Factors affecting the activity and stability of alkaline phosphatase in a marine pseudomonad.
Conditions optimum for the assay of alkaline phosphatase of marine pseudomonad B-16 (ATCC 19855) and for maintaining the activity of the enzyme have been determined. The pH for optimal activity of the cell-bound enzyme was 9.0, whereas that for the enzyme after its release from the cells exceeded 9.4. Release was effected by first washing the cells in 0.5 M NaCl and then suspending them in 0.5 M sucrose. In the absence of salts, the activity of the cell-bound enzyme decreased rapidly at 25 C and less rapidly at 4 C. This loss of activity could be arrested but not restored by adding Mg(2+). In the presence of Na(+), activity of the cell-bound enzyme dropped to about 50% of that prevailing initially, but in this case adding Mg(2+) restored enzyme activity completely. The activity of the enzyme after its release from the cells into 0.5 M sucrose was approximately 50% of that of the equivalent amount of enzyme in the original cells. This activity was relatively stable at both 25 and 4 C. Adding Mg(2+) to the released enzyme restored its activity to that of the cell-bound form. The synthesis of alkaline phosphatase by the cells was not affected by adding 50 mM inorganic phosphate to the growth medium. The K(m) of the released enzyme for p-nitrophenyl phosphate was found to be 6.1 x 10(-5) M.
D000469:Alkaline Phosphatase / Q000302:isolation & purification / Q000378:metabolism*; D002021:Buffers; D002473:Cell Wall / Q000201:enzymology; D002474:Cell-Free System; D003470:Culture Media; D004355:Drug Stability; D004794:Enzyme Repression; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D011549:Pseudomonas / Q000201:enzymology*; D012623:Seawater; D012964:Sodium / Q000494:pharmacology; D013053:Spectrophotometry; D013395:Sucrose; D013696:Temperature; D014325:Tromethamine / Q000494:pharmacology; D014871:Water Microbiology
D002021:Buffers; D003470:Culture Media; D010710:Phosphates; D014325:Tromethamine; D013395:Sucrose; D012964:Sodium; D000469:Alkaline Phosphatase; D008274:Magnesium