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A simple major protease, secreted into the medium during growth of Tetrahymenapyrijormis strain W, has been purified about 4000-fold by (NH,),SO, precipitation, ion-exchange chromatography, gel filtration and affinity chromatography on organomercurial-Sepharose. The purified protease was homogeneous as judged by polyacrylamide gel electrophoresis and was a nionomeric protein with a molecular weight of 22000-23000. Amino acid analysis showed that the enzyme was rich in acidic amino acids. In addition, the purified Tetrahymena protease consists of multiple forms with isoelectric point between pH 5.3 and 6.3. Optimum activity of the purified enzyme was in the pH range 6.5 -8.0 with a-N-benzoyl-m-arginine-p-nitroanilide and with azocasein, while it was in the lower pH range (4.5 -5.5) for denatured hemoglobins. The purified enzyme was inhibited by compounds effective against thiol proteases. Leupeptin and chymostatin were potent inhibitors but pepstatin was without effect. This enzyme is similar to cathepsin B and appears to be a major proteolytic enzyme in Tetrahymena.

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Candida utilis ribose 5-phosphate ketol isomerase has already been purified from cell-free extracts of C. utilis by the authors1) according to conventional pro cedures composed of ammonium sulfate precipitation, DEAE-cellulose (DE-23) column chromatography, Bio-Gel P-200 gel filtration and repeated preparative polyacrylamide gel electrophoresis.But, by this procedure, the enzyme yield was very low. So, in order to get a good yield of the enzyme preparation, purification procedure using affinity chro matography was tested. As an adsorbent for affinity chromatography, Sepharose 4B having p-mercuribenzo ate (p-MB) as a ligand and 6-amino-hexylamine as a spacer was used.Preparation of p-MB-6-aminohexyl-Sepharose 4B2) Sepharose 4B (25ml) suspended in distilled water (25ml) was activated by addition of cyanogen bromide (6.25g) at pH 11 -11.5. Cyanogen bromide-activated Sepharose 4B gel was swollen and washed successively with water (one liter), 0.1 M NaHCO3 (one liter) and finally with water (one liter). To the cyanogen bromi de-activated Sepharose 4B (25ml), 0.05 M 6-amino hexylamine (25ml) was added and adjusted pH at 10. After stirring at 4°C for 16 hr, it was filtered on a glass filter and successively washed with 0.1 M NaHCO3 and water. A half gram of p-MB to be coupled with 6-aminohexyl-Sepharose 4B was dissolved in 45ml of 40% dimethylformamide.To the mixture; 6-aminohexyl-Sepharose 4B (25ml) and 4 mm 1-ethyl-3-(3-dimethylaminopropyl) carbodi imide were added. After being adjusted pH to 6.0 with I N HCl, the mixture was kept at room temperature for 16 hr.After then, it was filtered with a glass filter and washed successively with 0.1 M NaHCO3 (one liter), 40 % dimethylformamide (500ml) and finally with water (one liter). Next, the column was washed successively with 0.1 % bovine serum albumin (250ml), 8 M urea in 0.1 M citrate buffer (pH 5.5, 250ml) to remove any trace of uncoupled p-MB, and then washed successively with 8 M urea in 0.1 M citrate buffer containing 50mm 2-mercaptoethanol (250ml), water (250ml), 40 % dime thylformamide (250 nil) and water (250 ml).Purification of C. utilis ribose 5-phosphate ketol isomerase by affinity chromatography on a p-MB-6-aminohexyl-Sepharose 4B was carried out in the fol lowing procedure. The adsorbent was equilibrated with 0.01 M Tris-HCl buffer (pH 7.3, hereafter referred as column buffer), and then 15.0 mg of partially purified ribose 5-phosphate ketol isomerase preparation (DE-23 eluate) dissolved in 70 ml of the column buffer, was applied to the column. After washing the column suc cessively with the column buffer and the column buffer containing 0.1 M NaCl, the column buffer containing 0.2 M NaCl was used to elute ribose 5-phosphate ketol isomerase from the column as shown in Fig. 1. Ribose 5-phosphate ketol isomerase (3.4mg) was recovered by this procedure. The purified enzyme preparation by affinity chromatographic method showed homogeneity on a polyacrylamide disc electrophoresis as shown in Fig. 2. The results of purification of ribose 5-pho sphate ketol isomera...