Cysteinyl peptides of pig heart NADP-dependent isocitrate dehydrogenase that are modified upon inactivation by N-ethylmaleimide

Smyth, Gerald E.; Colman, Roberta F.

doi:10.1016/s0021-9258(18)98565-0

Cited by 46 publications

(17 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cloning of the cDNA Encoding Porcine NADP-IDH by Polymerase Chain Reaction. The sequences for several cysteine-containing tryptic peptides of porcine NADP-IDH and a 30-residue sequence from the amino terminus of the intact polypeptide have previously been reported (Smyth & Colman, 1991). The amino-terminal sequence and the sequences of certain of the tryptic fragments were found to be homologous with discrete portions of the predicted amino acid sequence of yeast mitochondrial NADP-IDH (IDP1), and alignment of these peptides with the IDP1 protein sequence allowed putative mapping of their relative locations within the porcine protein.…”

Section: Resultsmentioning

confidence: 99%

“…Western Blot Analysis. For immunoblot analysis, yeast and porcine NADP-IDHs purified as previously described (Haselbeck & McAlister-Henn, 1991; Smyth & Colman, 1991) were electrophoresed on 10% polyacrylamide/sodium dodecyl sulfate gels and electroblotted to Immobilon poly(vinylidene difluoride) filters (Millipore). The filters were incubated with a 1:100 dilution of rabbit anti-yeast NADP-IDH antiserum and l25I-labeled protein A as previously described (Haselbeck & McAlister-Henn, 1991).…”

Section: Methodsmentioning

confidence: 99%

“…Comparison of amino acid sequences obtained from tryptic peptides of porcine mitochondrial NADP-IDH (Smyth & Colman, 1991) with the predicted amino acid sequence of 1 Abbreviations: IDH, isocitrate dehydrogenase; TCA, tricarboxylic acid cycle; PCR, polymerase chain reaction; NEM, /V-ethylmaleimide.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Isolation and sequence of a cDNA encoding porcine mitochondrial NADP-specific isocitrate dehydrogenase

1992

View full text Add to dashboard Cite

The cDNA for porcine mitochondrial NADP-specific isocitrate dehydrogenase was isolated from a lambda gt11 library using polymerase chain reaction. Translation of the DNA sequence gave a 413-residue amino acid sequence and a calculated molecular weight of 46,600 for the mature polypeptide. Previously determined peptide sequences for the amino terminus and for internal tryptic peptides were all contained within the translated sequence. The porcine protein was found to share 63% residue identity with yeast mitochondrial NADP-specific isocitrate dehydrogenase and to be immunoreactive with an antiserum against the yeast protein. Highly conserved regions include residues which have been implicated in substrate and cofactor binding in previous studies of the porcine enzyme. The two eucaryotic enzymes exhibit only minimal homology with the NADP-dependent isocitrate dehydrogenase from Escherichia coli, with the exception of a striking conservation of residues implicated in formation of the metal-isocitrate site of the procaryotic enzyme.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Isolation and sequence of a cDNA encoding porcine mitochondrial NADP-specific isocitrate dehydrogenase

1992

View full text Add to dashboard Cite

show abstract

“…Since the known amino a These are representative sequences and do not reflect the relative magnitude of peaks I and II. 4 In gas-phase sequence analysis, NEM-Cys can be detected asa doublet migrating on the HPLC column between the PTH derivatives of Pro and Met (Smyth & Colman, 1991).c An X at a given cycle indicates that the phenylthiohydantoin derivative did not migrate asa known PTH-amino acid derivative. The amount of derivative present was determined by measuring radioactivity recovered from sequencing and calculating the pmol of derivative using the specific radioactivity of [3,5-3H]-4-FSB.…”

Section: Resultsmentioning

confidence: 99%

Affinity labeling of glutathione S-transferase, isozyme 4-4, by 4-(fluorosulfonyl)benzoic acid reveals Tyr115 to be an important determinant of xenobiotic substrate specificity

Barycki

Colman

1993

Biochemistry

View full text Add to dashboard Cite

Incubation of 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, with the 4-4 isozyme of rat liver glutathione S-transferase at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme. The rate of inactivation exhibits a nonlinear dependence on 4-FSB concentration from 0.50 to 7.85 mM, with kmax = 0.082 min-1 and a KI of 1.95 mM. Nearly 1 mol of reagent/mol of enzyme subunit is incorporated when the enzyme is maximally inactivated. Protection against incorporation and inactivation is provided by bromosulfophthalein, a competitive inhibitor with respect to the hydrophobic substrate, 1-chloro-2,4-dinitrobenzene (CDNB), suggesting that the reaction occurs in the binding site of the xenobiotic substrate. Fractionation by high-performance liquid chromatography of a tryptic digest of inactivated enzyme yields a single, modified, 14-residue peptide containing Tyr115 as the altered amino acid. Modified and control enzymes have comparable affinities for glutathione, as indicated by fluorescence titration. In contrast, as distinguished from the control enzyme, modified enzyme does not adsorb to a column of an agarose-linked Cibacron Blue derivative, indicating that it has lost its ability to bind a hydrophobic substrate analogue. These results are supported by kinetic characteristics of modified and control enzymes: upon modification of the enzyme with 4-FSB, the apparent Km for glutathione is unchanged, while the apparent Km for CDNB increases dramatically from 193 to 1690 microM. When the reaction of 4-FSB with enzyme is monitored, the final percent residual activity is found to be dependent on the substrate used in the assay: 11% for CDNB, 20% for ethacrynic acid, 2.5% for trans-stilbene oxide, and 2% for trans-4-phenyl-3-butene-2-one. Analysis of the kinetics of modified enzyme suggests that Tyr115 of glutathione S-transferase, isozyme 4-4, contributes to xenobiotic substrate binding and, when certain types of substrates are employed, is involved in catalysis.

show abstract

“…An Applied Biosystems gas-phase protein (peptide) sequencer, Model 470, equipped with a Model 120 phenylthiohydantoin analyzer and a Model 900A computer, was used to determine the amino acid sequence of peptides. Cysteine modified by N-ethylmaleimide (NEM-Cys) was identified by the doublet migrating on the HPLC column of the sequencer between the PTH derivatives of Pro and Met (Smyth & Colman, 1991) and mB-Cys by a distinct peak appearing between PTH derivatives of Tyr and Pro (Hu & Colman, 1995). In addition, there is measurable fluorescence associated with the PTH derivatives of mB-Cys.…”

Section: Methodsmentioning

confidence: 99%

Resonance Energy Transfer between Sites in Rat Liver Glutathione S-Transferase, 1-1, Selectively Modified at Cysteine-17 and Cysteine-111

Colman

1997

Biochemistry

View full text Add to dashboard Cite

Monobromobimane (mBBr) can label both Cys111 and Cys17 of rat liver glutathione S-transferase, 1-1 (GST 1-1). However, selective modification of Cys111 was achieved by the maleimide-based sulfhydryl reagents N-ethylmaleimide (NEM) and fluorescein 5-maleimide (NFM). Incubation of GST 1-1 with 5 mM NEM for 30 min at pH 7.5 and 25 degrees C leads to the formation of modified enzyme with 92% residual activity toward 1-chloro-2,4-dinitrobenzene and completely blocks Cys111 from subsequent reaction with either NFM or mBBr. Reaction of GST 1-1 with 0.2 mM NFM under the same conditions affords a modified enzyme with only 14% residual activity even though NFM and NEM target the same Cys111. The results indicate that when the bulky fluorescein is covalently bound to Cys111, the ligand projects into both the xenobiotic binding site and the glutathione site. After NEM or NFM modification of GST 1-1, the enzyme was further modified by monobromobimane at Cys17 with loss of activity. Together with the only tryptophan (Trp20), fluorescein linked to Cys111 and bimane to Cys17 provide three fluorescent probes to study the solution structure of GST 1-1. Fluorescence spectral analysis suggests that Trp20 and bimane linked to Cys17 are located in a relatively hydrophobic environment, while fluorescein linked to Cys111 is located in a charged environment. These fluorescent probes constitute three sets of donor-acceptor pairs for the measurement of fluorescence energy transfer, and distances calculated from such measurements are 20 A between Trp20 and bimane at Cys17, 19 A between Trp20 and fluorescein at Cys111, and < 22 A between bimane at Cys17 and fluorescein at Cys111. Molecular modeling studies indicate that fluorescein lies between the two subunits, is surrounded by charged residues, and is extended into the xenobiotic binding site. They also suggest that mBBr must approach from the dimer interface in order to reach the reaction site at Cys17.

show abstract

Cysteinyl peptides of pig heart NADP-dependent isocitrate dehydrogenase that are modified upon inactivation by N-ethylmaleimide

Cited by 46 publications

References 29 publications

Isolation and sequence of a cDNA encoding porcine mitochondrial NADP-specific isocitrate dehydrogenase

Isolation and sequence of a cDNA encoding porcine mitochondrial NADP-specific isocitrate dehydrogenase

Affinity labeling of glutathione S-transferase, isozyme 4-4, by 4-(fluorosulfonyl)benzoic acid reveals Tyr115 to be an important determinant of xenobiotic substrate specificity

Resonance Energy Transfer between Sites in Rat Liver Glutathione S-Transferase, 1-1, Selectively Modified at Cysteine-17 and Cysteine-111

Contact Info

Product

Resources

About