title: Optimisation of calcium-dependent protease and cathepsin D assays in ostrich muscle and the effect of chemical and physical dry-curing parameters.
abstract: The best conditions for the assay of cathepsin D and Ca(2+)-dependent pro tease (CDP) activity in ostrich muscle was established in order to have a simple, rapid and reliable method for its determination. Measurements of A(280nm) of TCA-soluble peptides and amino acid digests of casein and haemoglobin were used for measuring proteolytic activity in muscle extracts. The best conditions for the reliable determination of cathepsin D activity were found to be the incubation of an enzyme extract for 1 hr at 55 °C in a reaction mixture containing 0.9% (w v ) haemoglobin in 50 mM sodium formate buffer, pH 3.7. Characterization of the assay system for CDPs, obtained after phenyl-Sepharose chromatography, indicated that proteolytic degradation of casein by CDPs was linear with time up to 30 min at 30 °C and up to 0.1 units of activity. The effect of NaCl, KCl, nitrate, ascorbic acid, phosphate, glucose and sucrose on ostrich muscle CDP and cathepsin D activities has been studied. Salt (NaCl and KCl) acts as a strong inhibitor of proteolytic activity. Sodium and potassium nitrates (in the range 0-1000 mg l(-1)) affected activity to varying degrees. CDP activity was enhanced by sodium nitrate concentrations below 700 mg l(-1) and unchanged by potassium nitrate. Cathepsin D activity was inhibited to some extent by sodium nitrate above 200 mg l(-1) and completely by potassium nitrate. Results showed that phosphate is an inhibitor of both activities. High concentrations of ascorbic acid (above 6 g l(-1)) inhibited cathepsin D activity. Glucose (up to 2g l(-1)) activated cathepsin D activity and inhibited CDP activity (up to 1 g l(-1)). Sucrose activated enzyme activities at very low concentrations (1 × 10(-3) M) and inhibited activities above 1 × 10(-3) M.
authors: Department of Biochemistry and Microbiology, University of Port Elizabeth, PO Box 1600, Port Elizabeth 6000, South Africa.
authors: F P
authors: FP
authors: van Jaarsveld
authors: R J
authors: RJ
authors: Naudé
authors: W
authors: W
authors: Oelofsen