| dc.contributor.author |
Labrou, NE |
en |
| dc.contributor.author |
Eliopoulos, E |
en |
| dc.contributor.author |
Clonis, YD |
en |
| dc.date.accessioned |
2014-06-06T06:43:10Z |
|
| dc.date.available |
2014-06-06T06:43:10Z |
|
| dc.date.issued |
1996 |
en |
| dc.identifier.issn |
02646021 |
en |
| dc.identifier.uri |
http://62.217.125.90/xmlui/handle/123456789/1057 |
|
| dc.relation.uri |
http://www.scopus.com/inward/record.url?eid=2-s2.0-0029922127&partnerID=40&md5=840af0f068361cfe7c31776a13f5d45f |
en |
| dc.subject.other |
adenosine diphosphate |
en |
| dc.subject.other |
dye |
en |
| dc.subject.other |
lysine |
en |
| dc.subject.other |
malate dehydrogenase |
en |
| dc.subject.other |
nicotinamide adenine dinucleotide |
en |
| dc.subject.other |
reduced nicotinamide adenine dinucleotide |
en |
| dc.subject.other |
animal tissue |
en |
| dc.subject.other |
article |
en |
| dc.subject.other |
cattle |
en |
| dc.subject.other |
competitive inhibition |
en |
| dc.subject.other |
controlled study |
en |
| dc.subject.other |
dialysis |
en |
| dc.subject.other |
enzyme active site |
en |
| dc.subject.other |
enzyme binding |
en |
| dc.subject.other |
enzyme inactivation |
en |
| dc.subject.other |
enzyme inhibition |
en |
| dc.subject.other |
enzyme subunit |
en |
| dc.subject.other |
gel filtration chromatography |
en |
| dc.subject.other |
heart mitochondrion |
en |
| dc.subject.other |
hydrolysis |
en |
| dc.subject.other |
molecular model |
en |
| dc.subject.other |
nonhuman |
en |
| dc.subject.other |
priority journal |
en |
| dc.subject.other |
reversed phase high performance liquid chromatography |
en |
| dc.subject.other |
scatchard plot |
en |
| dc.subject.other |
thin layer chromatography |
en |
| dc.subject.other |
Affinity Labels |
en |
| dc.subject.other |
Animals |
en |
| dc.subject.other |
Anthraquinones |
en |
| dc.subject.other |
Binding Sites |
en |
| dc.subject.other |
Cattle |
en |
| dc.subject.other |
Coloring Agents |
en |
| dc.subject.other |
Kinetics |
en |
| dc.subject.other |
Lysine |
en |
| dc.subject.other |
Malate Dehydrogenase |
en |
| dc.subject.other |
Mitochondria, Heart |
en |
| dc.subject.other |
Models, Molecular |
en |
| dc.subject.other |
Molecular Structure |
en |
| dc.subject.other |
NAD |
en |
| dc.subject.other |
Protein Conformation |
en |
| dc.subject.other |
Triazines |
en |
| dc.subject.other |
Animalia |
en |
| dc.subject.other |
Bos taurus |
en |
| dc.subject.other |
Bovinae |
en |
| dc.title |
Dye-affinity labelling of bovine heart mitochondrial malate dehydrogenase and study of the NADH-binding site |
en |
| heal.type |
journalArticle |
en |
| heal.publicationDate |
1996 |
en |
| heal.abstract |
The ability of the reactive dichlorotriazine dye Vilmafix Blue A-R (VBAR) to act as an affinity label for bovine heart L-malate dehydrogenase (MDH) was studied. VBAR binds specifically and irreversibly to MDH (k3, 0.16 min(-1); K(D) 14.4 μM). The inactivation of the NADH-dependent enzyme by VBAR is competitively inhibited by NAD+, NADH and ADP. Quantitatively inhibited MDH contained approx. 1 mol of dye per mol of active site. The inhibition is irreversible and activity cannot be recovered either on incubation with 10 mM NAD+, 10 mM NADH or 10 mM ADP, or by extensive dialysis or gel-filtration chromatography. Data obtained from high-performance gel-filtration chromatography and analysed by Scatchard plot suggested the presence of two coenzyme-binding sites per MDH dimer. Tryptic digestion of VBAR-labelled MDH followed by reverse-phase HPLC analysis revealed one VBAR-labelled peptide. It appears that each subunit features the same peptide bearing the modifying residue involved in MDH labelling. The pK(a) of the modifying residue is 8.05. Both total acid hydrolysis of VBAR-labelled MDH followed by HPLC and TLC analysis, and molecular-modelling studies suggest that the modifying residue is Lys-81 and/or Lys-217. |
en |
| heal.journalName |
Biochemical Journal |
en |
| dc.identifier.issue |
2 |
en |
| dc.identifier.volume |
315 |
en |
| dc.identifier.spage |
687 |
en |
| dc.identifier.epage |
693 |
en |