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We have developed a simple immunoaffinity chromatography procedure for the purification of a glycosyl-phosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) from bovine serum. The enzyme was initially purified by a procedure consisting of 9% polyethylene glycol precipitation, Q Sepharose anion-exchange chromatography, S-300 gel filtration, wheat germ lectin-Sepharose, hydroxylapatite agarose, zinc chelate matrix, Mono Q-high performance liquid chromatography (HPLC), and Superose 12 (gel filtration) HPLC. Using this purified material as immunogen, we generated a panel of monoclonal antibodies. A low affinity antibody was selected for the purification of catalytically active GPI-PLD from bovine serum by immunoaffinity chromatography, followed by wheat germ lectin-Sepharose and Mono Q-fast protein liquid chromatography. The latter method provides a simple purification procedure with an overall yield of 26%. The purified enzyme has an apparent molecular weight of about 100,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of about 5.6 by isoelectric focusing gel analysis. On Superose 12 HPLC, the material purified by the latter method elutes as a single peak with an apparent molecular weight of 200,000 as determined by protein standards. The enzyme activity is inhibited by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid or 1,10-phenanthroline. Phosphatidic acid is the only 3H-labeled product when [3H]myristate-labeled variant surface glycoprotein is hydrolyzed by the purified enzyme. Amino terminal sequence analysis of the intact 100-kDa protein reveals no strong homology to that of any other known protein. Twelve tryptic peptides derived from the intact protein have been subjected to amino acid sequence analysis. Two of them share sequence homology with each other and with the metal ion binding domains of members of the integrin family. Based upon these criteria, it appears that the purified enzyme is distinct from other phospholipases with specificity for inositol phospholipids.
K S Huang, and
S Li, and
W J Fung, and
J D Hulmes, and
L Reik, and
Y C Pan, and
M G Low
August 1989,
The Journal of biological chemistry,
K S Huang, and
S Li, and
W J Fung, and
J D Hulmes, and
L Reik, and
Y C Pan, and
M G Low
July 1993,
FEBS letters,
K S Huang, and
S Li, and
W J Fung, and
J D Hulmes, and
L Reik, and
Y C Pan, and
M G Low
October 1992,
Infection and immunity,
K S Huang, and
S Li, and
W J Fung, and
J D Hulmes, and
L Reik, and
Y C Pan, and
M G Low
January 1989,
The International journal of biochemistry,
K S Huang, and
S Li, and
W J Fung, and
J D Hulmes, and
L Reik, and
Y C Pan, and
M G Low
September 1982,
Journal of biochemistry,
K S Huang, and
S Li, and
W J Fung, and
J D Hulmes, and
L Reik, and
Y C Pan, and
M G Low
August 1993,
The Journal of biological chemistry,
K S Huang, and
S Li, and
W J Fung, and
J D Hulmes, and
L Reik, and
Y C Pan, and
M G Low
January 1997,
Annual review of biochemistry,
K S Huang, and
S Li, and
W J Fung, and
J D Hulmes, and
L Reik, and
Y C Pan, and
M G Low
December 1988,
Proceedings of the National Academy of Sciences of the United States of America,
K S Huang, and
S Li, and
W J Fung, and
J D Hulmes, and
L Reik, and
Y C Pan, and
M G Low
September 1991,
Biochimie,
K S Huang, and
S Li, and
W J Fung, and
J D Hulmes, and
L Reik, and
Y C Pan, and
M G Low
June 1989,
Journal of cell science,
