- Email: [email protected]
Protease activity from maize pollen
Pergamoa
Phymchemimy,
VoL 35. No. 4. pi. 85X%. 1994 0 1994 Elvvier sdcna Ltd Britia All righIS -cd 0031-9422191%OO+O.OJ
m0tsdLIIcirrpt
PROTEASE
ACTIVITY FROM MAIZE POLLEN
MAREK RADLOWSKI,* ANDRZEJ KALINOWSKI, Institute
of Plant
Genetics,
ZYGMUNT KR~LIKOWSKI~
and SLAWOMIR BARTKOWIAK
Polish Academy of Sciences, 60-479 Pozna6, Strzeszyfiska34, Poland; tPlant BreedingStation, 63-743 Smolice, Poland 30 July
(Received in reuisedjonn
Key Word I&x-Zea
mays;
Gramineae;
maize;
1993)
pollen; protease; purification of the enzyme.
Abstrati-Protease. activity from maize pollen has been purified by chromatography on DEAE-Sepharose CL 6B, Sephadex G-100 columns and an HPLC procedure. The final purification factor of the protease was cu 52. The single band after SDS-PAGE showed its M, to be 21000. The enzyme works at the optimum pH of 4.8 and in the temperature range 45-50”. The isolated protease appears to have a serine group at the active site. No loss in the activity of the enzyme was observed after storage for several months at - 18”.
INTRODUCT’ION
The plant proteolytic enzymes play the main role in such important processes as peptide degradation [l-3], posttranslational protein modification 143 and in the cell secretory system [S, 63. The activities of these enzymes influence such physiological functions as protein breakdown, protein translocation, plant germination, senescence processes and defence against pathogens [ 1-4, 7-101. A particularly interesting problem is the effect of proteases on the maturation and degradation of enzymatic regulators [l 11. The presence of proteases in pollen grains has been known for nearly 100 years [12]. Their activity in the pollen of different plants has been considered in refs [13-153. However, so far, we have not come across a case of high level purification of the enzyme from these sources. In our previous paper [ 163, we reported the presence of a peptidic inhibitor of the aminoacylation reaction in maize pollen. The inhibitor, after a short digestion with commercial pronase R, changed its M, and simultaneously showed stimulatory activity. The results suggested that such a transformation might occur in uiuo. Thus, it seemed necessary to isolate and purify the endogenous protease from this source as the first step in demonstrating the possibility of such a transformation of the effector during pollen germination. RESULTS AND DISCUSSION
Proteolytic
activity in crude extract from maize pollen
Little information has been available about the presence and the nature of proteolytic enzymes in pollen *Author to whom correspondence
should be addressed.
grains [12-15, 173. The amount of this plant material is limited. The protease activity was studied at first by a sensitive, qualitative method based on visualization of the enzyme in a polyacrylamide gel. A crude extract from maize pollen after isoelectric focusing separation was examined for proteolytic activity by the method of ref. [ 181. One region showed protease activity in the gel corresponding to the silver stained band and assays with fluorescein isothiocyanate-labelled casein (FTC-casein) as substrate showed that protease isolation from this source was possible. Protease assays
The protease activity detected in a crude extract from pollen grains of maize was low. The methods involving casein [19] and denaturated haemoglobin [20] as substrates lacked sensitivity. The methods with azocasein [21], azocoll [22] and radiolabelled proteins (peptides) [23, 241 as substrates were expensive. FTC-casein was chosen as substrate [253, as it is very sensitive, simple and inexpensive. In our experiments, only a limited amount of the substrate was hydrolysed in a linear way. The pH of the medium must be above 4.5 [26]. Protease purification
Sephadex G-25 column chromatography was used as the first step, as in the case of the purification of the low M, inhibitor [16]. After all the steps of purification, DEAE-Sepharose CL 6B, Sephadex G-100 column (Fig. l), HPLC chromatography and rechromatography, the fractions with the highest proteolytic activities were pooled, dialysed and concentrated with Aquacide. A summary is presented in Table 1. A total of five steps resulted in a 52-fold purification. The purification and specific activity were comparable with those from the 853
854
M. RAD~OWSKI er al. Table
Purification
step
Crude extract Sephadex G-25 DEAE-Sepharose Sephadex G-100 HPLC
CL 6B
1(A)
I
I. The puritkation
of protease
from pollen grains
of maize
Total protein
Specific activtty
mg
Uimg
Total activity U
Purification -fold
Yield %
133 121 4 0.8 0.13
12 12 253 291 611
1560 1460 912 226 76
1 1 22 25 52
100 99 58 14 5
0 6 4 2
8
0
,h s 5
:!
10 8
3
“0
PC
statin and diazoacetyl DL-norleucine methyl ester) or metalloproteinase (l,lO-phenanthroline) did not inhibit the purified protease from maize pollen. No influence of 5 mM dithiothreitol (DTT) and HgCl,, potential inhibitors of cysteine proteinase, was found. Only leupeptine (69% inhibition) and particularly phenylmethyl sulphony1 fluoride (PMSF, 86% inhibition) strongly affected the enzyme activity. DTT (5 mM), added to the test with PMSF, restores a great part of its proteolytic properties. It might be assumed that protease isolated from pollen grains of maize has a serine in its catalytic site. It has also been suggested that it is a different type to that purified by Abe et al. [30] from maize endosperm. pH and temperature optimum
0
20
40
60
80
loo
”
Fraction Fig. 1. Purification of protease from pollen grains of maize on DEAE-Sepharose CL 68 (A) and Sephadex G-100 (B) columns. Continuous line, A at 280nm. dashed line, NaCl gradient (O-O.35 M and 0.5 M), :1---O, proteolytic activity. Solid bars show collected fractions.
other plant tissues [21,26-281. The average yield (5%) is relatively high compared with that for other plant proteases [27, 291. PAGE of purified protease The homogeneity of the protease was demonstrated by nondenaturating PAGE. SDS-PAGE of the purified enzyme shows a single band corresponding to a M, of 21000. Both techniques led us to the conclusion that the protease is a monomer. A maize protease with a similar M, was purified by Abe et al. [30], but this cysteine enzyme was isolated from endosperm five days after germination. Inhibitor studies Inhibitors specific to each class of proteinase were employed because of their ability to influence the enzyme activity. The effecters for aspartic acid proteinases (pep-
The serine protease from pollen grains of maize shows a narrow pH range of activity (against FTC-casein as a substrate) with a maximum of ca 5.0. This is unexpected because the activities of most of the well-known serine proteases from plant sources have maximum values at a slightly alkaline pH [31, 321. The enzyme had optimum activity in the temperature range 45-50’. Ca2’, Zn2’, Mg2+, Mn2+ and Co’+ had no influence on the activity of the protease or on its thermal stability. The purified enzyme at pH 7.5 was stored at - 18” for three months without showing a decrease in proteolytic activity. The role of serine protease in pollen grains of maize is at present unclear. However, it is known that the germination of pollen in the Gramineae is rapid [33] and needs fast mobilization of protein synthesis [34, 351. We suppose that serine protease from pollen grains of maize might be engaged in such action. EXPERIMENTAL
Chemicals. The casein was from IEL, and fluorescein isothiocyanate was from BDH. L-lys-p-nitroanilide, N-benzoyl-tyr-p-nitroanilide, L-ala-ala-ala-p-nitroanilide, L-arg-pro-p-nitroanilide and t+-glu-gly-arg-p-nitroanhide were obtained from Sigma. Nitrocellulose membrane was purchased from Schleicher and Schull. Plant material. The pollen grains of maize, inbred line Co 255 were from the Plant Breeding Station, Smolice, Poland. Extraction. Pollen grains (15 g) were suspended in an ice bath, in 30 ml of a 100 mM Tris-HCI buffer, pH 7.5,
855
Protease activity from maize pollen 10% (v/v) glycerol, 1 mM MgCI,, 0.1 mM 2-mercaptoethanol, with 3 g of sterile quartz sand. The suspension was carefully ground with a pestle for 30 min and centrifuged at 14 000 g. The supernatant was passed through a Sartorius membrane filter (13 4OO),chromatographed on a Sephadex G-25 column (5 x 26 cm) and equilibrated with a 50 mM Tris-HCI buffer pH 7.5, 1 mM MgCI,, 0.1 mM 2-mercaptoethanol. The protein fr. after CC (V,) was used in further purification procedures. Purification. The protein, after the Sephadex G-25 column, was dialysed overnight against a 1OOmM NaOAc buffer pH 4.8,0.1 mM 2-mercaptoethanol (buffer A) and applied to a DEAE-Sepharose CL 6B column (2.6 x 32 cm) equilibrated with the same buffer. A linear gradient of NaCl (O-O.35 M and 0.5 M) was used as an elution agent (2 x 300 and 200 ml, respectively). The frs with the highest protease activity were pooled, dialysed against 2 x distilled H,O, coned with Aquacide and chromatographed on a Sephadex G-100 column (2.6 x 65 cm) equilibrated with buffer A. The frs showing proteolytic activity were collected and treated as before and purified by the HPLC procedure. Chromatography and rechromatography were carried out on a Spherogel TSK 4000 SW column (0.75 x 30 cm). Protein soln (30 pl) was applied and eluted with a 20 mM NaOAc buffer pH 4.8 at a flow rate of 0.8 ml min- ‘. The prepn was coned, divided into 0.25-ml portions and stored at - 18”. Substrate preparation. FTC-casein was prepd according to ref. [25]. The protein concn in stock soln was 0.82 mgml- ‘. Aliquots (5 ml) were stored at - 18”. Enzyme assays. The substrate soln (10 pl containing 8.2 pg of FTC-casein) and 20 pl 250 mM NaOAc buffer pH 4.8 were added to 20 pl of enzyme fr. and incubation was carried out at 37” for 1- 18 hr. When the incubation time was longer than 3 hr, 0.2% NaN, was added to the assay buffer to prevent bacterial growth. The reaction was stopped by the addition of I20 pl cold 5% trichloroacetic acid (TCA). After 30 min the mixt. was centrifuged and a 100~pl aliquot of the supernatant was mixed with 2.9 ml of a 500 mM Tri-HCI buffer pH 8.5. In the blank assays, 20 pl of a 100 mM NaOAc buffer, pH 4.8, in place of enzyme fr. (0% fluorescence) and 120~1 of H,O in place of TCA (100% fluorescence) were used [26]. The fluorescence was measured with an excitation of 490 nm and an emission of 525 nm. One unit (U) of protease activity was defined as the amount of enzyme digesting 1 pg of FTC-casein hr -I. Protein determination. Protein concn was determined according to the method of ref. [36]. PAGE, SDS-PAGE and isoelectric focusing. Nondenaturating PAGE was carried out according to ref. [37] on 7.5% gel. SDS-PAGE was performed on 13% gel according to ref. [38]. Isoelectric focusing in the pH range 4-9 was made as in the method of ref. [39], except that the gel was polymerized photochemically with riboflavin. In all cases the gels were silver stained by the method of ref. [40]. Visualization of protease activity on nitrocellulose membrane was performed according to ref. [ 181. The mixt. of L-lys-p-Np, N-benzoyl-L-tyr-p-Np, L-ala-ala-alap-Np, t_-arg-pro-p-Np and t_-glu-gly-arg-p-Np dissolved
in dimethyl sulphoxide (DMSO) and suspended in 200 mM Tris-HCI buffer pH 7.5 was used as a substrate soln. The effect ofpH and temperature on proteolytic actioity. The pH optimum of protease was determined with FTCcasein as a substrate. The enzyme activity was investigated using NaOAc buffers in the pH range 4.5-5.6 and NaPi buffers in the pH range 5.8-7.0. Blanks were made for each pH value. The influence of the temp. on enzyme activity was determined at pH 4.8 with FIC-casein as a substrate at 18-65”. The blanks containing the 100 mM NaOAc buffer pH 4.8 in place of enzyme were incubated at the same temps. Inhibitor studies. The experiments were performed according to the suggestions of ref. [41]. The tests were made in duplicate at pH 4.8. The enzyme solns were preincubated with an assay buffer and inhibitors for 1 hr at 25”. In cases when the inhibitor was dissolved in EtOH, a blank with the same vol. of EtOH without an inhibitor was made. The reaction was started by the addition of FTC-casein soln. The controls containing no inhibitors, and with inhibitors but without the enzyme, were treated in the same way as the assays studied. The inpuence ofmetal ions on the enzyme activity and its thermal stability. The effect of the divalent metal ions (final concn I mM) on purified protease was investigated at pH 4.8 with FTC-casein as a substrate at 37”. The controls without metal ions were treated as the assays. The thermal stability of the enzyme was studied as follows. The samples of protease from pollen grains were preincubated with metal ions (final concn 1 mM) for 1 hr at 25”. Next, the reaction carried out at 65” was started by addition of FTC-casein. In this experiment 2 kinds of blanks were made. The first, not containing the metal ions, preincubated as the assays and incubated at 65”, and the second with the metal ions preincubated as the assays and incubated at 37”. authors would like to thank Mrs E. Borzyszkowska and Mrs B. Wojtowicz for their valuable technical assistance. We would also like to thank MS Christina Griffiths for the reading and correction of the manuscript. Acknowledgements-The
REFERENCES
Moureaux, T. (1979) Phytochemistry 18, 1113. de Barros, E. G. and Larkins, B. A. (1990) Plant Physiol. 94, 297.
Harvey, B. M. R. and Oaks, A. (1974) Plant Physiol. 53, 453.
Lord, J. M. and
C. (1986) in Plant M. J., ed.), Vol. II, pp. 69-80. CRC Press, Boca Raton. 5. Chrispeels, M. J. (1991) A. Rev. Plant Physiol., Plant Molec. Biol. 42, 1. 6. Chrispeels, M. J. and Raikhel, N. V. (1992) Cell 68, 613. Proteolytic
Robinson,
Enzymes (Dalling,
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M. RALXC IWSKI
7. Preston, K. R. and Kruger, J. E. (1986) in Plum Proteolytic Enzymes (Dalling, M. J., ed.), Vol. II, pp. I- 18. CRC Press, Boca Raton. 8. Vance, C. P., Reibach, P. H. and Ellis, W. R. (1986) in Plant Proteolytic Enzymes (Dalling, M. J., ed.), Vol. 11, pp. 103-124. CRC Press, Boca Raton. 9. Dalling, M. J. and Nettleton, A. M. (1986) in Plant Proteofytic Enzymes (Dalling, M. J., ed.), Vol. II, pp. 125153. CRC Press, Boca Raton. 10. Boller, T. (1986) in Plant Proteotytic Enzymes (Dalling, M. J., ed.), Vol. I, pp. 67-96. CRC Press, Boca Raton. 11. McGrain, A. K., Chen, J. C., Wilson, K. A. and Tan-Wilson, A. L. (1992) Phytochemistry 31,42 1. 12. Kamman, 0. (1904) Beitr. Chem. Physio~. Path. 5,346. 13. Paton, J. B. (1921) Am. J. Botany 8, 471. 14. Makinen, Y. and Brewbaker, J. L. (1967) Physiol. Plant. 20, 477.
15. Stanley, R. G. and Linskens, H. F. (1985) in Pollen. Biologic, Biochemie, Gewinnung
16. 17.
18. 19.
und Verwendung.
Kapitel 14, pp. 221-256. Urs Freund Verlag D-8919 Greifen~rg/Ammers~. Radlowski, M., Bartkowiak, S. and Krolikowski, Z. (1993) Physiol. Plant. 87, 391. Bhalla, P. L., Singh, M. B. and Knox, R. B. (1986) in Plant Proteolytic Enzymes (Dalling, M. J., ed.), Vol. I, pp. 41-66. CRC Press, Boca Raton. Ohisson, B. G., Westrom, B. R. and Karlsson, B. W. ( 1987) Electrophoresis 8, 4 15. Kuboki, M., Ishii, H. and Kazama, M. (1987)
Biochim. Biophys. Acta 929, 154. 20. Kolaczkowska, M. K., Wieczorek, M. and Polanowski, A. (1983) Eur. J. Biochem. 132, 557. 21. Koehler, S. and Ho, T.-H. D. (1990) P/ant Physjo~. 94,
251. 22. Chavira, R. Jr, Burnett, T. J. and Hageman, J. H. (1984) Analyt. Biochem. 136, 446. 23. Sevier, E. D. (1976) Analyt. Biochem. 74, 592. 24. Rosen, J., Tomkinson, B., Petterson, G. and
ef al.
Zetterqvist, ii. (1991) J. Biof. Chem. 266, 3827. 25. Twining, S. S. (1984) Analyt. Biochem. 143, 30. 26. Heimgartner, U., Pietrzak, M., Geertsen, R., Brodelius, P., Da Silva Figueiredo, A. C. and Pais, M. S. S. (1990) Phytochemistry 29, 1405. 27. Koehler, S. M. and Ho, T.-H. D. (1988) Plant Physiol. 87.95. 28. Shannon, J. D. and Wallace, W. (1979) Eur. J. Biochem. 102, 399. 29. Holwerda, B. C. and Rogers, J. C. (1992) Plant Physiol. 99, 848. 30. Abe, M., Arai, S. and Fujimaki, M. (1977) Agric. Biol.
Chem. 41,893. 31. Barret, A. J. (1986) in P/ant Proteolyric Enzymes (Dalling, M. J.,ed.), Vol. I, pp. l-16. CRC Press, Boca Raton. 32. Ryan, C. and Walker-Simmons, M. (1981) in The Biochemistry of Plants (Marcus, A., ed.), pp. 183-195. Academic Press, New York. 33. Hoekstra, F. A. (I 983) in Pollen: Biology and fmpfications for Plant Breeding (Mulcahy, D. L. and Ottaviano, E., eds), pp. 35-41. Elsevier Biomedical, New York. 34. Stanley, R. G. (1971) in Pollen: Development and Physiology (Heslop-Harrison. J., ed.), pp. 13 1- 155. Butterworth, London. 35. Mascarenhas, J. P. (1971) in Pollen: Development and Physia~ogy (Hesiop-Harrison, J., ed.), pp. 201-222. Butterworth, London. 36. Bradford, M. M. (1976) Analyt. Biochem. 72, 248. 37. Davis, B. J. (1964) Ann. N. Y. Acad. Sci 121, 404. 38. Weber, K. and Osborn, M. (1969) J. Biol. Chem. 214, 4406. 39. Mulcahy,
D. L., Robinson, R. W., Ihara, M. and Kesseli. R. (1981) J. Hered. 72, 353. 40. Heukeshoven, J. and Dernick, R. (1985) Electrophoresis 6, 103. 41. Storey, R. D. and Wagner, chemistry 25, 2701.
F. W. (1986) Phyto-
Phymchemimy,
VoL 35. No. 4. pi. 85X%. 1994 0 1994 Elvvier sdcna Ltd Britia All righIS -cd 0031-9422191%OO+O.OJ
m0tsdLIIcirrpt
PROTEASE
ACTIVITY FROM MAIZE POLLEN
MAREK RADLOWSKI,* ANDRZEJ KALINOWSKI, Institute
of Plant
Genetics,
ZYGMUNT KR~LIKOWSKI~
and SLAWOMIR BARTKOWIAK
Polish Academy of Sciences, 60-479 Pozna6, Strzeszyfiska34, Poland; tPlant BreedingStation, 63-743 Smolice, Poland 30 July
(Received in reuisedjonn
Key Word I&x-Zea
mays;
Gramineae;
maize;
1993)
pollen; protease; purification of the enzyme.
Abstrati-Protease. activity from maize pollen has been purified by chromatography on DEAE-Sepharose CL 6B, Sephadex G-100 columns and an HPLC procedure. The final purification factor of the protease was cu 52. The single band after SDS-PAGE showed its M, to be 21000. The enzyme works at the optimum pH of 4.8 and in the temperature range 45-50”. The isolated protease appears to have a serine group at the active site. No loss in the activity of the enzyme was observed after storage for several months at - 18”.
INTRODUCT’ION
The plant proteolytic enzymes play the main role in such important processes as peptide degradation [l-3], posttranslational protein modification 143 and in the cell secretory system [S, 63. The activities of these enzymes influence such physiological functions as protein breakdown, protein translocation, plant germination, senescence processes and defence against pathogens [ 1-4, 7-101. A particularly interesting problem is the effect of proteases on the maturation and degradation of enzymatic regulators [l 11. The presence of proteases in pollen grains has been known for nearly 100 years [12]. Their activity in the pollen of different plants has been considered in refs [13-153. However, so far, we have not come across a case of high level purification of the enzyme from these sources. In our previous paper [ 163, we reported the presence of a peptidic inhibitor of the aminoacylation reaction in maize pollen. The inhibitor, after a short digestion with commercial pronase R, changed its M, and simultaneously showed stimulatory activity. The results suggested that such a transformation might occur in uiuo. Thus, it seemed necessary to isolate and purify the endogenous protease from this source as the first step in demonstrating the possibility of such a transformation of the effector during pollen germination. RESULTS AND DISCUSSION
Proteolytic
activity in crude extract from maize pollen
Little information has been available about the presence and the nature of proteolytic enzymes in pollen *Author to whom correspondence
should be addressed.
grains [12-15, 173. The amount of this plant material is limited. The protease activity was studied at first by a sensitive, qualitative method based on visualization of the enzyme in a polyacrylamide gel. A crude extract from maize pollen after isoelectric focusing separation was examined for proteolytic activity by the method of ref. [ 181. One region showed protease activity in the gel corresponding to the silver stained band and assays with fluorescein isothiocyanate-labelled casein (FTC-casein) as substrate showed that protease isolation from this source was possible. Protease assays
The protease activity detected in a crude extract from pollen grains of maize was low. The methods involving casein [19] and denaturated haemoglobin [20] as substrates lacked sensitivity. The methods with azocasein [21], azocoll [22] and radiolabelled proteins (peptides) [23, 241 as substrates were expensive. FTC-casein was chosen as substrate [253, as it is very sensitive, simple and inexpensive. In our experiments, only a limited amount of the substrate was hydrolysed in a linear way. The pH of the medium must be above 4.5 [26]. Protease purification
Sephadex G-25 column chromatography was used as the first step, as in the case of the purification of the low M, inhibitor [16]. After all the steps of purification, DEAE-Sepharose CL 6B, Sephadex G-100 column (Fig. l), HPLC chromatography and rechromatography, the fractions with the highest proteolytic activities were pooled, dialysed and concentrated with Aquacide. A summary is presented in Table 1. A total of five steps resulted in a 52-fold purification. The purification and specific activity were comparable with those from the 853
854
M. RAD~OWSKI er al. Table
Purification
step
Crude extract Sephadex G-25 DEAE-Sepharose Sephadex G-100 HPLC
CL 6B
1(A)
I
I. The puritkation
of protease
from pollen grains
of maize
Total protein
Specific activtty
mg
Uimg
Total activity U
Purification -fold
Yield %
133 121 4 0.8 0.13
12 12 253 291 611
1560 1460 912 226 76
1 1 22 25 52
100 99 58 14 5
0 6 4 2
8
0
,h s 5
:!
10 8
3
“0
PC
statin and diazoacetyl DL-norleucine methyl ester) or metalloproteinase (l,lO-phenanthroline) did not inhibit the purified protease from maize pollen. No influence of 5 mM dithiothreitol (DTT) and HgCl,, potential inhibitors of cysteine proteinase, was found. Only leupeptine (69% inhibition) and particularly phenylmethyl sulphony1 fluoride (PMSF, 86% inhibition) strongly affected the enzyme activity. DTT (5 mM), added to the test with PMSF, restores a great part of its proteolytic properties. It might be assumed that protease isolated from pollen grains of maize has a serine in its catalytic site. It has also been suggested that it is a different type to that purified by Abe et al. [30] from maize endosperm. pH and temperature optimum
0
20
40
60
80
loo
”
Fraction Fig. 1. Purification of protease from pollen grains of maize on DEAE-Sepharose CL 68 (A) and Sephadex G-100 (B) columns. Continuous line, A at 280nm. dashed line, NaCl gradient (O-O.35 M and 0.5 M), :1---O, proteolytic activity. Solid bars show collected fractions.
other plant tissues [21,26-281. The average yield (5%) is relatively high compared with that for other plant proteases [27, 291. PAGE of purified protease The homogeneity of the protease was demonstrated by nondenaturating PAGE. SDS-PAGE of the purified enzyme shows a single band corresponding to a M, of 21000. Both techniques led us to the conclusion that the protease is a monomer. A maize protease with a similar M, was purified by Abe et al. [30], but this cysteine enzyme was isolated from endosperm five days after germination. Inhibitor studies Inhibitors specific to each class of proteinase were employed because of their ability to influence the enzyme activity. The effecters for aspartic acid proteinases (pep-
The serine protease from pollen grains of maize shows a narrow pH range of activity (against FTC-casein as a substrate) with a maximum of ca 5.0. This is unexpected because the activities of most of the well-known serine proteases from plant sources have maximum values at a slightly alkaline pH [31, 321. The enzyme had optimum activity in the temperature range 45-50’. Ca2’, Zn2’, Mg2+, Mn2+ and Co’+ had no influence on the activity of the protease or on its thermal stability. The purified enzyme at pH 7.5 was stored at - 18” for three months without showing a decrease in proteolytic activity. The role of serine protease in pollen grains of maize is at present unclear. However, it is known that the germination of pollen in the Gramineae is rapid [33] and needs fast mobilization of protein synthesis [34, 351. We suppose that serine protease from pollen grains of maize might be engaged in such action. EXPERIMENTAL
Chemicals. The casein was from IEL, and fluorescein isothiocyanate was from BDH. L-lys-p-nitroanilide, N-benzoyl-tyr-p-nitroanilide, L-ala-ala-ala-p-nitroanilide, L-arg-pro-p-nitroanilide and t+-glu-gly-arg-p-nitroanhide were obtained from Sigma. Nitrocellulose membrane was purchased from Schleicher and Schull. Plant material. The pollen grains of maize, inbred line Co 255 were from the Plant Breeding Station, Smolice, Poland. Extraction. Pollen grains (15 g) were suspended in an ice bath, in 30 ml of a 100 mM Tris-HCI buffer, pH 7.5,
855
Protease activity from maize pollen 10% (v/v) glycerol, 1 mM MgCI,, 0.1 mM 2-mercaptoethanol, with 3 g of sterile quartz sand. The suspension was carefully ground with a pestle for 30 min and centrifuged at 14 000 g. The supernatant was passed through a Sartorius membrane filter (13 4OO),chromatographed on a Sephadex G-25 column (5 x 26 cm) and equilibrated with a 50 mM Tris-HCI buffer pH 7.5, 1 mM MgCI,, 0.1 mM 2-mercaptoethanol. The protein fr. after CC (V,) was used in further purification procedures. Purification. The protein, after the Sephadex G-25 column, was dialysed overnight against a 1OOmM NaOAc buffer pH 4.8,0.1 mM 2-mercaptoethanol (buffer A) and applied to a DEAE-Sepharose CL 6B column (2.6 x 32 cm) equilibrated with the same buffer. A linear gradient of NaCl (O-O.35 M and 0.5 M) was used as an elution agent (2 x 300 and 200 ml, respectively). The frs with the highest protease activity were pooled, dialysed against 2 x distilled H,O, coned with Aquacide and chromatographed on a Sephadex G-100 column (2.6 x 65 cm) equilibrated with buffer A. The frs showing proteolytic activity were collected and treated as before and purified by the HPLC procedure. Chromatography and rechromatography were carried out on a Spherogel TSK 4000 SW column (0.75 x 30 cm). Protein soln (30 pl) was applied and eluted with a 20 mM NaOAc buffer pH 4.8 at a flow rate of 0.8 ml min- ‘. The prepn was coned, divided into 0.25-ml portions and stored at - 18”. Substrate preparation. FTC-casein was prepd according to ref. [25]. The protein concn in stock soln was 0.82 mgml- ‘. Aliquots (5 ml) were stored at - 18”. Enzyme assays. The substrate soln (10 pl containing 8.2 pg of FTC-casein) and 20 pl 250 mM NaOAc buffer pH 4.8 were added to 20 pl of enzyme fr. and incubation was carried out at 37” for 1- 18 hr. When the incubation time was longer than 3 hr, 0.2% NaN, was added to the assay buffer to prevent bacterial growth. The reaction was stopped by the addition of I20 pl cold 5% trichloroacetic acid (TCA). After 30 min the mixt. was centrifuged and a 100~pl aliquot of the supernatant was mixed with 2.9 ml of a 500 mM Tri-HCI buffer pH 8.5. In the blank assays, 20 pl of a 100 mM NaOAc buffer, pH 4.8, in place of enzyme fr. (0% fluorescence) and 120~1 of H,O in place of TCA (100% fluorescence) were used [26]. The fluorescence was measured with an excitation of 490 nm and an emission of 525 nm. One unit (U) of protease activity was defined as the amount of enzyme digesting 1 pg of FTC-casein hr -I. Protein determination. Protein concn was determined according to the method of ref. [36]. PAGE, SDS-PAGE and isoelectric focusing. Nondenaturating PAGE was carried out according to ref. [37] on 7.5% gel. SDS-PAGE was performed on 13% gel according to ref. [38]. Isoelectric focusing in the pH range 4-9 was made as in the method of ref. [39], except that the gel was polymerized photochemically with riboflavin. In all cases the gels were silver stained by the method of ref. [40]. Visualization of protease activity on nitrocellulose membrane was performed according to ref. [ 181. The mixt. of L-lys-p-Np, N-benzoyl-L-tyr-p-Np, L-ala-ala-alap-Np, t_-arg-pro-p-Np and t_-glu-gly-arg-p-Np dissolved
in dimethyl sulphoxide (DMSO) and suspended in 200 mM Tris-HCI buffer pH 7.5 was used as a substrate soln. The effect ofpH and temperature on proteolytic actioity. The pH optimum of protease was determined with FTCcasein as a substrate. The enzyme activity was investigated using NaOAc buffers in the pH range 4.5-5.6 and NaPi buffers in the pH range 5.8-7.0. Blanks were made for each pH value. The influence of the temp. on enzyme activity was determined at pH 4.8 with FIC-casein as a substrate at 18-65”. The blanks containing the 100 mM NaOAc buffer pH 4.8 in place of enzyme were incubated at the same temps. Inhibitor studies. The experiments were performed according to the suggestions of ref. [41]. The tests were made in duplicate at pH 4.8. The enzyme solns were preincubated with an assay buffer and inhibitors for 1 hr at 25”. In cases when the inhibitor was dissolved in EtOH, a blank with the same vol. of EtOH without an inhibitor was made. The reaction was started by the addition of FTC-casein soln. The controls containing no inhibitors, and with inhibitors but without the enzyme, were treated in the same way as the assays studied. The inpuence ofmetal ions on the enzyme activity and its thermal stability. The effect of the divalent metal ions (final concn I mM) on purified protease was investigated at pH 4.8 with FTC-casein as a substrate at 37”. The controls without metal ions were treated as the assays. The thermal stability of the enzyme was studied as follows. The samples of protease from pollen grains were preincubated with metal ions (final concn 1 mM) for 1 hr at 25”. Next, the reaction carried out at 65” was started by addition of FTC-casein. In this experiment 2 kinds of blanks were made. The first, not containing the metal ions, preincubated as the assays and incubated at 65”, and the second with the metal ions preincubated as the assays and incubated at 37”. authors would like to thank Mrs E. Borzyszkowska and Mrs B. Wojtowicz for their valuable technical assistance. We would also like to thank MS Christina Griffiths for the reading and correction of the manuscript. Acknowledgements-The
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