Flow kinetics of a nucleoside phosphatase common to endoplasmic reticulum, Golgi apparatus, and plasma membrane of rat liver

Nucleoside mono-, di- and triphosphatase activities of highly purified endoplasmic reticulum (ER), Golgi apparatus, and plasma membrane fractions of rat liver were compared. The highest rates of hydrolysis were always in ER or plasma membrane. Golgi apparatus activity was intermediate between those of ER and plasma membrane. This relationship was true for both freshly isolated fractions and salt-extracted membranes. Detergent solubilization of the membranes, polyacrylamide gel electrophoresis of the solubilized proteins, and localization of the enzyme activities on the gel revealed bands of enzyme activity which had identical mobilities in all three membrane fractions as well as other bands of activity that occurred only in ER and to a lesser degree in the Golgi apparatus. Antibodies raised against one of the phosphatase bands of plasma membrane which was common to all three membrane fractions cross-reacted with the corresponding phosphatase band in ER and Golgi apparatus. The anti-nucleoside phosphatase was utilized in combination with pulse-chase techniques to investigate the flow kinetics of transfer of newly synthesized enzyme among different cell compartments. Label first appeared in nucleoside phosphatase within the ER. Maximum specific activity was observed at about 5 min after injection of label and was followed by rapid loss of label. This was followed by appearance of label in Golgi apparatus 15 to 25 min after injection of label and by subsequent rapid loss of label. Plasma membranes were labeled last with no evidence of either rapid accumulation of label or of rapid turnover. Flow of nucleoside phosphatase from its site of synthesis and insertion into the membrane at the endoplasmic reticulum to the plasma membrane via the Golgi apparatus is indicated but in a manner whereby a significant fraction of the protein may be processed (removed?) from the membrane concomitant with the flow process.