Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Flow cytometric measurements were made on acridine orange (AO) and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methyl-coumarin (CPM)-stained epididymal- and vas deferens-derived spermatozoal nuclei to follow the course of chromatin condensation and oxidation of free sulfhydryl groups, respectively, during passage through mouse and rat posttesticular reproductive tracts. Alterations of mouse and rat spermatozoal chromatin during transition from a testicular elongated spermatids to epididymal caput spermatozoa resulted in a threefold loss of DNA stainability with AO. Passage of spermatozoa from the caput to corpus epididymis was accompanied by an approximate 15% loss of DNA stainability, which was maintained at that level throughout passage into the vas deferens. AO stainability of epididymal spermatozoal nuclei was generally independent of -SH group stainability. CPM stainability of rat spermatozoal nuclei free -SH groups was 83%, 18%, and 11% of caput spermatozoal values for corpus, cauda epididymis, and vas deferens, respectively. Comparable values for mice were 69%, 20%, and 18%. CPM stainability was relatively homogeneous for these mouse and rat reproductive tract regions, except mouse corpus epididymis spermatozoal nuclei stained very heterogeneously. Rat spermatozoa detained by ligature up to 7 days in the caput, corpus, and cauda epididymi had CPM staining values equal to or below those of normal vas spermatozoa, indicating that disulfide (S-S) bonding is intrinsic to the spermatozoa and is independent of the epididymal environment. These data suggest that chromatin condensation and loss of spermatozoal DNA stainability during passage from the testis to the vas deferens are independent of S-S bonding.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1989
PMID:Flow cytometric analysis of rodent epididymal spermatozoal chromatin condensation and loss of free sulfhydryl groups. 248 18

The fluorogenic maleimide 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) has been shown to selectively form Michael adducts with hyperreactive sulfhydryls on the skeletal sarcoplasmic reticulum (SR) ryanodine receptor (RyR1) and triadin which are essential for normal Ca2+ channel function (Liu, G., Abramson, J.J., Zable, A.C., and Pessah, I.N. (1994) Mol. Pharmacol. 45, 189-200). The present report demonstrates a functionally important interaction between RyR1 and triadin which involves, in part, redox cycling of hyperreactive sulfhydryls in response to channel activation and inactivation. Nanomolar CPM is shown to selectively label RyR1 and triadin only in the presence of Ca2+ channel inhibitors (Mg2+, neomycin, ruthenium red, or anti-triadin antibody). Treatment of SR with channel activators (micromolar Ca2+, nanomolar ryanodine, or millimolar caffeine), 1) slows CPM labeling kinetics > 10-fold, 2) negates CPM labeling of channel-associated sulfhydryls, and 3) stabilizes a high molecular weight complex (HMWC) which appears on nonreducing SDS-polyacrylamide gel electrophoresis gels. The HMWC is positively identified as RyR1 and triadin by Western blot and immunoprecipitation analyses. High-affinity [3H]ryanodine-binding sites are immunoprecipitated by either anti-RyR1 or anti-triadin antibody dose dependently. 1,4-Naphthoquinone (< or = 40 pmol/micrograms protein) selectively oxidizes hyperreactive sulfhydryls on RyR1 and triadin, induces Ca2+ efflux from SR, and stabilizes the HMWC. The HMWC is reduced by beta-mercaptoethanol or dithiothreitol into its component RyR1 and triadin promoters. The results provide direct evidence for the existence of a functionally important complex between RyR1 and triadin whose stability is determined by the redox state of hyperreactive sulfhydryl moieties which are allosterically regulated by physiological and pharmacological channel ligands. The present results suggest a possible molecular mechanism by which localized transient changes in the redox state within the RyR1-triadin complex can signal information across the SR membrane.
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PMID:Molecular interaction between ryanodine receptor and glycoprotein triadin involves redox cycling of functionally important hyperreactive sulfhydryls. 780 31

The fluorogenic sulfhydryl probe 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) (1-50 nM) is used to characterize the functional role and location of highly reactive thiol groups on the ryanodine-sensitive Ca2+ release channel complex [i.e., ryanodine receptors (RyRs)] of skeletal and cardiac junctional sarcoplasmic reticulum (SR). The kinetics of forming fluorescent CPM adducts with junctional but not longitudinal SR membrane proteins (0.02-1 pmol of CPM/microgram of SR protein) are found to be markedly dependent on the presence of physiological and pharmacological modulators of the RyR Ca2+ channel. RyR agonists, micromolar Ca2+, and nanomolar ryanodine promote a slow SR thiol-CPM reaction, with an apparent rate constant k of 0.0021 +/- 0.0002 sec-1, and > 89% of the fluorescence is associated with the 110-kDa Ca2+ pump, which constitutes 68% of the protein in the SR preparations. However, in the presence of Ca2+ channel antagonists (millimolar Mg2+, millimolar Ca2+, or micromolar ryanodine), CPM rapidly forms adducts with a single class of highly reactive (hyperreactive) SR thiols (k = 0.025 +/- 0.002 sec-1). Nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CPM-labeled SR protein and Western blot analyses with antiryanodine or antitriadin antibodies reveal that the hyperreactive thiols labeled by CPM under conditions favoring channel closure are localized principally to the RyR protomer and triadin, which constitute < 6% of the protein in the SR preparation. Immunoprecipitation experiments with antiryanodine and antitriadin monoclonal antibodies confirm the location of CPM-labeled thiol groups on RyR and triadin, respectively. The results indicate that the RyR and triadin contain a small number of highly reactive cysteine residues that selectively conjugate with CPM only when channel closure is favored. It is shown that either 1) the redox state (sulfhydryl/disulfide status) or 2) the accessibility of the hyperreactive thiols on the RyR and triadin is determined by the conformational state of the channel. Covalent modification of hyperreactive thiols with nanomolar CPM inhibits both Ca(2+)-induced Ca2+ release and the gating activity of single channels reconstituted in bilayers, revealing the essential functional importance of hyperreactive thiols on channel-associated proteins. 1,4-Naphthoquinone (0.4-40 pmol/micrograms of protein) selectively oxidizes hyperreactive thiols on RyR and triadin and releases Ca2+ from SR vesicles, without inhibiting Ca(2+)-ATPase activity. The results provide direct evidence of the existence and functional role of hyperreactive cysteine residues on the RyR and triadin in regulating the gating of ryanodine-sensitive intracellular Ca2+ channels and strongly suggest that these important Ca2+ regulatory channels may be an important target for oxidative cell damage mediated by quinones.
Mol Pharmacol 1994 Feb
PMID:Direct evidence for the existence and functional role of hyperreactive sulfhydryls on the ryanodine receptor-triadin complex selectively labeled by the coumarin maleimide 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin. 811 70

Among the large family of fatty acid binding proteins, the liver L-FABP is unique in that it not only binds fatty acids but also interacts with sterols to enhance sterol transfer between membranes. Nevertheless, the mechanism whereby L-FABP potentiates intermembrane sterol transfer is unknown. Both fluorescence and dialysis data indicate L-FABP mediated sterol transfer between L-cell fibroblast plasma membranes occurs by a direct membrane effect: First, dansylated-L-FABP (DNS-L-FABP) is bound to L-cell fibroblast plasma membranes as indicated by increased DNS-L-FABP steady state polarization and phase resolved limiting anisotropy. Second, coumarin-L-FABP (CPM-L-FABP) fluorescence lifetimes were significantly increased upon interaction with plasma membranes. Third, dialysis studies with 3H-cholesterol loaded plasma membranes showed that L-FABP added to the donor compartment of the dialysis cell stimulated 3H-cholesterol transfer whether or not the dialysis membrane was permeable to L-FABP. However, L-FABP mediated intermembrane sterol transfer did require a sterol binding site on L-FABP. Chemically blocking the ligand binding site also inhibited L-FABP activity in intermembrane sterol transfer. Finally, L-FABP did not act either as an aqueous carrier or in membrane fusion. The fact that L-FABP interacted with plasma membrane vesicles and required a sterol binding site was consistent with a mode of action whereby L-FABP binds to the membrane prior to releasing sterol from the bilayer.
Mol Cell Biochem 1995 Nov 08
PMID:Liver fatty acid binding protein enhances sterol transfer by membrane interaction. 860 11

A highly purified preparation of a monoclonal antibody to vasoactive intestinal peptide (VIP) was analysed by gel filtration. Three peaks of VIP hydrolysing activity were observed, corresponding to the 150 kDa tetramer IgG, 80 kDa dimer of the heavy and light chains (H-L dimer) and 25 kDa L chain monomer. The hydrolytic activity of all three peaks was removed by adsorption on immobilized anti-mouse IgG. The specific activities (CPM hydrolysed/microgram protein) of the H-L dimer and the L chain monomer were more than 30-fold greater than of intact IgG. The presence of small amounts of the antibody fragments in unreduced IgG preparations was confirmed by electrophoresis of overloaded radiolabeled and unlabeled IgG preparations. Increased levels of the fragments were evident after prolonged incubation of a dilute solution of 125I-IgG at 37 degrees C. Iodoacetamide, a sulfhydryl alkylating reagent, suppressed the production of IgG fragments. Incubation of 125I-labeled L chain with unlabeled IgG resulted in incorporation of small amounts of the radioactivity into disulfide bonded 150 kDa IgG tetramer and 50 kDa L chain dimer fractions. These observations implicate disulfide reduction and exchange reactions as the mechanism underlying formation of the IgG fragments. Like the antibody fragments found in unreduced IgG, the L chain monomer and non-covalently associated H-L dimer isolated from reduced and alkylated IgG were capable of hydrolysing VIP. Hydrolysis of VIP by the recombinant L chain subunit was inhibited by excess IgG, suggesting that high affinity VIP binding by IgG limits its hydrolysis by the L chain. These observations suggest that small amounts of high activity antibody fragments may contribute to the catalytic characteristics of the anti-VIP IgG preparation.
Mol Immunol
PMID:Low level formation of potent catalytic IgG fragments mediated by disulfide bond instability. 876 Feb 70

Quinones undergo redox cycling and/or arylation reactions with key biomolecules involved with cellular Ca2+ regulation. The present study utilizes nanomolar quantities of the fluorogenic maleimide 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) to measure the reactivity of hyperreactive sulfhydryl moieties on sarcoplasmic reticulum (SR) membranes in the presence and absence of quinones by analyzing the kinetics of forming CPM-thioether adducts and localization of fluorescence by SDS-polyacrylamide gel electrophoresis. Doxorubicin, 1,4-naphthoquinone (NQ), and 1, 4-benzoquinone (BQ) are found to selectively and dose-dependently interact with a class of hyperreactive sulfhydryl groups localized on ryanodine-sensitive Ca2+ channels [ryanodine receptor (RyR)], and its associated protein, triadin, of skeletal type channels. NQ and BQ are the most potent compounds tested for reducing the rate of CPM labeling of hyperreactive SR thiols (IC50 = 0.3 and 1.8 microM, respectively) localized on RyR and associated protein. The reduced forms of quinone, tert-butylhydroquinone, and 5-imino-daunorubicin do not alter significantly the pattern or kinetics of CPM labeling up to 100 microM, demonstrating that the quinone group is essential for modulating the state of hyperreactive SR thiols. Nanomolar NQ is shown to enhance the association of [3H]ryanodine for its high-affinity binding site and directly enhance channel-open probability in bilayer lipid membrane in a reversible manner. By contrast, micromolar NQ produces a time-dependent biphasic action on channel function, leading to irreversible channel inactivation. These results provide evidence that nanomolar quinone selectively and reversibly alters the redox state of hyperreactive sulfhydryls localized in the RyR/Ca2+ channel complex, resulting in enhanced channel activation. The Ca2+-dependent cytotoxicities observed with reactive quinones formed at the microsomal surface by oxidative metabolism may be related to their ability to selectively modify hyperreactive thiols regulating normal functioning of microsomal Ca2+ release channels.
Mol Pharmacol 1999 May
PMID:Site-selective modification of hyperreactive cysteines of ryanodine receptor complex by quinones. 1022 May 60

Taxanes act by inhibiting microtubule dynamics; in this study, we have investigated mitochondria as an additional target of taxanes. We incubated isolated mitochondria in the presence of taxanes with or without stimulation of the mitochondrial respiratory state. Results showed that they rapidly induced the loss of deltapsim after stimulation of the respiratory state. To evaluate the binding of [14C]paclitaxel to isolated mitochondria, mitochondrial proteins were precipitated yielding 18.6 +/- 2.1 cpm/microg of protein. After stimulation of the respiratory state, binding of [14C]paclitaxel increased up to 163.2 +/- 46.7 cpm/microg of protein. CPM values after Bcl-2 immunoprecipitation was 62.8-fold higher than those of the control antibody, thereby indicating the involvement of Bcl-2 in paclitaxel binding. Then, we established a panel of A2780 cell lines resistant to increasing doses of paclitaxel alone or to high doses of paclitaxel/cyclosporin A (A2780 TC cells). In both cases, Bcl-2 expression was consistently down-regulated, whereas levels of other members of the Bcl-2 family, such as Bax and Bcl-x, did not change in paclitaxel-resistant cell lines. When A2780TC cells were stably transfected with a Bcl-2 construct, paclitaxel sensitivity was partially restored, thereby supporting a direct role of Bcl-2 down-regulation in the maintenance of drug-resistance. Finally, we examined Bcl-2 by immunohistochemistry in a small subset of ovarian cancer paclitaxel-resistant patients and we noticed that the protein is down-regulated in this clinical setting with respect to the expression levels found in drug-sensitive tumors. These findings demonstrate that Bcl-2 is an additional intracellular target of taxanes and that its down-regulation is involved in taxane resistance.
Mol Pharmacol 2003 Jul
PMID:Bcl-2 down-regulation is a novel mechanism of paclitaxel resistance. 1281 60

Human carboxypeptidase N (CPN), a member of the CPN/E subfamily of "regulatory" metallo-carboxypeptidases, is an extracellular glycoprotein synthesized in the liver and secreted into the blood, where it controls the activity of vasoactive peptide hormones, growth factors and cytokines by specifically removing C-terminal basic residues. Normally, CPN circulates in blood plasma as a hetero-tetramer consisting of two 83 kDa (CPN2) domains each flanked by a 48 to 55 kDa catalytic (CPN1) domain. We have prepared and crystallized the recombinant C-terminally truncated catalytic domain of human CPN1, and have determined and refined its 2.1 A crystal structure. The structural analysis reveals that CPN1 has a pear-like shape, consisting of a 319 residue N-terminal catalytic domain and an abutting, cylindrically shaped 79 residue C-terminal beta-sandwich transthyretin (TT) domain, more resembling CPD-2 than CPM. Like these other CPN/E members, two surface loops surrounding the active-site groove restrict access to the catalytic center, offering an explanation for why some larger protein carboxypeptidase inhibitors do not inhibit CPN. Modeling of the Pro-Phe-Arg C-terminal end of the natural substrate bradykinin into the active site shows that the S1' pocket of CPN1 might better accommodate P1'-Lys than Arg residues, in agreement with CPN's preference for cleaving off C-terminal Lys residues. Three Thr residues at the distal TT edge of CPN1 are O-linked to N-acetyl glucosamine sugars; equivalent sites in the membrane-anchored CPM are occupied by basic residues probably involved in membrane interaction. In tetrameric CPN, each CPN1 subunit might interact with the central leucine-rich repeat tandem of the cognate CPN2 subunit via a unique hydrophobic surface patch wrapping around the catalytic domain-TT interface, exposing the two active centers.
J Mol Biol 2007 Feb 16
PMID:Crystal structure of the human carboxypeptidase N (kininase I) catalytic domain. 1715 76

Lysine acetyltransferases (KATs) catalyze the acetylation of specific lysine residues in histone and nonhistone proteins. The enzymatic activities of KATs are involved in a broad spectrum of cellular processes. Thus far, the reaction of KAT catalysis has been studied by various bioanalytical methods such as radioisotopic labeling, spectrophotometric and fluorometric measurements, and antibody-dependent immunosorbent assays. In particular, the fluorescent method has the advantage of simplicity for implementation, fast assay speed, fine signal to noise ratio, and superior sensitivity. We describe here the technical protocols of using thiol-sensitive fluorogenic probes for the fluorescent analysis of enzymatic activities of KATs, with males on the first (MOF) as an exemplary KAT enzyme. 7-Diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) is selected as the KAT probe owing to its fast reaction kinetics with coenzyme A (CoA) and excellent fluorogenicity upon thiol conjugation. The fluorescence-based acetylation assay is well suited for both kinetic characterization of KAT catalysis and KAT inhibitor investigation.
Methods Mol Biol 2013
PMID:The fluorescence-based acetylation assay using thiol-sensitive probes. 2338 66

Chitin is an essential component of the peritrophic matrix (PM), which is a structure that lines the insect's gut and protects against mechanical damage and pathogens. Rhodnius prolixus (Hemiptera: Reduviidae) does not have a PM, but it has an analogous structure, the perimicrovillar membrane (PMM); chitin has not been described in this structure. Here, we show that chitin is present in the R. prolixus midgut using several techniques. The FTIR spectrum of the KOH-resistant putative chitin-material extracted from the midgut bolus showed peaks characteristic of the chitin molecule at 3500, 1675 and 1085 cm(1). Both the midgut bolus material and the standard chitin NMR spectra showed a peak at 1.88 ppm, which is certainly due to methyl protons in the acetamide a group. The percentages of radioactive N-acetylglucosamine (CPM) incorporated were 2 and 4% for the entire intestine and bolus, respectively. The KOH-resistant putative chitin-material was also extracted and purified from the N-acetylglucosamine radioactive bolus, and the radioactivity was estimated through liquid scintillation. The intestinal CHS cDNA translated sequence was the same as previously described for the R. prolixus cuticle and ovaries. Phenotypic alterations were observed in the midgut of females with a silenced CHS gene after a blood meal, such as retarded blood meal digestion; the presence of fresh blood that remained red nine days after the blood meal; and reduced trachea and hemozoin content compared with the control. Wheat germ agglutinin (a specific probe that detects chitin) labeling proximal to the intestine (crop and midgut) was much lower in females with a silenced CHS gene, especially in the midgut region, where almost no fluorescence signal was detected compared with the control groups. Midguts from females with a CHS gene silenced by dsRNA-CHS and control midguts pre-treated with chitinase showed that the chitin-derived fluorescence signal decreased in the region around the epithelium, the region facing the midgut and projections towards the intestinal lumen when evaluated microscopically. The relative reduction in CHS transcripts by approximately 80% using an RNAi assay supports the phenotypical alterations in the midgut observed using fluorescence microscopy assays. These data show that chitin is present in the R. prolixus midgut epithelium and in its surface projections facing the lumen. The CHS gene expression and the presence of chitin in the R. prolixus midgut may suggest a target for controlling Chagas disease vectors and addressing this public health problem.
Insect Biochem Mol Biol 2016 Feb
PMID:Chitin is a component of the Rhodnius prolixus midgut. 2591 Jun 79


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