Bacteriodes symbiosus pyruvate phosphate dikinase: purification and mechanism of action
Conference
·
· Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
OSTI ID:5020564
The PPDK dimer was found to co-purify with (5-50% contamination) and convert to a protein which appears to consist of a normal PPDK subunit and a fragmented subunit. The protein adduct can be separated from normal PPDK on Superose 6 FPLC columns. The normal PPDK migrates as a single protein band on SDS and PAGE gels, has no ATPase or myokinase activity and has a S.A. which varies with preparation and age between 5 and 20 units/mg. The protein adduct migrates as two equal sized bands on SDS and PAGE gels (in both cases one band co-migrates with normal PPDK). The adduct displays ca. 50% the activity of normal PPDK and a myokinase activity of ca. 0.4 units/mg. The protein on the PAGE gel of the protein adduct which migrates at the PPDK position shows only PPDK activity while the faster moving band shows only myokinase activity. Using pure normal PPDK they have begun to test the two possible chemical mechanisms for the first partial reaction of the bi (ATP, P/sub i/down arrow) bi (AMP, PP up arrow) uni (pyr down arrow) uni (PEP up arrow) reaction: one involving formation of E x ADP x PP while the other EPP x AMP x P/sub i/. They have tested for ADP formation using an equilibrium system containing 30..mu..M (/sup 14/C)-ATP, 5mM P/sub i/ and 50..mu..M PPDK. The amount of ADP found (ca. 4%) did not exceed the amount in the control reaction. The AMP:ATP ratio was found to be approx.1. Studies designed to test E x ADP x PP and EPP x AMP x P/sub i/ formation at steady state are in progress.
- Research Organization:
- Univ. of Maryland, College Park
- OSTI ID:
- 5020564
- Report Number(s):
- CONF-8606151-
- Conference Information:
- Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
- Country of Publication:
- United States
- Language:
- English
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