K. Ahn et al., SOLUBLE HUMAN ENDOTHELIN-CONVERTING ENZYME-1 - EXPRESSION, PURIFICATION, AND DEMONSTRATION OF PRONOUNCED PH SENSITIVITY, Archives of biochemistry and biophysics (Print), 359(2), 1998, pp. 258-268
Endothelin-converting enzyme-1 (ECE-1) is a type II integral membrane
protein that belongs to a family of metalloproteases which includes EC
E-S, neprilysin (neutral endopeptidase 24.11, EC 3.4.24.11), and Kell
blood group protein. ECE-1 cleaves its biologically inactive native su
bstrate, big endothelin-l, to generate a powerful vasoactive 21-amino
acid peptide, endothelin-1. ECE-1 consists of a short N-terminal cytop
lasmic tail, a transmembrane hydrophobic domain, and a large extracell
ular domain containing the catalytic site with a conserved Zn-binding
motif. We have constructed a secreted, soluble form of ECE-1 (solECE-1
) by fusing the cleavable N-terminal signal sequence of human alkaline
phosphatase in frame with the entire extracellular domain of ECE-1. S
table transfectant CHO cell lines expressing up to 6.1 mg of solECE-1
per liter culture medium were established and solECE-1 was purified to
homogeneity using three chromatographic steps with a 24% yield. SolEC
E-1 behaves as a dimer of 110-kDa subunits. SolECE-1 has a sharp pH op
timum, similar to the native form, ECE-la, but has a slightly more aci
dic pH optimum of 6.1-6.4 than that of 6.7-6.9 for ECE-la. At its opti
mal pH of 6.4, solECE-1 cleaved big ET-l:big ET-a:big ET-3 in a ratio
of 8.1:1:1.4, was inhibited by phosphoramidon with an IC50 value of 0.
35 +/- 0.05 mu M had a K-m value of 4.65 +/- 0.78 mu M for big ET-1, a
nd had a k(cat) value of 5.82 +/- 0.21 min(-1), all values comparable
to those for ECE-la at its optimal pH of 6.8. Phosphoramidon inhibitio
n of both ECE-la and solECE-1 is highly pH-dependent. At pH 5.8, phosp
horamidon inhibited ECE-la and solECE-1 with IC,, values of 14 and 33
nM, respectively, which are 49- and 1224-fold more potent than at pH 7
.2. SolECE-1 is highly glycosylated, similar to ECE-la. Deglycosylatio
n of solECE-1 by peptide N-glycosidase F shifted the apparent molecula
r weight of solECE-1 to approximately 80 kDa and the deglycosylated fo
rm(s) of solECE-1 preserved at least 72% of the activity of the glycos
ylated form. (C) 1998 Academic Press.