Purification and characterization of phosphofructokinase in bovine parotid gland.
1. Phosphofructokinase (PFK) was purified from bovine parotid gland to 750-fold with the specific activity of 67.5 units/mg protein by Cibacron Blue F3GA affinity chromatography, and TSK DEAE-5PW ion-exchange and TSK G4000SW size exclusion chromatographies on HPLC. 2. On gel-filtration, molecular weight of the native PFK was estimated to 400,000. 3. PFK was a heterotetramer composed of three kinds of subunit with molecular weights of 92,000 (C-type), 88,000 (M-type) and 86,000 (L-type), by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Densitometrically, relative amounts of C-, M- and L-type subunit were 1:1:2. 4. Under the physiological conditions of fructose 6-phosphate (Fru-6-P) and ATP concentrations and pH, PFK activity was suppressed and hardly detectable. 5. Fru-6-P relieved PFK from the ATP inhibition. 6. Fructose 2,6-bisphosphate (Fru-2,6-P2) and AMP activated PFK with a reduction of S0.5 for Fru-6-P and subunit cooperativity. Fru-2,6-P2 was more effective than AMP.
Fukushima
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E
Department of Physiology, Nihon University School of Dentistry, Matsudo, Chiba, Japan.
Sugiya
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H
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002384:Catalysis; D002417:Cattle; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005635:Fructosediphosphates / Q000494:pharmacology; D005636:Fructosephosphates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008970:Molecular Weight; D010306:Parotid Gland / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D005635:Fructosediphosphates; D005636:Fructosephosphates; D046911:Macromolecular Substances; D000249:Adenosine Monophosphate; C027618:fructose-6-phosphate; C027652:fructose 2,6-diphosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1