Synthesis and use of a chromogenic substrate analog for Ap4A catabolic enzymes

Preston N. Garrison, Carlos E. de la Peña, Larry D. Barnes

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)226-230
Number of pages5
JournalAnalytical Biochemistry
Volume210
Issue number2
DOIs
StatePublished - 1993

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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