Continuous spectrophotometric assays for three regulatory enzymes of the arginine biosynthetic pathway.

Continuous spectrophotometric assays for three regulatory enzymes of the arginine biosynthetic pathway. Research Abstract Details 

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Continuous spectrophotometric assays for three regulatory enzymes of the arginine biosynthetic pathway. Abstract Text:

Kentaro Takahara,Kinya Akashi,Akiho Yokota,

N-Acetylglutamate synthase (AGS), N-acetylglutamate kinase (AGK), and glutamate N-acetyltransferase (GAT) are the key enzymes in the synthesis of arginine that serves as an important precursor for the synthesis of protein, polyamines, urea, and nitric oxide. Current assays available for these three enzymes are laborious and time-consuming and do not allow continuous monitoring of enzyme activities. Here we established continuous enzyme assays for AGS, AGK, and GAT based on the coupling of AGS and GAT reactions to AGK followed by coupling of the AGK reaction to N-acetylglutamate 5-phosphate reductase (AGPR). The rate of AGPR-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate was monitored continuously as a change in absorbance at 340 nm using spectrophotometry. These methods were applied to kinetic analyses for Escherichia coli AGK, E. coli AGS, and Saccharomyces cerevisiae GAT, and the kinetic parameters obtained in the coupling assays showed nearly the same values as those obtained previously using discontinuous assays. The specificity of these coupled assays was confirmed by the lack of enzyme activity from extracts of E. coli AGS-, E. coli AGK-, and S. cerevisiae GAT-deletion mutants. Moreover, the coupled assay enabled us to measure AGS activity from mammalian liver mitochondrial extracts, known to be an important regulatory enzyme for the urea cycle. These coupled enzyme assays are rapid, highly sensitive, and reproducible.

Continuous spectrophotometric assays for three regulatory enzymes of the arginine biosynthetic pathway. Publishing Authors By Initials

K Takahara,K Akashi,A Yokota,

For similar natural sciences: time: time factors research abstracts see: natural sciences: time: time factors research

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Continuous spectrophotometric assays for three regulatory enzymes of the arginine biosynthetic pathway. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Analytical biochemistry

VOLUME: 368

Page Numbers: 138-47

Journal Abbreviation: Anal. Biochem.

ISSN: 0003-2697

DAY: 22

MONTH: 06

YEAR: 2007

Continuous spectrophotometric assays for three regulatory enzymes of the arginine biosynthetic pathway. Information

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LANGUAGE: eng

NlmUniqueID: 370535

Continuous spectrophotometric assays for three regulatory enzymes of the arginine biosynthetic pathway. Keywords Mesh Terms:

KEYWORDS: Time Factors

MESH TERMS: methods

Chemical & Substance for Abstract: Continuous spectrophotometric assays for three regulatory enzymes of the arginine biosynthetic pathway. Information

Substance Name: acetylglutamate kinase

Registry Number: EC 2.7.2.8

Grant and Affiliation Information for Continuous spectrophotometric assays for three regulatory enzymes of the arginine biosynthetic pathway.

AFFILIATION: Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.

Country: United States

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MEDLINETA: Anal Biochem

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