Mycopathologia vol. 56, 3, pag. 153-157, 1975
SOME PROPERTIES OF THE PROTEASE FROM ASPERGILLUS TERRICOLA
Nadejda A. ZAIKINA, Larisa K. SHATAEVA, N. P. ELINOV, & G. V. SAMSONOV Chemical Pharmaceutical Institute, Leningrad, USSR
Abstract
The protease from Aspergillus terricola (terrilytin) was isolated and purified by the ion exchange method and by gel chromatography. The data on its molecular weight, isoelectric point, N-terminal amino acids were described. The protease included polysaccharide which stabilized the enzyme activity under the storage and lyophylization. Polysaccharides isolated from different fungi and yeasts were found to stabilize and activate terrilytin rising the affinity of enzyme to substrate. Terrilytin was revealed to exibit the thrombolytic effect and expressive affinity to the fibrin in comparison with other substrates of high and low molecular weight. The enzyme showed the antigenic activity. From immune rabbit sera 7S y-globulins inhibiting proteolysis of caseine and 19S y-globulins activating the proteolysis of fibrin and fibrinogen by terrilytin were isolated by gel chromatography.
Introduction
Some proteases are isolated and identified at present from fungi of the genus Aspergillus of the following species: Asp. saitoi (23), Asp. oryzae (13,24), Asp. sojae (8), Asp. flavus (25). Some of them are used in the industry and in the medicine, the enzyme exibiting the fibrinolytic effect attracts the particular attention (2). This paper presents characteristics of a new neutral protease isolated from fungus Asp. terricola (9).
Materials and Methods
The enzyme was precipited from the cultural media by (NH4)zSO4 and purified on the ionexangers F A F and KMT and on biogel P-150 (14). The proteolytic activity was determined by Ancon method (1) and from the rate
of accumulation of c~-amine nitrogen in the reaction mixture (11). Thrombolytic activity was determined from the rate of the dissolution of a standard clot (0.1 ml of rabbit blood + 0.1 ml of thrombin solution) at 37 ~ when the volume ratio clot: enzyme solution was 1:1 (20). Polysaccharides were prepared from the cell extracts of the fungi and purified by the repeated precipitation by HC1 and acetone (26). Rabbits were immunized intravenous with 3 mg of the enzyme in the 0.5 ml of the saline receiving 4 injection in 5 days interval. They were bled in the 7 days after the last injection. The immunization was repeated for 2-3 months. Products of the protease autolysis were also used for the immunization. Rabbits were injected with 0.5 ml of the solution containing 7.1 millimole of e-amine nitrogen per 1 ml. The immunization schedule was the same as with the protease. Antibodies were determined by the complement fixation test (CFT) and by gelprecipitation test. Sera were fractioned on sephadex G-200 in 0.05 M tris buffer pH 8.0 containing 0.2 M NaC1 (7). Paper electrophoresis in veronal-medinal-borate buffer pH 8.6 was used for the analysis of serum proteins.
Results and Discussion
1. Physico-chemicalproperties of terrilytin The preparation precipited from the cultural media of the fungus by (NH4)zSO 4 and purified on the ionexangers F A F and KMT contained 2 enzymes - the neutral protease and acid amylase. These components were successfuly separated by gel chromatography on biogel P-150. The protease obtained (terrilytin) is a homogeneous enzyme according to ultracentrifugation, electrophoresis and coefficient of diffusion determination. Molecular weight of terrilytin is about 27,000, iso-electric point 4.6. The optimum of proteolytic activity and stability is at pH 6.5-7.0. N-terminal amino acids were determined in the 153
enzyme by dinitrophenilation, they were found to be alanine, glycine, serine, glutamic and aspartic acids. The purified terrilytin preparation was found to be separated by the gradient elution on carboxymethyl cellulose with the isolation of 2 fractions. The first fraction was polysaccharide and the second - protein with the same proteolytic and thrombolytic activity as the initial enzyme. The polysaccharide was hydrolyzed by acid with the formation of monosaccharides: mannose, glucose and traces ofxylose, which were identified by paper chromatography. The polysaccharide component of terrilytin reveals no important influence on the specific activity but essentially stabilizes the enzyme. Thus, the protein separated from polysaccharide was found to loss its activity under the storage and dialysis more rapidly than the initial enzyme. Lyophylization of pure protein results in the complete inactivation when the activity loss of the initial enzyme consists only in 12-16~. These findings confirm once more the hypothesis about the universal biological protective role of polysaccharides (5). The protease in solution was found to be in the equilibrium with the products of its autolysis. Their equilibrium concentration consists in (16 + 1) mole of a-amine nitrogen on 1 mole of protein at 4~ The products of terrilytin autolysis were separated from the native enzyme by gel chromatography. The superequilibrious addition of the products of autolysis to the terrilytin solution was found to stabilize the enzyme during heat denaturation (15). The products of terrilytin autolysis consist of polysaccharide and peptides. The polysaccharide composition is analogous to one described above. Peptides have the same N-terminal aminoacids as the native terrilytin and consist of the fractions with molecular weight from 600 to 3500, the average molecular weight is about 2000. Both the polysaccharide and peptide components of the autolysis product stabilize the native proteine structure. The following experiments revealed the ability of polysaccharides isolated from cell extracts of different fungi and yeasts to stabilize the purified terrilytin. Polysaccharides were found also to increase the thrombolytic activity of the enzyme. In these experiments the concentration of terrilytin was 2 mg/ml and of polysaccharide 20 mg/ml. The equal volumes of these ingredients were mixed, in control experiments polysaccharide solution was replaced by saline. The test solution was added to the clot immediately after mixing and after the 24 hr. exposure at 37 ~ All studied polysaccharides were found to activate terrilytin (table I). The effect was greater after the 24 hr. 154
Table I. The influence of polysaccharides on the activity of terdlytin
Polysacchadde from
Time of the thrombolysis (rain.) after the exposition of terrilytin and polysaccharide/ without exposition
Aspergillus terricola Candida albicans C tropicalis C pseudotropicalis Trichosporon condensatum T behrendii Saccharomyces cerevisiae Mucor racemosus.
Without polysaccharide
75 75 70 70 70 65 60 75 95
in 24 hr. 125 125 120 95 95 105 105 130 165
exposure with partial inactivation more rapid in the control than in the presence of polysaccharide. It is interesting that the polysaccharides from different species of fungi and yeasts were capable to activate terrilytin. These substances represent protein-polysaccharide complexes where the protein component is strongly attached to the polysaccharide and is not separated during repeated precipitation and gel chromatography. Thus, the substance from A. terricola consists of 31 ~ of reducing substances, 2.8 ~ of nitrogen and 8.6~ of phosphorous. It does not differ essentially from the composition of substances from the other fungi. For the more detailed investigation of the polysaccharide action on terrilytin activity the rate of the proteolysis of fibrinogen in the presence of polysaccharide and without it was compared. In these experiments the concentration of polysaccharide was 5 mg/ml and of terrilytin - 0 . 4 mg/ml. Concentration of fibrinogen changed from 5 to 30 mg/ml. Km was determined from the curve of the dependence of the reaction rate (by the accumulation of c~-amine nitrogen in the reaction mixture) from substrate concentration. Km was found to be 1.75 micromole/ml in the presence of polysaccharide from cell extract of Candida albicans and 5 micromole/ml without it. Therefore, these findings permit the supposition that the polysaccharide rises the affinity of enzyme for substrate. The following investigations of the biochemical properties of terrilytin were made with enzyme purified on biogel P-150, i.e. with the preparation containing protein and polysaccharide components because the protein separated from polysaccharide is comparatively unstable.
2. The effect of terrilytin on synthetic substrates and oligopeptides Terrilytin does not hydrolyse dipeptides ala-histidine, val-tyrosine, val-glycine, his-glycine, gly-alanine, glyleucine, gly-valine, gly-methionine, gly-phenil-alanine, tripeptides pro-gly-glycine, gly-gly-proline, ala-gly-glycine, and oligopeptides of/3-chain of insulin: leu-tyr-leu-val-cysglycine, tyr-leu-val-cys-glycineand leu-val-cys-glycine.The specific substrates of trypsin and chyrnotrypsin-benzoylarginine-methylic ether and benzoil-tyrosine-methylicether were not affected by terrilytin. The terrilytin was found to hydrolyse peptides asp-histidine, asp-glycine, pro-tyrosine, his-leucine, glu-asparagine, leu-valine, pro-leu-glycine, besides the enzyme hydrolyses asparagine and glutamine with the formation of amine acids. The hydrolysis rate of these substrates at 37~ consists from 1.4.10 -4 to 27.10-4 micromole of substrate hydrolysed in 1 sec. under enzyme concentration 0.8 micromole and substrate concentration 5 mg/ml (21). It is necessary to note that the hydrolysis rate of peptides by terrilytin is lower on 2-3 orders than the hydrolysis rate of specific low molecular weight substrates by the other proteases, for example, by trypsin, plasmin (18), 1-asparaginase (19).
3. The effect of terrilytin on native proteins The proteolysis of native proteins was studied at 37 ~ enzyme concentration 0.8 micromole and substrate concentration 10 mg/ml with the exeption of prothrombin: its concentration (0.2 mg/ml) was necessary because of the lability of the substance. Attempts to prepare more concentrated solutions led to its spontaneous conversion with the formation of thrombin. Amongs the proteins representing factors of blood coagulation fibrin was found to be hydrolyzed by terrilytin most extensively (table II). The rate of fibrin hydrolysis exceed considerably the rate of hydrolysis of the other
Table II, The rates of hydrolysis of proteins by terrilytin
soluble and unsoluble substrates as well as of low molecular weight substrates. The significant difference between the hydrolysis rate of fibrin and fibrinogen was confirmed when studing the preparation of these proteins released from plasminogen (3). It is possible to suppose that the increase of the hydrolysis rate of fibrin in comparison with fibrinogen correlates with the increase of the degree of ordering of structure and reciprocal orientation of polypeptides chains of the substrate (in this case-fibrin), forming a triplete helix (22).
4. Immunological properties of terrilytin The possibility of the application of terrilytin for the therapeutic purposes requires the studing of the antigenic activity of the enzyme and the investigation of the influence of specific antibodies on the proteolytic activity. The nonspecific serum inhibitors of proteases are known to complicate the investigation of the antibodies action on these enzymes (6, 12). The influence of nonspecific serum inhibitors must be studied carefully when using protease for the thrombolytic therapy (10). Intravenous injection of terrilytin produced in rabbits antibodies formation: at the end of the immunization sera titres run up to 1 : 1600 in CFT (Table III). The same sera reacted with the products of terrilytin autolysis but at a lower dilution. The products of autolysis showed antigenic activity which was weaker than that of the native enzyme: the titre 1 : 100 was observed in the reaction with homologous antigen only after the 2nd cycle of the immunization. The sera of rabbits immunized by products of autolysis reacted with terrilytin as well but the reaction was weakly positive at the low dilution of the sera. Table Ill. CFT with sera of rabbits immunized with terrilytin and products of its autolysis
Serum against Terrilytin
Substrate
Fibrin Fibrinogen Thrombin Prothrombin Insulin Collagen Elastin
Specific rate
140 33 8 4 60 19 17
mole ofa-NH 2 mole of enz. rain. Products of autolysis
Antigen
Dose of the antigen
Titre of the serum
Terrilytin Products of autolysis
1 : 100000
1 : 1600
1 : 1000
1 : 100
Terrilytin
1 : 100000
1 : 100-+
Products of autolysis
1 : 1000
1 : 100
Note: 4 rabbits were immunized with each antigen, the average data are presented. 155
The precipitation test was positive only with sera prepared against terrilytin with its homologous antigen. The sera prepared against the partly purified preparation containing protease and amylase gave two zone of the precipitation with homologous antigen and one zone with the individual protease. Sera against the highly purified terrilytin gave one zone of the precipitation with the both antigens. These findings confirm once more the homogeneity of the purified terrilytin preparation. The concentration of antigens in the precipitation test was 1 mg/ml, their doses in CFT were choosen on the basis of the anticomplementary properties of the antigens and verified in the 'square' reaction. When studying the influence of immune and normal sera on terrilytin activity, equal volumes of enzyme solution with the concentration 0.08 mg/ml in 0.1 M phosphate buffer pH 6.6 and the serum were mixed and 0.5 ml of this mixture was added to 1 ml of the substrate. Casein dissolved in 0.1 M.phosphate buffer pH 7.6, fibrin and fibrinogen suspended or dissolved, respectively, in saline were used as substrates. The concentration of substrates was 0.5 ~o. The results of the experiment was found to depend on the choosen substrate and on the concentration correlation of the components of the reaction. However the difference between the influence of immune and normal serum on the enzymatic activity hardly permits the interpretation because of the complex composition of the serum. Sermn is known to contain a number of proteins including different proteinases and their inhibitors both in a free state and in complexes. With the addition of the proteinase to the serum, the complex processes including both activation and destruction of active serum proteins can take place. Therefore, the following experiments were made with the isolated serum fractions prepared by gel filtration. This method permits to isolate 3 serum fractions, designated as P1, P2 and P3 (fig. 1). Under the electrophoresis the fractions P1 and P2 move as y-globulins and P3 contains y-globulins and albumins. The complement fixing antibodies were found in the fractions P1 and P2 and precipitins - in P2. The fractions P1 gave the positive CFT at the minimal concentration of prottein 0.240 mg/ml and P2 at the concentration 0.080 mg/ml. According to the certain data (7) fractions P1 and P2 contain 19S and 7S y-globulins, respectively. Unspecific inhibitors ofproteases were found in the fraction P3, although there is information (12) that 17S macroglobulins which can be present in the fraction P1 as admixture possess the antiprotease activity. Cel chromatography of normal sera resulted in the 156
P3
p/ o,~0o 4
Oj t ~
o
'
J, 3o
,
0o
~o
Fig. 1, Gel chromatography of the serum against terrilytin. 1 - absorbtion (fraction volume 2, 3 ml); 2 - concentration of protein of the complement fixing antibodies (mg/ml). J
preparation of the same 3 protein fractions, but the concentrations of the fraction P1 and P2 were considerably lower than in immune sera. These findings are possibly explained by the increase of y-globulins during the immunization due to antibodies formation (4). When studying the influence of the protein fractions on the terrilytin activity, the fractions were dialyzed against saline in order to remove the surplus of the salt in the lyophylized preparations, then they were diluted to the protein concentration 4 mg/ml and used as in the experiments with native sera. The activity of terrilytin in the presence of fractions was found to depend on the substrate (table IV). Immune Table IV. The influence of serum fractions on the activity of terrflytin
Serum
Proteolytic activity as per cent to Fraction / the controle in the presence of the fraction and substrate casein
fibrinogen
fibrin
Immune
P1 P2 P3
97 62 69
170 100 103
170 100 110
Normal
P1 P2 P3
103 101 89
105 108 105
110 100 102
Note: the average data of 4 experiments are presented
fraction P1 did not influence on the proteolysis of casein and activated the splitting of fibrin and fibrinogen. The fraction P2 contained antibodies that inhibited terrilytin activity on casein but no other substrate. Respective fractions of normal sera did not affect significantly the proteolytic activity of terrilytin. It is interesting that immune fraction P3 inhibited the activity of terrilytin more strongly than the same fraction from normal sera. The inability of fractions P1 and P2 from normal sera to affect t h e activity of terrilytin is evidence that these fractions contain 7-globulins without visible admixtures of the other proteins capable of changing the activity of the protease. Consequently, it is reasonable to assume that the change in proteolytic activity in the presence of the fractions of immune sera corresponds with the action of specific antibodies. Therefore, in sera of rabbits immunized with terrilytin both inhibiting and activating antibodies are formed. Moreover, during immunization nonspecific serum inhibitors of protease were found to increase. The final result of the influence of serum on the terrilytin activity depends on such experimental conditions as substrate used and correlates with the concentrations of ingredients in the reaction mixture. Analogous findings were obtained when investigating antibodies against a number of enzymes. (16, 17).
References 1. Anson, M. L. 1938. Estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J. Gen. Physiol., 22; 79-89. 2. Bergkvist, R. & P. O. Svard. 1964. The thrombolytic activity of a protease from Aspergillus oryzae. Acta Physiol. Scand., 60 (4): 36-71. 3. Blomback, B. & M. Blomback. 1956. Purification of bovine and human fibrinogen. Acta Chem. Scand, 10, 147. 4. Boyd, W. C. 1969. Fundamentals of immunology. Moscow. p. 5. Elinov, N. P. 1971. Biological functions of natural polysaccharides. Proc. Conf. Chem. Pharm. Inst. Leningrad: 5-7. 6. Fossum, K. 1970. Proteolytic enzymes and biological inhibitors. Acta Pathol. Microbiol. Scand., Sect. B. 78(5): 605-618. 7. Fukazava, Y. & T. Tsuchiya. 1969. Preparation of monospecific antisera for yeast antigens by immunological methods. Yeasts. Proc. I2 Syrup. on Yeasts. Bratislava: 553-558. 8. Hayashi, K., D. Fukushima & K. Mogi. 1967. Alkaline proteinase of Aspergillus sojae. Physico-chemical properties, amino acidcomposition, and molecular conformation. Agr. Biol. Chem. (Tokyo), 31(5): 642-3.
9. Imshenetskii, A. A., S. Z. Brodskaya & V. V. Korshunov. 1965. Action of fungal proteinases on blood thrombi. Dokl. Acad. Nauk. SSSR. 163(3):737-40. 10. Lindvall, S., O. Mangusson & K. Orth. 1969. On the ingibition of a fibrinolytic enzyme from Aspergillus oryzae by serum. Acta Chem. Scan&, 23: 2165-2174. 11. Moore, S. & W. H. Stein. 1948. Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem., 176; 367-88. 12. Mosolov, V. V. 1971. Proteolytic Enzymes. Moscow. 414p. 13. Nordwig, A. & W. F. Jahn. 1968. A collagenolytic enzyme from Aspergillus oryzae. Purification and properties. Eur. J. Biochem. 3, (4): 519-29. 14. Orlievskaya, O. V., L. K. Shataeva & G. V. Samsonov. 1971. Thrombolytic preparation 'terrilytin' and isolation of its individual enzymes. Prikl. Biokhim. Mikrobiol. 7(3): 355-9. 15. Orlievskaya, O. V., L. K. Shataeva & G. V. Samsonov. 1973. Denaturation and autolysis of protease from Aspergillus terricola. Prikl. Biokhim. Mikrobfol. 9 (I): 41-44. 16. Pelichova, H., T. Suzuki & B. Cinader. 1970. Enzyme activation by antibody. J. 2mmunol. 104(2): 195-202. 17. Pollok, M. P., J. Fleming & S. Petrie. 1967. Effect of specific antibodies on the biological activities of wild-type bacterial penicillinases and their mutationaUy altered analogs. Antibodies to Biological Active Molecules. I: 139-152. 18. Robbins, K. S., L. Summaria, D. H. Elwyn & G. H. Barlow. 1965. Further studies on the purification and characterization of human plasminogen and plasmin. J. Biol. Chem., 240(2): 541-50. 19. Roberts, J., G. Burson & J. M. Hill. 1968. New procedures for purification of L-asparaginase with high yield from Escherichia Coll. J. Bacteriol. 95(6): 2117-23. 20. Shataeva, L. K:&N.A. Zaikina. 1973. Kineticsofdissolving fibrin thrombi by terrilytine. Vopr. Med. Khimii. 19 : 11-14. 21. Shataeva, L. K., O. V. Orlievskaya, G. V. Chichkovskaya & G. V. Samsonov. 1973. The enzyme activity of terrilytine, Biochimia 38(6) : 1169-74. 22. Shataeva, L. K., O. V. Orlievskaya & G. V. Samsonov. 1969. Effect of the enzymic preparation terrilytin on the components of the blood coagulation system. Probl. Med. Enzimol., Mater. Vses. Simp. 2st. 1969 (Pub. 1970):278-87. 23. Ishima, E. & F. Yoshida. 1967. Conformation of aspergillopeptidase A in aqueous solution. 12. UV optical rotatory dispersion of aspergillopeptidase A. Biochim. Biophys. Acta, 147(2): 341-346. 24. Subramanian, A. R. & G. Kalnitsky. 1964. The magior alkaline proteinase of Aspergillus oryzae, aspergillopeptidase B. L Isolation in homogeneous form. Biochemistry, 3(12): 1861-67 . . . . . 25. Turkova, J., O. Mikeg, K. Gan6ev & M. Boublik. 1969. Isolation and characterization of alkaline proteinase of Aspergillus flavus. Biochim. Biophys. Acta. 178(1): 100-11. 26. Zaikina, N. A., N. P. Elinov, L. K. Shataeva & A. A. Domorad. 1970. Activation and stabilization of proteolytic enzymes by bacterial polysaccharides. Vopr. Med. Khimii. 16(4): 430434.
157
SOME PROPERTIES OF THE PROTEASE FROM ASPERGILLUS TERRICOLA
Nadejda A. ZAIKINA, Larisa K. SHATAEVA, N. P. ELINOV, & G. V. SAMSONOV Chemical Pharmaceutical Institute, Leningrad, USSR
Abstract
The protease from Aspergillus terricola (terrilytin) was isolated and purified by the ion exchange method and by gel chromatography. The data on its molecular weight, isoelectric point, N-terminal amino acids were described. The protease included polysaccharide which stabilized the enzyme activity under the storage and lyophylization. Polysaccharides isolated from different fungi and yeasts were found to stabilize and activate terrilytin rising the affinity of enzyme to substrate. Terrilytin was revealed to exibit the thrombolytic effect and expressive affinity to the fibrin in comparison with other substrates of high and low molecular weight. The enzyme showed the antigenic activity. From immune rabbit sera 7S y-globulins inhibiting proteolysis of caseine and 19S y-globulins activating the proteolysis of fibrin and fibrinogen by terrilytin were isolated by gel chromatography.
Introduction
Some proteases are isolated and identified at present from fungi of the genus Aspergillus of the following species: Asp. saitoi (23), Asp. oryzae (13,24), Asp. sojae (8), Asp. flavus (25). Some of them are used in the industry and in the medicine, the enzyme exibiting the fibrinolytic effect attracts the particular attention (2). This paper presents characteristics of a new neutral protease isolated from fungus Asp. terricola (9).
Materials and Methods
The enzyme was precipited from the cultural media by (NH4)zSO4 and purified on the ionexangers F A F and KMT and on biogel P-150 (14). The proteolytic activity was determined by Ancon method (1) and from the rate
of accumulation of c~-amine nitrogen in the reaction mixture (11). Thrombolytic activity was determined from the rate of the dissolution of a standard clot (0.1 ml of rabbit blood + 0.1 ml of thrombin solution) at 37 ~ when the volume ratio clot: enzyme solution was 1:1 (20). Polysaccharides were prepared from the cell extracts of the fungi and purified by the repeated precipitation by HC1 and acetone (26). Rabbits were immunized intravenous with 3 mg of the enzyme in the 0.5 ml of the saline receiving 4 injection in 5 days interval. They were bled in the 7 days after the last injection. The immunization was repeated for 2-3 months. Products of the protease autolysis were also used for the immunization. Rabbits were injected with 0.5 ml of the solution containing 7.1 millimole of e-amine nitrogen per 1 ml. The immunization schedule was the same as with the protease. Antibodies were determined by the complement fixation test (CFT) and by gelprecipitation test. Sera were fractioned on sephadex G-200 in 0.05 M tris buffer pH 8.0 containing 0.2 M NaC1 (7). Paper electrophoresis in veronal-medinal-borate buffer pH 8.6 was used for the analysis of serum proteins.
Results and Discussion
1. Physico-chemicalproperties of terrilytin The preparation precipited from the cultural media of the fungus by (NH4)zSO 4 and purified on the ionexangers F A F and KMT contained 2 enzymes - the neutral protease and acid amylase. These components were successfuly separated by gel chromatography on biogel P-150. The protease obtained (terrilytin) is a homogeneous enzyme according to ultracentrifugation, electrophoresis and coefficient of diffusion determination. Molecular weight of terrilytin is about 27,000, iso-electric point 4.6. The optimum of proteolytic activity and stability is at pH 6.5-7.0. N-terminal amino acids were determined in the 153
enzyme by dinitrophenilation, they were found to be alanine, glycine, serine, glutamic and aspartic acids. The purified terrilytin preparation was found to be separated by the gradient elution on carboxymethyl cellulose with the isolation of 2 fractions. The first fraction was polysaccharide and the second - protein with the same proteolytic and thrombolytic activity as the initial enzyme. The polysaccharide was hydrolyzed by acid with the formation of monosaccharides: mannose, glucose and traces ofxylose, which were identified by paper chromatography. The polysaccharide component of terrilytin reveals no important influence on the specific activity but essentially stabilizes the enzyme. Thus, the protein separated from polysaccharide was found to loss its activity under the storage and dialysis more rapidly than the initial enzyme. Lyophylization of pure protein results in the complete inactivation when the activity loss of the initial enzyme consists only in 12-16~. These findings confirm once more the hypothesis about the universal biological protective role of polysaccharides (5). The protease in solution was found to be in the equilibrium with the products of its autolysis. Their equilibrium concentration consists in (16 + 1) mole of a-amine nitrogen on 1 mole of protein at 4~ The products of terrilytin autolysis were separated from the native enzyme by gel chromatography. The superequilibrious addition of the products of autolysis to the terrilytin solution was found to stabilize the enzyme during heat denaturation (15). The products of terrilytin autolysis consist of polysaccharide and peptides. The polysaccharide composition is analogous to one described above. Peptides have the same N-terminal aminoacids as the native terrilytin and consist of the fractions with molecular weight from 600 to 3500, the average molecular weight is about 2000. Both the polysaccharide and peptide components of the autolysis product stabilize the native proteine structure. The following experiments revealed the ability of polysaccharides isolated from cell extracts of different fungi and yeasts to stabilize the purified terrilytin. Polysaccharides were found also to increase the thrombolytic activity of the enzyme. In these experiments the concentration of terrilytin was 2 mg/ml and of polysaccharide 20 mg/ml. The equal volumes of these ingredients were mixed, in control experiments polysaccharide solution was replaced by saline. The test solution was added to the clot immediately after mixing and after the 24 hr. exposure at 37 ~ All studied polysaccharides were found to activate terrilytin (table I). The effect was greater after the 24 hr. 154
Table I. The influence of polysaccharides on the activity of terdlytin
Polysacchadde from
Time of the thrombolysis (rain.) after the exposition of terrilytin and polysaccharide/ without exposition
Aspergillus terricola Candida albicans C tropicalis C pseudotropicalis Trichosporon condensatum T behrendii Saccharomyces cerevisiae Mucor racemosus.
Without polysaccharide
75 75 70 70 70 65 60 75 95
in 24 hr. 125 125 120 95 95 105 105 130 165
exposure with partial inactivation more rapid in the control than in the presence of polysaccharide. It is interesting that the polysaccharides from different species of fungi and yeasts were capable to activate terrilytin. These substances represent protein-polysaccharide complexes where the protein component is strongly attached to the polysaccharide and is not separated during repeated precipitation and gel chromatography. Thus, the substance from A. terricola consists of 31 ~ of reducing substances, 2.8 ~ of nitrogen and 8.6~ of phosphorous. It does not differ essentially from the composition of substances from the other fungi. For the more detailed investigation of the polysaccharide action on terrilytin activity the rate of the proteolysis of fibrinogen in the presence of polysaccharide and without it was compared. In these experiments the concentration of polysaccharide was 5 mg/ml and of terrilytin - 0 . 4 mg/ml. Concentration of fibrinogen changed from 5 to 30 mg/ml. Km was determined from the curve of the dependence of the reaction rate (by the accumulation of c~-amine nitrogen in the reaction mixture) from substrate concentration. Km was found to be 1.75 micromole/ml in the presence of polysaccharide from cell extract of Candida albicans and 5 micromole/ml without it. Therefore, these findings permit the supposition that the polysaccharide rises the affinity of enzyme for substrate. The following investigations of the biochemical properties of terrilytin were made with enzyme purified on biogel P-150, i.e. with the preparation containing protein and polysaccharide components because the protein separated from polysaccharide is comparatively unstable.
2. The effect of terrilytin on synthetic substrates and oligopeptides Terrilytin does not hydrolyse dipeptides ala-histidine, val-tyrosine, val-glycine, his-glycine, gly-alanine, glyleucine, gly-valine, gly-methionine, gly-phenil-alanine, tripeptides pro-gly-glycine, gly-gly-proline, ala-gly-glycine, and oligopeptides of/3-chain of insulin: leu-tyr-leu-val-cysglycine, tyr-leu-val-cys-glycineand leu-val-cys-glycine.The specific substrates of trypsin and chyrnotrypsin-benzoylarginine-methylic ether and benzoil-tyrosine-methylicether were not affected by terrilytin. The terrilytin was found to hydrolyse peptides asp-histidine, asp-glycine, pro-tyrosine, his-leucine, glu-asparagine, leu-valine, pro-leu-glycine, besides the enzyme hydrolyses asparagine and glutamine with the formation of amine acids. The hydrolysis rate of these substrates at 37~ consists from 1.4.10 -4 to 27.10-4 micromole of substrate hydrolysed in 1 sec. under enzyme concentration 0.8 micromole and substrate concentration 5 mg/ml (21). It is necessary to note that the hydrolysis rate of peptides by terrilytin is lower on 2-3 orders than the hydrolysis rate of specific low molecular weight substrates by the other proteases, for example, by trypsin, plasmin (18), 1-asparaginase (19).
3. The effect of terrilytin on native proteins The proteolysis of native proteins was studied at 37 ~ enzyme concentration 0.8 micromole and substrate concentration 10 mg/ml with the exeption of prothrombin: its concentration (0.2 mg/ml) was necessary because of the lability of the substance. Attempts to prepare more concentrated solutions led to its spontaneous conversion with the formation of thrombin. Amongs the proteins representing factors of blood coagulation fibrin was found to be hydrolyzed by terrilytin most extensively (table II). The rate of fibrin hydrolysis exceed considerably the rate of hydrolysis of the other
Table II, The rates of hydrolysis of proteins by terrilytin
soluble and unsoluble substrates as well as of low molecular weight substrates. The significant difference between the hydrolysis rate of fibrin and fibrinogen was confirmed when studing the preparation of these proteins released from plasminogen (3). It is possible to suppose that the increase of the hydrolysis rate of fibrin in comparison with fibrinogen correlates with the increase of the degree of ordering of structure and reciprocal orientation of polypeptides chains of the substrate (in this case-fibrin), forming a triplete helix (22).
4. Immunological properties of terrilytin The possibility of the application of terrilytin for the therapeutic purposes requires the studing of the antigenic activity of the enzyme and the investigation of the influence of specific antibodies on the proteolytic activity. The nonspecific serum inhibitors of proteases are known to complicate the investigation of the antibodies action on these enzymes (6, 12). The influence of nonspecific serum inhibitors must be studied carefully when using protease for the thrombolytic therapy (10). Intravenous injection of terrilytin produced in rabbits antibodies formation: at the end of the immunization sera titres run up to 1 : 1600 in CFT (Table III). The same sera reacted with the products of terrilytin autolysis but at a lower dilution. The products of autolysis showed antigenic activity which was weaker than that of the native enzyme: the titre 1 : 100 was observed in the reaction with homologous antigen only after the 2nd cycle of the immunization. The sera of rabbits immunized by products of autolysis reacted with terrilytin as well but the reaction was weakly positive at the low dilution of the sera. Table Ill. CFT with sera of rabbits immunized with terrilytin and products of its autolysis
Serum against Terrilytin
Substrate
Fibrin Fibrinogen Thrombin Prothrombin Insulin Collagen Elastin
Specific rate
140 33 8 4 60 19 17
mole ofa-NH 2 mole of enz. rain. Products of autolysis
Antigen
Dose of the antigen
Titre of the serum
Terrilytin Products of autolysis
1 : 100000
1 : 1600
1 : 1000
1 : 100
Terrilytin
1 : 100000
1 : 100-+
Products of autolysis
1 : 1000
1 : 100
Note: 4 rabbits were immunized with each antigen, the average data are presented. 155
The precipitation test was positive only with sera prepared against terrilytin with its homologous antigen. The sera prepared against the partly purified preparation containing protease and amylase gave two zone of the precipitation with homologous antigen and one zone with the individual protease. Sera against the highly purified terrilytin gave one zone of the precipitation with the both antigens. These findings confirm once more the homogeneity of the purified terrilytin preparation. The concentration of antigens in the precipitation test was 1 mg/ml, their doses in CFT were choosen on the basis of the anticomplementary properties of the antigens and verified in the 'square' reaction. When studying the influence of immune and normal sera on terrilytin activity, equal volumes of enzyme solution with the concentration 0.08 mg/ml in 0.1 M phosphate buffer pH 6.6 and the serum were mixed and 0.5 ml of this mixture was added to 1 ml of the substrate. Casein dissolved in 0.1 M.phosphate buffer pH 7.6, fibrin and fibrinogen suspended or dissolved, respectively, in saline were used as substrates. The concentration of substrates was 0.5 ~o. The results of the experiment was found to depend on the choosen substrate and on the concentration correlation of the components of the reaction. However the difference between the influence of immune and normal serum on the enzymatic activity hardly permits the interpretation because of the complex composition of the serum. Sermn is known to contain a number of proteins including different proteinases and their inhibitors both in a free state and in complexes. With the addition of the proteinase to the serum, the complex processes including both activation and destruction of active serum proteins can take place. Therefore, the following experiments were made with the isolated serum fractions prepared by gel filtration. This method permits to isolate 3 serum fractions, designated as P1, P2 and P3 (fig. 1). Under the electrophoresis the fractions P1 and P2 move as y-globulins and P3 contains y-globulins and albumins. The complement fixing antibodies were found in the fractions P1 and P2 and precipitins - in P2. The fractions P1 gave the positive CFT at the minimal concentration of prottein 0.240 mg/ml and P2 at the concentration 0.080 mg/ml. According to the certain data (7) fractions P1 and P2 contain 19S and 7S y-globulins, respectively. Unspecific inhibitors ofproteases were found in the fraction P3, although there is information (12) that 17S macroglobulins which can be present in the fraction P1 as admixture possess the antiprotease activity. Cel chromatography of normal sera resulted in the 156
P3
p/ o,~0o 4
Oj t ~
o
'
J, 3o
,
0o
~o
Fig. 1, Gel chromatography of the serum against terrilytin. 1 - absorbtion (fraction volume 2, 3 ml); 2 - concentration of protein of the complement fixing antibodies (mg/ml). J
preparation of the same 3 protein fractions, but the concentrations of the fraction P1 and P2 were considerably lower than in immune sera. These findings are possibly explained by the increase of y-globulins during the immunization due to antibodies formation (4). When studying the influence of the protein fractions on the terrilytin activity, the fractions were dialyzed against saline in order to remove the surplus of the salt in the lyophylized preparations, then they were diluted to the protein concentration 4 mg/ml and used as in the experiments with native sera. The activity of terrilytin in the presence of fractions was found to depend on the substrate (table IV). Immune Table IV. The influence of serum fractions on the activity of terrflytin
Serum
Proteolytic activity as per cent to Fraction / the controle in the presence of the fraction and substrate casein
fibrinogen
fibrin
Immune
P1 P2 P3
97 62 69
170 100 103
170 100 110
Normal
P1 P2 P3
103 101 89
105 108 105
110 100 102
Note: the average data of 4 experiments are presented
fraction P1 did not influence on the proteolysis of casein and activated the splitting of fibrin and fibrinogen. The fraction P2 contained antibodies that inhibited terrilytin activity on casein but no other substrate. Respective fractions of normal sera did not affect significantly the proteolytic activity of terrilytin. It is interesting that immune fraction P3 inhibited the activity of terrilytin more strongly than the same fraction from normal sera. The inability of fractions P1 and P2 from normal sera to affect t h e activity of terrilytin is evidence that these fractions contain 7-globulins without visible admixtures of the other proteins capable of changing the activity of the protease. Consequently, it is reasonable to assume that the change in proteolytic activity in the presence of the fractions of immune sera corresponds with the action of specific antibodies. Therefore, in sera of rabbits immunized with terrilytin both inhibiting and activating antibodies are formed. Moreover, during immunization nonspecific serum inhibitors of protease were found to increase. The final result of the influence of serum on the terrilytin activity depends on such experimental conditions as substrate used and correlates with the concentrations of ingredients in the reaction mixture. Analogous findings were obtained when investigating antibodies against a number of enzymes. (16, 17).
References 1. Anson, M. L. 1938. Estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J. Gen. Physiol., 22; 79-89. 2. Bergkvist, R. & P. O. Svard. 1964. The thrombolytic activity of a protease from Aspergillus oryzae. Acta Physiol. Scand., 60 (4): 36-71. 3. Blomback, B. & M. Blomback. 1956. Purification of bovine and human fibrinogen. Acta Chem. Scand, 10, 147. 4. Boyd, W. C. 1969. Fundamentals of immunology. Moscow. p. 5. Elinov, N. P. 1971. Biological functions of natural polysaccharides. Proc. Conf. Chem. Pharm. Inst. Leningrad: 5-7. 6. Fossum, K. 1970. Proteolytic enzymes and biological inhibitors. Acta Pathol. Microbiol. Scand., Sect. B. 78(5): 605-618. 7. Fukazava, Y. & T. Tsuchiya. 1969. Preparation of monospecific antisera for yeast antigens by immunological methods. Yeasts. Proc. I2 Syrup. on Yeasts. Bratislava: 553-558. 8. Hayashi, K., D. Fukushima & K. Mogi. 1967. Alkaline proteinase of Aspergillus sojae. Physico-chemical properties, amino acidcomposition, and molecular conformation. Agr. Biol. Chem. (Tokyo), 31(5): 642-3.
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