Characterization of soluble vs membrane-bound human placental 5'-nucleotidase.
Three forms of 5'-nucleotidase purified from human placenta (two membrane-bound forms, one sensitive and one resistant to cleavage by phosphatidylinositol-specific phospholipase C, as well as a soluble form) had the same molecular weight before (73,000 Da) and after (56,000 Da) digestion with N-glycosidase F and showed similar amino acid compositions, N-terminal amino acid sequences, and KMs for IMP (9.6 to 11.9 microM). Thus, these three forms of 5'-nucleotidase appear to have very similar structures. The form sensitive to phosphatidylinositol-specific phospholipase C contained nearly 1 mol myo-inositol/mol of protein as determined by mass spectrometry, indicating a glycosyl phosphatidylinositol membrane anchor. Soluble 5'-nucleotidase contained a similar quantity of myo-inositol, suggesting that it was previously membrane-anchored via glycosyl phosphatidylinositol. The form resistant to phosphatidylinositol-specific phospholipase C contained less myo-inositol, leaving open the possibility of a third form of 5'-nucleotidase with a conventional transmembrane anchor.
Klemens
M R
MR
Oklahoma Medical Research Foundation, Oklahoma City 73104.
Sherman
W R
WR
Holmberg
N J
NJ
Ruedi
J M
JM
Low
M G
MG
Thompson
L F
LF
D015720:5'-Nucleotidase / Q000032:analysis*; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000494:pharmacology; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D010920:Placenta / Q000187:drug effects / Q000201:enzymology*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D012995:Solubility
D010716:Phosphatidylinositols; D012333:RNA, Messenger; D015720:5'-Nucleotidase; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D006026:Glycoside Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase