| RRC ID |
39894
|
| Author |
Fukasawa KM, Fukasawa K, Higaki K, Shiina N, Ohno M, Ito S, Otogoto J, Ota N.
|
| Title |
Cloning and functional expression of rat kidney dipeptidyl peptidase II.
|
| Journal |
Biochem J
|
| Abstract |
Dipeptidyl peptidase II (DPP II; EC 3.4.14.2) from rat kidney was purified to a specific activity of 65.4 micromol/min per mg of protein for Lys-Ala-beta-naphthylamide. The N-terminal and partial amino acid sequences of the enzyme were determined. The peptide sequences were used to identify expressed sequence tag (EST) clones. By using the cDNA fragment of one of the EST clones as a probe, we isolated a cDNA clone with 1710 bp encoding DPP II from a rat kidney cDNA library. The cDNA of rat DPP II contained an open reading frame of 1500 bp, coding for a protein of 500 amino acids. The first 10 residues of the purified enzyme matched the deduced protein sequence starting with residue 37, suggesting the presence of a signal peptide. The mature enzyme (464 residues) had a calculated molecular mass of 51400 Da, which was lower than the value (about 60000 Da) determined by SDS/PAGE; and the deduced amino acid sequence showed six potential N-glycosylation sites. The deduced amino acid sequence of rat DPP II shared high similarity with quiescent-cell proline dipeptidase (78% identity) and prolyl carboxypeptidase (38% identity) and bore the putative catalytic triad (Ser, Asp, His) conserved in serine peptidase families. We transiently transfected COS-7 cells with pcDNA3.1 containing the cloned cDNA and obtained the overexpression of an immunoreactive protein (of about 60000 Da). The transfected cells showed Lys-Ala-methylcoumarinamide-hydrolysing activity that was 50 times higher than the control cells.
|
| Volume |
353(Pt 2)
|
| Pages |
283-90
|
| Published |
2001-1-15
|
| DOI |
10.1042/0264-6021:3530283
|
| PMID |
11139392
|
| PMC |
PMC1221570
|
| MeSH |
Amino Acid Sequence
Animals
Base Sequence
COS Cells
Cloning, Molecular
DNA, Complementary / chemistry
DNA, Complementary / isolation & purification
DNA, Complementary / metabolism
Dipeptidases / metabolism
Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / genetics*
Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / isolation & purification
Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / metabolism
Female
Hydrogen-Ion Concentration
Kidney / enzymology*
Kinetics
Molecular Sequence Data
RNA, Messenger / chemistry
RNA, Messenger / isolation & purification
RNA, Messenger / metabolism
Rats
Rats, Wistar
Sequence Alignment
Transfection
|
| IF |
4.097
|
| Times Cited |
18
|
|
WOS Category
|
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| Resource |
| Human and Animal Cells |
COS-7(RCB0539) |
