Linked Life Data

2517378

Source:http://linkedlifedata.com/resource/pubmed/id/2517378

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:dateCreated
1990-5-21
pubmed:abstractText
The results presented herein describe the development of a new method for the purification of thymidylate synthase from amethopterin-resistant L. casei. This method includes three chromatographic steps: batchwise CM-Sephadex, Q-Sepharose, and 10-formylfolate-Sepharose affinity steps, and the whole procedure can be performed in three days. Additionally, the procedure has consistently produced enzyme with a specific activity of 3.0 to 3.2. The first step, the batchwise purification of L. casei cell free extracts on CM-Sephadex, resulted in a 120-fold purification with 115% recovery of the activity applied to the resin. The Q-Sepharose purification step yielded a 1.5-fold purification producing enzyme with a specific activity of 2.4 units/mg. As the final step in the sequence, 10-formylfolate-Sepharose affinity chromatography yielded enzyme with a specific activity of 3.0-3.2 units/mg. The entire procedure provided a 230-fold purification with 67% recovery of the activity originally present in the cell free extract. However, the purified enzyme required extensive dialysis to achieve maximal activity. The data presented also clearly demonstrate the resolution of thymidylate synthase on the 10-formylfolate affinity column into forms which have different specific activity values and FdUMP binding ratios. Generally, enzyme with low activity leaches off this affinity matrix first, while enzyme with high activity elutes off last. These results are in accord with the sensitivity of the dimeric thymidylate synthase active site cysteines to oxidization. Consequently, when one of the two active site cysteines is oxidized, the enzyme loses the ability to bind FdUMP at this active site and its specific activity is proportionally lower. Thus, pure thymidylate synthase protein exists as a mixture of heterogeneous activity states. This condition is best examined by performing native PAGE on enzyme that has been incubated with excesses of FdUMP and CH2H4folate.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:issn
0065-2571
pubmed:author
pubmed:issnType
Print
pubmed:volume
29
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
189-207
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1989
pubmed:articleTitle
A rapid, reliable, and convenient method for the purification of thymidylate synthase from amethopterin-resistant Lactobacillus casei.
pubmed:affiliation
Department of Chemistry, University of South Carolina, Columbia 29208.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.