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PubMed1988

Purification and affinity labeling of dihydropyridine receptor from rabbit skeletal muscle membranes.

Undegraded dihydropyridine (DHP)-receptor (putatively a voltage-gated Ca2+ channel) has been purified as a 340-kDa protein complex to approximately 80% homogeneity (2.4 nmol of DHP-receptor per mg of protein) from rabbit skeletal muscle by a rapid purification protocol. Transverse-tubule membranes were prepared in high yield by Ribi-press treatment. The DHP-receptor complex was solubilized in 1% digitonin followed by a two-step chromatographic purification procedure. The equilibrium dissociation constant of [3H](+)-PN200-110 binding (Kd; 0.9 nM) was not significantly changed by solubilization or purification. The purified DHP-receptor is composed of two subunits with apparent molecular masses of 148 kDa and 195 kDa migrating in polyacrylamide gels under nonreducing conditions as a single moiety of approximately 300 kDa. The 195-kDa subunit was affinity-labeled with [3H]azidopine in both transverse-tubule membranes and purified DHP-receptor preparations. The subunit can be degraded by high-energy irradiation to a 26-kDa peptide and by proteolysis to a 32-kDa peptide. Thus, it is probably due to proteolytic cleavage and/or photolysis that neither purification nor affinity-labeling studies have previously identified a DHP-receptor subunit of comparable molecular mass (195 kDa).

Affinity Labels↗
PubMed1984

Nitrendipine and isoproterenol induce phosphorylation of a 42,000 dalton protein that co-migrates with the affinity labeled calcium channel regulatory subunit.

Slow inward calcium channels in canine cardiac membranes were affinity labeled with the calcium channel analogue, [3H]o-NCS [2,6 dimethyl-3,5-dicarbomethoxy-4-(2- isothiocyanatophenyl )-1, 4-dihydropyridine], in the presence and absence of cold o-NCS or nicardipine. A major specifically labeled peak was identified with Mr 42,000 on NaDodSO4 polyacrylamide gels. In parallel experiments the effects of the calcium channel antagonist, nitrendipine and a variety of other chemical mediators were tested for their ability to stimulate protein phosphorylation in cardiac membranes. These data demonstrate that both nitrendipine and isoproterenol induce the phosphorylation of a 42,000 dalton protein via a kinase endogenous to the cardiac membranes and that the effects of isoproterenol are attenuated by carbachol.

Affinity Labels↗
PubMed1989

Selection and characterization of a mutant of the cloned gene for mandelate racemase that confers resistance to an affinity label by greatly enhanced production of enzyme.

The plasmid pSCR1 containing the gene for mandelate racemase (EC 5.1.2.2) from Pseudomonas putida (ATCC 12633) allows Pseudomonas aeruginosa (ATCC 15692) to grow on (R)-mandelate as its sole carbon source [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540]; the chromosome of the P. aeruginosa host apparently does not contain the gene for mandelate racemase but does contain genes for the remaining enzymes in the mandelate pathway and enables growth on (S)-mandelate as carbon source. However, in the presence of alpha-phenylglycidate, an active-site-directed irreversible inhibitor (affinity label) of mandelate racemase, P. aeruginosa transformed with pSCR1 can utilize (S)-mandelate but not (R)-mandelate as carbon source. This inhibition of growth on (R)-mandelate provides a metabolic selection for mutants that are resistant to alpha-phenylglycidate. When (R)-mandelate is used as carbon source and alpha-phenylglycidate is present, a few colonies of P. aeruginosa transformed with pSCR1 grow slowly and appear on plates after several days. The plasmid isolated from these cells confers resistance to alpha-phenylglycidate on newly transformed cells of P. aeruginosa. This resistance to the affinity label is not due to a mutation within the primary structure of the enzyme. A single base change (C----A) located 87 bp upstream of the initiation codon for the gene for mandelate racemase was detected in three independent isolates of alpha-phenylglycidate-resistant colonies and appears responsible for a 30-fold increase in the amount of mandelate racemase encoded by the gene contained in the plasmid.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1986

Dynamic aspects of affinity labelling as revealed by alkylation and phosphorylation of pancreatic ribonuclease with reactive deoxyribodinucleotide derivatives.

Affinity labelling of pancreatic RNase with 4-(N-2-chloroethyl-N-methylamino)benzylamide and (N----P) N-methylimidazolide of d(pTpA) results in the formation of monomodified enzyme derivatives retaining partially enzymatic activity. These data together with some cases described in the literature are considered as suggesting the dynamic nature of the enzyme-reagent complex represented by a set of states differing in the probability of intra-complex reaction. In particular, modification may proceed in a low probability state with an especially favorable mutual orientation of reagent and some protein residue remote from the active site of the enzyme resulting in the removal of the covalently attached reagent moiety from the active center.

Affinity Labels↗
PubMed1999

Affinity labeling of delta opioid receptors by an enkephalin-derivative alkylating agent, DSLET-Mal.

Opioid binding properties of Tyr-D-Ser-Gly-Phe-Leu-Thr-NH-NH-Gly-Mal (DSLET-Mal), a novel enkephalin-framed affinity label, was determined in rat brain membranes. In competition studies the ligand showed high affinity for the delta opioid sites, labelled by [(3)H][Ile(5,6)]deltorphin II (K(i) = 8 nM), whereas its binding to the mu ([(3)H]DAMGO) and kappa ([(3)H]EKC) sites was weaker. Preincubation of the rat brain membranes with DSLET-Mal at micromolar concentrations resulted in a wash-resistant and dose-dependent inhibition of the [(3)H][Ile(5,6)]deltorphin II binding sites (96% blocking at 10 microM concentration). Intracerebroventricular (ICV) administration of DSLET-Mal reduced the density of delta opioid receptors and had no effect on mu and kappa receptors, as determined by saturation binding studies. [Ile(5, 6)]deltorphin II-stimulated [(35)S]GTPgammaS binding was determined in membrane preparations of different brain areas of the ICV-treated animals. In both frontal cortex and hippocampus DSLET-Mal significantly decreased G protein activation by the delta agonist, having no effect on DAMGO stimulated [(35)S]GTPgammaS binding. DSLET-Mal had qualitatively similar effects on both receptor binding and G protein activation. These characteristics of the compound studied suggest that DSLET-Mal can serve as an affinity label for further studies of the delta-opioid receptors.

Affinity Labels↗
PubMed1987

Synthesis and characterization of an affinity label for brain receptors to psychotomimetic benzomorphans: differentiation of sigma-type and phencyclidine receptors.

Brain sigma-type receptors and phencyclidine receptors are thought to mediate the psychotomimetic effects of benzomorphans and phencyclidine in humans. Recently, we reported the characterization of a selective sigma receptor ligand, 1,3-di-o-tolyl-guanidine (DTG), that shows negligible crossreactivity with phencyclidine receptors. Here we describe the synthesis and characterization of an isothiocyanate derivative of DTG, di-o-tolyl-guanidine-isothiocyanate (DIGIT). Guinea pig brain membranes treated with nanomolar doses of DIGIT followed by extensive washing exhibit a dose dependent reduction of [3H]DTG and (+)[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [+)[3H]3-PPP) binding to sigma receptors. Binding of radiolabelled ligands for phencyclidine, mu-opioid, benzodiazepine and dopamine-D2 receptors is not affected by membrane treatment with DIGIT, indicating specificity of the affinity label for sigma-type receptors. Treatment of DIGIT-derivatized membranes with 2 M NaCl does not result in recovery of sigma binding activity, suggesting that DIGIT's interaction with sigma receptors is not of an ionic nature. Equilibrium saturation binding experiments show that the inhibition of [3H]DTG binding to sigma receptors by DIGIT pretreatment of membranes is attributable to an irreversible reduction in the affinity (increase in Kd) of sigma receptors for DTG. The finding that sigma receptors are irreversibly modified by DIGIT whereas phencyclidine receptors are not affected suggests that sigma receptors are physically separate from phencyclidine receptors. The availability of a selective affinity label for the sigma receptor should facilitate the purification of the receptors and the characterization of sigma-type pharmacological effects in vivo and in vitro.

Affinity Labels↗
PubMed1988

Affinity labeling of Escherichia coli DNA polymerase I by 5'-fluorosulfonylbenzoyladenosine. Identification of the domain essential for polymerization and Arg-682 as the site of reactivity.

Preincubation of Escherichia coli DNA polymerase I (pol I) with 5'-fluorosulfonylbenzoyladenosine (5'-FSBA) results in an irreversible inactivation of DNA polymerase activity with concomitant covalent binding of 5'-FSBA to enzyme. pol I-associated 3'-5' exonuclease activity, however, remains unaffected. Kinetic studies of inactivation indicate that the degree of inactivation is directly proportional to the concentration of 5'-FSBA and increases linearly with time. The presence of the metal chelate form of dNTP substrates or template primer, but not the template or primer alone, protects the enzyme from inactivation by 5'-FSBA. A complete inactivation of polymerase activity occurs when 2 mol of 5'-FSBA are covalently linked to 1 mol of enzyme, suggesting two sites of modification. Tryptic peptide mapping of 5'-FSBA-treated enzyme revealed the presence of two distinct peptides containing the affinity label, confirming the presence of two reactive sites in the enzyme. However, we find that only one of the two sites is essential for the polymerase activity since, in the presence of substrate dNTP or template primer during preincubation of enzyme with 5'-FSBA, incorporation of the affinity label is reduced by only 1 mol. Peptide analysis of dNTP or template primer-protected enzyme further revealed that a peptide eluting at 35 min from the C-18 matrix was protected from the 5'-FSBA reaction. It is therefore concluded that this peptide contains the domain essential for polymerase activity. Staphylococcus aureus V-8 protease digestion, amino acid composition, and sequence analysis of this peptide revealed this domain to span residues 669 to 687 in the primary amino acid sequence of pol I, and arginine 682 was found to be the site of 5'-FSBA reactivity.

Adenosine↗
PubMed1985

Covalent affinity labeling, detergent solubilization, and fluid-phase characterization of the rabbit neutrophil formyl peptide chemotaxis receptor.

The formyl peptide chemotaxis receptor of rabbit neutrophils and purified rabbit neutrophil plasma membranes has been identified by several affinity labeling techniques: covalent affinity cross-linking of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys (125I-hexapeptide) to the membrane-bound receptor with either dimethyl suberimidate or ethylene glycol bis(succinimidyl succinate) and photoactivation of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-N epsilon-[6-[(4-azido-2-nitrophenyl)amino]hexanoyl]Lys(125I-PAL). These techniques specifically identify the receptor as a polypeptide that migrates as a broad band on sodium dodecyl sulfate-polyacrylamide electrophoresis, with Mr 50 000-65 000. The receptor has been solubilized in active form from rabbit neutrophil membranes with the detergents 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin and from whole cells with CHAPS. Chemotaxis receptor activity was measured by the ability of the solubilized membrane material to bind 125I-hexapeptide or fMet-Leu-[3H]Phe with gel filtration or rapid filtration through poly(ethylenimine)- (PEI) treated filters as assay systems. 125I-PAL was specifically cross-linked to the same molecular weight material in the CHAPS and digitonin solubilized extract, but no specific labeling of the receptor was seen when membranes were extracted with Nonidet P-40 and Triton X-100. Therefore, although a large number of detergents are able to solubilize the receptor, it appears that some release the receptor in an inactive form. The ligand binding characteristics of fMet-Leu-[3H]Phe to the CHAPS-solubilized receptor shared properties with the membrane-bound formyl peptide receptor, both of which showed curvilinear, concave-upward Scatchard plots. Computer curve fitting with NONLIN and statistical analyses of the binding data indicated that for both the membrane-bound and solubilized receptors a two saturable sites model fitted the data significantly better (p less than 0.01) than did a one saturable site model. The characteristics of the two saturable sites model for the soluble receptor were a high-affinity site with a KD value of 1.25 +/- 0.45 nM and a low-affinity site with a KD value of 19.77 +/- 3.28 nM. A total of 35% of the two sites detected was of the higher affinity. In addition, a Hill coefficient of 0.61 +/- 0.12 was observed.

Affinity Labels↗
PubMed1976

Affinity labelling of phenylalanyl-tRNA synthetase from E. coli MRE-600 by E. coli tRNAphe containing photoreactive group.

The photoinduced reaction of phenylalanyl-tRNA synthetase (E.C.6.1.1.20) from E.coli MRE-600 with tRNAphe containing photoreative p-N3-C6H4-NHCOCH2-group attached to 4-thiouridine sU8 (azido-tRNAphe) was investigated. The attachment of this group does not influence the dissociation constant of the complex of Phe-tRNAphe with the enzyme, however it results in sevenfold increase of Km in the enzymatic aminoacylation of tRNAphe. Under irradiation at 300 nm at pH 5.8 the covalent binding of [14C]-Phe-azido-tRNAphe to the enzyme takes place 0.3 moles of the reagent being attached per mole of the enzyme. tRNA prevents the reaction. Phenylalanine, ATP,ADP,AMP, adenosine and pyrophosphate (2.5 xx 10(-3) M) don't affect neither the stability of the tRNA-enzyme complex nor the rate of the affinity labelling. The presence of the mixture of either phenylalanine or phenylalaninol with ATP as well as phenylalaninol adenylate exhibits 50% inhibition of the photoinduced reaction. Therefore, the reaction of [14C]-Phe-azido-tRNA with the enzyme is significantly less sensitive to the presence of the ligands than the reaction of chlorambucilyl-tRNA with the reactive group attached to the acceptor end of the tRNA studied in 1. It has been concluded that the kinetics of the affinity labelling does permit to discriminate the influence of the low molecular weight ligands of the enzyme on the different sites of the tRNA enzyme interaction.

Adenine Nucleotides↗
PubMed1987

Studies on the functional topography of Escherichia coli RNA polymerase. Highly selective affinity labelling by analogues of initiating substrates.

RNA polymerase was treated in the presence of promoter-containing templates with 16 affinity reagents, derivatives on NMPs, NDPs and NTPs with reactive substituents at the terminal phosphate. This treatment was followed by addition of a pyrimidine [alpha-32P]NTP. Due to 'catalytic competence' of some of the residues of the affinity reagents bound covalently near the active center at the first stage, active-center-catalyzed synthesis of a phosphodiester bond occurred, and radioactive residues with the general formula -pNpN (where p = radioactive phosphate) appeared covalently attached to the enzyme. Such affinity labelling was super-selective because affinity reagent residues bound outside the active center were not elongated and thus remained non-radioactive. Labelling took place only when the combination of the reagent and [alpha-32P]NTP corresponded to the sequence of nucleotides of the promoter. With reagents having short 'arms', only the beta subunit was labelled; the targets were His and/or Lys residues. With reagents having longer 'arms', the sigma subunit was also labelled.

Affinity Labels↗
PubMed1979

Antibiotic effects on the photoinduced affinity labeling of Escherichia coli ribosomes by puromycin.

The effect of ribosomal antibiotics on the photoinduced affinity labeling of Escherichia coli ribosomes by puromycin [Cooperman, B.S., Jaynes, E.N., Brunswick, D.J., & Luddy, M.A. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1974; Jaynes, E.N. Jr., Grant, P.G., Giangrande, G., Wieder, R., & Cooperman, B.S. (1978) Biochemistry 17, 561] has been studied. Although blasticidin S, sparsomycin, lincomycin, and erythromycin are essentially without effect, major changes are seen on addition of either chloramphenicol or tetracycline. The products of photoincorporation have been characterized by one- and two-dimensional gel electrophoresis and by specific immunoprecipitation with antibodies to ribosomal proteins. In the presence of chloramphenicol, protein S14 becomes the major labeled protein. In the presence of tetracycline, L23 remains the major labeled protein, but the yield of labeled ribosomes is enormously increased, and the labeling is more specific for L23. These results are discussed in terms of the known modes of action of these antibiotics and the photoreactivity of tetracycline.

Affinity Labels↗
PubMed2002

Relationships between effects of smoking, gender, and alcohol dependence on platelet monoamine oxidase-B: activity, affinity labeling, and protein measurements.

BACKGROUND: Many studies have reported apparent associations between platelet monoamine oxidase (MAO) activity and susceptibility to alcoholism and other psychiatric conditions. Alcohol-dependent individuals generally exhibit lower platelet MAO activity compared with controls, and on this basis, platelet MAO has been proposed as a potential genetic marker for predisposition to alcoholism. However, several lines of evidence also suggest that MAO activity is reduced in both the brain and platelets of smokers. Many alcohol-dependent individuals are also tobacco users, and few studies have attempted to dissociate the effect of alcohol and tobacco use on MAO activity. METHODS: Platelet MAO-B activity in 629 subjects recruited as part of the WHO/ISBRA Study of State and Trait Markers of Alcohol Use and Dependence was assayed by using a high throughput fluorescence assay. Platelet MAO-B protein concentrations were measured by analysis of immunoblots probed with a polyclonal antibody selective for MAO. Quantitative measurements of affinity labeling of platelet MAO were made by using the selective MAO-B catalytic site antagonist [3H]Ro 19-6327. RESULTS: Multiple regression analysis revealed that subjects' gender, cigarette smoking, lifetime alcohol dependence, and recruitment site each contributed independently to the variance in platelet MAO activity levels. Female subjects had significantly higher MAO activity levels than males, whereas heavy smokers had significantly lower MAO activity levels than nonsmokers. Immunoblot measurement of platelet MAO-B protein demonstrated that females had significantly higher MAO-B protein concentrations. Platelet MAO-B protein concentrations did not differ significantly between smokers and nonsmokers but were lower in subjects with a diagnosis of lifetime alcohol dependence (DSM-IV) compared with subjects who were never alcohol dependent. Measurements of affinity labeling by [3H]Ro 19-6327 of platelet MAO correlated significantly with MAO activity levels (i.e., the lower MAO-B activity in smokers was mirrored by lower levels of [3H]Ro 19-6327 binding). CONCLUSIONS: In this international population, gender, cigarette smoking, lifetime history of alcohol dependence, and recruitment site were associated with lower platelet MAO activity levels. Differences in MAO activity associated with gender and lifetime alcohol dependence can be attributed largely to differences in MAO-B protein concentration, whereas those associated with smoking behavior may be the result of binding of an inhibitor contained in cigarette smoke to platelet MAO-B at catalytic site of MAO.

Adolescent↗
PubMed1983

Affinity labeling of peptidyl transferase center using the 3' terminal pentanucleotide from amino acyl-tRNA.

Affinity labeling of proteins in the peptidyl transferase center of eukaryotic ribosomes can be carried out using as a probe p-nitrophenylcarbamyl-amino acyl-tRNA. However, when the reactive p-nitrophenylcarbamyl group is in the amino terminal of the 3' end pentanucleotide derived from amino acyl-tRNA by ribonuclease T1, covalent binding does not take place. An interpretation of the results suggests that the 3' terminal fragment binds to an RNA rich part of the ribosome, which probably forms the P-site in the peptidyl transferase center.

Acyltransferases↗
PubMed1987

[Highly selective affinity labeling of DNA-dependent RNA-polymerase of bacteriophage T7].

T7 phage RNA polymerase was affinity labelled in the presence of its promoter by treatment with an ATP gamma-derivative (a phosphoamide obtained from 4-(N-chloroethyl, N-methyl)aminobenzylamine, or one of esters obtained from 2-methoxy-4-formylphenol, 4-formylphenol, and 2[N-(4-formylphenyl), N-methyl]-aminoethanol) followed by addition of [alpha-32P]GTP. The most efficient labelling took place with the alkylating phosphoamide reagent.

Affinity Labels↗
PubMed1976

Affinity labeling of a previously undetected essential lysyl residue in class I fructose bisphosphate aldolase.

The affinity label N-bromoacetylethanolamine phosphate (BrAcNHEtOP) has been used previously at pH 6.5 to identify His-359 of rabbit muscle aldolase as an active site residue. We now find that the specificity of the reagent is pH-dependent. At pH 8.5, alkylation with 14C-labeled BrAcNHEtOP abolishes both fructose-1,6-P2 cleavage activity and transaldolase activity. The stoichiometry of incorporation, the kinetics of inactivation, and the protection against inactivation afforded by a competitive inhibitor or dihydroxyacetone phosphate are consistent with the involvement of an active site residue. A comparison of 14C profiles obtained from chromatography on the amino acid analyzer of acid hydrolysates of inactivated and protected samples reveals that inactivation results from the alkylation of lysyl residues. The major peptide in tryptic digests of the inactivated enzyme has been isolated. Based on its amino acid composition and the known sequence of aldolase, Lys-146 is the residue preferentially alkylated by the reagent. Aldolase modified at His-359 is still subject to alkylation of lysine; thus Lys-146 and His-359 are not mutually exclusive sites. However, aldolase modified at Lys-146 is not subject to alkylation of histidine. One explanation of these observations is that modification of Lys-146 abolishes the binding capacity of aldolase for substrates and substrate analogs (BrAcNHEtOP), whereas modification of his-359 does not. Consistent with this explanation is the ability of aldolase modified at His-359 to form a Schiff base with substrate and the inability of aldolase modified at Lys-146 to do so. Therefore, Lys-146 could be one of the cationic groups that functions in electrostatic binding of the substrate's phosphate groups.

Affinity Labels↗
PubMed1991

Peptidyl (acyloxy)methyl ketones and the quiescent affinity label concept: the departing group as a variable structural element in the design of inactivators of cysteine proteinases.

(Acyloxy)methyl ketones, of general structure Z-[AA2]-[AA1]-CH2OCOAr, are potent inactivators of the cysteine proteinase cathepsin B. These reagents have been designed as affinity labels in which the dipeptidyl moiety serves as an affinity group (complementary to the S1 and S2 sites of the enzyme), while the (acyloxy)methyl ketone unit (-COCH2OCOR), containing a weak leaving group in the form of a carboxylate nucleofuge, functions as the potentially reactive entity that labels the enzyme. The inhibition is time dependent, active site directed, and irreversible. The apparent second-order rate constant kinact/Kinact, which characterizes the inhibition of cathepsin B by this series, spans several orders of magnitude and in certain cases exceeds 10(6) M-1 s-1. The activity of this series of inhibitors was found to be exquisitely sensitive to the nature of the carboxylate leaving group as well as the affinity group. A strong dependence of second-order inactivation rate on leaving group pKa was uncovered for Z-Phe-Ala (acyloxy)methyl ketones [log(k/K) = 1.1 (+/- 0.1) X pKa + 7.2 (+/- 0.4); r2 = 0.82, n = 26]. Heretofore in constructing affinity labels the choice of leaving group was quite restricted. The aryl carboxylate group thus offers considerable variation as a design element in that both its binding affinity and reactivity can be controlled by substituent effects. Specific peptidyl (acyloxy)methyl ketones thus represent prime examples of highly potent, chemically stable enzyme inhibitors with variable structural elements in both the affinity and departing groups.

Affinity Labels↗
PubMed1984

Reactivation of human placental 17 beta, 20 alpha-hydroxysteroid dehydrogenase: affirmation of affinity labeling principles.

Human placental 17 beta, 20 alpha-hydroxysteroid dehydrogenase was completely inactivated by the affinity alkylator, 3-bromoacetoxy-1,3,5(10)-estratrien-17-one (estrone 3-bromoacetate). The inactivated enzyme was then reactivated to 100% of the enzyme activity by base-catalyzed hydrolysis of the steroidalester-enzyme conjugate. After the reactivated enzyme was repurified by dialysis, re-inactivation studies were performed on it. The reactivated enzyme could not be re-inactivated by the original alkylator, estrone 3-bromoacetate. However, 16 alpha-bromoacetoxyestradiol-17 beta 3-methyl ether caused a loss of reactivated enzyme activity at a rate comparable to that for the native enzyme. These observations demonstrate that a specific amino acid modification within the enzyme active site was produced by estrone 3-bromoacetate alkylation and suggest that the conformation of the active center was essentially unaltered. Thus, these successful reactivation studies of 17 beta, 20 alpha-hydroxysteroid dehydrogenase affirm the specificity of affinity labeling. This methodology also offers a new tool to investigate the steroid binding regions of macromolecular proteins.

20-Hydroxysteroid Dehydrogenases↗
PubMed1987

Affinity labeling of the galactose/N-acetylgalactosamine-specific receptor of rat hepatocytes: preferential labeling of one of the subunits.

The galactose/N-acetylgalactosamine-specific receptor (also known as asialoglycoprotein receptor) of rat hepatocytes consists of three subunits, one of which [43 kilodalton (kDa)] exists in a greater abundance (up to 70% of total protein) over the two minor species (52 and 60 kDa). When the receptor on the hepatocyte membranes was photoaffinity labeled with an 125I-labeled high-affinity reagent [a triantennary glycopeptide containing an aryl azide group on galactosyl residues; Lee, R. T., & Lee, Y. C. (1986) Biochemistry 25, 6835-6841], the labeling occurred mainly (51-80%) on one of the minor bands (52 kDa). Similarly, affinity-bound, N-acetylgalactosamine-modified lactoperoxidase radioiodinated the same 52-kDa band preferentially. In contrast, both the photoaffinity labeling and lactoperoxidase-catalyzed iodination of the purified, detergent-solubilized receptor resulted in a distribution of the label that is comparable to the Coomassie blue staining pattern of the three bands; i.e., the 43-kDa band was the major band labeled. These and other experimental results suggest that the preferential labeling of the minor band and inefficient labeling of the major band on the hepatocyte membrane resulted from a specific topological arrangement of these subunits on the membranes. We postulate that in the native, membrane-bound state of the receptor, the 52-kDa minor band is topologically prominent, while the major (43 kDa) band is partially masked. This partial masking may result from a tight packing of the receptor subunits on the membranes to form a lattice work [Hardy, M. R., Townsend, R. R., Parkhurst, S. M., & Lee, Y. C. (1985) Biochemistry 24, 22-28].

Affinity Labels↗