Asymmetric reassociation of calf spleen NAD+ glycohydrolase into liposomes.
NAD+ glycohydrolase (NAD+ nucleosidase, EC 3.2.2.6) can be solubilized from calf spleen microsomes (microsomal fractions) by steapsin or by detergents to yield respectively a hydrophilic (i.e. water-soluble) and a hydrophobic form of the enzyme. The detergent-solubilized enzyme was successfully reassociated into phosphatidylcholine liposomes either by a cholate-dialysis or by a gel-filtration procedure. In both cases the incorporation of NAD+ glycohydrolase was found to be completely asymmetric, i.e. the active site of the enzyme was exposed only at the outer surface of the vesicles. By contrast, as judged by flotation experiments, the hydrophilic form of NAD+ glycohydrolase could not be reassociated into liposomes. These results are in agreement with the hypothesis that calf spleen NAD+ glycohydrolase is an amphipathic protein. When incorporated into large unilamellar vesicles composed of phosphatidylcholine, NAD+ glycohydrolase was not found to catalyse vectorial transfer of NAD+ by transglycosidation with nicotinamide as acceptor.
Muller
H M
HM
Laboratoire de Chimie Enzymatique (UA 1182), Université Louis Pasteur, Strasbourg, France.
Schuber
F
F
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002417:Cattle; D008081:Liposomes / Q000378:metabolism*; D009699:N-Glycosyl Hydrolases / Q000493:pharmacokinetics*; D009244:NAD+ Nucleosidase; D010713:Phosphatidylcholines / Q000378:metabolism; D013154:Spleen / Q000201:enzymology*
D008081:Liposomes; D010713:Phosphatidylcholines; D009699:N-Glycosyl Hydrolases; D009244:NAD+ Nucleosidase