Preparation of homogeneous pig liver thioltransferase by a thiol:disulfide mediated pI shift.
Identifieur interne : 001347 ( Main/Exploration ); précédent : 001346; suivant : 001348Preparation of homogeneous pig liver thioltransferase by a thiol:disulfide mediated pI shift.
Auteurs : Z R Gan ; W W WellsSource :
- Analytical biochemistry [ 0003-2697 ] ; 1987.
Descripteurs français
- KwdFr :
- Acides aminés (analyse), Animaux (MeSH), Chromatographie (méthodes), Cinétique (MeSH), Concentration en ions d'hydrogène (MeSH), Disulfures (MeSH), Focalisation isoélectrique (MeSH), Foie (enzymologie), Glutarédoxines (MeSH), Masse moléculaire (MeSH), Oxidoreductases (isolement et purification), Oxydoréduction (MeSH), Protein-disulfide reductase (glutathione) (MeSH), Suidae (MeSH), Thiols (MeSH), Électrophorèse sur gel de polyacrylamide (MeSH).
- MESH :
- analyse : Acides aminés.
- enzymologie : Foie.
- isolement et purification : Oxidoreductases.
- méthodes : Chromatographie.
- Animaux, Cinétique, Concentration en ions d'hydrogène, Disulfures, Focalisation isoélectrique, Glutarédoxines, Masse moléculaire, Oxydoréduction, Protein-disulfide reductase (glutathione), Suidae, Thiols, Électrophorèse sur gel de polyacrylamide.
English descriptors
- KwdEn :
- Amino Acids (analysis), Animals (MeSH), Chromatography (methods), Disulfides (MeSH), Electrophoresis, Polyacrylamide Gel (MeSH), Glutaredoxins (MeSH), Hydrogen-Ion Concentration (MeSH), Isoelectric Focusing (MeSH), Kinetics (MeSH), Liver (enzymology), Molecular Weight (MeSH), Oxidation-Reduction (MeSH), Oxidoreductases (isolation & purification), Protein Disulfide Reductase (Glutathione) (MeSH), Sulfhydryl Compounds (MeSH), Swine (MeSH).
- MESH :
- chemical , analysis : Amino Acids.
- enzymology : Liver.
- chemical , isolation & purification : Oxidoreductases.
- methods : Chromatography.
- Animals, Disulfides, Electrophoresis, Polyacrylamide Gel, Glutaredoxins, Hydrogen-Ion Concentration, Isoelectric Focusing, Kinetics, Molecular Weight, Oxidation-Reduction, Protein Disulfide Reductase (Glutathione), Sulfhydryl Compounds, Swine.
Abstract
An enzyme catalyzing thiol-disulfide exchange, thioltransferase, was purified to homogeneity from pig liver. By taking advantage of the relatively large pI shift of the enzyme between its reduced and disulfide forms, the purification procedure, which included a heat step, ammonium sulfate precipitation, Sephadex G-75 and G-50 gel chromatography, and two CM-Sepharose chromatography separations, resulted in a 32% overall yield. The purified enzyme was demonstrated to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography. The protein had a Mr of approximately 11,000 and, in the reduced form, a pI of 6.4. The amino acid composition of the enzyme was similar to that of rat liver thioltransferase and calf thymus glutaredoxin and the N-terminus of the protein was blocked. The optimal pH for the enzyme activity was 9.0. The plots of thioltransferase activity as a function of S-sulfocysteine, 2-hydroxyethyl disulfide, and reduced glutathione concentrations did not display Michaelis-Menten kinetics. The enzyme was very sensitive to a sulfhydryl alkylating reagent. Preincubation of the enzyme with its disulfide substrates prevented the inactivation of the enzyme by iodoacetic acid while the other substrate, GSH, did not provide such protection. The results suggest that the active center of thioltransferase is cysteine dependent.
DOI: 10.1016/0003-2697(87)90036-4
PubMed: 3605592
Affiliations:
Links toward previous steps (curation, corpus...)
Le document en format XML
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By taking advantage of the relatively large pI shift of the enzyme between its reduced and disulfide forms, the purification procedure, which included a heat step, ammonium sulfate precipitation, Sephadex G-75 and G-50 gel chromatography, and two CM-Sepharose chromatography separations, resulted in a 32% overall yield. The purified enzyme was demonstrated to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography. The protein had a Mr of approximately 11,000 and, in the reduced form, a pI of 6.4. The amino acid composition of the enzyme was similar to that of rat liver thioltransferase and calf thymus glutaredoxin and the N-terminus of the protein was blocked. The optimal pH for the enzyme activity was 9.0. The plots of thioltransferase activity as a function of S-sulfocysteine, 2-hydroxyethyl disulfide, and reduced glutathione concentrations did not display Michaelis-Menten kinetics. The enzyme was very sensitive to a sulfhydryl alkylating reagent. Preincubation of the enzyme with its disulfide substrates prevented the inactivation of the enzyme by iodoacetic acid while the other substrate, GSH, did not provide such protection. The results suggest that the active center of thioltransferase is cysteine dependent.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">3605592</PMID><DateCompleted><Year>1987</Year><Month>07</Month><Day>24</Day></DateCompleted><DateRevised><Year>2019</Year><Month>06</Month><Day>28</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0003-2697</ISSN><JournalIssue CitedMedium="Print"><Volume>162</Volume><Issue>1</Issue><PubDate><Year>1987</Year><Month>Apr</Month></PubDate></JournalIssue><Title>Analytical biochemistry</Title><ISOAbbreviation>Anal Biochem</ISOAbbreviation></Journal><ArticleTitle>Preparation of homogeneous pig liver thioltransferase by a thiol:disulfide mediated pI shift.</ArticleTitle><Pagination><MedlinePgn>265-73</MedlinePgn></Pagination><Abstract><AbstractText>An enzyme catalyzing thiol-disulfide exchange, thioltransferase, was purified to homogeneity from pig liver. By taking advantage of the relatively large pI shift of the enzyme between its reduced and disulfide forms, the purification procedure, which included a heat step, ammonium sulfate precipitation, Sephadex G-75 and G-50 gel chromatography, and two CM-Sepharose chromatography separations, resulted in a 32% overall yield. The purified enzyme was demonstrated to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography. The protein had a Mr of approximately 11,000 and, in the reduced form, a pI of 6.4. The amino acid composition of the enzyme was similar to that of rat liver thioltransferase and calf thymus glutaredoxin and the N-terminus of the protein was blocked. The optimal pH for the enzyme activity was 9.0. The plots of thioltransferase activity as a function of S-sulfocysteine, 2-hydroxyethyl disulfide, and reduced glutathione concentrations did not display Michaelis-Menten kinetics. The enzyme was very sensitive to a sulfhydryl alkylating reagent. Preincubation of the enzyme with its disulfide substrates prevented the inactivation of the enzyme by iodoacetic acid while the other substrate, GSH, did not provide such protection. The results suggest that the active center of thioltransferase is cysteine dependent.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Gan</LastName><ForeName>Z R</ForeName><Initials>ZR</Initials></Author><Author ValidYN="Y"><LastName>Wells</LastName><ForeName>W W</ForeName><Initials>WW</Initials></Author></AuthorList><Language>eng</Language><GrantList CompleteYN="Y"><Grant><GrantID>AM 32930</GrantID><Acronym>AM</Acronym><Agency>NIADDK NIH HHS</Agency><Country>United States</Country></Grant></GrantList><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013487">Research Support, U.S. Gov't, P.H.S.</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Anal Biochem</MedlineTA><NlmUniqueID>0370535</NlmUniqueID><ISSNLinking>0003-2697</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D000596">Amino Acids</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D004220">Disulfides</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D054477">Glutaredoxins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D013438">Sulfhydryl Compounds</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 1.-</RegistryNumber><NameOfSubstance UI="D010088">Oxidoreductases</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 1.8.4.2</RegistryNumber><NameOfSubstance UI="D011490">Protein Disulfide Reductase (Glutathione)</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000596" MajorTopicYN="N">Amino Acids</DescriptorName><QualifierName UI="Q000032" MajorTopicYN="N">analysis</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D000818" MajorTopicYN="N">Animals</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D002845" MajorTopicYN="N">Chromatography</DescriptorName><QualifierName UI="Q000379" MajorTopicYN="N">methods</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004220" MajorTopicYN="N">Disulfides</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D004591" MajorTopicYN="N">Electrophoresis, Polyacrylamide Gel</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D054477" MajorTopicYN="N">Glutaredoxins</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D006863" MajorTopicYN="N">Hydrogen-Ion Concentration</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D007525" MajorTopicYN="N">Isoelectric Focusing</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D007700" MajorTopicYN="N">Kinetics</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D008099" MajorTopicYN="N">Liver</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="Y">enzymology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D008970" MajorTopicYN="N">Molecular Weight</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010084" MajorTopicYN="N">Oxidation-Reduction</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010088" MajorTopicYN="N">Oxidoreductases</DescriptorName><QualifierName UI="Q000302" MajorTopicYN="Y">isolation & purification</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D011490" MajorTopicYN="Y">Protein Disulfide Reductase (Glutathione)</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D013438" MajorTopicYN="N">Sulfhydryl Compounds</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D013552" MajorTopicYN="N">Swine</DescriptorName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>1987</Year><Month>4</Month><Day>1</Day></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>1987</Year><Month>4</Month><Day>1</Day><Hour>0</Hour><Minute>1</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>1987</Year><Month>4</Month><Day>1</Day><Hour>0</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">3605592</ArticleId><ArticleId IdType="pii">0003-2697(87)90036-4</ArticleId><ArticleId IdType="doi">10.1016/0003-2697(87)90036-4</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list></list><tree><noCountry><name sortKey="Gan, Z R" sort="Gan, Z R" uniqKey="Gan Z" first="Z R" last="Gan">Z R Gan</name><name sortKey="Wells, W W" sort="Wells, W W" uniqKey="Wells W" first="W W" last="Wells">W W Wells</name></noCountry></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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