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At least 487 records · Page 27Linked to original sources
PubMed1993

Interaction of ATP analogs with yeast 3-phosphoglycerate kinase. Affinity labeling of the hinge region.

Yeast 3-phosphoglycerate kinase is inactivated by incubation with pyridoxal 5'-diphospho-5'-adenosine (AdoP2Pxy) [Tamura, J. K., Rakov, R. D. & Gross, R. L. (1986) J. Biol. Chem. 261, 4126-4133). Incorporation of 1 mol affinity label/mol enzyme was sufficient for complete inactivation of 3-phosphoglycerate kinase. The substrate ATP affords substantial protection against inactivation. Partial protection is afforded by the substrate glycerate 3-phosphate. When AdoP2Pxy-modified phosphoglycerate kinase was reduced with [3H]NaBH4 and subjected to trypsin hydrolysis, only one radioactive peptide was isolated by reverse-phase high-performance liquid chromatography. The amino acid composition and sequence analysis of the purified radioactive peptide revealed that it spans residues 379-403 of the enzyme and Lys385 specifically reacted with the affinity label. This peptide represents the hinge region between the two domains of the protein, where the active site is also located. The fluorescence intensity of enzyme-bound AdoP2Pxy is enhanced when glycerate 3-phosphate is added, suggesting exposure of the fluorescent probe to a more hydrophobic environment. Another fluorescent analog, anthraniloyl-dATP (ant-dATP), which carries the fluorescent reporter group on the ribose ring, binds to the enzyme at two distinct sites with Kd values of 6 +/- 2 microM and 25 +/- 3 microM, as determined by steady-state anisotropy measurements. Bound ant-dATP was displaced from the enzyme by glycerate 3-phosphate and ATP, as monitored by the fluorescence anisotropy. These results suggest that both fluorescent ATP analogs bind to the active site, which is at the hinge region of the enzyme. Model-building studies showed that when AdoP2Pxy is built into the open form of the enzyme, as described in X-ray studies, the pyridoxyl group of AdoP2Pxy cannot reach Lys385 for Schiff-base formation. Labeled Lys385 is on a beta-turn immediately following helix XII, which was suggested to interact with the nucleotide and become ordered at the active site of 3-phosphoglycerate kinase [Watson, H. C., Walker, N. P. C., Shaw, P. J., Bryant, T. N., Wendell, P. L., Fothergill, L. A., Perkins, R. E., Conroy, S. C., Dobson, M. J., Tuite, M. F., Kinesman, A. J. & Kinesman, S. M. (1982) EMBO J. 1, 1635-1640]. The results presented here suggest that binding of substrates cause significant structural changes in the enzyme.

Adenosine Diphosphate↗
PubMed1996

Covalent modification of protein kinase C isozymes by the inactivating peptide substrate analog N-biotinyl-Arg-Arg-Arg-Cys-Leu-Arg-Arg-Leu. Evidence that the biotinylated peptide is an active-site affinity label.

We recently reported that the peptide substrate analog Arg-Lys-Arg-Cys-Leu-Arg-Arg-Leu (RKRCLRRL) irreversibly inactivates the protein kinase C (PKC) isozymes alpha, beta, and gamma in a dithiothreitol-sensitive manner by an active site-directed mechanism. We hypothesized that the inactivation mechanism entailed covalent complex formation between the PKC isozyme and the inactivator peptide. In this report, N-biotinylated analogs of RKRCLRRL that inactivate Ca2+-dependent PKC activity were designed and tested for their ability to covalently label PKC isozymes. A purified PKC isozyme mixture (alpha, beta, gamma, epsilon, zeta) was incubated with the N-biotinylated peptides and then subjected to denaturing gel electrophoresis, transferred to nitrocellulose, and probed for avidin-reactive species. The Ca2+-dependent PKC subfamily members PKC-alpha, -beta, and -gamma comigrated at 82 kDa and were distinguished by isozyme-specific immunoprecipitation. N-Biotinyl-RRRCLRRL covalently labeled all of the isozymes examined. When the isozymes were denatured prior to incubation with the N-biotinylated peptides, no labeling was observed. Inactivation of the Ca2+-dependent PKC subfamily by the N-biotinylated peptides was associated with covalent labeling of the 82-kDa PKC subspecies. The concentration dependence curves observed with N-biotinyl-RRRCLRRL were similar for inactivation and covalent labeling. The rank order of potency of three N-biotinylated peptides was the same for the inactivation and covalent labeling. Both the inactivation and covalent labeling were dithiothreitol-sensitive, and they were each subject to protection by MgATP and a peptide substrate analog. The covalent label was mapped to the catalytic domain of PKC by limited proteolysis of the modified enzyme. These results provide evidence that the N-biotinylated inactivator peptides are active-site affinity labels of PKC. The inactivator peptides most likely function by S-thiolating the active-site Cys residue conserved in PKC. This is the first report to demonstrate covalent labeling of PKC by a peptide substrate analog.

Animals↗
PubMed1975

Synthesis and use of affinity-labeling steroids for interceptive purposes.

Synthesis of 17 beta-bromoacetoxy-19-nortestosterone was carried out by reaction of 19-nortestosterone with bromoacetic acid in the presence of dicyclohexycarbodiimide. The steroid was capable of alkylating cysteine, methionine, and histidine under physiologic conditions, indicating its potential as an affinity-labeling steroid. 17beta-Bromoacetoxy-19-nortestosterone interrupted postimplantation pregnancy in the rat when administered into the lumen of the uterus at low doses or subcutaneously at higher doses. Exogenous gonadotropins or steroids in dosages sufficient to maintain pregnancy do not prevent the interceptive action of this steroid. Animals whose first pregnancies were interrupted by this steroid had a subsequent normal pregnancy. The mode of action may be via covalent bonding to the progesterone receptor resulting in exclusion of endogenous progesterone.

Abortifacient Agents↗
PubMed1983

The labelling of an axonal membrane component with 4-(N-maleimido) benzyltrimethylammonium, a reagent capable of affinity-labelling the alpha-subunit of the nicotinic acetylcholine receptor.

Membrane vesicles, isolated from crustacean axons, were treated, following disulfide reduction, with 3H-NEM or with 3H-MBTA. SDS polyacrylamide gel electrophoresis showed that exposure to NEM (a nonspecific thiol reagent) resulted in the labelling of several peptide bands, while with MBTA only a single band with a molecular weight of 50,000 was labelled. Reaction with MBTA (believed to be a specific label for the nicotinic acetylcholine receptor) could be largely prevented by preincubation with d-tubocurarine or bromacetylcholine.

Affinity Labels↗
PubMed1997

Investigations using photo affinity labeled analogues confirm the binding between sCD4 and the PND of HIV-1, MN.

In previous studies we demonstrated that a synthetic peptide corresponding to the sequence in the (307-330) region of the gp120 principal neutralizing domain of the HIV-1 MN strain is able to bind sCD4 in an affinity chromatography assay and to enhance CD4 expression, CD4 affinity for gp120, and HIV-1 infection. This paper describes a photo affinity labeling experiment, designed to confirm the gp120 peptide-CD4 interaction and to locate the binding site of the synthetic peptide on the CD4 molecule. To this end two specifically marked analogues of the peptide patterned on the (307-330) region of HIV-MN-gp120, in which the TyrI residue is replaced with Phe(p-N3) or Phe(p-NO2), have been synthesized. Irradiation of CD4 solutions in the presence of both analogues produced a new component, the mass value of which confirms the formation of a covalent bond between the peptide and the protein.

Binding Sites↗
PubMed1988

Pyruvate carboxylase from Pseudomonas citronellolis: shape of the enzyme, and localization of its prosthetic biotin group by electron microscopic affinity labeling.

Pseudomonas citronellolis is known to contain a pyruvate carboxylase with an alpha 4 beta 4 composition. All the other pyruvate carboxylases investigated so far are made up of four seemingly identical subunits. Nevertheless, this exceptional pyruvate carboxylase exhibits a size and overall shape similar to other pyruvate carboxylases. Electron microscopic affinity labeling with avidin revealed that the prosthetic biotin groups (one per alpha beta unit, i.e. four per enzyme particle) are located close to the inter-unit junctions of pairs of alpha beta units making up the enzyme. This position of the prosthetic biotin groups is very similar to the location of the biotin in the other carboxylases.

Biotin↗
PubMed1982

Affinity labelling and characterization of the ppp(A2'p)nA-dependent endoribonuclease from different mammalian sources.

The ppp(A2'p)nA-dependent endoribonucleases from a number of different mammalian sources have been investigated. The enzyme from reticulocyte lysates shows optimal activity of 50-150 mM KCl and requires the presence of Mg2+. Whilst the enzyme is inactivated after passage of reticulocyte lysates through Sephadex columns in the absence of ATP, it retains full activity provided ATP is included in the column buffer. The activity of the partially purified nuclease was unaffected by the addition of reticulocyte RNase inhibitor, which, in contrast, effectively inhibited other endogenous endonucleases. The ppp(A2'p)nA-dependent Rnase co-purified with a ppp(A2'p)nA-binding protein and with a protein which could be specifically covalently labelled with an oxidised radioactive analogue of ppp(A2'p)nA. This covalent labelling could be carried out either with the partially purified RNase or in crude extracts from rabbit reticulocytes, mouse Krebs and Ehrlich ascites tumour cells and human lymphoblastoid (Daudi) or HeLa cells. In each case the affinity labelled protein migrated to a position corresponding to a apparent molecular weight of about 85 000 on electrophoresis on dodecylsulphate/polyacrylamide gels. In all cases labelling could be prevented by the addition of an excess of unlabelled ppp(A2'p)nA but not, for example, by a similar excess of the biologically inactive dimer ppp(A2'p)'A. It is concluded that the RNase and ppp(A2'p)nA binding activities are likely to reside in the same molecule.

Adenosine Triphosphate↗
PubMed2002

Affinity labeling of the guanine nucleotide binding site of transducin by pyridoxal 5'-phosphate.

Transducin (T), a guanine nucleotide binding regulatory protein composed of alpha-, beta-, and gamma-subunits, serves as an intermediary between rhodopsin and cGMP phosphodiesterase during signaling in the visual process. Pyridoxal 5'-phosphate (PLP), a reagent that has been used to modify enzymes that bind phosphorylated substrates, was probed here as an affinity label for T. PLP inhibited the guanine nucleotide binding activity of T in a concentration dependent manner, and was covalently incorporated into the protein in the presence of [3H]NaBH4. Approximately 1 mol of 3H was bound per mol of T. GTP and GTP analogs appreciably hindered the incorporation of 3H to T, suggesting that PLP specifically modified the protein active site. Interestingly, PLP modified both the alpha- and beta-subunits of T. Moreover, PLP in the presence of GDP behaved as a GTP analog, since this mixture was capable of dissociating T from T:photoactivated rhodopsin complexes.

Animals↗
PubMed1990

[Replication complex of tick-borne encephalitis complex. I. Identification of a nuclear fraction protein responsible for the initiation of RNA synthesis using affinity labeling].

The pig embryo kidney cells infected by tick-borne encephalitis virus were fractionated into nuclear-associated, cytoplasmic and membrane fractions. The main part of the virus replicase activity was associated with the nuclei. The replication complex is able to synthesize full-length viral RNAs in vitro. To identify proteins involved in the initiation of the replication at the late stages of the infection, the highly specific affinity labelling technique was used. It was shown that with aldehyde-containing derivatives of ATP, ADP and AMP and [alpha-32P]GTP the target of labelling is a polypeptide having molecular weight about 69 kDa. The same protein is immunostained with TBE virus specific antibodies after blotting onto nitrocellulose. The conclusion is made that nonstructural protein NS3 takes part in virus replication at the late stage of the infection.

Animals↗
PubMed1976

Glycosylation of hemoglobin in vitro: affinity labeling of hemoglobin by glucose-6-phosphate.

To determine the mechanism for the formation of hemoglobin A1c (Hb A1c) in vivo, we incubated human hemoglobin with glucose and metabolites of glucose. [14C]Glucose-6-phosphate (G6P) reacted readily with deoxyhemoglobin, and formed a covalent linkage. The reaction rate was considerably reduced in the presence of carbon monoxide or 2,3-diphosphoglycerate (2,3-DPG). Purified G6P hemoglobin had a lowered oxygen affinity and decreased reactivity with 2,3-DPG compared to Hb A. G6P behaved as a 2,3-DPG analog and reacted specifically at the NH2-terminal amino group of the beta chain. In contrast, the interaction of hemoglobin with glucose was much slower, and was unaffected by carbon monoxide or 2,3-DPG. Neither glucose-1-phosphate, fructose-6-phosphate, nor fructose-1,6-diphosphate formed a reaction product with hemoglobin. G6P behaves as an affinity label with the phosphate group forming electrostatic bonds at the 2,3-DPG binding site and the aldehvde group reacting with the NH2-terminal amino group of the beta chain. Thus, G6P hemoglobin may be an intermediate in the conversion of Hb A to Hb A1c.

Amino Acid Sequence↗
PubMed1989

Human placental Fc gamma receptor. Studies on affinity labeling of the receptor with IgG.

The aim of the presented work was to find out whether the Fc gamma receptor from human placental syncytiotrophoblast plasma membranes in its native membrane-bound state is composed of more subunit chains than previously found in our laboratory in the purified receptor. The chains might be lost during the purification procedure with the use of various chaotropic reagents, e.g. acidic or alkaline buffers, detergents. To study this problem, affinity labeling technique and bifunctional crosslinking reagents were used to covalently link IgG with the Fc gamma receptor. The reagents used were: noncleavable dimethylsuberimidate (DMS), cleavable dimethyl 3,3-dithiobispropionimidate (DTBP), and photoactivable sulfosuccinimidyl 6-/4' azido-2'nitrophenylamino/hexanoate (sulfo-SANPAH). Human 125I-IgG were crosslinked to the membrane-bound receptor or unlabeled IgG was crosslinked to 3H-labeled placental membranes. When 125I-IgG were used in the presence of an excess of unlabeled IgG, recovery of radioactivity after crosslinking was much lower, indicating that human IgG and the placental Fc gamma receptor were involved in the formation of crosslinking of ligand-receptor complexes. The products of crosslinking were analyzed in SDS-PAGE. In the absence of reducing agents, high molecular weight products were present which did not enter the gel or formed broad diffused bands at the top of the gel. Therefore, the products of crosslinking were analyzed under reducing conditions. Analysis by SDS-PAGE demonstrated a major polypeptide band Mr of 160,000 in soluble products of crosslinking of IgG to the placental Fc gamma receptor, regardless of which noncleavable crosslinker was used. The protein is built either of two molecules of the receptor subunit chain, Mr of 60,000 and one IgG heavy chain (Mr of 56,000) or of two IgG heavy chains and one molecule of the receptor chain. The presence of this protein in control samples was not observed at all or its content was markedly lower. The same effect was also observed when DTBP, a cleavable crosslinking reagent was used. In this case, 125I-IgG heavy and light chains or the receptor subunit chains were found after SDS-PAGE under reducing conditions. The results presented in this paper do not suggest a presence of additional chains in the placental Fc gamma receptor others than described in our previous paper concerning the subunit structure of this receptor.

Cross-Linking Reagents↗
PubMed1974

Identification by photo-affinity labeling of the proteins in Escherichia coli ribosomes involved in elongation factor G-dependent GDP binding.

Guanosine diphosphate esterified at the beta-phosphate group with the photolabile 4-azidophenol [1-(4-azidophenyl)-2-(5'-guanyl)pyrophosphate] was found to inhibit GDP binding to the ribosomes of E. coli. UV-irradiation of the fusidic acid-stabilized complex among ribosomes, elongation factor G, and the azidophenyl-GDP, results in selective attachment of the photo-affinity label to the proteins L5, L11, L13, L18, and L30. In addition, a substantial reduction of the amount of L16 present in irradiated ribosomes was found. Except for L13, no significant reaction of azidophenyl-GDP with the ribosomal proteins was observed when fusidic acid was omitted from the irradiation mixture. The results strongly suggest that L5, L11, L18, and L30 are involved in GDP binding. The possibility of a transient binding of GDP to L13 followed by migration to the actual GTPase site is discussed.

Azides↗
PubMed1990

Bombesin receptor in membranes from Swiss 3T3 cells. Binding characteristics, affinity labelling and modulation by guanine nucleotides.

Bombesin-like neuropeptides, including mammalian gastrin-releasing peptide (GRP), are potent mitogens for Swiss 3T3 cells. In this study, we have characterized the bombesin receptor in membrane preparations from these cells. Addition of Mg2+ during cell homogenization was essential to preserve 125I-GRP binding activity in the resulting membrane preparation. The effect of Mg2+ was concentration dependent, with a maximum at 5 mM. Specific binding of 125I-GRP was saturable; Scatchard analysis indicated a single class of high-affinity sites of Kd = (2.1 +/- 0.3) x 10(-10) M at 15 degrees C and Kd = (1.9 +/- 0.4) x 10(-10) M at 37 degrees C, and a maximum binding capacity of 580 +/- 50 fmol/mg of protein (15 degrees C) or 604 +/- 40 fmol/mg of protein (37 degrees C). The kinetically derived dissociation constant was 1.5 x 10(-10) M. 125I-GRP binding was inhibited in a concentration-dependent manner by various peptides containing the highly conserved C-terminal heptapeptide of the bombesin family, including bombesin, GRP, neuromedin B and the 8-14 fragment of bombesin. In contrast, a variety of structurally unrelated mitogens and neuropeptides had no effect. The cross-linking agent ethyleneglycolbis(succinimidylsuccinate) covalently linked 125I-GRP to a single Mr 75 000-85 000 protein in membrane preparations of 3T3 cells. Affinity labelling of this molecule was specific and dependent on the presence of Mg2+ during membrane preparation. Finally, the non-hydrolysable GTP analogue guanosine-5'-[gamma-thio]triphosphate (GTP[S]) caused a concentration-dependent inhibition of 125I-GRP binding and cross-linking to 3T3 cell membranes [concentration giving half-maximal inhibition (IC50) approximately 0.2 microM]. The inhibitory effect was specific (GMP, ATP or ATP[S] had no effect at 10 microM) and was due to an increase in Kd from (1.7 +/- 0.2) x 10(-10) M to (4.3 +/- 0.6) x 10(-10) M in the presence of 10 microM-GTP[S]. This modulation of ligand affinity and cross-linking implies that the bombesin receptors that mediate mitogenesis in Swiss 3T3 cells are coupled to a guanine-nucleotide-binding-protein signal-transduction pathway.

Animals↗
PubMed1975

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12. Specific inactivation of the homoserine dehydrogenase activity by the affinity label, 2-amino-4-oxo-5-chloropentanoic acid.

2-Amino-4-oxo-5-chloropentanoic acid inactivates specifically the homoserine dehydrogenase activity of the bifunctional enzyme, aspartokinase I--homoserine dehydrogenase I. The aspartokinase activity remains essentially untouched and retains its threonine sensitivity. The inactivation of the dehydrogenase requires the covalent binding of one equivalent of the analogue per subunit. Alkylation does not affect the tetrameric state of the protein. The alkylating agent, a substrate analogue, meets the qualitative and quantitative requirements of an affinity label.

Alcohol Oxidoreductases↗
PubMed1980

[Affinity labeling and differential labeling of leucine aminopeptidase with diazopeptide inhibitors].

Specific modification of amino acid residues in the active center of LAP by the substrate-like diazonium peptide L-Phe(pN2+)-L-Phe was investigated using the technic of affinity and differential labeling. Prelabeling of the enzyme was performed with the diastereomer D-Phe(pN2+)-L-Phe in the presence of the competitive inhibitor L-Thr(But)-L-Phe-L-Phe (Ki 9.4 x 10(-5) mol/l). The amino derivative L-Phe(pNH2)-L-Phe is a substrate of the LAP with Km 1.2 x 10(-4) mol/l and D-Phe(pNH2)-L-Phe a non-competitive inhibitor with Ki 4.0 x 10(-3) mol/l. The decrease of activity by covalent modification of tyrosine and histidine side chains was irreversible and affects only the catalytic step, whereas the substrate binding was unimpaired. The affinity labeling as well as the differential labeling in presence of Thr(But)-Phe-Phe showed that up to 12 potential covalent modification sites of LAP can be protected.

Affinity Labels↗
PubMed1990

Subcloning, characterization, and affinity labeling of Escherichia coli glycinamide ribonucleotide transformylase.

Glycinamide ribonucleotide transformylase (GAR TFase; EC 2.1.2.2) has been purified 70-fold to apparent homogeneity from Escherichia coli harboring an expression vector encoding the purN gene product, GAR TFase. The protein is a monomer of Mr 23,241 and catalyzes a single reaction. Steady-state kinetic parameters for the enzyme have been obtained. The structural requirements for cofactor utilization have been investigated and found to parallel those of the multifunctional avian enzyme. The enzyme was inactivated with the affinity label N10-(bromoacetyl)-5,8-dideazafolate in a stoichiometric and active-site-specific manner. The ionization state of the cofactor analogue in the enzyme-cofactor complex appears to require the dissociation of the proton at N3 of the pyrimidine within the complex.

Acyltransferases↗
PubMed1988

Nucleotide binding and affinity labelling support the existence of the phosphate-binding subsite p2 in bovine pancreatic ribonuclease A.

When the reaction of bovine pancreatic ribonuclease A with 6-chloropurine riboside 5'-monophosphate was carried out in the presence of several natural mononucleotides, a decrease of 25-75% was found in the amount of the reaction product derivative II (the main product of the reaction which has the nucleotide label at the alpha-NH2 group of Lys-1). The efficiency of inhibition followed the order 3'-AMP greater than 5'CMP approximately equal to 5'AMP greater than 3'CMP. Previous studies indicate that this order reflects the extent of occupancy of p2, a phosphate-binding subsite adjacent to the catalytic centre. This finding suggests that derivative II is the result of affinity labelling and that the phosphate group of the halogenated nucleotide binds to p2 before the reaction takes place. The dissociation constants and stoichiometry of the interaction between native enzyme, derivative II and derivative E (homologous to derivative II, but labelled with a nucleoside instead of a nucleotide) with 3'AMP and 5'AMP at several pH values were also determined. Although in general one strong binding site was found, no strong binding occurs between 3'AMP and derivative II. It is concluded that the phosphate of the label occupies the same site p2, as the phosphate of 3'AMP. Finally, the pH dependence for the binding of 3'AMP and 5'AMP to RNAase A indicates that they bind to different protein groups. The results presented support the structure of the active site of ribonuclease A postulated previously (Parés, X., Llorens, R., Arús, C. and Cuchillo, C.M. (1980) Eur. J. Biochem. 105, 571-579).

Adenosine Monophosphate↗