Human placental alkaline phosphatase. An improved purification procedure and kinetic studies.
An improved method for the purification of human placental alkaline phosphatase is described. The partially purified enzyme from Sigma was further purified by successive Concanavalin A-Sepharose and Q-Sepharose chromatography. The whole procedure may be completed in one working day. Highly purified enzyme was obtained with a 39% yield. The intrinsic fluorescence of the enzyme decreased at elevated temperature. The conformation of the enzyme molecule was studied by the fluorescence quenching technique. Upward Stern-Volmer plots were obtained for the quenching data which suggested that, in addition to collisional quenching, static quenching was involved in the quenching mechanism. The dynamic and static quenching constants were found to be 0.7 +/- 0.16 M-1 and 0.44 +/- 0.1 M-1, respectively, using acrylamide as the quenching agent. The corresponding values were 0.43 +/- 0.23 M-1 and 0.84 +/- 0.18 M-1, respectively, with KI as the quenching agent. Mg2+ and PO4(3-) induced protein conformational changes which altered both the dynamic and static quenching constants. Mg2+ was found to be a non-essential activator for the placental alkaline-phosphatase-catalyzed hydrolysis of 4-nitrophenyl phosphate. At pH 9.8, Mg2+ increased Vmax by 1.2-fold without affecting the Kd of the substrate. The tetranitromethane-modified enzyme showed slower migration toward the anode on electrophoresis and increased Kd for Mg2+.
Chang
T C
TC
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Huang
S M
SM
Huang
T M
TM
Chang
G G
GG
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013774:Tetranitromethane / Q000494:pharmacology
D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D000469:Alkaline Phosphatase; D008274:Magnesium; D013774:Tetranitromethane