Characterization of a recombinant soluble form of human placental leucine aminopeptidase/oxytocinase expressed in Chinese hamster ovary cells.
Serum levels of human placental leucine aminopeptidase/oxytocinase (P-LAP) increase with gestation. cDNA cloning of P-LAP revealed that the enzyme is a type II membrane-bound protein containing the consensus HEXXH(X)18E motif found in the M1 family of zinc-metallopeptidase proteins. In this study, a recombinant soluble form of P-LAP found in maternal serum was expressed in Chinese hamster ovary cells, purified to homogeneity and then characterized. Although N-terminal sequencing revealed a four-amino-acid deletion, the purified enzyme was active and was shown to be a zinc-containing homodimeric protein with molecular mass of 280 kDa in solution. Using artificial substrates, it was shown that the enzyme has broad specificity and is inhibited by several compounds known as aminopeptidase inhibitors. Subsequently, sequential N-terminal amino-acid liberation of several peptide hormones by the enzyme was monitored and structures of the products were determined. Among the hormones having a cysteine residue at their N-terminal end and intramolecular disulfide bonds, it was found that vasopressin and oxytocin, but not calcitonin and endothelins, were cleaved by the enzyme. Because the molecular properties of oxytocinase so far reported often conflict, our results provide an initial biochemical and enzymatic characterization of moleculary defined P-LAP/oxytocinase.
Matsumoto
H
H
The Institute of Phyical and Chemical Research (RIKEN), Wako-shi, Saitama, Japan.
Rogi
T
T
Yamashiro
K
K
Kodama
S
S
Tsuruoka
N
N
Hattori
A
A
Takio
K
K
Mizutani
S
S
Tsujimoto
M
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D010122:Cystinyl Aminopeptidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004220:Disulfides; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010121:Oxytocin / Q000737:chemistry / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D012995:Solubility; D013004:Somatostatin / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014667:Vasopressins / Q000737:chemistry / Q000378:metabolism
D004220:Disulfides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D014667:Vasopressins; D010121:Oxytocin; D013004:Somatostatin; D010122:Cystinyl Aminopeptidase; C105964:leucyl-cystinyl aminopeptidase