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KINETIC STUDIES ON ALKALINE PHOSPHATASE FROM ECHINOPLUTEI 1
Author(s) -
Hsiao Sidney C.
Publication year - 1965
Publication title -
limnology and oceanography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.7
H-Index - 197
eISSN - 1939-5590
pISSN - 0024-3590
DOI - 10.4319/lo.1965.10.suppl2.r129
Subject(s) - chemistry , alkaline phosphatase , absorbance , michaelis–menten kinetics , hydrolysis , arrhenius equation , chromatography , reaction rate constant , activation energy , phosphomonoesterase , analytical chemistry (journal) , nuclear chemistry , enzyme , phosphatase , kinetics , enzyme assay , biochemistry , organic chemistry , physics , quantum mechanics
Using mass culture of the sea urchin Tripneustes gratilla (L.) eggs, a method was worked out for extracting fairly large quantities of the phosphomonoesterase alkaline phosphatase. The extracted enzyme showed a single ultraviolet absorbance band after elution from a Dowex 2 column, and the material that absorbed most strongly at this wave length contained alkaline phosphatase. It showed two characteristic bands in starch‐gel electrophoresis. When 6.25 mM p‐nitrophenyl phosphate was hydrolyzed in glycine buffer at 38C, the optimum p H was 10.5. The hydrolysis conformed to a first order reaction, and the reaction constant was 0.00864 min ‐1 . The optimum temperature for enzymatic action was 25–30C, coinciding with the range of fluctuation of the animal's ambient temperature. The temperature–activity curve showed a close conformity to the Arrhenius equation, and the activation energy was 11,250 cal/mole. The value of K m (Michaelis constant) obtained at p H 10.5 (at 25C) is 2.197 × 10 ‐6 M of p‐NPP/liter.