Characterization of recombinant human renin: kinetics, pH-stability, and peptidomimetic inhibitor binding.
AuthorsHolzman TF, Chung CC, Edalji R, Egan DA, Martin M, Gubbins EJ, Krafft GA, Wang GT, Thomas AM, Rosenberg SH
JournalJ Protein Chem
PubMed ID1799412
The kinetic behavior and pH-stability of recombinant human renin was analyzed using a new fluorogenic substrate based on the normal P6-P3' renin cleavage sequence in human angiotensinogen. The design of this fluorogenic substrate makes possible, for the first time, direct monitoring of the kinetics of proteolytic conversion of prorenin to ... More
Recombinant human prorenin from CHO cells: expression and purification.
AuthorsHolzman TF, Chung CC, Edalji R, Egan DA, Gubbins EJ, Rueter A, Howard G, Yang LK, Pederson TM, Krafft GA
JournalJ Protein Chem
PubMed ID1963533
The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 ... More
Application of a fluorogenic substrate in the assay of proteolytic activity and in the discovery of a potent inhibitor of Candida albicans aspartic proteinase.
AuthorsCapobianco JO, Lerner CG, Goldman RC
JournalAnal Biochem
PubMed ID1514700
A fluorescent method for monitoring the activity of the secreted Candida carboxyl (aspartic) proteinase (EC 3.4.23.6) was developed using a fluorogenic substrate based on resonance energy transfer. The fluorescent assay was used to monitor proteinase production, purification, and inhibition. The Km for the fluorogenic substrate, 4-(4-dimethylaminophenylazo)benzoyl-gamma-aminobutyryl-Ile-His-Pro - Phe-His-Leu-Val-Ile-His-Thr- [5-(2-aminoethyl)amino]naphthalene-1-sulfonic acid, ... More
A continuous fluorescence assay of renin activity.
AuthorsWang GT, Chung CC, Holzman TF, Krafft GA
JournalAnal Biochem
PubMed ID8512070
A sensitive fluorescence assay that employs a new fluorogenic peptide substrate has been developed to continuously measure the proteolytic activity of human renin. The substrate, DABCYL-gaba-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-EDANS, has been designed to incorporate the renin cleavage site that occurs in the N-terminal peptide of human angiotensinogen. The assay relies upon resonance energy ... More
Real-time imaging of renin release in vitro.
AuthorsPeti-Peterdi J, Fintha A, Fuson AL, Tousson A, Chow RH
JournalAm J Physiol Renal Physiol
PubMed ID15082450
Renin release from juxtaglomerular granular cells is considered the rate-limiting step in activation of the renin-angiotensin system that helps to maintain body salt and water balance. Available assays to measure renin release are complex, indirect, and work with significant internal errors. To directly visualize and study the dynamics of both ... More