Abstract
Avian myeloblastosis virus (AMV) DNA polymerase is inactivated by preincubation with pyridoxal 5'-phosphate. This inactivation is relatively specific since various pyridoxal-5'-P analogs cause no inactivation. This effect is reversible but can be made irreversible by reduction with sodium borohydride; the reduced pyridoxal-5'-P adduct exhibits a new absorbance maximum at 325 nm and a fluorescence emission at 392 nm when excited at 325 nm. The evidence presented suggests the formation of a Schiff base between pyridoxal-5'-P and a nucleophilic residue of AMV DNA polymerase. The presence of a deoxynucleoside 5'-triphosphate (dTTP) protected the enzyme from inactivation. Reduction of the pyridoxal-5'-P enzyme complex in the presence or absence of a deoxynucleoside 5'-triphosphate showed that the alpha subunit possesses five reactive amino groups, one of which is essential for catalytic activity; the beta subunit has three reactive amino groups which are not involved in the deoxynucleoside binding site.
Highlights
Avian myeloblastosis virus (AMV) DNA polymerase is inactivated by preincubation with pyridoxal 5’-phosphate
As an active site-directed reagent. This reagent is useful because it possesses a phosphate group which can direct it to a triphosphate binding site in the active center of the DNA polymerase and a reactive aldehyde group which can form an adduct with the e-amino group of lysine [7, 10]
Complete inhibition was observed only with pyridoxaM’P, both the aldehyde and the phosphate groups were required for complete inhibition
Summary
BioResearch, specific activities (Curies/mmol) were: dl”l’P, 17.3; ATP, 13; dGTP 8.5. Corp.; sodium borohydride (NaBH,) was obtained from Fisher Scientific; Hepes buffer was purchased from Calbiochem. The virus was concentrated and purified as described [12]. Enzymes - AMV DNA polymerase was assayed essentially as described [13]. To each reaction 0.2 mg of bovine serum albumin, 0.2 mg of yeast RNA, and 10 pmol of pyrophosphate were added followed by cold trichloroacetic acid to final concentration of. 4”, acid-insoluble material was collected on Whatman GF/C filters. AMV DNA polymerase was purified as described by Kacian et al [14]. The purified fraction of AMV DNA polymerase showed two distinct bands on sodium dodecyl sulfate gel electrophoresis When the enzyme was assayed after reacting with pyridoxal-5’-P the assay mixture contained the same concentration of pyridoxal-5’-P as the preincubation mixture; these conditions prevented dissociation of the complex
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