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Structural and mechanistic investigations into a DNA polymerase from Drosophila melanogaster embryos

Thesis/Dissertation ·
OSTI ID:5253459
A procedure for isolating DNA polymerase ..cap alpha.. (DNAP..cap alpha..) from Drosophila melanogaster embryos is described. A novel affinity chromatographic step exploits the differential binding affinity exhibited by this enzyme for poly A and poly G agarose. DNAP..cap alpha.. isolated from embryos of 9 hour average age appears identical to an enzyme previously described. A potentially larger form of the enzyme is isolated from 2.5 hour average age embryos. Two independent methods were used to demonstrate that DNAP..cap alpha.. obeys a rigidly ordered substrate binding mechanism with template-primer binding being prerequisite to dNTP binding. One method, utilizing alternative pathway kinetics, is described here for the first time. Pyridoxal-5-phosphate (PLP) was found to inhibit DNAP..cap alpha.. reversibly, at low stoichiometry and with a saturation effect, all criteria for an affinity label. Furthermore, PLP inhibition is dependent on pH and MgCl/sub 2/ concentration in the range of optimal DNAP activity. From protection experiments with normal substrates and dideoxyterminated primers and from the effects of substrates and PLP on initial velocities, it was conclusively shown that PLP inhibits DNAP by binding at two different sites. A procedure for the isolation of a pyridoxal kinase from Lactobacillus casei, optimal reaction conditions of the purified enzyme and its use in the synthesis of /sup 32/P PLP are all described.
Research Organization:
New York Univ., NY (USA)
OSTI ID:
5253459
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
ALKALINE EARTH METAL COMPOUNDS
ANIMALS
ARTHROPODS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL PATHWAYS
CHEMICAL BONDS
CHLORIDES
CHLORINE COMPOUNDS
DAYS LIVING RADIOISOTOPES
DIPTERA
DNA POLYMERASES
DROSOPHILA
EMBRYOS
ENZYME ACTIVITY
ENZYME INHIBITORS
ENZYMES
FLIES
FRUIT FLIES
HALIDES
HALOGEN COMPOUNDS
INSECTS
INVERTEBRATES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LACTOBACILLUS
LIGHT NUCLEI
MAGNESIUM CHLORIDES
MAGNESIUM COMPOUNDS
MICROORGANISMS
NUCLEI
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
OXYGEN COMPOUNDS
PH VALUE
PHOSPHATES
PHOSPHORUS 32
PHOSPHORUS COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
POLYMERASES
PURIFICATION
RADIOISOTOPES
REACTION KINETICS
STOICHIOMETRY
TRACER TECHNIQUES
TRANSFERASES