TY - JOUR
T1 - Two rat intestinal alkaline phosphatase isoforms with different carboxyl- terminal peptides are both membrane-bound by a glycan phosphatidylinositol linkage
AU - Engle, M. J.
AU - Mahmood, A.
AU - Alpers, D. H.
PY - 1995
Y1 - 1995
N2 - Two cDNAs encode rat intestinal alkaline phosphatases having completely different carboxyl-terminal peptides; one is hydrophobic and fulfills the consensus requirements for glycan phosphatidylinositol linkage, and the other is neither hydrophobic nor hydrophilic, but contains a small amino acid domain (-NSASS-) just distal to a region of 17 threonine residues. Constructs were created using 80% of the amino-terminal portion of one alkaline phosphatase and the carboxyl-terminal portions of each of the isoforms. Both of the carboxyl-terminal peptides supported glycan phosphatidylinositol linkage as demonstrated by the following criteria: 1) plasma membrane targeting in transfected COS-1 cells, 2) release of transfected alkaline phosphatase by phosphatidylinositol-specific phospholipase C, 3) appearance of the trypanosome variable glycoprotein cross-reacting determinant after phospholipase C treatment, 4) ethanolamine incorporation into newly synthesized enzyme, 5) loss of phospholipase C release after mutation of the ω and ω + 2 positions in the putative linkage site, -NSA-, and 6) evidence of surface membrane localization by immunofluorescence using antibody against rat intestinal alkaline phosphatase. These data demonstrate that a predicted hydrophobic carboxyl-terminal sequence is not essential for glycan phosphatidylinositol linkage. Moreover, because both isomers are membrane- bound, the origin of soluble enzyme in the serum is likely to arise from the action of serum phosphatidylinositol-specific phospholipase D.
AB - Two cDNAs encode rat intestinal alkaline phosphatases having completely different carboxyl-terminal peptides; one is hydrophobic and fulfills the consensus requirements for glycan phosphatidylinositol linkage, and the other is neither hydrophobic nor hydrophilic, but contains a small amino acid domain (-NSASS-) just distal to a region of 17 threonine residues. Constructs were created using 80% of the amino-terminal portion of one alkaline phosphatase and the carboxyl-terminal portions of each of the isoforms. Both of the carboxyl-terminal peptides supported glycan phosphatidylinositol linkage as demonstrated by the following criteria: 1) plasma membrane targeting in transfected COS-1 cells, 2) release of transfected alkaline phosphatase by phosphatidylinositol-specific phospholipase C, 3) appearance of the trypanosome variable glycoprotein cross-reacting determinant after phospholipase C treatment, 4) ethanolamine incorporation into newly synthesized enzyme, 5) loss of phospholipase C release after mutation of the ω and ω + 2 positions in the putative linkage site, -NSA-, and 6) evidence of surface membrane localization by immunofluorescence using antibody against rat intestinal alkaline phosphatase. These data demonstrate that a predicted hydrophobic carboxyl-terminal sequence is not essential for glycan phosphatidylinositol linkage. Moreover, because both isomers are membrane- bound, the origin of soluble enzyme in the serum is likely to arise from the action of serum phosphatidylinositol-specific phospholipase D.
UR - http://www.scopus.com/inward/record.url?scp=0029077570&partnerID=8YFLogxK
U2 - 10.1074/jbc.270.20.11935
DO - 10.1074/jbc.270.20.11935
M3 - Article
C2 - 7744844
AN - SCOPUS:0029077570
VL - 270
SP - 11935
EP - 11940
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 20
ER -