Linked Life Data

10415082

Source:http://linkedlifedata.com/resource/pubmed/id/10415082

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1999-8-30
pubmed:abstractText
We developed a method to determine dihydrofolate reductase (DHFR) activity at pH 7.4 (37 degrees C) by monitoring its product, tetrahydrofolate (H(4)folate), using HPLC with electrochemical detection. After the assay mixture was deproteinized by 0.5 M perchloric acid, the H(4)folate concentration was measured. Using sodium ascorbate at 20 mM, H(4)folate was stable in our assay system. The enzyme activity was also stable. The detection limit of this method was less than 1 nM of H(4)folate in the enzyme assay system, which was 1/100 lower than those for the NADPH-spectrophotometric assay, which is commonly used for analysis of DHFR activity. This value of 1 nM allowed us to control the conversion from dihydrofolate (H(2)folate) to H(4)folate less than 10% of initial substrate concentrations during assay, when we used a concentration around K(m) values reported for DHFR from various sources. The rate of reduction showed a linearity at concentrations around the K(m). The reduction rate must be evaluated exactly around the K(m), in order to obtain an accurate profile of Michaelis-Menten kinetics. This assay method has a sensitivity high enough to determine the reduction rate at H(2)folate concentrations around K(m). In addition, the assay procedure is very simple. Therefore, our method may be useful for studying DHFR.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
0003-2697
pubmed:author
pubmed:copyrightInfo
Copyright 1999 Academic Press.
pubmed:issnType
Print
pubmed:day
1
pubmed:volume
272
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
143-8
pubmed:dateRevised
2003-11-14
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Assay of dihydrofolate reductase activity by monitoring tetrahydrofolate using high-performance liquid chromatography with electrochemical detection.
pubmed:affiliation
Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, 183-0057, Japan.
pubmed:publicationType
Journal Article