemical application of these inhibitors into more complex molecular environments where the possibilities of competing reactions are greatly increased. In dealing with the current state and potential developments for the in vivo use of affinity-labeling reagents of cysteine proteinases, the presently known variety of cysteinyl proteinases had to be considered. Therefore this chapter has, at the same time, attempted to survey these proteinases with respect to specificity and gene family. The continual discovery of new proteinases will increase the complexity of this picture. At present the lysosomal cysteine proteinases cathepsins B and L and the cytoplasmic calcium-dependent proteinases are reasonable goals for a fairly complete metabolic clarification. The ability of investigators to inactivate individual members of this family in vivo, possibly without complications due to concurrent inactivation of serine proteinases by improvements in reagent specificity, is increasing. Among the cysteine proteinases, at least those of the papain super family, hydrophobic interactions in the S2 and S3 subsites are important and some specificity has been achieved by taking advantage of topographical differences among members of this group. Some of this has probably involved surface differences removed from the regions involved in proteolytic action. The emerging cysteine proteinases include some which, in contrast to the papain family, have a pronounced specificity in S1 for the binding of basic side chains, familiar in the trypsin family of serine proteinases. At least a potential conflict with serine proteinases can be avoided by choice of a covalent bonding mechanism. The departing group region, has not been exploited. As a sole contributor to binding, this region may be rather limited as a source of specificity.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity Labels↗
PubMed1992

Endoglin is a component of the transforming growth factor-beta receptor system in human endothelial cells.

Endoglin, a dimeric membrane glycoprotein expressed at high levels on human vascular endothelial cells, shares regions of sequence identity with betaglycan, a major binding protein for transforming growth factor-beta (TGF-beta) that co-exists with TGF-beta receptors I and II in a variety of cell lines but is low or absent in endothelial cells. We have examined whether endoglin also binds TGF-beta and demonstrate here that the major TGF-beta 1-binding protein co-existing with TGF-beta receptors I and II on human umbilical vein endothelial cells is endoglin, as determined by specific immunoprecipitation of endoglin affinity-labeled with 125I-TGF-beta. Furthermore, endoglin ectopically expressed in COS cells binds TGF-beta 1. Competition affinity-labeling experiments showed that endoglin binds TGF-beta 1 (KD approximately 50 pM) and TGF-beta 3 with high affinity but fails to bind TGF-beta 2. This difference in affinity of endoglin for the TGF-beta isoforms is in contrast to beta-glycan which recognizes all three isoforms. TGF-beta however is binding with high affinity to only a small fraction of the available endoglin molecules, suggesting that some rate-limiting event is required to sustain TGF-beta binding to endoglin.

Antigens, CD↗
PubMed1998

Purinergic receptors in human blood platelets: chemical modification and cloning investigations.

Platelet aggregation is important for maintaining normal hemostasis. However, aberrant platelet aggregation plays a major role in acute coronary artery diseases, myocardial infarction, unstable angina, and stroke. ADP is one of the earliest and most important platelet agonists. ADP induces platelet aggregation, shape change, secretion, influx and intracellular mobilization of Ca2+, and inhibition of the adenylyl cyclase stimulated by prostaglandins. Binding of ADP to purinergic receptor(s) is required for elicitation of the ADP-induced platelet responses. But the platelet ADP receptor(s) has not been purified, largely due to the unavailability of the reagents that can be used to selectively label the platelet ADP receptor. The ADP receptor responsible for the ADP-induced platelet aggregation and inhibition of stimulated adenylyl cyclase activity has not been cloned due to difficulties in screening responsive clones generated from a cDNA library. Since the purified ADP-receptor protein is not available, antibodies that can be used as alternative tools to purify the ADP receptor or screen the clones expressing the receptor could not be made. In addition, the problem may be compounded by the low copy number and the susceptibility of the receptor to proteolysis. Therefore, signal transduction mechanisms underlying biochemical transformations in ADP-induced platelet responses remain less well defined and/less well understood. In the past decade efforts have been made to identify a platelet ADP receptor(s) by photoaffinity as well as affinity labeling by the ADP-affinity analogs. More recently efforts have been directed to clone the platelet ADP receptors. These investigations, however, have not produced definite results. The purpose of this review is to examine the results obtained by the photoaffinity- and affinity-labeling investigations and cloning experiments to identify a platelet ADP receptor(s).

Blood Platelets↗
PubMed1983

Reconstitution of glucose transport using human erythrocyte band 3.

Band 3 and the diffuse component of zone 4.5, designated band 4.5.B, have been separately prepared from human erythrocyte membranes and incorporated into the membranes of 150 nm 1-palmitoyl-2-oleoyl phosphatidylcholine vesicles. The rates of glucose influx into these vesicles were measured under zero-trans conditions. Both sets of vesicles exhibited substrate-saturable transport which was inhibited by phloretin. However, the specific activity of the band 3 vesicles, 292 mumol X min-1 X (mg protein)-1, was more than twice that of the band 4.5.B vesicles, and the turnover number of transporters in the band 3 vesicles was at least 4-fold greater than those in the 4.5.B vesicles. Very little background density was visible in the band 4.5 region of erythrocyte membranes protected from degradation. In unprotected membranes, band 4.5.B was abundantly present, could be purified, and had glucose transport activity. Previously we have shown (Biochemistry 19, 1205 (1980] that maltosyl isothiocyanate, an affinity label for the glucose transporter, labelled a single 100 000 Mr protein of the intact erythrocyte membrane. Based upon the results of both affinity labelling and reconstitution we suggest that the native glucose transporter is a component of band 3, and that band 4.5.B contains a partially active fragment of the native transporter.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1975

Synthesis of a chemically reactive analog of the nonsense codon U-G-A. Its reaction with ribosomes of Escherichia coli.

Nitrophenylated 5'-uridylic acid could be employed as primer in a polyribonucleotide nucleotidyltransferase (Micrococcus luteus) reaction to yield 5'-nitrophenylated Pu-G-A. After reduction of the nitrophenyl moiety and subsequent bromoacetylation, a 5'-bromoacetamido-phenyl-phosphorylated U-G-A was obtained, which could be used as an affinity label for the ribosomal binding site of the nonsense codon. If freshly prepared active ribosomes were employed in the incubation mixtures, the U-G-A analog reacted exclusively with one protein, which is tentatively identified as protein S18. Exposure of ribosomes to low temperatures gave rise to a reaction of the U-G-A label with another protein, which was identified as protein S4, the ram gene product. The results of the affinity labeling experiments with the chemically reactive U-G-A derivative are very similar to that obtained with a corresponding derivative of the initiation codon A-U-G (O. Pongs and E. Lanka (1975) Proc. Natl. Acad. Sci. U.S.A. 75, 1505-1509), which suggests that 70S ribosomes have one preferential codon binding site.

Codon↗
PubMed1989

Localization of a portion of the active site of two rat liver glutathione S-transferases using a photoaffinity label.

The glutathione S-transferases are a family of dimeric enzymes that catalyze the reaction between GSH and a variety of electrophiles. Two closely related isozymes, referred to as YaYa and YcYc, were purified from rat liver. A radiolabeled azido derivative of glutathione (S-(p-azidophenacyl)[3H]glutathione) was prepared and used to label covalently the active site of the above two glutathione S-transferases. The noncovalently bound affinity label was a competitive inhibitor of glutathione S-transferase YaYa toward both 1-chloro-2,4-dinitrobenzene and GSH. The covalently labeled enzymes no longer bound to a GSH-affinity column, and covalent labeling was reduced in the presence of GSH and S-(dinitrophenyl)glutathione. These results suggest that the affinity label was binding at the active site. The covalently labeled enzymes were digested with trypsin, and the labeled peptides were purified by HPLC and then sequenced. A single-labeled peptide was identified in the tryptic digest of the YaYa isozyme, whereas two labeled peptides were present in the tryptic digest of YcYc. The Ya peptide sequence was identical with the published deduced sequence of amino acids between residues 212 and 218 and the sequences of the two peptides purified from Yc were identical with the deduced sequence of amino acids between 91 and 110 and 206 and 218. Hence, the Ya peptide and the smaller peptide purified from Yc came from the same region of the Ya and Yc subunits. This common region and a second region of the Yc subunit appear to form a portion of the active site of these two forms of glutathione S-transferase.

Affinity Labels↗
PubMed1999

Phase II study of interferon-enhanced 131I-labeled high affinity CC49 monoclonal antibody therapy in patients with metastatic prostate cancer.

Adjuvant Interferon (IFN) was given to increase tumor antigen expression and enhance localization with 131I-labeled CC49 radioimmunotherapy in a Phase II trial for hormone resistant metastatic prostate cancer. Patients received four doses of alpha-IFN (3 x 10(6) IU) s.c. on alternate days, from day -5 to day +1 of 75 mCi/m2 131I-CC49 treatment. Toxicity was well tolerated, with the majority of patients experiencing transient grade 3 or 4 neutropenia and/or thrombocytopenia (maximal at 4-6 weeks). The absorbed dose was >25 Gy in four of eight tumors visualized, which represents an increase of >20 fold over whole body radiation dose. Two patients had radiographic minor responses by 6 weeks post-therapy, whereas five of six patients experiencing pain had symptom relief without radiographic changes. The protocol provided modest antitumor effects (pain relief in five of six patients and two minor radiographic responses). This study suggests that the addition of IFN enhanced tumor uptake and antitumor effects as compared to a prior Phase II trial of 131I-CC49 alone.

Aged↗
PubMed1977

Production of BALB/c anti-idiotypic antibodies against the BALB/c myeloma protein 315 does not require an intact ligand-binding site.

To determine whether the ligand-binding site of the BALB/c myeloma protein 315 is essential for the anti-idiotypic response in syngeneic animals, 17 BALB/c mice were immunized with M315 that had been affinity-labeled with bromo-acetyl-DNP-L-lysine (BADL). Essentially all the active sites of M315 were blocked by the affinity label. Fourteen mice produced antibodies that reacted with an idiotypic determinant localized in the Fv fragment of M315, but this idiotype was not part of the DNP-lysine-binding site, and it was absent from L315 and H315 chains. Two groups of BALB/c mice were immunized with nonaffinity-labeled M315, to determine whether the same idiotype was recognized with this immunogen. All animals in the group that received the most prolonged immunization produced antibodies that could be divided in two populations: about 75% were directed against the site-associated idiotype, and the rest reacted with the nonsite idiotype. The other group produced antibodies exclusively specific for the site. Thus, the site-associated idiotype of M315 is not essential for the antibody response of BALB/c mice against M315, and M315 carries at least two different idiotypes that can be recognized by B cells of syngeneic animals.

Animals↗
PubMed1988

Monoclonal antibody to the human glucose transporter that differentiates between the glucose and nucleoside transporters.

A monoclonal antibody to the glucose transporter has been prepared with band 4.5 (Mr 45,000-65,000) from human erythrocyte ghosts as antigen. This antibody, designated 7F7.5, is of the IgG2b type. The antibody bound exclusively to proteins in the band 4.5 region of immunoblots of human erythrocyte ghosts separated on sodium dodecyl sulfate-polyacrylamide gels. Immobilized 7F7.5 antibody removed glucose transport activity from solubilized alkaline-treated ghosts. The material that was eluted from the immobilized antibody matrix migrated primarily in the band 4.5 region of electrophoretic gels and bound the antibody in immunoblots. To test the specificity of the antibody, glucose and nucleoside transporters in alkaline-treated human erythrocyte ghosts were affinity labeled with [3H]cytochalasin B and [3H]-S-(nitrobenzyl)thioinosine (NBMPR), respectively. Both of these transporters are band 4.5 proteins and "copurify" by DEAE-cellulose chromatography. A filter paper assay was developed to assess the presence of the labeled transporters. Immobilized 7F7.5 antibody bound 99% of the labeled glucose transporter. In contrast, only 3% of the specifically labeled nucleoside transporter bound to the immobilized antibody. Furthermore, the antibody did not remove nucleoside transport or NBMPR binding activities from detergent solution. The antibody recognized two tryptic fragments, Mr 23,000 and 18,000, which contain the cytochalasin B binding site of the glucose transporter. By immunoblot, the monoclonal antibody recognized the glucose transporter in cultured human IM9 lymphocytes, synovial cells, and HBL 100 mammary cells but not cells of murine or rat origin. These results indicate that the glucose and nucleoside transporters are distinct proteins which can be distinguished by monoclonal antibody 7F7.5. The method developed to quantitate covalently labeled glucose and nucleoside transporters should have broad applicability as a rapid and easy method for determining the recovery of affinity-labeled membrane proteins in detergent solution during purification. Because of the location of the epitope, the antibody itself should prove to be a valuable tool in establishing the molecular basis for the function and regulation of the glucose transporter.

Antibodies, Monoclonal↗
PubMed1982

Chemical modification A probe of the structure and function of the subunits of DPN-dependent isocitrate dehydrogenase.

DPN-dependent isocitrate dehydrogenase is composed of three distinct types of subunits: alpha, beta, and gamma which have molecular weights of about 40,000 but differ in isoelectric points. The relationship of subunit diversity to function was probed by use of chemical modification. 3-Bromo-2-ketoglutarate, a substrate and affinity label for the active site of isocitrate dehydrogenase, was shown to cause significant modification of all types of subunits. The substrate affinity label, 3-ene-2-keto-glutarate, labels each of the subunits equally. Approximately equal labeling of subunits was also found upon modification by cyanate of an essential lysyl residue in the isocitrate binding site. When enzyme was inactivated by a carbodiimide in the presence of glycine ethyl ester, both glutamate and aspartate residues reacted, and labeling of each type of subunit occurred. These studies suggest that the structurally distinct subunits of DPN-dependent isocitrate dehydrogenase are functionally similar and each type of subunit contains a substrate binding site.

Animals↗
PubMed1995

Bee venom phospholipase A2 is recognized by the macrophage mannose receptor.

A high affinity and a specific binding site for bee venom PLA2 was found on the surface of J774E macrophages. The binding sites for bee venom PLA2 are entirely different from the binding sites for pancreatic and snake venom PLA2 as revealed by competition experiments. Binding and uptake of bee venom PLA2 by J774E macrophages was shown to be competed by mannose-BSA, glucose-BSA, N-acetylglucosamine-BSA, but not by galactose-BSA, indicating that the binding of bee venom PLA2 is probably mediated by macrophage mannose receptor. An affinity labeling experiment revealed that the bee venom PLA2 specifically binds to a single polypeptide with a mass of approximately 180 kDa. Moreover, the affinity labeled protein component, i.e., the binding site, was not detected in the presence of excess mannose-BSA, suggesting that mannose-BSA and the bee venom PLA2 bind to the same site on macrophages. These observations were further supported by the binding of bee venom PLA2 to cells which are known to express the mannose receptor and by specific binding of bee venom PLA2 to the purified mannose receptor. These data confirm that bee venom PLA2 binding to macrophages is mediated through the mannose receptor.

Animals↗
PubMed1988

Insulin-like growth factor I and insulin potentiate luteinizing hormone-induced androgen synthesis by rat ovarian thecal-interstitial cells.

We tested the hypothesis that insulin-like growth factor I (IGF-I) and insulin play a role in androgen production by rat ovarian thecal-interstitial cells. Collagenase/DNase-dispersed rat ovarian thecal-interstitial cells obtained from immature hypophysectomized Sprague-Dawley rats were cultured at a concentration of 10(6) cells/ml in serum-free medium in the presence of increasing concentrations of LH, IGF-I, or insulin. The medium was replaced every 48 h, and the androsterone concentration in the culture supernatants was used as an index of androgen production. In the absence of added hormones (control) androsterone levels were consistently less than 0.1 ng/ml. Increasing concentrations of LH stimulated androsterone synthesis in a dose-dependent manner. IGF-I, in the absence of LH, did not significantly increase androsterone levels above control values. However, when combined with 10 ng/ml LH, IGF-I increased androsterone synthesis above levels seen with LH alone in a dose-related fashion: for example, the peak androsterone levels seen with LH and 100 ng/ml (13 nM) IGF-I at 96 h of culture were significantly greater than the peak level seen with 10 ng/ml LH alone (302 +/- 71 vs. 17 +/- 7 ng/ml; P less than 0.0125). Similarly, while insulin alone did not increase androsterone synthesis above control values, androsterone concentrations were increased by insulin in combination with 10 ng/ml LH; a peak value of 240 +/- 67.7 ng/ml was observed at 96 h of culture with 100 ng/ml (18 mM) insulin (P less than 0.025 vs. LH alone) Androsterone levels were slightly less with insulin than with IGF-I, but this difference was not significant. The combination of IGF-I and insulin did not increase levels of androsterone synthesis above those observed with each hormone alone. IGF-I bound to a high affinity binding site on ovarian cell monolayer cultures with an apparent binding affinity of 1.3 x 10(-9) M. Insulin also competed for binding with radiolabeled IGF-I in a dose-dependent manner, but the affinity of insulin was approximately 500-fold less; half-maximal inhibition of [125I] IGF-I binding occurred with an insulin concentration of approximately 300 nM (or approximately 1700 ng/ml). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of thecal-interstitial cell monolayers affinity labeled with radiolabeled IGF-I in the absence and presence of unlabeled hormone revealed proteins with characteristics of type I IGF receptors. Affinity labeling to a protein of a relative molecular mass of approximately 45,000 was also noted, probably representing IGF carrier proteins synthesized by thecal-interstitial cell monolayers.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgens↗
PubMed1992

Y2 receptor proteins for peptide YY and neuropeptide Y. Characterization as N-linked complex glycoproteins.

Affinity labeling using [125I-Tyr36]PYY and homobifunctional affinity crosslinking reagents of the rabbit Y2 receptor for peptide YY(PYY) results in specifically labeled proteins of both M(r) = 50,000 to 60,000 and M(r) = 96,000 to 115,000 [1,2]. In this work the glycoprotein nature of affinity labeled Y2 receptor proteins were investigated by enzymatic deglycosylation using neuraminidase, endoglycosidase F (endo F), N-glycosidase F (PNGase F), and O-glycanase treatment. Only N-glycosidase F and neuraminidase increased the electrophoretic mobility of the radiolabeled receptor bands, whereas all other glycosidases did not. PNGase F treatment of both radiolabeled receptor bands electroeluted from gel slices reduced the apparent molecular mass of by 16-17 kDa units, that is M(r) = 96,000 to 79,000 and M(r) = 60,000 to 44,000, indicating removal of N-linked oligosaccharide chains of similar size from both species. Neuraminidase treatment caused slight increases in the electrophoretic mobilities suggesting the presence of terminal sialic residues. It is concluded that the Y2 binding proteins are N-linked complex (sialo)glycoproteins with a minimal core protein size of M(r) = 44,000. Furthermore, based on this sensitivity pattern of the glycosidases, the Asn-linked carbohydrate may be of the tri- or tetra-antennary complex type containing terminal sialic acid residues.

Affinity Labels↗