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  • Title: Synthesis and use of a chromogenic substrate analog for Ap4A catabolic enzymes.
    Author: Garrison PN, de la Peña CE, Barnes LD.
    Journal: Anal Biochem; 1993 May 01; 210(2):226-30. PubMed ID: 7685562.
    Abstract:
    ATP was coupled with 5-bromo-4-chloro-3-indolyl phosphate using a water-soluble carbodiimide to yield 5-bromo-4-chloro-3-indolyl tetraphospho-5'-adenosine (BCIp4A) which is an analog of diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A). BCIp4A is a chromogenic substrate for three different types of Ap4A catabolic enzyme in alkaline phosphatase-coupled reactions. Ap4A phosphorylase I from Saccharomyces cerevisiae was used as a model enzyme to demonstrate that BCIp4A stains for enzymic activity in polyacrylamide gels under nondenaturing conditions. A yeast colony assay was developed to detect Ap4A phosphorylase I activity in situ using BCIp4A as a chromogenic substrate. Ap4A phosphorylase I was assayed in situ in yeast transformed with a multicopy plasmid containing APA1, the gene encoding Ap4A phosphorylase I. BCIp4A should facilitate screening of genomic or cDNA libraries for genes encoding Ap4A catabolic enzymes.
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