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[30a] The acid protease of Mucor miehei
[30a]
THE ACID PROTEASE OF M. miehei
459
hydrolyzed by mucor rennin: Z-L-Glu-L-Tyr-0H, A-L-Glu-L-Phe-0H, Z-L-Phe-L-Tyr-OH, Z-L-Phe-L-Leu-OH, Z-L-Tyr-L-Leu-OH, L-Leu-GlyOH, L-Leu-Gly-Gly-0H, and Z-Gly-Pro-L-Leu-Gly-0H. Altogether, the specificity of mucor rennin appears to be similar to that of pepsin and calf rennin but somewhat narrower than that of Aspergillus saitoi and Streptomyces griseus proteases.
[ 3 0 a ] T h e A c i d P r o t e a s e of M u c o r rniehei By MARTIN OTTESEN and W. RICKERT A milk-clotting acid protease which resembles pepsin, rennin, and the protease from Mucor pusiUus, 1 has recently been isolated from a strain of Mucor miehei, CBS number 370.65. 2 The enzyme was obtained from commercially available culture concentrate TM by ammonium sulfate fractionation, batchwise adsorption on DEAE-Sephadex, SE-Sephadex, and DEAE-Sephadex chromatography and gel filtration. Although the enzyme has not as yet been crystallized, it appeared homogeneous by paper electrophoresis within the pH range 4.0-8.0, by free moving boundary electrophoresis in the pH range 5.2-3.2, and during ultracentrifugation within the pH range 5.0-8.0. The isoelectric point was approximately 4.2 in acetare buffer, and the sedimentation coefficient, s°0.w, 3.35 Svedberg units. Boundary spreading in the ultracentrifuge indicated a value for the diffusion coefficient, D O 20,W, of approximately 7.6 X 10-7 cm 2 see-~. From this value, and an estimate of 0.72 for the partial specific volume, a molecular weight of around 38,000 was indicated. Optical rotatory dispersion measurements indicated the Moffitt parameter bo to be close to zero in agreement with the find!ngs of other acid proteases. The amino acid composition expressed as moles per 100,000 grams of protein was as follows: Asp~lo-m Thr72_,3 Ser88-91 Glu81-62 Pro4~-~ Glys~-84 Ala6~-66 1/2Cys9-1o Val~3-~4 Mete5_16 Ileu~6~, Leu4~-5o Tyr48-49 Phe~2-54 TrypT-8 Lys~2-23 His4_5 Arg15-~6. Although no covalently bound phosphorus was detected in the enzyme preparation, the Mucor miehei protease did have a 6% carbohydrate content made up of hexosamine and neutral hexoses. Since neither heat denaturation nor exposure of the enzyme to 8 M urea diminished the carbohydrate content, it appeared to be covalently bound to the enzyme. The carbohydrate content, the sedimentation coefficient, and the amino i j. Yu, G. Tamura, and K. Ariraa, Biochim. Biophys. Acla 171, 138 (1969). 1~"Rennilase," Novo Industry A/S, Copenhagen, Denmark. 2M. Ottesen and W. Rickert, Compt. Rend. Tray. Lab. Carlsberg 37, 301 (1970).
460
PROTEASES RELEASING COOH-TERMINAL AMINO ACIDS
[31]
acid composition distinguished the M u c o r miehei protease from the M u c o r pusillus protease described by Arima et al. 1 M u c o r miehei protease was remarkably stable. After 8 days of incubation at 38 ° between pH 3.0 and 6.0 more than 90% of the activity was retained, and in 8 M urea at pH 6 no detectable loss in activity occurred after 11 hours incubation. The pH optimum for the enzyme was close to 4, when the B-chain of oxidized insulin was used as substrafe. Splittings of the substrate were observed at the bonds Phe(1)Val (2), Leu (15)-Tyr (16), Tyr (16)-Leu (17), Phe (24)-Phe (25), and Phe (25)-Tyr(26). This indicated that among the bonds cleaved by both pepsin and rennin, only those bonds involving aromatic residues were hydrolyzed by the M u c o r miehei protease. 3 J W. Rickert, Campt. Rend. Tray. Lab. Carlsberg 38, 1 (1970).
[31] Bovine Procarboxypeptidase and Carboxypeptidase A
B y PmLIP H. P~TRA
Bovine Procarboxypeptidase A Bovine procarboxypeptidase A (PCP-A) occurs in the pancreas as a zymogen from which the active form can be generated by limited proteolysis.The existence of the zymogen in vivo was firstinferred by Anson I in 1935 and was, established 20 years later by Keller, Cohen, and Neurath. s,3 Some of the properties of this early preparation of the zymogen have already been reviewed.* In recent years, much work has been directed toward the elucidation of the activation processS-9;however, up to the present, the definition of the molecular events describing the generation of carboxypeptidase A activity is incomplete. The reasons for this difficultycan be stated as follows. First, bovine procarboxypeptidase A, unlike trypsinogen and chymotrypsinogen, exists in aggregate forms. 1M. L. Anson, Sc/ence 81, 467 (1935). 2p. j. Keller, E. Cohen, and H. Neurath, J. Biol. Chem. 223, 457 (1956). IP. J. Keller, E. Cohen, and It. Neurath, J. Biol. Chem. 230, 905 (1958).
4H. Neurath, Vol. II, p. 77. 5H. Neurath, Proc. Intern. Corr. Biochem. 7th, Tokyo, 1967. ' H. Neurath, R. A. Bradshaw, P. H. P~tra, and K. A. Walsh, Phil. Trans. Proc. Roy. Soe. (London) B 257, 159-176 (1970). ~J. H. Freisheim, K. A. Walsh, and H. Neurath, Biochemistry 6, 3020 (1967). 8j. H. Freisheim, K. A. Walsh, and H. Neurath, Biochemistry 6, 3010 (1967). 'P. Piras and B. L. VaJlee, Biochemistry 6, 348 (1967).
THE ACID PROTEASE OF M. miehei
459
hydrolyzed by mucor rennin: Z-L-Glu-L-Tyr-0H, A-L-Glu-L-Phe-0H, Z-L-Phe-L-Tyr-OH, Z-L-Phe-L-Leu-OH, Z-L-Tyr-L-Leu-OH, L-Leu-GlyOH, L-Leu-Gly-Gly-0H, and Z-Gly-Pro-L-Leu-Gly-0H. Altogether, the specificity of mucor rennin appears to be similar to that of pepsin and calf rennin but somewhat narrower than that of Aspergillus saitoi and Streptomyces griseus proteases.
[ 3 0 a ] T h e A c i d P r o t e a s e of M u c o r rniehei By MARTIN OTTESEN and W. RICKERT A milk-clotting acid protease which resembles pepsin, rennin, and the protease from Mucor pusiUus, 1 has recently been isolated from a strain of Mucor miehei, CBS number 370.65. 2 The enzyme was obtained from commercially available culture concentrate TM by ammonium sulfate fractionation, batchwise adsorption on DEAE-Sephadex, SE-Sephadex, and DEAE-Sephadex chromatography and gel filtration. Although the enzyme has not as yet been crystallized, it appeared homogeneous by paper electrophoresis within the pH range 4.0-8.0, by free moving boundary electrophoresis in the pH range 5.2-3.2, and during ultracentrifugation within the pH range 5.0-8.0. The isoelectric point was approximately 4.2 in acetare buffer, and the sedimentation coefficient, s°0.w, 3.35 Svedberg units. Boundary spreading in the ultracentrifuge indicated a value for the diffusion coefficient, D O 20,W, of approximately 7.6 X 10-7 cm 2 see-~. From this value, and an estimate of 0.72 for the partial specific volume, a molecular weight of around 38,000 was indicated. Optical rotatory dispersion measurements indicated the Moffitt parameter bo to be close to zero in agreement with the find!ngs of other acid proteases. The amino acid composition expressed as moles per 100,000 grams of protein was as follows: Asp~lo-m Thr72_,3 Ser88-91 Glu81-62 Pro4~-~ Glys~-84 Ala6~-66 1/2Cys9-1o Val~3-~4 Mete5_16 Ileu~6~, Leu4~-5o Tyr48-49 Phe~2-54 TrypT-8 Lys~2-23 His4_5 Arg15-~6. Although no covalently bound phosphorus was detected in the enzyme preparation, the Mucor miehei protease did have a 6% carbohydrate content made up of hexosamine and neutral hexoses. Since neither heat denaturation nor exposure of the enzyme to 8 M urea diminished the carbohydrate content, it appeared to be covalently bound to the enzyme. The carbohydrate content, the sedimentation coefficient, and the amino i j. Yu, G. Tamura, and K. Ariraa, Biochim. Biophys. Acla 171, 138 (1969). 1~"Rennilase," Novo Industry A/S, Copenhagen, Denmark. 2M. Ottesen and W. Rickert, Compt. Rend. Tray. Lab. Carlsberg 37, 301 (1970).
460
PROTEASES RELEASING COOH-TERMINAL AMINO ACIDS
[31]
acid composition distinguished the M u c o r miehei protease from the M u c o r pusillus protease described by Arima et al. 1 M u c o r miehei protease was remarkably stable. After 8 days of incubation at 38 ° between pH 3.0 and 6.0 more than 90% of the activity was retained, and in 8 M urea at pH 6 no detectable loss in activity occurred after 11 hours incubation. The pH optimum for the enzyme was close to 4, when the B-chain of oxidized insulin was used as substrafe. Splittings of the substrate were observed at the bonds Phe(1)Val (2), Leu (15)-Tyr (16), Tyr (16)-Leu (17), Phe (24)-Phe (25), and Phe (25)-Tyr(26). This indicated that among the bonds cleaved by both pepsin and rennin, only those bonds involving aromatic residues were hydrolyzed by the M u c o r miehei protease. 3 J W. Rickert, Campt. Rend. Tray. Lab. Carlsberg 38, 1 (1970).
[31] Bovine Procarboxypeptidase and Carboxypeptidase A
B y PmLIP H. P~TRA
Bovine Procarboxypeptidase A Bovine procarboxypeptidase A (PCP-A) occurs in the pancreas as a zymogen from which the active form can be generated by limited proteolysis.The existence of the zymogen in vivo was firstinferred by Anson I in 1935 and was, established 20 years later by Keller, Cohen, and Neurath. s,3 Some of the properties of this early preparation of the zymogen have already been reviewed.* In recent years, much work has been directed toward the elucidation of the activation processS-9;however, up to the present, the definition of the molecular events describing the generation of carboxypeptidase A activity is incomplete. The reasons for this difficultycan be stated as follows. First, bovine procarboxypeptidase A, unlike trypsinogen and chymotrypsinogen, exists in aggregate forms. 1M. L. Anson, Sc/ence 81, 467 (1935). 2p. j. Keller, E. Cohen, and H. Neurath, J. Biol. Chem. 223, 457 (1956). IP. J. Keller, E. Cohen, and It. Neurath, J. Biol. Chem. 230, 905 (1958).
4H. Neurath, Vol. II, p. 77. 5H. Neurath, Proc. Intern. Corr. Biochem. 7th, Tokyo, 1967. ' H. Neurath, R. A. Bradshaw, P. H. P~tra, and K. A. Walsh, Phil. Trans. Proc. Roy. Soe. (London) B 257, 159-176 (1970). ~J. H. Freisheim, K. A. Walsh, and H. Neurath, Biochemistry 6, 3020 (1967). 8j. H. Freisheim, K. A. Walsh, and H. Neurath, Biochemistry 6, 3010 (1967). 'P. Piras and B. L. VaJlee, Biochemistry 6, 348 (1967).