Synthesis and use of a chromogenic substrate analog for Ap4A catabolic enzymes.
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.
Garrison
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Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
de la Peña
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CE
Barnes
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LD
D017766:Acid Anhydride Hydrolases; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D002863:Chromogenic Compounds; D004247:DNA / Q000235:genetics; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D016681:Genome, Fungal; D007211:Indoles / Q000138:chemical synthesis* / Q000737:chemistry; D009713:Nucleotidyltransferases / Q000032:analysis* / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods
D002863:Chromogenic Compounds; D015226:Dinucleoside Phosphates; D007211:Indoles; C081548:5-bromo-4-chloro-3-indolyltetraphospho-5'-adenosine; C020733:diadenosine tetraphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D009713:Nucleotidyltransferases; C044740:diadenosine 5,5'''-P(1),P(4)-tetraphosphate alpha,beta-phosphorylase; D010744:Phosphoric Monoester Hydrolases; D017766:Acid Anhydride Hydrolases; C020023:bis(5'-nucleosyl)tetraphosphatase (asymmetrical)