Selective inactivation of an extra-cytoplasmic acid phosphatase of yeast-like cells of Sporothrix schenckii by sodium fluoride.
Suspensions of intact, yeast-like cells of Sporothrix schenckii exhibited an acid phosphatase (EC 3.1.3.2) activity against p-nitrophenyl phosphate of about 5 IU (g dry wt)-1, without recourse to membrane perturbation. This extra-cytoplasmic acid phosphatase was reversibly and competitively inhibited by orthophosphate (Ki = 2 mM at pH 5) but unaffected by L(+)-tartrate (in contradistinction to some of the cytoplasmic acid phosphatases of the same organism). Inactivation by NaF of the extra-cytoplasmic isoenzyme was irreversible and followed first order kinetics; sensitivity to NaF was decreased by the presence of citrate, phosphate or substrate. Neither Km (0.3 mM at pH 5) nor Vmax for this enzyme in acetate buffer was greatly affected by pH in the range 3-5 but the first order rate constant for inactivation by NaF was strongly dependent on pH (maximum at pH 3.5). Crude cell-free extracts of yeast cells had nine electrophoretically distinct acid phosphatase activity bands and, on the basis of the pattern of inhibitors, the extra-cytoplasmic activity was identified as Y-I, an isoenzyme that barely penetrates standard polyacrylamide gel electropherograms. Additional evidence for the assignment came from selective inactivation of this isoenzyme by short treatments of intact cells with NaF under conditions that did not allow penetration of the plasma membrane by the inhibitor and did not kill the cells.
Arnold
W N
WN
Department of Biochemistry, University of Kansas Medical Center, Kansas City, 66103.
Sakai
K H
KH
Mann
L C
LC
D000135:Acid Phosphatase / Q000037:antagonists & inhibitors*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010710:Phosphates / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology*; D013175:Sporothrix / Q000201:enzymology*; D013644:Tartrates / Q000494:pharmacology
D010710:Phosphates; D013644:Tartrates; D012969:Sodium Fluoride; D000135:Acid Phosphatase; C018279:sodium phosphate; C029768:tartaric acid