Biochemical Characterization of Secreted Proteases During Growth in Tetrahymena pyriformis WH‐14: Comparison of Extracellular with Intracellular Proteases

Abstract

Tetrahymena pyriformis strain WH-14 secreted large quantities of intracellular proteases into its culture medium during growth. Extracellular enzymes were purified to homogeneity from cell-free medium by ammonium sulfate precipitation, CM-Sephadex column chromatography, gel filtration, and DEAE-cellulose column chromatography. The DEAE-cellulose eluates were separated into four peaks (P-I, P-II, P-III, and P-IV), each of which exhibited a different specific activity toward azocasein and alpha-N-benzoyl-DL-arginine-rho-nitroanilide (Bz-Arg-Nan). These four forms of the protease showed similarity in amino acid composition, molecular weight (21,000-24,000), and antigenic reactivity. They had pH optima at neutral range. P-I showed the highest specificity to azocasein whereas P-IV was most effective toward the synthetic substrates. The Km values for hydrolysis of Bz-Arg-Nan were 2.4, 1.6, 1.3 and 1.4 mM for P-I, P-II, P-III, and P-IV, respectively, and the corresponding Kcat/Km values were 5.0, 9.4, 28.5, and 114.3 S-1 . M-1. These properties of secreted proteases were compared with those of intracellular proteases purified by the same procedure except for the initial Triton X-100 extraction. There were similarities in specific activity toward two substrates, molecular weight, Km, pH optima, and antigenic reactivity between the proteases from two sources, providing evidence that the intracellular proteases may be secreted into the extracellular medium without modification.

Prolonged elevation of insulin content in the unicellular tetrahymena after insulin treatment: induction of insulin production or storage?

“…These enzymes are also secreted into the extracellular medium without any modi®cation. 31 Protein digestion occurs in Tetrahymena over broad ranges of pH, 27 and with regard to protein digestion, the cell reacts in much the same way as mammalian cells. 32 This allows us to suppose that insulin present in the medium can be digested both extra-and intracellularly.…”

An Analysis of Intracellular Proteolytic Activities of Tetrahymena pyriformis GL

“…Proteinase fractions were separated on DEAEcellulose as described by Banno et al (1982), except that only (NH4)$04 fractionation of their preceding purification steps was retained. This abbreviated procedure does not affect the proteinase separation (Banno et al, 1982).…”

“…Proteinase fractions were separated on DEAEcellulose as described by Banno et al (1982), except that only (NH4)$04 fractionation of their preceding purification steps was retained. This abbreviated procedure does not affect the proteinase separation (Banno et al, 1982). The protein precipitated from cell lysates by (NH4)2S04 between 30% and 60% saturation was collected by centrifugation at 26000g for 10 min at 4 "C and dissolved in 0.01 M-potassium phosphate buffer, pH 6.7.…”

“…Fractions (3 ml) were collected. Those containing proteinase activity analogous to fractions P-I, P-I1 and P-IV (Banno et al, 1982) were collected immediately and after application of buffer containing 0.1 M-KCl and 0.3 M-KCl, respectively. Subcellular fractionation.…”

“…The proteolytic system of the ciliate Tetrahymena pyriformis is probably that most studied amongst protozoan species (Dickie & Liener, 1962;Levy et al, 1976;Banno et al, 1982Banno et al, , 1983. Both acid proteinase and neutral proteinase activities have been described but it is not at all clear how many different types of enzyme are responsible for these.…”