Comparison of the catalytic activities of three isozymes of carnitine palmitoyltransferase 1 expressed in COS7 cells.
The enzyme carnitine palmitoyltransferase 1 (CPT1) catalyzes the transfer of an acyl group from acyl-CoA to carnitine to form acylcarnitine, and three isozymes of it, 1a, 1b, and 1c, have been identified. Interestingly, the 1c isozyme was reported to show no enzymatic activity, but it was not clearly demonstrated whether this inactivity was due to its dysfunction or due to its poor expression. In the present study, we (a) expressed individual CPT1 isozymes in COS7 cells, (b) evaluated quantitatively their expression levels by Western blotting using the three bacterially expressed CPT1 isozymes as standards, and (c) evaluated their catalytic activities. With these experiments, we successfully demonstrated that the absence of the enzymatic activity of the 1c isozyme was due to its dysfunction. In addition, experiments on the preparation of standard CPT1 isozymes revealed that the 1c isozyme did not show the standard relationship between migration in an SDS-PAGE gel and molecular size. We further tried to determine why the 1c isozyme was inert by preparing chimeric CPT1 between 1a and 1c, but no clear conclusion could be drawn because one of the chimeric CPT1s was not sufficiently expressed.
Institute for Genome Research, University of Tokushima, Kuramoto-cho-3, Tokushima, 770-8503, Japan.
Takuya
T
Hada
Takenori
T
Yamamoto
Atsushi
A
Yamamoto
Kazuto
K
Ohkura
Naoshi
N
Yamazaki
Yoshiharu
Y
Takiguchi
Yasuo
Y
Shinohara
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002334:Carnitine O-Palmitoyltransferase / Q000096:biosynthesis* / Q000235:genetics; D002522:Chlorocebus aethiops; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology
D000214:Acyl Coenzyme A; D007527:Isoenzymes; D002334:Carnitine O-Palmitoyltransferase