Details

Autor(en) / Beteiligte

• Garrison, P.N.
• Delapena, C.E.
• Barnes, L.D.

Titel

Synthesis and Use of a Chromogenic Substrate Analog for Ap4A Catabolic Enzymes

Ist Teil von

• Analytical biochemistry, 1993-05, Vol.210 (2), p.226-230

Ort / Verlag

San Diego, CA: Elsevier Inc

Erscheinungsjahr

1993

Quelle

Elsevier Journal Backfiles on ScienceDirect (DFG Nationallizenzen)

Beschreibungen/Notizen

• 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 (BClp4A) which is an analog of diadenosine 5′,5′″-P1,P4-tetraphosphate (Ap4A). BClp4A 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 BClp4A 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 BClp4A 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. BClp4A should facilitate screening of genomic or cDNA libraries for genes encoding Ap4A catabolic enzymes.