NADP phosphatase as a marker in free-flow electrophoretic separations for cisternae of the Golgi apparatus midregion.
Based on cytochemical analysis, the enzyme NADP phosphatase is most concentrated in the so-called intercalary cisternae from the mid-region of the Golgi apparatus stack. Using free-flow electrophoresis to separate different Golgi regions of rat liver Golgi apparatus, the NADP phosphatase activity, based on estimation of the rate of release of inorganic phosphate from NADP under standard conditions, was similarly localized to membrane fractions from the center of electrophoretic separations. Peak specific activities for both a putative cis marker (NADH-cytochrome c reductase) and an established trans marker (galactosyltransferase) coincided with minima in NADP phosphatase activity, in agreement with the cytochemical observations. The pattern of distribution of enzyme activity for NADP phosphatase differed from that of both acid phosphatase and glucose-6-phosphatase. The pH optimum was 5.0, the Km for NADP was 0.6 mM and a corresponding production of NAD and inorganic phosphorus was shown. Taken together with other markers for free-flow electrophoresis separation, the NADP phosphatase will provide considerable utility as a specific marker to help identify intercalary cisternae of the mammalian Golgi apparatus and to monitor electrophoretic separations.
D000818:Animals; D004586:Electrophoresis; D005952:Glucose-6-Phosphatase / Q000302:isolation & purification; D005964:Glucosyltransferases / Q000302:isolation & purification; D006056:Golgi Apparatus / Q000032:analysis / Q000201:enzymology* / Q000648:ultrastructure; D008099:Liver / Q000648:ultrastructure; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D008854:Microscopy, Electron; D009243:NAD / Q000032:analysis; D009245:NADH Dehydrogenase / Q000302:isolation & purification; D009249:NADP / Q000032:analysis; D009708:Nucleotidases / Q000302:isolation & purification*; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification; D051381:Rats
D009243:NAD; D009249:NADP; D009245:NADH Dehydrogenase; D005964:Glucosyltransferases; C022699:sucrose synthase; C021896:2'-nucleotidase; D009708:Nucleotidases; D010744:Phosphoric Monoester Hydrolases; C022394:phosphoserine phosphatase; D005952:Glucose-6-Phosphatase