Identification and characterization of carboxyl ester hydrolase as a phospholipid hydrolyzing enzyme of zymogen granule membranes from rat exocrine pancreas.
Salt-washed (0.6 m NaCl) zymogen granule membranes (ZGM) of rat pancreatic acinar cells were utilized to identify and characterize membrane protein(s) responsible for phospholipase and lysophospholipase activities. Five major bands were identified in salt-washed ZGM by Coomassie Brilliant Blue. A 70-kDa protein with enzymatic activity was retained in significant quantities after several washes with 0.6 M NaCl but could be displaced from ZGM by 2 m NaCl or by 100 mg/ml heparin. By contrast, GP2, an integral membrane protein, was not displaced under these conditions. These findings suggest that the enzyme is a peripheral membrane protein of ZGM. Renaturation of ZGM proteins following electrophoresis revealed that the 70-kDa protein possessed phospholipase activity. Identification of the 70-kDa protein as a membrane-associated carboxyl ester hydrolase was based upon: (a) the use of a specific polyclonal antiserum, (b) N-terminal sequence, (c) two-dimensional gel analysis, (d) enzymatic characterization, and (e) co-localization to an area of a non-reducing gel containing significant phospholipase activity. Other ZGM proteins, namely GP2 and GP3, could not be demonstrated to possess phospholipase activity under the experimental conditions employed. Our finding that carboxyl ester hydrolase from ZGM exhibits PLA1 and lysophospholipase activities represents the first identification and characterization of a protein responsible for phospholipase activity in secretory granule membranes.
Withiam-Leitch
M
M
Department of Pharmacology and Toxicology, State University of New York, School of Medicine and Biomedical Sciences, Buffalo 14214.
Rubin
R P
RP
Koshlukova
S E
SE
Aletta
J M
JM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism*; D006868:Hydrolysis; D008245:Lysophospholipase / Q000378:metabolism; D008297:Male; D010179:Pancreas / Q000201:enzymology*; D010741:Phospholipases A / Q000378:metabolism; D054466:Phospholipases A1; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D004792:Enzyme Precursors; D010743:Phospholipids; D002265:Carboxylic Ester Hydrolases; D010741:Phospholipases A; D054466:Phospholipases A1; D008245:Lysophospholipase; D002118:Calcium