Enzyme kinetics in mammalian cells. 3. Regulation of activities of galactokinase, galactose-1-phosphate uridyl transferase and uridine diphosphogalactose-4-epimerase in human erythrocytes.

Abstract

AbstractThe sequential enzyme assay as previously described has been used to study various effects on the three enzymes in human red cells involved in the phosphorylation of galactose: galactokinase, galactose‐1‐phosphate uridyl transferase and uridine diphospho‐galactose‐4‐epimerase. Enzyme activities in undiluted lysates appear to reflect the respective activities in whole cells. Added extracellular Gal‐1‐P, G‐1‐P, UDPGal and UPDG do not affect enzyme activities in whole cells. The kinase and transferase enzymes do not appear to be associated with the membrane fraction of the red cells. Galactokinase activity is inhibited by G‐6‐P and Gal‐1‐P, but not by glucose, G‐1‐P, UDPG, UDPGal, UTP or NAD+. It is inhibited by ATP and ADP in high concentration. Galactose‐1‐phosphate uridyl transferase activity is inhibited by G‐1‐P, G‐6‐P, UDPG, UDPGal, ATP, and ADP. It is not affected by UTP, NAD+, or galactose. Uridine diphospho‐galactose‐4‐epimerase activity is inhibited by UDPG, ATP, ADP, UTP and NADH. It is stimulated by NAD+ and possibly by Gal‐1‐P. It is unaffected by G‐1‐P, G‐6‐P. The rates of the three reactions decrease with decreasing temperature. The activities of transferase and epimerase are inactivated at the same rate, the kinase activity is inactivated more slowly. Dilution experiments indicate the presence in lysates of a pool of UDPG (or, possibly UDPGal) which regulates the activities transferase and the epimerase enzymes. Results of dilution experiments suggest that the radioactive product of the transferase enzyme is different from commercially available UDPGal‐u‐14C. ATP, UTP and UDPG interact with some substance(s) in the red cell lysate to cause a time dependent inactivation of the epimerase. These interactions are the result of glucose metabolism.