Structural features of GPI-specific phospholipase D revealed by proteolytic fragmentation and Ca2+ binding studies.
Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in plasma and is potentially capable of degrading the anchor utilized by many cell surface proteins. The goal of this work was to study structural features of the GPI-PLD that might be involved in regulation of its activity. Trypsin cleaved the 100-110 kDa GPI-PLD polypeptide into three major fragments (two of approximately 40 kDa and a carboxyl-terminal fragment of 30 kDa) which were relatively resistant to further proteolysis. Pretreatment of the GPI-PLD with chelators resulted in complete degradation. During the cleavage process the GPI-PLD enzymatic activity increased approximately 3-4-fold but no other major change in its properties (e.g. inhibition by chelators and lipids, thermal stability, oligomerization, etc.) was observed. Intact or trypsinized GPI-PLD bound 45Ca2+ (approximately 5.5 ions/molecule GPI-PLD; Kd approximately 16.1 microM as determined by equilibrium dialysis) which could not be blocked by the addition of other divalent metal ions. However, inhibition of enzymatic activity by divalent cation chelators appeared to involve removal of bound Zn2+ rather than Ca2+. A metal analysis of GPI-PLD revealed approximately 5 and 10 atom/molecule of calcium and zinc, respectively. The data suggest that the predicted integrin E-F hand-like sites in GPI-PLD are functional but not directly involved in enzymatic activity.
Li
J Y
JY
Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Hollfelder
K
K
Huang
K S
KS
Low
M G
MG
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010739:Phospholipase D / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D014357:Trypsin / Q000378:metabolism
D010739:Phospholipase D; C065017:glycoprotein phospholipase D; D014357:Trypsin; D002118:Calcium