Biochemical and Biophysical Research Communications
Biochemical characterization of prostasin, a channel activating protease☆
Section snippets
Reagents
All reagents were from Sigma–Aldrich unless otherwise stated. α1-antitrypsin, α1-antichymotrypsin, α1-antitrypsinPDX, and soybean trypsin inhibitor (SBTI) were from Calbiochem. Recombinant placental bikunin (HAI-2A) was obtained from R&D Systems.
Cloning
The cDNA sequence of human prostasin (Accession No. NM_002773) was isolated from a human testis cDNA library (Clontech) by PCR with sense primer, 5′-ATGGCCCAGAAGGGGGTC-3′, and antisense primer, 5′-GTGCTCGCTGAGCCATGG-3′. The gene was modified by PCR to
Protein expression
The mature protease domain of prostasin was secreted into the insect culture medium at a level of 0.8–1.2 mg/L over 72 h. The expression of this active form of prostasin was low in comparison to what was achieved with the prostasin pro-enzyme under similar conditions; however, the pro-form was not active without digestion by trypsin in vitro (data not shown). Auto-activation of the pro-enzyme was not observed with prolonged incubation at 4 or 37 °C. The purified active protein migrated as a single
Discussion
The mature epithelial sodium channel (ENaC) is a heterotetramer composed of two α-subunits, one β-subunit, and one γ-subunit [24], [25], [26] although an alternative stoichiometry has been proposed [27]. The α-, β-, and γ-subunits are homologous, and each consists of a large extracellular domain flanked on each side by a transmembrane domain and a short intracellular amino- or carboxy-terminal domain [28]. ENaC is responsible for transepithelial Na+ transport and thereby fluid homeostasis in a
Acknowledgments
We thank Dr. Ian Hunt for advice and Mr. Reynand Pacoma for technical assistance.
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Abbreviations: acc, 7-amino-4-carbamoylmethyl coumarin; acmc, 7-amino-3-carbamoylmethyl-4-methyl coumarin; amc, 7-amino-4-methyl coumarin; CAP, channel activating protease; DTT, dithiothreitol; ENaC, epithelial sodium channel; hCAP, human channel activating protease; Nle, norleucine; NBS, non-binding surface; PNGaseF, peptide N-glycosidase F; SBTI, soybean trypsin inhibitor.