The effects of phospholipids on the properties of hepatic 5'-nucleotidase.
Arrhenius plots of 5'-nucleotidase activity in microsomes or plasma membranes from rat liver exhibited transitions at approximately 35 degrees C. The enzyme was purified from homogenates after solubilization in 2% Triton X-100 and 1% sodium deoxycholate. After the initial steps of the purification, the enzyme was recovered in membranes, as judged by both thin section and freeze-fracture electron microscopy, which contained sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. The purest fractions of 5'-nucleotidase were enriched approximate 3,000-fold, consisted of similar membranes, but only contained sphingomyelin. Thermal transitions were detected in Arrhenius plots of 5'-nucleotidase after detergent solubilization, in the membranes which contained the three phospholipids, but not in the purified fraction which contained only sphingomyelin; transitions were also detected after reassociation of the purified enzyme with microsomal or plasma membrane lipids and phosphatidylcholine but not with phosphatidylethanolamine. Phosphatidylcholines containing specific fatty acids all affected the energy of activation of 5'-nucleotidase, and the detergent Sarkosyl, which has been shown to dissociate phospholipids from 5'-nucleotidase (Evans, W. H., and Gurd, J. W. (1973) Biochem. J. 133, 189-199), caused a marked decrease in the stability of the enzyme to heating. Inhibition of 5'-nucleotidase by concanavalin A followed by reactivation with alpha-methyl-D-mannoside resulted in linear Arrhenius plots of 5'-nucleotidase activity in membrane fractions, and in lower transition temperatures for the detergent, solubilized enzyme. It is concluded that in situ, 5'-nucleotidase interacts with both sphingomyelin and phosphatidylcholine; the first apparently influences the stability of the enzyme and the second, the energy of activation. In addition, the lipid environment of the enzyme seems to be altered as a result of lectin binding.
D015720:5'-Nucleotidase; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D003208:Concanavalin A / Q000494:pharmacology; D004789:Enzyme Activation; D005614:Freeze Fracturing; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008563:Membrane Lipids / Q000032:analysis; D008854:Microscopy, Electron; D008862:Microsomes, Liver / Q000201:enzymology; D009708:Nucleotidases / Q000378:metabolism*; D010743:Phospholipids / Q000032:analysis / Q000494:pharmacology*; D051381:Rats; D013816:Thermodynamics
D008563:Membrane Lipids; D010743:Phospholipids; D003208:Concanavalin A; D009708:Nucleotidases; D015720:5'-Nucleotidase