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Quantitative traits are shaped by networks of pleiotropic genes . To understand the mechanisms that maintain genetic variation for quantitative traits in natural populations and to predict responses to artificial and natural selection, we must evaluate pleiotropic effects of underlying quantitative trait genes and define functional allelic variation at the(More)
The NADH and NADPH specific FMN oxidoreductases from Beneckea harveyi have been purified to homogeneity as judged by single bands on sodium dodecyl sulfate gel electrophoresis. The overall purification for the NADH specific enzyme is 3000-fold and 4000-fold for the NADPH specific enzyme from a crude extract. The final step in the purification procedure is(More)
The study of enzymes sequestered in artificial or biological systems is generally conducted by indirect methodology with macroscopic measurements of reactants in the bulk medium. This paper describes a new approach with firefly luciferase to monitor ATP concentration directly in the microenvironment of enzymes producing or consuming ATP. Upon addition of(More)
Studies of the three human creatine phosphokinase (EC isoenzymes, MM, MB, and BB, show that important differences exist in substrate dependency of the reaction rates. A method was developed to study these properties in which the ATP formed in the reverse reaction was measured by means of firefly luciferase. With substrate conditions at which the(More)
Firefly luciferase, containing an average of seven free sulfhydryls per two 50 000-dalton polypeptides, was modified by various sulfhydryl reagents. The differential reactivities of the sulfhydryls in luciferase protected by substrates allow one to define three categories of these groups: Class SH-III contains three sulfhydryls that are not involved in(More)
A new procedure for measuring binding of tRNA to aminoacyl-tRNA synthetases is described. The purified isoleucyl-tRNA synthetase from Escherichia coli can be covalently bound to activated Sepharose with retention of approximately 40% of the original enzymatic activity. If crude tRNA is passed through a small column of enzyme-Sepharose, isoleucyl-tRNA is(More)
A simple, rapid, and sensitive bioluminescence method for measuring primary bile acids has been developed and validated. The method is based on enzymatic dehydrogenation of bile acids using a bacterial 7 alpha-hydroxysteroid dehydrogenase that is co-immobilized on Sepharose 4B beads with NADH:FMN oxidoreductase and a bacterial luciferase. The assay is(More)
NADH : FMN oxidoreductase and bacterial luciferase have been efficiently coimmobilized onto Sepharose 4B. This luminescent immobilized enzyme system can be used to assay NADH. The assay is rapid and sensitive with a lower limit of detection of 0.2 pmol/assay tube. The intra-assay precision was 3.5% at 2 × 10(-5) M and 5.8% at 2 × 10(-6) M NADH. Light(More)