PubMed · 4107434
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
A R Henderson, J King. 1971-11-20. Too many tests?. https://doi.org/10.1016/s0140-6736(71)91315-8
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
We report here a new pH-indicator-based assay for galactosyltransferase. The method is simple and fast, requires no specialized equipment, labeled substrate, or other expensive materials, and is thus expected to have broad applications including automated high-throughput screening. The method is based upon the detection of absorbance change of a pH indicator, phenol red, in response to proton release that accompanies the galactosyltransferase-catalyzed galactose transfer. The assay was used to compare three galactosyltransferases in our collection. As demonstrated here, subtle differences in substrate specificity were readily discerned with this sensitive method. All three enzymes accept both N-acetylglucosamine and glucose as acceptor but the relative activity varies with the origin of the enzyme. The method was demonstrated to be useful in the initial characterization of recombinant galactosyltransferase from crude cell extract. Optimal metal cofactor Mn(2+) concentration and temperature were determined with the method. Overall, the method offers a great improvement over current methods in reducing time and material consumption. It is the first pH-sensitive method for galactosyltransferase. The principles of using pH indicator in galactosyltransferase assay should be applicable to other glycosyltransferase enzymes.
Explore the source record for details and available documents.
A simple and accurate method for determination of enzymatic activity of the NAD-dependent DNA ligase of Thermus thermophilus HB8 has been developed that requires no radiolabeled substrates. lambda-DNA digested with BstEII provides two substrate DNA molecules (fragments 1 and 4) containing 12 base pair cohesive ends that are stably annealed at the assay temperature of 45 degrees C. One cohesive end unit is defined as the amount of enzyme required to achieve 50% ligation of fragment 1 in 15 min at 45 degrees C. Percent ligation is determined by analysis of reaction products, produced in reactions containing serial dilutions of enzyme, separated by agarose gel electrophoresis and photographed using a digital imaging device. Imaging software quantifies the amounts of fragment 1 and non-substrate fragment 7 present in the each lane (reaction). The latter is used to normalize the amount of fragment 1. This normalization process corrects for variations in sample loading, electrophoretic artifacts, and optical distortion of the gel image. A negative control containing no enzyme allows calculation of percent substrate ligated into product. Unit activity is then calculated from a dose-response curve in which percent of fragment 1 ligated is plotted against the log(10) of the enzyme dilution factor.