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PubMedDate not supplied

Affinity labeling in the presence of the reduced diphosphopyridine nucleotide NADH identifies peptides associated with the activities of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase/isomerase.

OBJECTIVE: We sought to identify peptides associated with activity in the primary structure of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase/isomerase (3 beta-HSD/isomerase). METHODS: Purified human placental 3 beta-HSD/isomerase was affinity-radioalkylated by 2 alpha-bromo [2'-14C]acetoxyprogesterone (2 alpha-[14C]BAP) in the presence or absence of the reduced diphosphopyridine nucleotide, NADH. NADH protected both 3 beta-HSD and isomerase from inactivation by 2 alpha-[14C]BAP. Tryptic peptides of unprotected and NADH-protected radioalkylated enzyme were purified by high-pressure liquid chromatography. The amino acid sequence of each radiolabeled peptide was determined and localized within the cDNA-derived primary structure of the enzyme. RESULTS: According to the sequence analyses, NADH shifted radioalkylation by 2 alpha-[14C]BAP away from the Arg-250 peptide (251GQFYYISDDTPHQSYDNLNYTLSK274) and toward the Lys-135 tryptic peptide (136EIIQNGHEEEPLENTWPAPYPHSK159). Based on amino acid analysis to quantitate radioactivity incorporated per nmol peptide, NADH decreased the radiolabeling of His262 in the Arg-250 peptide by 8.2-fold. His142 in the Lys-135 peptide was radiolabeled by 2 alpha-[14C]BAP only in the presence of NADH. CONCLUSIONS: We have previously reported that the substrate pregnenolone blocks the inactivation of 3 beta-HSD by 2 alpha-[14C]BAP through the protection of His262 in the Arg-250 peptide. Protection by NADH against the inactivation of isomerase as well as 3 beta-HSD is evidence that 2 alpha-[14C]BAP binds at the active sites of both enzyme activities. Because the same Arg-250 peptide has been affinity-alkylated in studies that targeted each of the two activities, we propose that the 3 beta-HSD and isomerase reactions are catalyzed in this region of the enzyme protein.

Amino Acid Sequence↗
PubMed1988

The receptor site for the bee venom mast cell degranulating peptide. Affinity labeling and evidence for a common molecular target for mast cell degranulating peptide and dendrotoxin I, a snake toxin active on K+ channels.

The mast cell degranulating peptide (MCD) and dendrotoxin I (DTXI) are two toxins, one extracted from bee venom, the other one from snake venom, that are thought to act on voltage-sensitive K+ channels. Binding sites for the two toxins have been solubilized. The solubilized sites were stable and retained their high affinity for 125I-DTXI and 125I-MCD (Kd approximately equal to 100 pM). Interactions were found between MCD and DTXI binding sites in the solubilized state, establishing that the two different toxins act on the same protein complex. This conclusion was strengthened by the observations (i) that conditions of solubilization that eliminated 125I-MCD binding activity also eliminated 125I-DTX binding activity while both types of activities were preserved in the presence of K+ or Rb+ and (ii) that binding components for the two types of toxins had similar sedimentation coefficients and copurified in partial purifications. A component of the receptor protein for 125I-MCD has been identified; it has a Mr of 77,000 +/- 2000. This polypeptide was similar to or identical in molecular weight with that which serves as a receptor for DTXI (Mr 76,000 +/- 2000).

Animals↗
PubMed2000

Fluorescence characterization of structural transitions at the strong actin binding motif in skeletal myosin affinity labeled at cysteine 540 with novel spectroscopic cysteaminyl mixed disulfides.

We have synthesized the luminescent and fluorescent lanthanide chelate S-(2-nitro-5-thiobenzoic acid)cysteaminyldiethylenetriaminepentaacetate-5-[(2-aminoethyl)am ino ]naphthalene-1-sulfonic acid as well as the fluorescent analogue S-(2-nitro-5-thiobenzoic acid)cysteaminyl-5-carboxyfluorescein using the procedure we recently described [Bertrand, R., Capony, J.-P., Derancourt, J., and Kassab, R. (1999) Biochemistry 38, 11914-11925]. Both mixed disulfides react with the skeletal myosin motor domain (S-1) as actin site-directed agents and label exclusively and stoichiometrically Cys 540 in the hydrophobic strong actin binding helix-loop-helix motif, causing only a 1.9-2.4-fold decrease in the V(max) for acto-S-1 ATPase. The covalently attached cysteaminyl probe side chain spans maximally 17 and 8 A, respectively, and the fluorophores have different polarity, volume, and flexibility. Thus, they may provide complementary spectroscopic information on the environmental properties of this critical actin binding region. Here, we have analyzed by extrinsic fluorescence spectroscopy S-1 derivatized with the fluorescein label or with the Tb(3+) or Eu(3+) chelate of the other label to assess the conformational transitions precisely occurring at this site upon interaction with F-actin, nucleotides, or phosphate analogues. For either label, specific spectral changes of significant amplitude were obtained, identifying at least two major structural states. One was mediated by rigor binding of F-actin in the absence or presence of MgADP. It was abolished by MgATP, and it was not produced by the binding of nonpolymerizable G-actin. A modeling of the corresponding changes in the intensity and lambda(max) of the fluorescence emission spectra, achieved using the fluorescent adducts of 2-mercaptoethanol in varying concentrations of dimethylformamide, illustrates the predicted apolar nature of the strong acto-S-1 interface. A second state was promoted by the binding of ATP, AMP-PNP, ADP.AlF4, ADP. BeFx, or PP(i). It should be prevalent in the weak acto-S-1 binding complexes. The accompanying fluorescence intensity reduction, observed with each label, in both the absence and presence of F-actin, would result from a specific modification by these ligands of the probe orientation and/or solvent accessibility as suggested by acrylamide quenching experiments. It could represent the spectral manifestation of the predicted allosteric linkage from the ATPase site to the strong actin binding site of S-1 that modulates the acto-S-1 affinity. Our study offers the basis necessary for further detailed spectroscopic investigations on the conformational dynamics in solution of the stereospecific and hydrophobic actin binding motif during the skeletal cross-bridge cycle.

Actins↗
PubMed1982

Different classes of nucleotide binding sites in the (Na+ + K+)-ATPase studied by affinity labeling and nucleotide-dependent SH-group modifications.

The ATP analog 6-[(3-carboxy-4-nitrophenyl)thiol]-9-beta-D-ribofuranosylpurine 5'-triphosphate (Nbs6ITP) is slowly hydrolyzed at pH 7.4 by the (Na+ + K+)-ATPase, whereas it binds covalently at pH 8.5 and inhibits the enzyme irreversibly. Time courses of irreversible inhibition could only be fitted to a model in which the enzyme can exist in two slowly interchangeable states, one of which is enzymatically active and binds Nbs6ITP first reversibly and then covalently. Arguments that the covalent binding occurs at a low affinity nucleotide binding site are: (a) similarity of the Ki Nbs6ITP for the reversible and the irreversible inhibition and of K0.5 for ATP protection; (b) stoichiometry of covalent Nbs6ITP binding per alpha subunit of 0.8; and (c) change of complex substrate dependence of the enzyme to a Michaelis-Menten type after Nbs6ITP modification. This change in kinetics and the finding that the Nbs6ITP inactivation at a low affinity nucleotide binding site is increased by micromolar ADP concentrations indicates that the (Na+ + K+)-ATPase contains two different nucleotide binding sites. Since studies of nucleotide effects on enzyme inactivation by 5,5'-dithiobis(2-nitrobenzoic acid) did not confirm the hypothesis of an SH-group in a nucleotide binding site, Nbs6ITP may bind to another functional group, e.g. to an OH-group of tyrosine.

Adenosine Triphosphate↗
PubMed1999

[Synthesis of tumor affinity label Yb-169- and Y-90- porphyrin complexes and animal experimental investigations with various Yb-169-porphyrins].

AIM: It should be shown, that it is possible to insert radioactive isotopes of Yb and Y into some selected porphyrins. Besides, first informations about the biodistribution of Yb-169-por-phyrin-complexes should be obtained. METHODS: Carrier added radioactive isotopes were used for the synthesis of the metal porphyrin complexes. The animal experiments were done with mamma carcinoma bearing mice. The activity of the organs was determined 5 and 24 h after i.v. injection in a well counter. RESULTS: Four Yb-169-porphyrin complexes and Y-90-porphyrin complexes could be synthesized in non-carrier-free form. This was verified by absorption spectra, TLC and HPLC. Depending on the complex, the average tumour/background ratios were between 2 and 20. CONCLUSION: The synthesized radioactive metal-porphyrin complexes showed a clear tumour-affinity which could be used for tumour scintigraphy or perhaps therapy if the synthesis is improved (goal: reduction of carrier, other radionuclides).

Animals↗
PubMed2004

Total synthesis of 34-hydroxyasimicin and its photoactive derivative for affinity labeling of the mitochondrial complex I.

The asymmetric total synthesis of the 34-hydroxyasimicin and its 3-(4-benzoylphenyl)propionate ester was achieved by means of a convergent synthetic strategy. This ester, which contains eight asymmetric centers, represents the first photoaffinity-labeling agent that is derived from an Annonaceous acetogenin. The key transformations in the synthesis include the Sharpless asymmetric dihydroxylation reaction, the Wittig olefination reaction, an oxidative cyclization reaction with rhenium(vii) oxide, the Williamson etherification reaction, and a palladium-catalyzed cross-coupling reaction. Use of the target molecule for photoaffinity-labeling studies of bovine mitochondrial NADH-ubiquinone oxidoreductase (Complex I) may shed light on the structure/function of this intricate enzyme and on the origin of the high antitumor activity exhibited by the Annonaceous acetogenins.

Animals↗
PubMed1977

Reexamination of carbodiimide as a possible affinity label for the acetylcholine receptor at the frog neuromuscular junction.

The effects of water-soluble carbodiimide were examined at the frog neuromuscular junction. Acetylcholine sensitivity was measured using a fluid electrode technique and intracellular recording of miniature end-plate potentials. The carbodiimide blocked synaptic sensitivity by a reversible, curare-like action. Irreversible blockade was also observed, probably due to covalent binding. The conditions of reaction and irreversibility suggest that several different residues may be attacked. The inability of cholinergic antagonists to protect the receptor from attack indicates that nonspecific sites, and not the acetylcholine binding site, are involved.

Acetylcholine↗
PubMed1976

Identification of components of the streptomycin-binding center of E. coli MRE 600 ribosomes by photo-affinity labelling.

The [H3]-labelled photo-activated analog of streptomycin (photo-Sm) is obtained as a result of the streptomycin reaction with 2-nitro, 4-azidobenzoylhydrazide and subsequent reduction with NaBH34. The analog retains the functional activity of the initial antibiotic as judged by two criteria: (1) it binds only to the 30S subparticle of ribosomes and (2) it inhibits the factor-free ("non-enzymatic") PCMB-stimulated polyU-dependent system of translation (Gavrilova and Spirin, 1971). After irradiation of the reaction mixture containing photo-Sm and either the 30S or 50S subparticles of ribosomes under similar conditions, the analog covalently binds chiefly to the 30S subparticle. Irradiation of the photo-Sm mixture with whole 70S ribosomes leads to a uniform distribution of a covalently bound label among the subparticles. A comparison of the effects obtained allows the conclusion that the analog is located on the interface of the ribosomal subparticles. In the 30S subparticle the photo-Sm attacks mainly the protein component (more than 95% of all the covalently bound label). The proteins labelled by photo-reaction are identified as S7 (main), S14 (additional) and S16/S17 (minor).

Binding Sites↗