Calf spleen NAD+ glycohydrolase: solubilization, purification, and properties of the intact form of the enzyme.
NAD+ glycohydrolase was solubilized from calf spleen microsomes with emulphogene, a nonionic detergent, and purified to apparent homogeneity by ion-exchange chromatographies and by affinity chromatography on Affi-Gel blue gel. In contrast to the hydrosoluble form of the enzyme, which can be obtained by a proteolytic treatment of the microsomes with steapsin, the intact form of NAD+ glycohydrolase is characterized by its high hydrophobicity; i.e., the enzyme interacts very strongly with hydrophobic gels such as octyl-Sepharose and partitions into Triton X-114-rich phases. The apparent M(r) of the intact form of calf spleen NAD+ glycohydrolase is about 30 kDa, as compared to 24 kDa for the hydrosoluble form. This difference in molecular mass could account for the polypeptide moiety which allows the anchoring of the enzyme to the membranes. Both forms of the enzyme are strongly adsorbed by immobilized concanavalin A gels and biospecifically eluted with alpha-methylmannoside; this glycoprotein (mannosylated) nature of NAD+ glycohydrolase is in agreement with the previous demonstration that it is an ectoenzyme.
Laboratoire de Chimie Bioorganique (CNRS URA 1386), Faculté de Pharmacie, Illkirch, France.
H
H
Muller-Steffner
I
I
Schenherr-Gusse
C
C
Tarnus
F
F
Schuber
D000818:Animals; D002384:Catalysis; D002417:Cattle; D002846:Chromatography, Affinity; D017261:Glycosylphosphatidylinositols; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism*; D009244:NAD+ Nucleosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013154:Spleen / Q000201:enzymology
D017261:Glycosylphosphatidylinositols; D009243:NAD; D009244:NAD+ Nucleosidase