An isozyme of microsomal carboxyesterases, carboxyesterase Sec, is secreted from rat liver into the blood.
It is generally believed that liver carboxyesterases are localized exclusively in the endoplasmic reticulum (ER), mostly in the lumen, loosely bound to the inner side of the membrane. A cDNA clone, clone (8-1/2-1) supposed to code for one of the isozymes, carboxyesterase E1, was isolated by Takagi et al. [J. Biochem. 104, 801-806 (1988)]. However, the protein coded by clone (8-1/2-1) had no consensus ER retention signal at its carboxy terminus, and the mechanism of its retention by ER lumen was unclear. When clone (8-1/2-1) was expressed in COS cells in this study, the plasmid-coded protein was secreted into the medium. When the carboxy terminal portion of the clone (8-1/2-1)-coded protein was replaced with the corresponding region of another carboxyesterase, pI 6.1 esterase, which had the HVEL sequence at the carboxy terminus, the chimeric protein was retained in the COS cells. We searched for a secretory form carboxyesterase in rat blood immunochemically using polyclonal antibodies to carboxyesterase E1, and detected a cross-reacting protein with a molecular weight of 68 kDa. The molecular weight was decreased by endoglycosidase F treatment but not by endoglycosidase H treatment, indicating that the protein carries complex type sugar chains. In addition, the cross-reacting protein was labeled with [3H] diisopropylfluorophosphate (DFP), suggesting that the protein has an esterase-type active center serine.(ABSTRACT TRUNCATED AT 250 WORDS)
Murakami
K
K
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka.
Takagi
Y
Y
Mihara
K
K
Omura
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D051381:Rats; D014162:Transfection
D007527:Isoenzymes; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; C469448:Ces1d protein, rat