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PubMed1983

Affinity labeling of tubulin's exchangeable guanosine 5'-triphosphate binding site.

Tubulin requires GTP for maximal rate and extent of polymerization into microtubules. The localization of the guanine nucleotide in the microtubule was examined by preparing affinity probes that would permit tubulin polymerization prior to their covalent coupling to amino acids in tubulin's exchangeable GTP binding site. Two different hydrolyzable GTP analogues with modified ribose moieties, 3'-p-azidobenzoyl)-GTP and the periodate oxidation product of GTP, 2-(guanylformylmethoxy)-3-(triphospho)propanal, were isolated by thin-layer chromatography and high-voltage electrophoresis and identified by ultraviolet and infrared spectroscopy. The analogues bind to the tubulin molecule and promote polymerization. After tubulin polymerization and isolation of microtubules, the [3H]GTP analogues were covalently coupled to tubulin by NaBH4 reduction or UV irradiation. The microtubules possessed about 1 mol of acid-precipitable 3H-labeled nucleotide/mol of tubulin dimer. Separation of the subunits showed that the nucleotide analogues were associated with both alpha and beta subunits of tubulin in nearly equal amounts. The binding of analogues to both alpha and beta subunits was saturable and competitive with GTP. Cyanogen bromide cleavage of both alpha and beta subunits showed that the 3H-labeled nucleotide was associated with a single molecular weight species of similar size (approximately 10 000) from each subunit. Two-dimensional electrophoresis of chymotryptic peptides from both (alpha and beta) cyanogen bromide fragments showed that the 3H-labeled nucleotide was associated with a peptide of nearly identical migration properties from both subunits. These results suggest that a similar peptide segment of both alpha- and beta-tubulin has the ability to bind GTP. Furthermore, this peptide was localized to the amino-terminal one-third of the tubulin molecule.

Affinity Labels↗
PubMed1988

[3H]Ro 16-6491, a selective probe for affinity labelling of monoamine oxidase type B in human brain and platelet membranes.

[3H]Ro 16-6491 [N-(2-aminoethyl)-p-chlorobenzamide HCl], a reversible "mechanism-based" inhibitor of monoamine oxidase (MAO) type B, binds selectively and with high affinity to the active site of MAO-B in brain and platelet membranes. Under normal conditions, the binding of [3H]Ro 16-6491 is fully reversible. However, [3H]Ro 16-6491 could be irreversibly bound (covalently) to membranes by the addition of the reducing agent NaBH3CN to the sample and adjusting to pH 4.5 with acetic acid. No irreversible labelling occurred in the absence of NaBH3CN and at neutral pH. The presence of the irreversible MAO-B inhibitor l-deprenyl completely abolished the irreversible labelling of the membranes by [3H]Ro 16-6491. The selective inactivation of MAO-B, e.g., by l-deprenyl prevented the covalent incorporation of [3H]Ro 16-6491 whereas selective inhibition of the MAO-A by clorgyline was without effect. The covalent linkage to membranes of unlabelled Ro 16-6491 and Ro 19-6327 (a selective and reversible MAO-B inhibitor closely related to Ro 16-6491) after the addition of NaBH3CN at pH 4.5 irreversibly inactivated MAO-B activity whereas MAO-A activity was unaffected. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of labelled membranes showed that [3H]Ro 16-6491 was incorporated into a single polypeptide with a molecular mass identical to the one labelled by [3H]pargyline (58 kilodaltons). Our results indicate that the polypeptide that is covalently labelled by [3H]Ro 16-6491 corresponds to one of the two MAO-B subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed2005

Affinity labeling mu opioid receptors with novel radioligands.

1. A series of novel opiate ligands based upon 6alpha-naloxamine have been examined in opioid receptor binding assays. 2. Coupling an ethylamine spacer alone to 6-alpha-naloxamine gave a compound with relatively poor affinity for mu opioid receptors compared to naloxone, although it retained high affinity for kappa1 opioid receptors. Coupling a benzoyl group significantly increased the affinity. The presence at the 4-position of the benzoyl moiety of an amino-(NalAmiBen) or an azido-substituent (NalAziBen) did not significantly effect the affinity at mu receptors. However, iodinating the benzoyl moiety at the 3-position increased the affinity of the derivatives. 3. Two compounds were radiolabeled and evaluated in receptor binding assays. Both radioligands labeled sites in CHO cells stably transfected with the mouse MOR-1 clone. The amino coupound [125I]NalAmiBen and the azido derivative [125I]NalAziBen reversibly bound to membranes from CHO cells transfected with MOR-1 with high affinity in the dark. Exposure of [125I]NalAmiBen to UV did not alter the reversibility of binding, but exposure of [125I]NalAziBen to UV light led to the covalent coupling of the radioligand to the receptor. When run on SDS-PAGE, [125I]NalAziBen binding showed a band at approximately 70-80 kDa. A control corresponding to nonspecific binding failed to reveal any labeling. No bands were observed from membranes labeled with [125I]NalAmiBen.

Affinity Labels↗
PubMed1999

Affinity labeling of the androgen receptor with nonsteroidal chemoaffinity ligands.

We synthesized a series of potential chemoaffinity ligands for the androgen receptor (AR) by means of structural modifications of bicalutamide, a known nonsteroidal antiandrogen used in the treatment of hormone-dependent prostate cancer. We determined AR binding affinities of these ligands, identified chemoaffinity ligands by exchange assays, and confirmed irreversible binding to the AR by Scatchard analyses. AR binding affinity was determined in a competitive binding assay with a radiolabeled high-affinity AR ligand, [3H]mibolerone ([3H]MIB). For exchange assays, AR were incubated with an excess of each ligand, and then adsorbed onto hydroxyapatite (HAP). HAP-bound AR then were incubated with [3H]MIB to determine the remaining exchangeable specific binding sites. To determine the concentration of binding sites (Bmax), using Scatchard analysis, AR were incubated with a fixed concentration of ligand and increasing [3H]MIB concentrations. The ligands showed a wide range of AR binding affinities. In the exchange assays, three isothiocyanate derivatives of R-bicalutamide, the p-isothiocyanate (R-4), the p-thio-isothiocyanate (R-6), and the m-isothiocyanate (R-3), reduced exchangeable specific binding of [3H]MIB by 85, 84, and 50%, respectively. The S-isomer of p-thio-isothiocyanate (S-6), which showed 700-fold lower AR binding affinity than R-6, did not reduce exchangeable specific binding of [3H]MIB. In Scatchard analyses, the isothiocyanate derivatives R-3, R-4, and R-6 showed significant and progressive reduction in Bmax at increasing concentrations. The results indicate that initial specific reversible AR binding was required for subsequent covalent labeling, and that R-3, R-4, and R-6 bound the AR specifically and irreversibly. These isothiocyanate derivatives of R-bicalutamide are the first specific chemoaffinity ligands for the AR, and will provide valuable tools for the molecular characterization of the ligand binding domain of the AR.

Affinity Labels↗
PubMed1988

Affinity labeling of the ATP-binding site of type II calmodulin-dependent protein kinase by 5'-p-fluorosulfonylbenzoyl adenosine.

Modification of the type II calmodulin-dependent protein kinase by 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) resulted in a time-dependent inactivation of the enzyme. The reaction followed pseudo-first-order kinetics and showed a nonlinear dependence on reagent concentration. The rate of inactivation was sensitive to Mg2+- and calmodulin-induced conformational changes on the enzyme. However, the enhancing effects of these ligands were not additive; indeed, the kinetic parameters of the Mg2+-stimulated inactivation reaction with FSBA (Kinact = 2.4 mM; kappa max = 0.12 min-1) were almost unaffected by the simultaneous addition of calmodulin (Kinact = 1.5 mM; kappa max = 0.086 min-1). Protection from inactivation by FSBA was provided by Mg2+-ADP which is consistent with modification of the catalytic site. An analysis of the protective effect of Mg2+-ADP in the absence (Kd = 590 microM) and presence (Kd = 68 microM) of calmodulin demonstrated that binding of the modulator protein to the enzyme increases the affinity of the protein kinase for nucleotides. Modification by FSBA resulted in labeling of both Tyr and Lys residues but only labeling of Lys was decreased by Mg2+-ADP which is consistent with the hypothesis that a conserved Lys residue is important in nucleotide binding to the protein kinase. However, the kinetic results of the inactivation reaction suggest that this Lys is not involved in mediating the calmodulin-promoted increase in the affinity of the enzyme for Mg2+-nucleotide complexes.

Adenosine↗
PubMed1993

Characterization of the cysteine proteinases of the common liver fluke Fasciola hepatica using novel, active-site directed affinity labels.

The excreted/secreted proteinases of adult and juvenile Fasciola hepatica maintained in vitro were found to hydrolyse the fluorogenic substrates Cbz-Phe-Arg- and Cbz-Arg-Arg-NHMec. This activity was demonstrated to have a classical cysteine proteinase inhibitor profile, with turn-over of both substrates being blocked by pre-incubation with E64 and peptidyl diazomethanes. The Cbz-Arg-Arg-NHMec hydrolysing activity of the mature fluke exhibited an alkaline stability not characteristic of its mammalian lysosomal counterparts. Further, the biotinylated affinity reagents biotin-Phe-Ala-CHN2 and biotin-Phe-Cys(SBzyl)-CHN2 were used to label and characterize these cysteine proteinases in terms of apparent molecular weight and subsite specificity. Adult fluke media were found to contain four species of molecular weights 66, 58, 50 and 25-26 kDa; juvenile media contained three species of molecular weights 66, 54 and 25-26 kDa. The major 25-26 kDa cysteine proteinase common to both stages was shown to have a subsite specificity similar to that of mammalian cathepsin B.

Affinity Labels↗
PubMed1992

Discriminative affinity labelling of opioid receptors by enkephalin and morphiceptin analogues containing 3-nitro-2-pyridinesulphenyl-activated thiol residues.

The thiol groups of leucinthiol, cysteamine and cysteine incorporated into opioid peptides enkephalin and morphiceptin were activated by the 3-nitro-2-pyridinesulphenyl (Npys) group to form mixed disulphides highly reactive to a free thiol. Enkephalin analogues containing Npys-leucinthiol or -cysteine at positions 4, 5 and 6 exhibited high affinities for both mu and delta receptors, while morphiceptin analogues containing Npys-cysteine at positions 4 and 5 showed relatively weak affinity only for mu receptors. When these S-activated opioid peptides were incubated with rat brain membrane preparations, it was found, by binding assay using radiolabelled and non-labelled [D-Ala2,MePhe4,Gly-ol5]enkephalin, that they label mu opioid receptors in a dose-dependent manner. The concentrations required to label half of the receptors were 0.2-2 microM for enkephalins and 10-30 microM for morphiceptins. These results suggested that the thiol group labelled by S-activated enkephalins and morphiceptins is present in the ligand binding site of receptor protein, but not in GTPase-binding protein.

Affinity Labels↗
PubMed2006

New role for an old probe: affinity labeling of oxylipid protein conjugates by N'-aminooxymethylcarbonylhydrazino d-biotin.

Free radicals, electrophiles, and endogenous reactive intermediates are generated during normal physiological processes and are capable of modifying DNA, lipids, and proteins. However, elevated levels of oxidative modifications of proteins by reactive species are implicated in the etiology and pathology of oxidative stress-mediated diseases, neurodegeneration, and aging. A mass spectrometry-based approach is reported that aids to the identification and characterization of carbonyl-modified proteins. The method uses N'-aminooxymethylcarbonylhydrazino d-biotin, a biotinylated hydroxylamine derivative that forms an oxime derivative with the aldehyde/keto group found in oxidatively modified proteins. In this paper, the method is demonstrated for one class of carbonyl-modified proteins, namely, oxylipid peptide and protein conjugates formed by Michael addition-type conjugation reactions of alpha,beta-unsaturated aldehydic lipid peroxidation products with nucleophilic peptide side chains. This new application of an "old" probe, which has been used for the detection of abasic sites in DNA strands, introduces a biotin moiety into the oxylipid peptide conjugate. The biotin-modified oxylipid peptide conjugate is then amenable to enrichment using avidin affinity capture. The described method represents an attractive alternative to hydrazine-based derivatization methods for oxidized peptides and proteins because the reduction step necessary for the transformation of the hydrazone bond to the chemically more stable hydrazine bond can be omitted. Tandem mass spectrometry of the labeled oxylipid peptide conjugates indicates that the biotin moiety is at least partially retained on the fragment ion during the collisionally induced dissociation experiments, a prerequisite for the use of automated database searching of uninterpreted tandem mass spectra. The reported approach is outlined for the detection, identification, and characterization of oxylipid peptide conjugates, but the labeling chemistry may also be applicable to other carbonyl-modified proteins.

Affinity Labels↗
PubMed1998

Autonomous folding of the recombinant large cytoplasmic loop of sarcoplasmic reticulum Ca2+-ATPase probed by affinity labeling and trypsin digestion.

Recombinant large cytoplasmic loop (LCL, residues 329-740) of sarcoplasmic reticulum Ca2+-ATPase, expressed in and purified from Escherichia coli, comprises most of the active site and binds ATP [Moutin, M.-J., Cuillel, M., Rapin, C., Miras, R., Anger, M., Lompré, A.-M. & Dupont, Y. (1994) J. Biol. Chem. 269, 11147-11154]. In this study, we show that fluorescein-5' isothiocyanate (FITC) specifically labels the same lysine residue as in the native Ca2+-ATPase (Lys515), with similar kinetics and pH dependence. ATP blocks the reaction with the lysine residue, but at higher concentrations compared with those for the native pump, in agreement with the lower ATP-binding affinity found previously. Graded tryptic digestion of LCL shows that favored cleavage is at the T1 site and that the N-terminal 75% of LCL are resistant to trypsin, as is native Ca2+-ATPase. Other experiments reveal differences to the native pump. (a) FITC derivatizes some -SH groups of LCL. (b) The C-terminal 25% of the polypeptide is susceptible to end-clipping by trypsin. (c) 2',3'-O-(2,4,6-trinitrophenyl)-ATP fails to specifically label the LCL (on the equivalent of Lys492), although it binds tightly (KD = 1.3 microM) and (d) Glutaraldehyde does not specifically cross-link LCL (between the equivalent of Lys492 and Arg678). These results could be explained by a flexible and loose structure of the hinge region of LCL (C-terminal 25%). Anchoring this region in the membrane and/or interaction with the missing beta-strand domain may be required for its compact folding and proper interaction with the rest of LCL. The results suggest that the N-terminal 75% of LCL expressed in E. coli folds autonomously to a fairly stable unit and native-like structure, encompassing the phosphorylation and central ATP binding sections. The hinge region does not appear to be part of the FITC-binding site but constitutes portions of the 2',3'-O-(2,4,6-trinitrophenyl)-ATP and, probably, ATP-binding site.

Adenosine Triphosphate↗
PubMed1990

Affinity labeling of spinach phosphoribulokinase subsequent to S-methylation at Cys16.

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme, S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparent Kd of 3-4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [14C]reagent per mole of enzyme subunit. Amino acid analysis of the [14C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.

Adenosine Triphosphate↗
PubMed1999

Affinity labelling with MgATP analogues reveals coexisting Na+ and K+ forms of the alpha-subunits of Na+/K+-ATPase.

To test the hypothesis that Na+/K+-ATPase works as an (alpha beta)2-diprotomer with interacting catalytic alpha-subunits, tryptic digestion of pig kidney enzyme, that had been inactivated with substitution-inert MgATP complex analogues, was performed. This led to the demonstration of coexisting C-terminal Na+-like 80-kDa as well as K+-like 60-kDa peptides and N-terminal 40-kDa peptides of the alpha-subunit. To localize the ATP binding sites on tryptic peptides, studies with radioactive MgATP complex analogues were performed: Co(NH3)4-8-N3-ATP specifically modified the E2ATP (low affinity) binding site of Na+/K+-ATPase with an inactivation rate constant (k2) of 12 x 10-3.min-1 at 37 degrees C and a dissociation constant (Kd) of 207 +/- 28 microm. Tryptic digestion of the [gamma32P]Co(NH3)4-8-N3-ATP-inactivated and photolabelled alpha-subunit (Mr = 100 kDa) led, in the absence of univalent cations, to a K+-like C-terminal 60-kDa fragment which was labelled in addition to an unlabelled Na+-like C-terminal 80-kDa fragment. Tryptic digestion of [alpha32P]-or [gamma32P]Cr(H2O)4ATP - bound to the E1ATP (high affinity) site - led to the labelling of a Na+-like 80-kDa fragment besides the immediate formation of an unlabelled K+-like N-terminal 40-kDa fragment and a C-terminal 60-kDa fragment. Because a labelled Na+-like 80-kDa fragment cannot result from an unlabelled K+-like 60-kDa fragment, and because unlabelled alpha-subunits did not show any catalytic activity, the findings are consistent with a situation in which Na+- and K+-like conformations are stabilized by tight binding of substitution-inert MgATP complex analogues to the E1ATP and E2ATP sites. Hence, all data are consistent with the hypothesis that ATP binding induces coexisting Na+ and K+ conformations within an (alphabeta)2-diprotomeric Na+/K+-ATPase.

Adenosine Triphosphate↗
PubMed1988

Characterization, purification, and affinity labeling of the brain [3H]glibenclamide-binding protein, a putative neuronal ATP-regulated K+ channel.

Sulfonylurea and particularly glibenclamide are potent blockers of ATP-regulated K+ channels in insulin-secreting cells. A very good correlation exists between binding of sulfonylurea to brain and insulinoma cell membranes. The [3H]glibenclamide-binding component from pig brain microsomes was solubilized with digitonin with a complete retention of its properties of interaction with glibenclamide and other sulfonylureas. A four-step purification was achieved that used (i) hydroxylapatite chromatography, (ii and iii) affinity chromatographies on ADP-agarose and wheat germ agglutinin-agarose columns, and (iv) a final chromatographic step on a mixture of AMP-agarose/GMP-agarose/hydroxylapatite. This procedure led to a 2500-fold purification. NaDodSO4/polyacrylamide gel electrophoresis of the purified material in reducing and nonreducing conditions showed that the sulfonylurea-binding component is made of a single major polypeptide chain of Mr 150,000 +/- 10,000. Direct photoaffinity labeling of the receptor with [3H]glibenclamide at different steps of the purification also showed that radioactivity was specifically incorporated into a polypeptide of Mr 150,000 +/- 5000, thus confirming the subunit structure indicated by the purification.

Adenosine Triphosphate↗
PubMed1987

Affinity labeling of a novel cholecystokinin-binding protein in rat pancreatic plasmalemma using new short probes for the receptor.

Previous biochemical characterizations of the cholecystokinin (CCK) receptor have used the "long" probe 125I-Bolton-Hunter-CCK-33 since it was the only CCK analogue with high affinity and high specific radioactivity which possessed an amino group available for chemical cross-linking. These studies have consistently identified a major binding protein of approximately 81 kilodaltons and have identified several minor proteins which were obtained under different cross-linking conditions and in different laboratories. Because the receptor-binding region of CCK-33 (carboxyl-terminal heptapeptide) is so far removed from the radiolabel and from available amino groups (positions 1 and 11), this probe carries potential for proteolytic cleavage of label from receptor and for labeling "near neighbors" instead of the binding site. We therefore designed two "short" probes for the CCK receptor. 125I-Bolton-Hunter-Lys-Gly-CCK-8 has an epsilon-amino group available for cross-linking. 125I-Tyr-[Thr28,Nle31]CCK-25-33 has an alpha-amino group for cross-linking and has the major advantage of being labeled by oxidative means, unique for CCK derivatives. Both radioiodinated decapeptides were purified by reverse-phase high pressure liquid chromatography to yield specific radioactivity of 2,000 Ci/mmol; demonstrated saturable, specific, and high affinity binding to rat pancreatic plasma membranes; and retained full biological activity to stimulate amylase secretion. Using a variety of cross-linking methods, these probes each identified the same Mr = 85,000-95,000 protein in rat pancreatic plasmalemma, and CCK-8 competed for this labeling in a concentration-dependent manner (IC50 = 1 nM). No change in apparent mobility of this band was observed under reducing or nonreducing conditions, suggesting lack of covalent attachment to other subunits. The Mr = 85,000-95,000 species migrated differently on sodium dodecyl sulfate gels than any of the components previously identified using 125I-Bolton-Hunter-CCK-33, confirming the novel nature of this binding protein. These short probes should be very useful for further characterization of CCK receptors on this and other tissues.

Affinity Labels↗
PubMed1977

Affinity labeling of rat-kidney gamma-glutamyl transpeptidase.

The reaction of gamma-glutamyl transpeptidase from rat kidney with a glutamine analog, 6-diazo-5-oxo-L-norleucine, resulted in irreversible inactivation of the enzyme. The concentration of this reagent giving a half-maximum rate of inactivation was 6 mMat pH 7.5. The inactivation was prevented by the presence of reduced glutathione in a competitive fashion, which indicates the active-site-directed nature of this reagent. The rate of inactivation was greatly accelerated in the presence of maleate, which is known to enhance the glutaminase activity of this enzyme. The presence of maleate increased the maximum velocity of the inactivation, but did not affect the affinity of the enzyme for 6-diazo-5-oxo-L-norleucine. Inactivation of the enzyme with 6-diazo-5-oxo-L-[6=14C]norleucine as well as with 6-diazo-5-oxo-L[1,2,3,4,5-14C]norleucine resulted in a stoichiometric incorporation of radioactivity into the enzyme protein via covalent linkage. The amount of radioactivity incorporated was 1 mol 14C label/248000 g enzyme protein. A native enzyme preparation showing a single protein band on polyacrylamide gel electrophoresis gave four distinct bands upon sodium dodecylsulfate/polyacrylamide gel electrophoresis. Upon sodium dodecylsulfate/polyacrylamide gel electrophoresis of the 14C-labeled enzyme, only the band moving the fastest towards the anode was found to contain radioactivity. This finding indicates that this protein band represents the catalytic component of the enzyme.

Affinity Labels↗
PubMed1988

Affinity labeling of calmodulin-binding proteins in skeletal muscle sarcoplasmic reticulum.

125I-Calmodulin (125I-CaM) binding to sarcoplasmic reticulum (SR) membranes isolated from skeletal muscle cells was investigated, and the CaM receptors associated with the membrane were identified by using the photoaffinity cross-linker methyl-4-azidobenzimidate or the chemical cross-linker dithiobis-N-hydroxysuccinimidyl propionate. Exogenous CaM binds to CaM-depleted membranes in a Ca2+- or Mg2+-dependent way. When both cations are added together to the reaction medium, the stimulatory effects appear to be additive, suggesting that Ca2+ and Mg2+ act by two distinct mechanisms. The Ca2+/Mg2+-dependent binding of CaM is specific since it is inhibited by unlabeled CaM or by trifluoperazine. Furthermore, it is saturable and shows one class of high affinity binding sites with a KD of about 52 nM and a beta max of about 5 pmol/mg of protein. The sensitivity of Ca2+ is expressed in two steps reaching half-saturation at free Ca2+ concentrations of about 1.6 x 10(-7) and 3 x 10(-5) M, respectively. On the other hand, the sensitivity to Mg2+ is expressed in one step with a half-saturation Mg2+ concentration of about 2 x 10(-3) M. Electrophoretic analysis in a polyacrylamide gradient and subsequent autoradiography demonstrated a major CaM-binding protein of about 60 kDa and five minor CaM receptors of about 148, 125, 41, 33, and 23 kDa, respectively. The major labeled protein (60 kDa) probably represents the CaM-dependent component involved in Ca2+ release from SR, whereas the others represent a previously unrecognized class of CaM receptors in skeletal SR.

Affinity Labels↗
PubMed1989

Fetal lamb 3 beta, 20 alpha-hydroxysteroid oxidoreductase: dual activity at the same active site examined by affinity labeling with 16 alpha-(bromo[2'-14C]acetoxy)progesterone.

3 beta,20 alpha-Hydroxysteroid oxidoreductase was purified to homogeneity from fetal lamb erythrocytes. The Mr 35,000 enzyme utilizes NADPH and reduces progesterone to 4-pregnen-20 alpha-ol-3-one [Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1] and 5 alpha-dihydrotestosterone to 5 alpha-androstane-3 beta, 17 beta-diol [Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1]. 5 alpha-Dihydrotestosterone competitively inhibits (Ki = 102 microM) 20 alpha-reductase activity, suggesting that both substrates may be reduced at the same active site. 16 alpha-(Bromoacetoxy)progesterone competitively inhibits 3 beta- and 20 alpha-reductase activities and also causes time-dependent and irreversible losses of both 3 beta-reductase and 20 alpha-reductase activities with the same pseudo-first order kinetic t1/2 value of 75 min. Progesterone and 5 alpha-dihydrotestosterone protect the enzyme against loss of the two reductase activities presumably by competing with the affinity alkylating steroid for the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase. 16 alpha-(Bromo[2'-14C]acetoxy) progesterone radiolabels the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase wherein 1 mol of steroid completely inactivates 1 mol of enzyme with complete loss of both reductase activities. Hydrolysis of the 14C-labeled enzyme with 6 N HCl at 110 degrees C and analysis of the amino acid hydrolysate identified predominantly N pi-(carboxy[2'-14C]methyl)histidine [His(pi-CM)].(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1986

Binding characteristics and affinity labeling of protein constituents of the human IM-9 lymphoblast receptor for substance P.

The neuropeptide substance P (SP) stimulates human T-lymphocyte function in vitro. Human blood T-lymphocytes and cultured human IM-9 B-lymphoblasts express 7,000-10,000 and 25,000-30,000 substance P receptors per cell, respectively. The specific binding of 125I-SP is retained in IM-9 lymphoblast membranes solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) at a detergent-to-protein ratio of 1.0. In addition, specific and reversible SP binding to soluble IM-9 cell membrane proteins is demonstrated by gel filtration. The saturation of binding of 125I-SP to both intact and solubilized IM-9 cell membranes attained a steady state after 40-50 min at 4 degrees C. Scatchard analysis of the concentration dependence of 125I-SP binding to IM-9 cell membranes revealed a KD of 0.87 +/- 0.8 nM (mean +/- S.D., n = 4), which is similar to that observed in intact cells, and a density of receptors of 21 +/- 3 fmol/mg of membrane protein (mean +/- S.D.). Binding of 125I-SP to solubilized membranes demonstrated a KD of 0.75 +/- 0.33 nM (mean +/- S.D., n = 3) and a density of receptors of 3.7 +/- 1.5 fmol/mg of membrane protein (mean +/- S.D., n = 3). Affinity cross-linking of 125I-SP by disuccinimidyl suberate to intact IM-9 cells and membranes revealed specifically labeled proteins of Mr 58,000 and 33,000 in cells, and 58,000, 33,000, and 16,000 in membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Competitive effects of substituent peptides of SP on cross-linking and 125I-SP binding to membranes demonstrated that the SP receptor recognized the carboxyl-terminal domain of the peptide. Membranes from cells preincubated in vitro for 12 h at 37 degrees C with 10(-8) M SP demonstrated a decrease in SP receptor density to 13 +/- 2 fmol/mg (mean +/- S.D., n = 2), and a parallel diminution in the specific labeling of membrane proteins of Mr 58,000 and 33,000. These observations suggest that solubilization in CHAPS preserves the binding characteristics of the IM-9 lymphoblast receptor for SP, and that affinity cross-linking techniques identify by sodium dodecyl sulfate-polyacrylamide gel electrophoresis membrane proteins that are specifically labeled by SP.

Affinity Labels↗
PubMed1970

Affinity labeling of the heavy and light chains of a myeloma protein with anti-2,4-dinitrophenyl activity.

A mouse myeloma protein with high affinity for 2,4-dinitrophenyl (Dnp) ligands was reacted with the bromoacetyl derivatives of N-Dnp-ethylenediamine and (epsilon)-N-Dnp-L-lysine. Up to 1.4 sites per protein molecule were covalently labeled. The labeling reactions were essentially completely blocked by a large excess of Dnp ligands that do not combine covalently (e.g., (epsilon)-Dnp-L-lysine). Analyses of the labeled protein revealed that the bromoacetyl derivative of N-Dnp-ethylenediamine reacted exclusively with tyrosyl in the light chain, while the derivative of (epsilon)-Dnp-L-lysine reacted exclusively with lysyl in the heavy chain. The findings support the conclusion that chains are involved in forming specific combining sites.

Amino Acid Sequence↗