An Analysis of Intracellular Proteolytic Activities of Tetrahymena pyriformis GL

Abstract

1977Five different assay conditions were used to analyse the intracellular proteolytic system of Tetrahymena pyriformis GL. Acid proteinase activity was measured using hide powder azure at pH 4 and neutral proteinase activity was measured using hide powder azure and azocasein at pH 8 and two peptide nitroanilides at pH 7. Differences between the activities were apparent from their sensitivity to proteinase inhibitors although all were likely to be due to cysteine proteinases. Indeed all were enhanced by the presence of 1 mM-DTT. The neutral activities could be resolved into at least three forms by ion-exchange chromatography, each form having a different specificity for the substrates used. The acid proteinase activity was similarly separated into different fractions. The intracellular activity of acid proteinase responded differently from that of the neutral activities to culture age or resuspension of cells in fresh medium and starvation buffer. Furthermore, analysis on sucrose density gradients showed that although the neutral activities had a similar subcellular distribution to one another this differed from that of the acid proteinase activity. This suggests that the physiological roles of the acid and neutral proteinases may differ. PAGE using gels containing haemoglobin to detect proteinases was less successful than with other species of protozoa. However, it did reveal multiple forms of acid proteinase.

The in vitro inhibition of proteases from Cryptobia salmositica Katz by a monoclonal antibody (MAb‐001) against a glycoprotein on the pathogenic haemoflagellate

“…Proteolytic activity was detected using azocasein as the substrate, as previously described by North & Walker (1984) with slight modifications (Zuo & Woo 1997a). Briefly, a volume (10 µl) of protease sample (containing 100 µg protein of parasite lysate or 10 µg protein of partially purified protease) was mixed with 0.5 ml of azocasein (10 mg ml -1 ) and 0.1 M phosphate buffer (pH 5.0) was added up to 1 ml.…”

Proteases in pathogenic and nonpathogenic haemoflagellates, Cryptobia spp. (Sarcomastigophora:Kinetoplastida), of fishes

“…Proteolytic activities were assayed using azocasein (AZC) (North & Walker 1984) and hide powder azure (HPA) (North & Whyte 1984) as substrates with slight modifications. Briefly, 100 p1 of the cell lysate (containing 100 pg protein) was incubated with 0.5 m1 of either AZC (Sigma, 10 mg ml-l) or 0.5 m1 HPA (Sigma, 10 mg ml-l) and 0.5 m1 buffer at 37°C.…”

“…The activity is given in unit (1 pg substrate protein hydrolysed per hour per mg protein of the lysate). An increase of 1 absorbency unit was caused by the hydrolysis of 0.71 mg AZC and 3.4 mg HPA under the reaction conditions (North & Walker 1984, North & Whyte 1984. The buffers used to determine pH effects on protease activity were 0.1 M phosphate buffer (pH 3.0 to 7.0) and 0.1 M Tris-HC1 buffer (pH 7.5 to 9.0).…”