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PubMed1987

Photoaffinity labelling of the TSH receptor on FRTL5 cells.

An investigation of the properties of TSH receptors on FRTL5 cells using affinity labelling with a 125I-labelled photoactive derivative of TSH is described. Our studies suggest that FRTL5 cells contain 2 principal types of cell surface TSH receptors. One form, probably a precursor, consists of a single polypeptide chain (Mr 120,000) with an intrachain loop of amino acids formed by a disulphide bridge. The other type of receptor consists of a water-soluble A chain (Mr 55,000) linked to an amphiphilic B chain (Mr 35,000) by a disulphide bridge. The 2 chain structure is probably derived from the single chain 120,000 protein by enzymatic cleavage of peptide sequences within the loop of amino acids formed by the intrachain disulphide bridge.

Affinity Labels↗
PubMed1994

Mapping of the immunophilin-immunosuppressant site of interaction on calcineurin.

The interaction of the immunosuppressive complexes cyclosporin A-cyclophilin A and FK506 binding protein-FK506 with the Ca(2+)- and calmodulin-dependent protein phosphatase calcineurin has been investigated by means of photoaffinity labeling and chemical cross-linking. Photolabeling of purified bovine brain calcineurin with the affinity label [O-[4-[4-(1-diazo-2,2,2-trifluoroethyl)benzoyl]aminobutanoyl]-D- serine8]cyclosporin in the presence of cyclophilin A results, in addition to the labeling of cyclophilin itself, in the transfer of some of the chemical probe to both the catalytic subunit A and the regulatory subunit B of calcineurin. Chemical cross-linking studies with disuccinimidyl suberate in the presence of either cyclophilin A, B, or C in complex with cyclosporin A or FK506 binding protein-FK506 result on the other hand in the apparently exclusive and strictly immunosuppressant-dependent formation of covalent immunophilin-calcineurin B subunit products. Cross-linking of immunophilins to calcineurin B subunit requires the presence of subunit A. In the present study, using a set of recombinant maltose-binding protein fusion products representing different stretches of the catalytic subunit A, we were able to map the minimal calcineurin A sequence necessary for immunophilin-ligand-calcineurin B interaction to occur.

Affinity Labels↗
PubMed2005

(-)-7'-Isothiocyanato-11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM841), a high-affinity electrophilic ligand, interacts covalently with a cysteine in helix six and activates the CB1 cannabinoid receptor.

The CB1 cannabinoid receptor has been shown to play important physiological roles in the central nervous system, as well as peripherally, and is a target for development of therapeutic medications. To gain insight on the ligand binding site(s) and structural features of activation, we designed and synthesized (-)-7'-isothiocyanato-11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM841), a classical cannabinoid affinity label that incorporates an isothiocyanate substituent as an electrophilic reactive group capable of interacting irreversibly with a suitably located and properly oriented nucleophilic amino acid residue at or near the binding site. To obtain evidence for the site of covalent attachment of AM841, C6.47, identified in part by interactive ligand docking, was mutated to serine, alanine, and leucine to reduce or eliminate the nucleophilic character. Wild-type (WT) and mutant CB1 receptors were evaluated for their abilities to recognize a series of cannabinergic ligands. Each bound comparably to WT, excluding C6.47L, which displayed a reduced affinity for 3H-labeled (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol (CP55940), AM841, 11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM4056), and (-)-7'-bromo-11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM4043) and an improvement in affinity for (-)-trans-delta9-tetrahydrocannabinol (delta9-THC). The affinity of 3H-labeled [2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](naphthyl)methanone (WIN55212-2) was unchanged across all mutants. It is noteworthy that AM841 was shown to bind irreversibly to WT CB1 but exhibited no covalent attachment with the mutants and behaved as an agonist suggesting irreversible attachment to C6.47 maintains CB1 in its active state. The evidence presented identifies C6.47 as the site of covalent bond formation with AM841 and combined with the binding data fully supports the molecular modeling. These studies present the first report of tandem applications of affinity labeling, site-directed mutagenesis, and interactive ligand docking for CB1.

Affinity Labels↗
PubMed1998

Analysis of the molecular mechanism of substrate-mediated inactivation of leukotriene A4 hydrolase.

The bifunctional leukotriene A4 hydrolase catalyzes the final step in the biosynthesis of the proinflammatory leukotriene B4. During exposure to the substrate leukotriene A4, a labile allylic epoxide, the enzyme is gradually inactivated as a consequence of the covalent binding of leukotriene A4 to the active site. This phenomenon, commonly referred to as suicide inactivation, has previously been rationalized as a mechanism-based process in which the enzyme converts the substrate to a highly reactive intermediate within an activated enzyme-substrate complex that partitions between covalent bond formation (inactivation) and catalysis. To further explore the molecular mechanism of the self-inactivation of leukotriene A4 hydrolase by leukotriene A4, we prepared and analyzed mutated forms of the enzyme that were either catalytically incompetent or fully active but resistant toward substrate-mediated inactivation. These mutants were treated with leukotriene A4 and leukotriene A4 methyl and ethyl esters and subjected to differential peptide mapping and enzyme activity determinations, which showed that inactivation and/or covalent modification can be completely dissociated from catalysis. Our results, together with recent findings described in the literature, argue against a mechanism-based model for suicide inactivation. We conclude that the collected data on the substrate-mediated inactivation of leukotriene A4 hydrolase best conforms to an affinity-labeling mechanism.

Affinity Labels↗
PubMed1981

Selective labeling of the delta subunit of the acetylcholine receptor by a covalent local anesthetic.

A radioactive photoaffinity derivative of the potent local anesthetic trimethisoquin, 5-azido[3H]trimethisoquin, was used to label the acetylcholine receptor from Torpedo marmorata electric organ. The product labeled the 66 000-dalton (delta) subunit of the receptor with the selectivity expected for an affinity label of the site for noncompetitive blockers. That is, the labeling was enhanced by cholinergic agonists and inhibited by other noncompetitive blockers. The 40 000-dalton (alpha)( subunit of the receptor was labeled in a manner consistent with the attachment of 5-azido[3H]trimethisoquin to an acetylcholine binding site as the incorporation of radioactivity into the alpha chain was inhibited by cholinergic agonists and antagonists, such as carbamylcholine, d-tubocurarine, and alpha-bungarotoxin. The reversible binding of [3H]phencyclidine, a potent noncompetitive blocker, to acetylcholine receptor rich membranes resembled qualitatively and quantitatively the 5-azido[3H]trimethisoquin labeling of the delta subunit and was inhibited by the prior covalent labeling of the membranes with nonradioactive 5-azidotrimethisoquin. Thus, 5-azido[3H]-trimethisoquin labels at least a portion of the binding site for noncompetitive blockers at the level of the delta subunit. The functional significance of this site and the use of 5-azidotrimethisoquin in the study of acetylcholine receptor structure and function are discussed.

Acetylcholine↗
PubMed1984

Identification of the lysine and tyrosine peptides labeled by 5'-p-fluorosulfonylbenzoyladenosine in the NADH inhibitory site of glutamate dehydrogenase.

5'-p-Fluorosulfonylbenzoyladenosine (5'-FSBA) is a specific affinity label for the inhibitory NADH site of bovine liver glutamate dehydrogenase. Reaction of the enzyme with 5'-FSBA results in the loss of inhibition by high concentrations of NADH with covalent attachment of 0.53 sulfonylbenozyladenosine/subunit, i.e. modification of three subunits of the hexameric enzyme. Equal amounts of N epsilon-(4-carboxybenzenesulfonyl)lysine (Lys-(CBS] and O-(4-carboxybenzenesulfonyl)tyrosine (Tyr-(CBS] are found throughout the course of the reaction (Saradambal, K. V., Bednar, R. A., and Colman, R. F. (1981) J. Biol. Chem. 256, 11866-11872). Modified enzyme, prepared by incubating 2 mg/ml glutamate dehydrogenase with 0.3 mM 3H-labeled 5'-FSBA at pH 8 for 1 h, was carboxymethylated and digested with thermolysin. Two nucleosidyl peptides were isolated by a combination of chromatography on phenyl boronate-agarose, high-performance liquid chromatography in ammonium bicarbonate and high-performance liquid chromatography in trifluoroacetic acid. By comparison of the amino acid analysis and NH2-terminal residue of each isolated peptide with the known amino acid sequence of the enzyme, the peptides were identified as Leu-Gly-Arg-Lys(CBS) and Ile-Gly-His-Tyr(CBS)-Asp. These sequences correspond to residues 417-420 and 187-191, respectively. Lys-420 and Tyr-190 of glutamate dehydrogenase react with 5'-FSBA, and both are apparently located in the NADH inhibitory site.

Adenosine↗
PubMed1989

Aspartyl peptide labeled by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate in the allosteric ADP site of pig heart NAD+-dependent isocitrate dehydrogenase.

The nucleotide affinity label 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate (2-BDB-TADP) reacts covalently with pig heart NAD+-dependent isocitrate dehydrogenase with a limiting value of 75% inactivation and loss of ADP activation concomitant with incorporation of about 1 mol of reagent/mol of average enzyme subunit (Huang, Y.-C., Bailey, J. M., and Colman, R. F. (1986) J. Biol. Chem. 251, 14100-14107). Complete protection against the functional changes is provided by ADP + Mn2+, and reagent incorporation is decreased to about 0.5 mol/mol of average enzyme subunit. We have now identified the critical modified peptide by comparison of the peptides labeled by 2-BDB-TADP at pH 6.8 in the absence and presence of ADP + Mn2+. After removal of excess reagent, modified enzyme was treated with [3H]NaBH4 to reduce the keto groups of the reagent and introduce a radioactive tracer into the reagent which is covalently linked to the protein. Following carboxymethylation and digestion with trypsin, the specific modified peptide was isolated using two successive high performance liquid chromatography steps: 1) 0.1% trifluoroacetic acid with an acetonitrile gradient; and 2) 20 mM ammonium acetate, pH 5.8, with an acetonitrile gradient. Gas phase sequencing gave the modified peptide Leu-Gly-Asp-Gly-Leu-Phe-Leu-Gln in which aspartic acid is the target of 2-BDB-TADP. Isolation of the corresponding tryptic peptide from unmodified enzyme yielded the sequence Leu-Gly-Asp-Gly-Leu-Phe-Leu-Gln-CmCys-CmCys-Lys. Isocitrate dehydrogenase is composed of three distinct subunits (alpha, beta, and gamma), separable by chromatofocusing in urea and identified by analytical gel isoelectric focusing. The evidence indicates that the specific peptide labeled by 2-BDB-TADP, which is at or near the ADP site, can be derived from the gamma subunit.

Adenosine Diphosphate↗
PubMed1990

[Highly selective labeling of the E. coli RNA-polymerase promoter complex with reactive derivatives of oligonucleotide primers of various specificity].

A technique of highly selective affinity labelling, which includes covalent modification of the enzyme-T7A2 promoter complex with reactive oligonucleotide derivatives and subsequent elongation of the attached oligonucleotide residue with a radioactive substrate was used to study the product-binding site of E. coli RNA polymerase. Different oligonucleotides complementary to the T7A2 promoter (with lengths ranging from 2 to 8 residues) containing 5'-terminal phosphorylating, alkylating or aldehyde groups were used for the labelling. The procedure resulted in labelling DNA and beta-, beta'- or sigma-subunits of the enzyme, which are therefore believed to contact with growing RNA in the course of initiation. Consideration of the labelling patterns as a functions of the oligonucleotide's length as well as of the structure and chemical specificity of the reactive groups led to a tentative topographic scheme of the RNA polymerase product-binding region.

Affinity Labels↗
PubMed2003

Glutathione s-transferase omega 1-1 is a target of cytokine release inhibitory drugs and may be responsible for their effect on interleukin-1beta posttranslational processing.

Stimulus-induced posttranslational processing of human monocyte interleukin-1beta (IL-1beta) is accompanied by major changes to the intracellular ionic environment, activation of caspase-1, and cell death. Certain diarylsulfonylureas inhibit this response, and are designated cytokine release inhibitory drugs (CRIDs). CRIDs arrest activated monocytes so that caspase-1 remains inactive and plasma membrane latency is preserved. Affinity labeling with [(14)C]CRIDs and affinity chromatography on immobilized CRID were used in seeking potential protein targets of their action. Following treatment of intact human monocytes with an epoxide-bearing [(14)C]CRID, glutathione S-transferase (GST) Omega 1-1 was identified as a preferred target. Moreover, labeling of this polypeptide correlated with irreversible inhibition of ATP-induced IL-1beta posttranslational processing. When extracts of human monocytic cells were chromatographed on a CRID affinity column, GST Omega 1-1 bound selectively to the affinity matrix and was eluted by soluble CRID. Recombinant GST Omega 1-1 readily incorporated [(14)C]CRID epoxides, but labeling was negated by co-incubation with S-substituted glutathiones or by mutagenesis of the catalytic center Cys(32) to alanine. Peptide mapping by high performance liquid chromatography-mass spectrometry also demonstrated that Cys(32) was the site of modification. Although S-alkylglutathiones did not arrest ATP-induced IL-1beta posttranslational processing or inhibit [(14)C]CRID incorporation into cell-associated GST Omega 1-1, a glutathione-CRID adduct effectively demonstrated these attributes. Therefore, the ability of CRIDs to arrest stimulus-induced IL-1beta posttranslational processing may be attributable to their interaction with GST Omega 1-1.

Adenosine Triphosphate↗
PubMed1992

Suggested mechanism for the modulation of the activity of NAD(P)H:quinone acceptor oxidoreductase (DT-diaphorase) by menadione: interpretation of the effect of menadione on 5'-[p-(Fluorosulfonyl)benzoyl]adenosine labeling of rat liver NAD(P)H:quinone acceptor oxidoreductase.

5'-[p-(Fluorosulfonyl)benzoyl]adenosine (5'FSBA) was previously shown to be an active site-directed affinity label of rat liver NAD(P)H:quinone acceptor oxidoreductase [Mol. Pharmacol. 35:818-822 (1989)]. Our recent study revealed that menadione, the substrate of this quinone reductase, had a magnifying effect on inactivation of the enzyme by 5'-FSBA. The dissociation constant for the initial reversible enzyme-inhibitor complex was significantly lower and the rate of inactivation was increased when menadione was present during the incubation. However, [14C]5'FSBA labeling was reduced in the presence of menadione. These results are presented and a possible mechanism for the enzyme is discussed.

Adenosine↗
PubMed1979

Alkylation of estradiol 17 beta-dehydrogenase from human placenta with 3-chloroacetylpyridine--adenine dinucleotide phosphate.

3-Chloroacetylpyridine--adenine dinucleotide phosphate is both active as a hydride acceptor and inactivates estradiol 17 beta-dehydrogenase. This coenzyme analogue behaves like an affinity label. The inactivation kinetics are discussed in relation to those observed with 3-chloroacetylpyridine--adenine dinucleotide. The pH dependence of the rate of inactivation, in combination with determination of the number of reactive cysteine residues, pointed to the alkylation of one cysteine residue/subunit. The stoichiometry was one molecule of dinucleotide per subunit and no cooperativity was detected. When 14C-labeled dinucleotide was used, the 14C label was found mainly in one peptide, accounting for 90% of the incorporated radioactivity, whereas in previous work it had been shown that 3-chloroacetylpyridine--adenine dinucleotide is an affinity reagent which labels three peptides.

17-Hydroxysteroid Dehydrogenases↗
PubMed1988

Structural determinants of ligand recognition by type I insulin-like growth factor receptors: use of semisynthetic insulin analog probes.

We undertook a systematic analysis of the structural determinants necessary for ligand recognition by the type I insulin-like growth factor (IGF) receptor by investigating the binding of semisynthetic insulin analogs to IGF receptors from human placental cell membrane fragments. Analogs were prepared by synthetic and semisynthetic methods. Three groups of insulin analogs were synthesized: the first group contained insulin analogs modified at the amino-terminal position of the insulin A chain and included acetyl-insulin and human proinsulin; the second group included analogs in which B chain residues B26-B30 [despentapeptide insulin (DPI)], B25-B30 (deshexapeptide insulin), and B24-B30 (desheptapeptide insulin) were removed; the third group contained insulin analogs in which B chain residues B26-B30 were removed (DPI) and phenylalanine(B25) substituted with other amino acids, including alanine, serine, leucine, and tyrosine. Half-maximal inhibition of binding of radiolabeled IGF-I to placental cell membrane fragments was used as an index of relative binding affinity (K1/2). To determine further if semisynthetic insulin analogs bound to the type I IGF receptor, placental membrane fragments were affinity labeled with radiolabeled IGF-I in the presence and absence of submaximal concentrations of unlabeled hormone, insulin, or semisynthetic analogs, and the labeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Insulin had a 500 times lower affinity for the type I IGF receptor than IGF-I [K1/2 = 140 +/- 69 nM (mean +/- SD)] whereas proinsulin and acetyl insulin had a more than 100 times lower affinity than insulin for this receptor type. Removal of insulin B chain amino acid residues 26-30 (DPI) did not negatively affect the binding of the insulin-derived peptide and actually increased the apparent affinity of ligand-receptor association approximately 2-fold. However, further removal of phenylalanine(B25) (deshexapeptide insulin) and phenylalanine(B24) (desheptapeptide insulin) decreased the binding of ligand to the type I IGF receptor progressively by several orders of magnitude. Substitution of phenylalanine(B25) of DPI with tyrosine, a substitution that actually increased the homology of this analog to IGF-I, resulted in a 4- to 5-fold increase in the relative apparent affinity of the analog for the type I IGF receptor (K1/2 = 31 +/- 4 nM). On the other hand, substitution of phenylalanine(B25) with alanine, serine, and leucine decreased the relative apparent binding affinity approximately 2- to 8-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity Labels↗
PubMedDate not supplied

Synthesis and characterization by 1H and 13C nuclear magnetic resonance spectroscopy of 17 alpha-cyano, 17 alpha-aminomethyl, and 17 alpha-alkylamidomethyl derivatives of 5 alpha-dihydrotestosterone and testosterone.

17 alpha-Aminomethyl, 17 alpha-acetamidomethyl, and 17 alpha-hemiglutaramidomethyl derivatives of dihydrotestosterone and testosterone have been prepared by hydrocyanation of 3,3'-(ethylenedioxy)-5 alpha-androstan-17-one and 3,3'-ethylenedioxyandrost-5-en-17-one, reduction of the corresponding acetylated 17 alpha-cyanohydrins with lithium aluminium hydride, and acylation of the resulting 17 alpha-aminomethyl derivatives with either acetic anhydride or the mono acid chloride of glutaric acid mono methyl ester. Saponification of the 17 alpha-hemiglutaramidomethyl methyl esters gave the corresponding hemiglutaramido derivatives, while acid hydrolysis of the 3-ethylene ketal group of 17 alpha-acetamidomethyl and 17 alpha-hemiglutaramidomethyl derivatives regenerated the 3-oxo and 3-oxo-4-ene functions. The 17 alpha-configuration of 17-substituted steroids was determined by 1H and 13C NMR and confirmed by comparing with NMR data for 17 alpha- and 17 beta-cyano-17-hydroxyandrost-4-en-3-one, 17 beta-cyano-3,3'-(ethylenedioxy)androst-5-en-17-ol, 17 alpha-alkynyl, and 17 alpha-hexanoic derivatives of dihydrotestosterone and testosterone, of known 17-configurations. Several ambiguous assignments of 13C NMR signals of 17 alpha-substituted steroids and unsubstituted 17 beta-hydroxy or 17-oxo precursors have been resolved using steroid analogs deuterated at positions C5-7, or C16 for androstane derivatives, and at positions C6-7, or C7 for androstene derivatives. 17 alpha-Aminomethyl and 17 alpha-alkylamidomethyl derivatives of dihydrotestosterone and testosterone are useful intermediates for the access to potential ligands of androgen-binding proteins necessary for affinity chromatography purification or affinity-labeling experiments.

Acetamides↗
PubMed1994

Photoaffinity labeling of lipoproteins in human cerebrospinal fluid with a heterobifunctional derivative of galactosylsphingosine.

The binding of a galactosylceramide (GalCer) analog to proteins in normal human cerebrospinal fluid (CSF) was examined by photo-affinity labeling using a radioiodolabeled 2-(p-azido-salicylamido) ethyl-1,3-dithiopropionate (ASD) derivative of galactosylsphingosine (GalSph) as a probe. The affinity-bound peptides appeared at 66, 36 and 28 kDa as radiolabeled bands. The latter two peptides, 36 and 28 kDa, were independently identified by immunostaining of the isolated peptides using biotinylated GalSph, and immobilized Avidin, and by immunoprecipitation of the photolabeled peptide, to apolipoproteins (ALPs) E and A-I, respectively. The direct binding of the GalCer analog to these apo-peptides suggested that the existence in the body fluids or the transfer between the fluids and cells of the glycolipid was related to high density lipoprotein (HDL) constructions.

Affinity Labels↗
PubMed1992

Nerve growth factor-activated protein kinase N. Characterization and rapid near homogeneity purification by nucleotide affinity-exchange chromatography.

Protein kinase N (PKN) is a protein kinase rapidly activated by nerve growth factor (NGF) and other agents in PC12 pheochromocytoma and additional cell types. PKN is selectively inhibited by purine analogs, and this property has served both as a diagnostic for PKN activity and to establish its apparent involvement in certain pathways of the NGF mechanism of action. The present work has focused on further characterization, identification, and purification of NGF-activated PKN. We show here that PKN can be substantially enriched by elution from ion exchange resins with ATP. We exploited this novel technique (nucleotide affinity exchange chromatography) to devise two alternative isolation schemes for PKN. One utilizes sequential chromatographic steps and provides a preparation that is apparently 60% homogeneous for PKN and represents a total enrichment of approximately 10,000-fold. The other is a single column procedure and includes prewashes with NAD. This method yields material that is about 5-10% homogeneous for PKN, requires about 1 h, and can be applied to multiple samples in parallel. The ATP elution technique furthermore distinguishes NGF-regulated from basal PKN activity and thereby suggests the presence of distinct PKN isoforms. The applications of sucrose gradient centrifugation, gel filtration chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/silver staining, affinity labeling with 8-azido-ATP/SDS-PAGE, and autophosphorylation (after SDS-PAGE, blotting and renaturation) all indicate that PKN has an apparent molecular mass of 45-47 kDa and is mainly monomeric in solution. These and additional properties appear to distinguish PKN from many previously described protein kinases.

Adenosine Triphosphate↗
PubMed1990

Template primer-dependent binding of 5'-fluorosulfonyl-benzoyldeoxyadenosine by Escherichia coli DNA polymerase I. Identification of arginine 682 as the binding site and its implication in catalysis.

We have labeled a template primer-dependent substrate deoxynucleoside triphosphate binding domain in Escherichia coli DNA polymerase I using an affinity labeling analogue of dATP, the 5'-fluorosulfonylbenzoyldeoxyadenosine (FSBdA). Using enzyme-template primer complex as a test system, we find that FSBdA-mediated inactivation occurs only when the template in the enzyme-template primer complex is poly(dT).(dA)10. A ribonucleotide analogue, 5'-fluorosulfonylbenzoyladenosine (FSBA) is not an effective inactivator under these conditions. In the absence of template primer, however, deoxyribonanalogue (FSBdA) irreversibly inactivates polymerase activity with characteristics similar to those reported for FSBA (Pandey, V.N., and Modak, M.J. (1988) J. Biol. Chem. 263, 6068). Binding stoichiometric studies in the presence and absence of template primer revealed that only 1 mol of FSBdA is incorporated per mol of enzyme which results in complete inactivation. The site of FSBdA action was investigated by comparative tryptic peptide mapping, followed by amino acid composition analysis of the modified peptide. Arginine 682 was found to be the target of FSBdA reactivity. We therefore conclude that the domain containing Arg-682 plays a major role in template-dependent dNTP binding and polymerization.

Adenosine↗
PubMed1989

Purification and characterization of angiotensin-binding protein from porcine liver cytosolic fraction.

A protein that binds angiotensins with high affinity was found in porcine liver cytosol, purified to apparent homogeneity and characterized. The protein was named soluble angiotensin-binding protein (sABP) to distinguish it from angiotensin II receptors present on plasma membranes. Purification of the protein was achieved by a combination of ammonium sulfate fractionation, hydrophobic chromatography, ion-exchange chromatography, hydroxylapatite column chromatography and Mono Q ion-exchange chromatography. Specific angiotensin-binding activity, as measured using 125I-angiotensin II, was enriched more than 3400-fold. SDS/polyacrylamide gel electrophoresis of the purified sABP yielded a single 75-kDa protein band, in good agreement with the molecular mass estimated by affinity labeling. sABP was very similar to the angiotensin II receptor in its sensitivity to reducing agents and in its affinities for angiotensin analogues ([Sar1, Ala8]angiotensin II greater than angiotensin III greater than angiotensin II greater than angiotensin I), suggesting a possible similarity between the ligand-binding sites of sABP and the angiotensin II receptor. To obtain a clue to its physiological role(s), we examined the tissue distribution of sABP and found that this protein is widely distributed not only in the peripheral organs but also in the brain.

Affinity Labels↗
PubMed1981

The specificity of cathepsin B.

Peptidyl chloromethyl ketones, largely derived from arginine, inactivate cathepsin B (beef spleen) at rates that vary 300 fold according to sequence, but the residue in the P1 position is not responsible for this variation since homoarginine or nitroarginine in this position provide inhibitors as good or better than those containing arginine. Peptidyl chloromethyl ketones containing hydrophobic residues such as phenylalanine or valine in the P2 and P3 position are the most effective inhibitors of the group. Cystamine (bis-aminoethyl disulfide) inactivates cathepsin B by formation of a mixed disulfide. Derivatives of cystamine containing phenylalanine, such as bis-N,N'-Phe-cystamine and bis-N,N'-Ala-Ala-Phe-cystamine are more effective and represent a new class of affinity labels for cathepsin B. Immobilized peptidyl cystamine derivatives can be used for the purification of cathepsin B by covalent affinity chromatography. Cathepsin B from beef spleen has a pronounced carboxydipeptidase action on glucagon as described for the human liver enzyme. This action can be conveniently followed by high pressure liquid chromatography.

Affinity Labels↗