Amino Acid Sequence Deduced from a Rat Kidney cDNA Suggests It Encodes the Zn-Peptidase Aminopeptidase N

Dipeptidyl peptidase II (DPP II; EC 3.4.14.2) from rat kidney was purified to a specific activity of 65.4µmol/min per mg of protein for Lys-Ala-β-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 1710bp encoding DPP II from a rat kidney cDNA library. The cDNA of rat DPP II contained an open reading frame of 1500bp, 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 51400Da, which was lower than the value (about 60000Da) 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 60000Da). The transfected cells showed Lys-Ala-methylcoumarinamide-hydrolysing activity that was 50 times higher than the control cells.

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The amino acid sequence of the fluorescein isothiocyanate reactive site of lamb and rat kidney Na+- and K+-dependent ATPase

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(84)90605-3 ◽

1984 ◽

Vol 125 (2) ◽

pp. 767-773 ◽

Cited By ~ 74

Author(s):

Terence L. Kirley ◽

Earl T. Wallick ◽

Lois K. Lane

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Rat Kidney ◽

Fluorescein Isothiocyanate ◽

Reactive Site ◽

Dependent Atpase

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Cloning and expression of mouse legumain, a lysosomal endopeptidase

Biochemical Journal ◽

10.1042/bj3350111 ◽

1998 ◽

Vol 335 (1) ◽

pp. 111-117 ◽

Cited By ~ 80

Author(s):

Jinq-May CHEN ◽

Pam M. DANDO ◽

Richard A. E. STEVENS ◽

Mara FORTUNATO ◽

Alan J. BARRETT

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Cathepsin B ◽

Rat Kidney ◽

Cloning And Expression ◽

Human Embryonic Kidney ◽

Pig Kidney ◽

Naturally Occurring ◽

293 Cells ◽

Mouse Tissues

Legumain, a recently discovered mammalian cysteine endopeptidase, was found in all mouse tissues examined, but was particularly abundant in kidney and placenta. The distribution in subcellular fractions of mouse and rat kidney showed a lysosomal localization, and activity was detectable only after the organelles were disrupted. Nevertheless, ratios of legumain activity to that of cathepsin B differed considerably between mouse tissues. cDNA encoding mouse legumain was cloned and sequenced, the deduced amino acid sequence proving to be 83% identical to that of the human protein [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090–8098]. Recombinant mouse legumain was expressed in human embryonic kidney 293 cells by use of a vector containing a cytomegalovirus promoter. The recombinant enzyme was partially purified and found to be an asparagine-specific endopeptidase closely similar to naturally occurring pig kidney legumain.

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