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Noncatalytic protein component of elastase from horse leucocytes. A protein with regulatory function
Int. J. Biochem. Vol. 20, No. 1, pp. 105-108, 1988
0020-711X/88 $3.00+0.00 Copyright © 1988 PergamonJournals Ltd
Printed in Great Britain. All rights reserved
NONCATALYTIC PROTEIN COMPONENT OF ELASTASE FROM HORSE LEUCOCYTES. A PROTEIN WITH REGULATORY FUNCTION* J. POTEMPA,l E. KORZUS,l J. SILBERRING2 and A. DUBINl qnstitute of Molecular Biology, Jagiellonian University, Cracow and 2Institute of Pharmacology, Polish Academy of Sciences, Cracow, Poland (Received 23 March 1987)
Abstract--l. Noncatalytic protein component (NPC), a strongly acidic protein (pH = 4.5) was separated from native horse leucocyte elastase 1. 2. This protein reduces elastinolytic properties of elastases: 1 and 2A probably by decreasing their isoelectric points. 3. A possible regulatory role of this protein may be inferred from a higher affinity of elastase ! to NPC rather than to elastin.
INTRODUCTION In distinction to other mammals (Ashe and Zimmerman, 1982) the extract of horse leucocyte granules exhibits only elastase activity (Dubin et al., 1986) for which three serine proteinases l, 2A and 2B of well defined properties are responsible (Dubin et al., 1976; Koj et al., 1976; Ardelt et al., 1976; Potempa, 1982; Potempa et al., 1986). Native elastase l (Mr 49,000, pH 5.4) occurs in the leucocyte granule extract as a complex of two proteins, one of which is catalytically active (modified elastase l, Mr 30,000, pH 8.2 and 9.18) and the second one exhibits no enzymatic activity (noncatalytic protein component, M, 20,000, pI 4.5) (Potempa, 1982). Thus a question can be raised: what is the physiological function of this noncatalytic protein component of elastase l? METHODS
Elastases were purified from the horse leucocyte granules: elastase 1 (native and modified) by the method of Potempa (1982) and elastases 2A and 2B by the method of Dubin et al. (1976). The amounts of active enzymes in the preparations were determined using the procedure of Beatty et al. (1980) based on stoichiometry of proteinases inactivation by human ~:proteinase inhibitor (Dubin eta[., 1986) standardized with bovine trypsin which had been titrated with p-nitrophenyl guanidinobenzoate. Esterolytic activity was determined with N-tertbutyloxycarbonyl-L-alanine-p-nitrophenyl ester (BOC-AIaONp, Fluka, Buchs, AG) using procedures modified from Visser and Blout (1972). Lung elastin from healthy horses was isolated by Lansing's method according to Reilly and Travis (1980). The influence of noncatalytic protein component (NPC) of native elastase 1 on adsorption of modified elastase 1 and 2A on the horse lung elastin has been studied by two methods: (1) 30/~1 of elastase (0.2 nmol) or 30 #1 of the mixtures: elastase (0.2 nmol) and noncatalytic component (0.2 nmol) preincubated first for 2 rain at 20°C were added to 270/~1 of *Supported by grant 04.01.2. I0 from the Polish Academy of Sciences. I05
0.05 M Tris-HC1 buffer, pH 7.4 containing 2 mg of suspended horse lung elastin, shaken for 5 min and centrifuged. (2) Elastin and elastase were mixed, shaken and centrifuged as in method (1). The pellet was washed for 5 min with 300/~1 of 0.05 M Tris-HCl buffer pH 7.4 and than with the almve buffer containing 0.1, 0.2, 0.3, 0.4.and 0.5 nmol of noncatalytic protein component. The esterolytic activity was estimated in all supernatants. Monospecific antiserum to NPC was obtained in rabbits according to the procedure of Harbo¢ and Ingild (1973). Crossed immunoelectrophoresis was performed according to Wecke's procedure (1973) and isoelectric focusing was carried out as described by Svensson (1962).
RESULTS AND DISCUSSION Elastase 1 was isolated from the granular extract of the horse blood leucocytes as a complex consisting of two proteins; one of them is a catalytically inactive (noncatalytic protein component, NPC) and exhibits high electropboretic mobility in alkaline buffers while the second one is an enzyme with two pH maxima at 8.5 and 9.15 (Potempa, 1982). This complex of native elastase 1 may be dissociated during Cibacron Bluc-Sepharose chromatography (Potempa, 1982). The noncatalytic protein component of the native elastase 1 is localized mainly in the granular fraction of leucocytes. This was documented by immunodiffusion technique in agarose gel, containing antibodies against NPC. The quantity of NPC (approx. 0.25 pg/cell) is comparable with the concentration of both elastases 1 and 2A (total 0.3 pg/cell), thus the major part of NPC is bound to these enzymes, as shown also in Fig. IA. In order to confirm that the presence of NPC is not an artifact produced during preparation of granules, we also applied the polyacrylamide gel electrophoresis, combined with immunoelectrophoresis in the presence of monospecific antiserum against NPC isolated from elastase 1 (Fig. 1). As shown in this figure, the major part of NPC is bound to the elastase (Fig. 1A) and partial purification of the enzyme causes slight release of the NPC. This latter fragment
106
J. POTEMPA et al.
binds to the unknown granular component giving an additional immunoprecipitable peak (Fig. IB). The described noncatalytic component exhibits isoelectric point at pH 4.5 (Fig. 2) and reacts with modified elastase 1, giving the native form of the enzyme (Potempa, 1982). In addition, this protein also forms complexes with the two remaining leucocyte granular elastases 2A and 2B. These enzymes have isoelectric points at pH 8.8 and above 10, respectively (Dubin et aL, 1976). As shown in Fig. 3a, two peaks at pH 8.8 (solid triangles) and 5.3 (open circles) belong to free and NPC-bound elastase 2A, respectively, while the third peak at pH 8.6 (circles) corresponds to the NPC-2B complex. Thus, as expected, noncatalytic protein component decreases isoelectric point of the enzyme after complex formarion. Since pH of free elastase 2B is above 10, the enzyme activity could not be demonstrated in Fig. 3 in the experiment with purified elastase. The presence of noncatalytic protein component in the two peaks observed in the second experiment was additionally confirmed by immunodiffusion (Fig. 3b). All known proteolytic enzymes which degrade elastin, are basic proteins with high isoelectric points, and the electrostatic forces are involved in binding of the elastases with their physiological substrates (Gertler, 1971). The noncatalytic protein component, when bound to the horse leucocyte elastases, decreases their isoelectric points (see Fig. 3 and Potempa, 1982). For that reason we investigated the influence of NPC on the estero- and elastinolytic activities. The technique of agarose-elastin plates (Fig. 4) demonstrated that NPC causes the loss of eleastinolytic activity of elastases 1 and 2A but not of elastase 2B. These results could be expected, since NPC complexes with elastases 1 and 2A exhibit identical, low isoelectric point around pH 5.3, while the value for NPC-elastase 2B is somewhat higher, around pH 8.5. To elucidate the importance of isoelectric point for enzyme activity, we tested also adsorption of the investigated enzymes on the horse lung elastin. Preincubation of enzymes 1 and 2A and NPC considerably reduced their adsorption on elastin as compared with the free enzymes (Fig. 5). On the other hand, addition of NPC to the elastase, that was previously adsorbed on elastin, causes release of the enzyme (Fig. 6). This fact suggests a higher affinity of the enzyme to NPC rather than to elastin. Of the horse leucocyte proteinases, only native proteinase 1 does not fulfill the major condition characterizing elastases, but it becomes again a basic protein after NPC dissociation. Taking this into account, one may suggest a specific regulatory role of
the noncatalytic protein component derived from proteinase 1. Although heparin also forms complexes with horse leucocyte proteinases the interaction appears to be less specific and has negligible effect on enzyme activity (Dubin et al., 1976).
REFERENCES
Ardelt W., Koj A., Chudzik J. and Dubin A. (1976) Inactivation of some pancreatic and leucocyte elastases by peptide chloromethyl ketones and alkyl isocyanates. FEBS Lett. 67, 156-160. Ashe B. M. and Zimmerman M. (1982) Comparison of the neutral proteinases from polymorphonuclear leukoeytes of several animal species. Biochem. Int. 5, 487-494. Beatty K., Bieth J. and Travis J. (1980) Kinetics of association of serine proteinases with native and oxidized a~-proteinase inhibitor and at-antichymotrypsin. J. biol. Chem. 255, 393 !-3934. Dubin A., Koj A. and Chudzik J. (1976) Isolation and some molecular parameters of elastase-like neutral proteinases from horse blood leucocytes. Biochem. J. 153, 389-396. Dubin A., Potempa J., Schnebli H. P. and Koj A. (1986) Comparison of specificity of human and horse leucocyte proteinases with synthetic peptide substrates. Folia Histochem. Cytobiol. 24, 157-162. Dubin A., Potempa J., Kurdowska A., Pajdak W. and Koj A. (1986) Comparison of antiproteolytic activities of ,q-proteinase inhibitors from the plasma of some mammalian species. Comp. Biochem. Physiol. 83, 375--380. Gertler A. (1971) The non-specific electrostatic nature of the adsorption of elastase and other basic proteins on elastin. Eur. J. Biochem. 20, 541-546. Harboe N. and Ingild A. (1973) Immunization, isolation of immunoglobulins, estimation of antibody titre. Scand. J. Immunol. 2, 161-164. Koj A., Chudzik J. and Dubin A. (1976) Substrate specificity and modification of the active centre of elastase-like neutral proteinases from horse blood leucocytes. Biochem. J. 153, 397-402. Potempa J. (1982) Cibacron Blue---induced modification of neutral proteinase from horse blood leukocytes. Acta biol. reed. germ. 41, 47-52. Potempa J., Korzus E., Dubin A. and Silberring J. (1986) Elastinolytic activity of horse leukocyte proteinase. Comparison with elastases from human leukocytes and porcine pancreas. Folia Histochem. CytobioL 24, 149-156. Reilly C. F. and Travis J. (1980) The degradation of human lung elastin by neutrophil proteinases. Biochim. biophys. Acta 621, 147-157. Svensson H. (1962) Isoelectric fractionation, analysis and characterization of ampholytes in natural pH gradients. Archs Biochem. Biophys. 1, 132-138. Visser L. and Blout E. R. (1972) The use of p-nitrophenyl N-tert-butyloxycarbonyl-L-alanine as substrate for elastase. Biochim. biophys. Acta 268, 257-260. Weeke B. (1973) Rocket immunoelectrophoresis. Scand. J. Immunol. (Suppl. 1) 2, 37-46.
A
i
Fig. i. Crossed immunoelectrophoresis of the successive steps of elastase 1 purification. Separation in the first direction (in 7.5% polyacrylamide gel, pH 8.3) was run from right to the left; second direction (in 1% w/v agarose with antiserum against NPC)--from bottom to top. The gel, stained for protein is placed instead of the gel used during immunoelectrophoresis. A---Crude extract of lysosomal granules (100/zg); B---partially purified elastase 1 (40/zg); C--native elastase I (20 pg); D~noncatalytic protein component (lO#g). I
|
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pH
pH
6.0
60 I D
5.5
5.5 I
5.0
5.0
4.5
4.5 0
4.0
4.0
2
4
6
8
10
12
14
16
Fig. 2. Electrophoretic analysis of the noncatalytic protein component of native elastase l from horse blood leucocytes. Isoelectric focusing in 5% (w/v) polyacrylamide gel--first direction from left to fight (gel load: 2/zg of protein). Crossed immunoelectrophoresis in 1% (w/v) agarose--seeond direction (from bottom to top). Part of the preparation was run separately and then was stained with Coomassie Brilliant Blue R-250. 107
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10
20
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40
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4
50
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Fig. 3. a. Isoelectric focusing of purified horse leucocyte elastases 2 (column load: 3 mg of proteins). Proteolytic activity was determined with casein as a substrate; A--proteolytic activity in first experiment, where only elastases were separated; O--proteolytic activity in the second experiment, where mixtures of elastases (3 mg) with NPC were separated, b. Radial immunodiffusion in 1% (w/v) agarose. Central well: antiserum against NPC; well a: noncatalytic protein component (0.5 #g); well b: fraction No. 20, pH = 5.3; well c: fraction No. 42, pH = 8.5; well d, e, f: purified elastases 2A and 2B.
ELASTASE 1
Fig. 4. The influence of noncatalytic protein component on the elastinolytic activity of horse leucocyte proteinases as measured against homologous elastin in 0.05 M Tris-HCl buffer, pH 8.0, containing 0.05% NaN 3. Incubation time: 62 hr at 37°C; agarose concentration: 1.5% (w/v). Elastin concentration: 0.3% (w/v). a--Modified elastase 1 (0.1nmol); ~ l a s t a s e 2A (0.1nmol); c---elastase 2B (0.1 nmol); d--mixture of the modified elastase 1 (0.1 nmol) with NPC (0.3 nmol); e---mixture of the elastase 2A (0.1 nmol) with NPC (0.3 nmol); f--mixture of elastase 2B (0.1 nmol) with NPC (0.6 nmol).
ELASTASE 2
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[~-FREE
ENZYME
[~-ENZYME CONPLEXED WITH NONCATALYTIC PROTEIN COMPONENT
Fig. 5. The influence of NPC (0.2 nmol) on adsorption of elastase 1 (0.2 nmol) and elastase 2A (0.2 nmol) on the horse lung elastin.
A
B
C
O
E
F
G
Fig. 6. Desorption of elastase 1 (0.2 nmol) adsorbed on the horse lung elastin by various amounts of NPC. A---Control of enzyme adsorption; B--washing with buffer. Desorption by NPC: C~0.1nmol; D--0.2nmol; E ~ . 3 n m o l ; F - 0,4 nmol; G--0.5 nmol.
108
0020-711X/88 $3.00+0.00 Copyright © 1988 PergamonJournals Ltd
Printed in Great Britain. All rights reserved
NONCATALYTIC PROTEIN COMPONENT OF ELASTASE FROM HORSE LEUCOCYTES. A PROTEIN WITH REGULATORY FUNCTION* J. POTEMPA,l E. KORZUS,l J. SILBERRING2 and A. DUBINl qnstitute of Molecular Biology, Jagiellonian University, Cracow and 2Institute of Pharmacology, Polish Academy of Sciences, Cracow, Poland (Received 23 March 1987)
Abstract--l. Noncatalytic protein component (NPC), a strongly acidic protein (pH = 4.5) was separated from native horse leucocyte elastase 1. 2. This protein reduces elastinolytic properties of elastases: 1 and 2A probably by decreasing their isoelectric points. 3. A possible regulatory role of this protein may be inferred from a higher affinity of elastase ! to NPC rather than to elastin.
INTRODUCTION In distinction to other mammals (Ashe and Zimmerman, 1982) the extract of horse leucocyte granules exhibits only elastase activity (Dubin et al., 1986) for which three serine proteinases l, 2A and 2B of well defined properties are responsible (Dubin et al., 1976; Koj et al., 1976; Ardelt et al., 1976; Potempa, 1982; Potempa et al., 1986). Native elastase l (Mr 49,000, pH 5.4) occurs in the leucocyte granule extract as a complex of two proteins, one of which is catalytically active (modified elastase l, Mr 30,000, pH 8.2 and 9.18) and the second one exhibits no enzymatic activity (noncatalytic protein component, M, 20,000, pI 4.5) (Potempa, 1982). Thus a question can be raised: what is the physiological function of this noncatalytic protein component of elastase l? METHODS
Elastases were purified from the horse leucocyte granules: elastase 1 (native and modified) by the method of Potempa (1982) and elastases 2A and 2B by the method of Dubin et al. (1976). The amounts of active enzymes in the preparations were determined using the procedure of Beatty et al. (1980) based on stoichiometry of proteinases inactivation by human ~:proteinase inhibitor (Dubin eta[., 1986) standardized with bovine trypsin which had been titrated with p-nitrophenyl guanidinobenzoate. Esterolytic activity was determined with N-tertbutyloxycarbonyl-L-alanine-p-nitrophenyl ester (BOC-AIaONp, Fluka, Buchs, AG) using procedures modified from Visser and Blout (1972). Lung elastin from healthy horses was isolated by Lansing's method according to Reilly and Travis (1980). The influence of noncatalytic protein component (NPC) of native elastase 1 on adsorption of modified elastase 1 and 2A on the horse lung elastin has been studied by two methods: (1) 30/~1 of elastase (0.2 nmol) or 30 #1 of the mixtures: elastase (0.2 nmol) and noncatalytic component (0.2 nmol) preincubated first for 2 rain at 20°C were added to 270/~1 of *Supported by grant 04.01.2. I0 from the Polish Academy of Sciences. I05
0.05 M Tris-HC1 buffer, pH 7.4 containing 2 mg of suspended horse lung elastin, shaken for 5 min and centrifuged. (2) Elastin and elastase were mixed, shaken and centrifuged as in method (1). The pellet was washed for 5 min with 300/~1 of 0.05 M Tris-HCl buffer pH 7.4 and than with the almve buffer containing 0.1, 0.2, 0.3, 0.4.and 0.5 nmol of noncatalytic protein component. The esterolytic activity was estimated in all supernatants. Monospecific antiserum to NPC was obtained in rabbits according to the procedure of Harbo¢ and Ingild (1973). Crossed immunoelectrophoresis was performed according to Wecke's procedure (1973) and isoelectric focusing was carried out as described by Svensson (1962).
RESULTS AND DISCUSSION Elastase 1 was isolated from the granular extract of the horse blood leucocytes as a complex consisting of two proteins; one of them is a catalytically inactive (noncatalytic protein component, NPC) and exhibits high electropboretic mobility in alkaline buffers while the second one is an enzyme with two pH maxima at 8.5 and 9.15 (Potempa, 1982). This complex of native elastase 1 may be dissociated during Cibacron Bluc-Sepharose chromatography (Potempa, 1982). The noncatalytic protein component of the native elastase 1 is localized mainly in the granular fraction of leucocytes. This was documented by immunodiffusion technique in agarose gel, containing antibodies against NPC. The quantity of NPC (approx. 0.25 pg/cell) is comparable with the concentration of both elastases 1 and 2A (total 0.3 pg/cell), thus the major part of NPC is bound to these enzymes, as shown also in Fig. IA. In order to confirm that the presence of NPC is not an artifact produced during preparation of granules, we also applied the polyacrylamide gel electrophoresis, combined with immunoelectrophoresis in the presence of monospecific antiserum against NPC isolated from elastase 1 (Fig. 1). As shown in this figure, the major part of NPC is bound to the elastase (Fig. 1A) and partial purification of the enzyme causes slight release of the NPC. This latter fragment
106
J. POTEMPA et al.
binds to the unknown granular component giving an additional immunoprecipitable peak (Fig. IB). The described noncatalytic component exhibits isoelectric point at pH 4.5 (Fig. 2) and reacts with modified elastase 1, giving the native form of the enzyme (Potempa, 1982). In addition, this protein also forms complexes with the two remaining leucocyte granular elastases 2A and 2B. These enzymes have isoelectric points at pH 8.8 and above 10, respectively (Dubin et aL, 1976). As shown in Fig. 3a, two peaks at pH 8.8 (solid triangles) and 5.3 (open circles) belong to free and NPC-bound elastase 2A, respectively, while the third peak at pH 8.6 (circles) corresponds to the NPC-2B complex. Thus, as expected, noncatalytic protein component decreases isoelectric point of the enzyme after complex formarion. Since pH of free elastase 2B is above 10, the enzyme activity could not be demonstrated in Fig. 3 in the experiment with purified elastase. The presence of noncatalytic protein component in the two peaks observed in the second experiment was additionally confirmed by immunodiffusion (Fig. 3b). All known proteolytic enzymes which degrade elastin, are basic proteins with high isoelectric points, and the electrostatic forces are involved in binding of the elastases with their physiological substrates (Gertler, 1971). The noncatalytic protein component, when bound to the horse leucocyte elastases, decreases their isoelectric points (see Fig. 3 and Potempa, 1982). For that reason we investigated the influence of NPC on the estero- and elastinolytic activities. The technique of agarose-elastin plates (Fig. 4) demonstrated that NPC causes the loss of eleastinolytic activity of elastases 1 and 2A but not of elastase 2B. These results could be expected, since NPC complexes with elastases 1 and 2A exhibit identical, low isoelectric point around pH 5.3, while the value for NPC-elastase 2B is somewhat higher, around pH 8.5. To elucidate the importance of isoelectric point for enzyme activity, we tested also adsorption of the investigated enzymes on the horse lung elastin. Preincubation of enzymes 1 and 2A and NPC considerably reduced their adsorption on elastin as compared with the free enzymes (Fig. 5). On the other hand, addition of NPC to the elastase, that was previously adsorbed on elastin, causes release of the enzyme (Fig. 6). This fact suggests a higher affinity of the enzyme to NPC rather than to elastin. Of the horse leucocyte proteinases, only native proteinase 1 does not fulfill the major condition characterizing elastases, but it becomes again a basic protein after NPC dissociation. Taking this into account, one may suggest a specific regulatory role of
the noncatalytic protein component derived from proteinase 1. Although heparin also forms complexes with horse leucocyte proteinases the interaction appears to be less specific and has negligible effect on enzyme activity (Dubin et al., 1976).
REFERENCES
Ardelt W., Koj A., Chudzik J. and Dubin A. (1976) Inactivation of some pancreatic and leucocyte elastases by peptide chloromethyl ketones and alkyl isocyanates. FEBS Lett. 67, 156-160. Ashe B. M. and Zimmerman M. (1982) Comparison of the neutral proteinases from polymorphonuclear leukoeytes of several animal species. Biochem. Int. 5, 487-494. Beatty K., Bieth J. and Travis J. (1980) Kinetics of association of serine proteinases with native and oxidized a~-proteinase inhibitor and at-antichymotrypsin. J. biol. Chem. 255, 393 !-3934. Dubin A., Koj A. and Chudzik J. (1976) Isolation and some molecular parameters of elastase-like neutral proteinases from horse blood leucocytes. Biochem. J. 153, 389-396. Dubin A., Potempa J., Schnebli H. P. and Koj A. (1986) Comparison of specificity of human and horse leucocyte proteinases with synthetic peptide substrates. Folia Histochem. Cytobiol. 24, 157-162. Dubin A., Potempa J., Kurdowska A., Pajdak W. and Koj A. (1986) Comparison of antiproteolytic activities of ,q-proteinase inhibitors from the plasma of some mammalian species. Comp. Biochem. Physiol. 83, 375--380. Gertler A. (1971) The non-specific electrostatic nature of the adsorption of elastase and other basic proteins on elastin. Eur. J. Biochem. 20, 541-546. Harboe N. and Ingild A. (1973) Immunization, isolation of immunoglobulins, estimation of antibody titre. Scand. J. Immunol. 2, 161-164. Koj A., Chudzik J. and Dubin A. (1976) Substrate specificity and modification of the active centre of elastase-like neutral proteinases from horse blood leucocytes. Biochem. J. 153, 397-402. Potempa J. (1982) Cibacron Blue---induced modification of neutral proteinase from horse blood leukocytes. Acta biol. reed. germ. 41, 47-52. Potempa J., Korzus E., Dubin A. and Silberring J. (1986) Elastinolytic activity of horse leukocyte proteinase. Comparison with elastases from human leukocytes and porcine pancreas. Folia Histochem. CytobioL 24, 149-156. Reilly C. F. and Travis J. (1980) The degradation of human lung elastin by neutrophil proteinases. Biochim. biophys. Acta 621, 147-157. Svensson H. (1962) Isoelectric fractionation, analysis and characterization of ampholytes in natural pH gradients. Archs Biochem. Biophys. 1, 132-138. Visser L. and Blout E. R. (1972) The use of p-nitrophenyl N-tert-butyloxycarbonyl-L-alanine as substrate for elastase. Biochim. biophys. Acta 268, 257-260. Weeke B. (1973) Rocket immunoelectrophoresis. Scand. J. Immunol. (Suppl. 1) 2, 37-46.
A
i
Fig. i. Crossed immunoelectrophoresis of the successive steps of elastase 1 purification. Separation in the first direction (in 7.5% polyacrylamide gel, pH 8.3) was run from right to the left; second direction (in 1% w/v agarose with antiserum against NPC)--from bottom to top. The gel, stained for protein is placed instead of the gel used during immunoelectrophoresis. A---Crude extract of lysosomal granules (100/zg); B---partially purified elastase 1 (40/zg); C--native elastase I (20 pg); D~noncatalytic protein component (lO#g). I
|
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pH
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5.5
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4.5
4.5 0
4.0
4.0
2
4
6
8
10
12
14
16
Fig. 2. Electrophoretic analysis of the noncatalytic protein component of native elastase l from horse blood leucocytes. Isoelectric focusing in 5% (w/v) polyacrylamide gel--first direction from left to fight (gel load: 2/zg of protein). Crossed immunoelectrophoresis in 1% (w/v) agarose--seeond direction (from bottom to top). Part of the preparation was run separately and then was stained with Coomassie Brilliant Blue R-250. 107
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pH 11 10 °
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10
20
30
Fraction
40
f
4
50
no.
Fig. 3. a. Isoelectric focusing of purified horse leucocyte elastases 2 (column load: 3 mg of proteins). Proteolytic activity was determined with casein as a substrate; A--proteolytic activity in first experiment, where only elastases were separated; O--proteolytic activity in the second experiment, where mixtures of elastases (3 mg) with NPC were separated, b. Radial immunodiffusion in 1% (w/v) agarose. Central well: antiserum against NPC; well a: noncatalytic protein component (0.5 #g); well b: fraction No. 20, pH = 5.3; well c: fraction No. 42, pH = 8.5; well d, e, f: purified elastases 2A and 2B.
ELASTASE 1
Fig. 4. The influence of noncatalytic protein component on the elastinolytic activity of horse leucocyte proteinases as measured against homologous elastin in 0.05 M Tris-HCl buffer, pH 8.0, containing 0.05% NaN 3. Incubation time: 62 hr at 37°C; agarose concentration: 1.5% (w/v). Elastin concentration: 0.3% (w/v). a--Modified elastase 1 (0.1nmol); ~ l a s t a s e 2A (0.1nmol); c---elastase 2B (0.1 nmol); d--mixture of the modified elastase 1 (0.1 nmol) with NPC (0.3 nmol); e---mixture of the elastase 2A (0.1 nmol) with NPC (0.3 nmol); f--mixture of elastase 2B (0.1 nmol) with NPC (0.6 nmol).
ELASTASE 2
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[~-FREE
ENZYME
[~-ENZYME CONPLEXED WITH NONCATALYTIC PROTEIN COMPONENT
Fig. 5. The influence of NPC (0.2 nmol) on adsorption of elastase 1 (0.2 nmol) and elastase 2A (0.2 nmol) on the horse lung elastin.
A
B
C
O
E
F
G
Fig. 6. Desorption of elastase 1 (0.2 nmol) adsorbed on the horse lung elastin by various amounts of NPC. A---Control of enzyme adsorption; B--washing with buffer. Desorption by NPC: C~0.1nmol; D--0.2nmol; E ~ . 3 n m o l ; F - 0,4 nmol; G--0.5 nmol.
108