- Email: [email protected]
The Commonly Used Mg2+–Enolate Assay Can Lead to Underestimation of Thiolase Activity
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NOTES & TIPS
tation method (7) and a silica matrix method (8) by testing respective yields and qualities of nucleic acids isolated from clinical samples (Fig. 3). We made a nucleic acid extract from DBS and added heparin to one half of the extract to a concentration that was twice as much as required for antiagglutination of equivalent amounts of whole blood absorbed in the DBS according to Sigma product specification (No. 210-6). Each half of the extract was further divided equally into several parts that were then subjected to nucleic acid isolation. As with the phenol extraction method, the cellulose matrix method was able to isolate nucleic acids as small as 50 nt as well as large genomic DNA which appeared to be less sheared than that isolated by the silica method. Furthermore, only the cellulose matrix method (using PEGcellulose as a precipitant) was able to separate heparin from DNA efficiently so that the isolated DNA could be completely digested by AluI restriction enzyme and amplified in the polymerase chain reaction (PCR). We have used the cellulose matrix method to purify nucleic acids from thousands of millimeter punches of DBS for molecular diagnostic studies. Additionally, we have isolated genomic DNA from blood and sputum, plasmid DNA from bacteria, and RNA from plants. We were also able to isolate HIV RNA from DBS for viral load studies. The functionality of the cellulose matrix can be attributed to the secondary fibrils. The presence of the secondary fibrils on the cellulose fiber surfaces suggests that free-moving colloidal cellulosic chains may be present (9). Thus nucleic acid molecules can coaggregate with or be adsorbed to the hydrophilic cellulosic chains under precipitating conditions and, therefore, reversibly bind to the cellulose matrix. On the other hand, the physical structures of the secondary fibrils may function as microfilters to retain precipitated nucleic acids. Acknowledgments. We thank D. Pan for technical assistance and G. F. Grady for critical review of the manuscript. This project was supported by NICHD Contract NO1-HD-5-3228.
REFERENCES 1. Yang, R. C. A., Lis, J., and Wu, B. (1979) Methods Enzymol. 65, 176 –182. 2. Vogelstein, B., and Gillespie, D. (1979) Proc. Natl. Acad. Sci. USA 76, 615– 619. 3. Semancik, J. S. (1986) Virology 155, 39 – 45. 4. Comeau, A., Hsu, H. W., Schwerzlar, M., Mushinsky, G., Walter, E., Hofman, L., and Grady, G. F. (1993) J. Pediatr. 123, 252–258. 5. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156 –159. 6. Chomczynski, P. (1989) United States Patent 4,843,155. 7. Strauss, W. M. (1989) in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seid-
man, J. G., Smith, J. A., and Struhl, K., Eds.),Vol. I, pp. 2.2.1– 2.2.3, Wiley, New York. 8. Boom, R., Sol, C. J. A., Salimans, M. M. M., Jansen, C. L., Wertheim-van Dillen, P. M. E., and van der Noordaa. J. (1990) J. Clin. Microbiol. 28, 495–503. 9. Kremer, R. D., and Tabb, D. (1990) Am. Lab. February, 136 – 143.
The Commonly Used Mg 21–Enolate Assay Can Lead to Underestimation of Thiolase Activity Vasily D. Antonenkov, Paul P. Van Veldhoven, and Guy P. Mannaerts Katholieke Universiteit Leuven, Departement Moleculaire Celbiologie, Afdeling, Farmacologie, Campus Gasthuisberg (O & N), Herestraat 49, B-3000 Leuven, Belgium Received August 18, 1998
Thiolases are ubiquitous enzymes that catalyze the thiolytic cleavage of different 3-oxoacyl-CoAs. 1 Acetoacetyl-CoA-specific thiolases use acetoacetyl-CoA as the sole substrate (1– 4), whereas 3-oxoacyl-CoA thiolases possess a broad substrate specificity and act on short-, medium-, and long-straight-chain 3-oxoacylCoAs (1, 5–11). A common method for the measurement of thiolase activity is UV registration (in the range of 303 to 313 nm) of the disappearance of the Mg 21 – enolate complex (Fig. 1, peak 1) during the thiolytic cleavage of 3-oxocompounds in the presence of Mg 21 (1, 3–5, 7–10). However, the true substrate for the thiolases is believed to be the oxo-tautomer and not the chelated or free enol form of 3-oxoacyl-CoAs (3). Thus, thiolase activity measurements in the presence of Mg 21 are based on the assumption that (i) Mg 21 has no direct inhibitory effect on the thiolases; (ii) Mg 21 does not suppress thiolase activity as a result of oxo-tautomer depletion; and (iii) the Mg 21 – enolate complex dissociation and enol to oxo-form conversion are not rate limiting in the cleavage assay. Although most authors did not find indications that Mg 21 would adversely affect thiolase activity with substrates of different chain length (5, 7–10), Middleton (3) and Lazarow (6) found that Mg 21 strongly inhibits the cleavage of acetoacetyl-CoA in rat liver cytosol and peroxisomes, respectively. This discrepancy prompted us to investigate the effect of Mg 21 on the cleavage activities of purified peroxisomal 3-oxoacylCoA thiolase A (thiolase A) and sterol carrier protein 1
Abbreviations used: CoA, coenzyme A; SCP, sterol carrier protein. Analytical Biochemistry 267, 418 – 420 (1999) Article ID abio.1998.3016 0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
NOTES & TIPS
FIG. 1. Absorption spectrum of 3-oxooctanoyl-CoA before and after incubation with purified peroxisomes. 3-Oxooctanoyl-CoA (25 mM) was incubated for 10 min at 25°C with a purified peroxisomal fraction (25 mg protein) in the presence of 25 mM MgCl 2 and 70 mM CoA in 50 mM Tris–HCl buffer, pH 8.0. The blank cuvette did not contain peroxisomes and Mg 21. Absorption spectrum before (—) and after (- - -) incubation.
2/3-oxoacyl-CoA thiolase (SCP-2/thiolase), using an alternative method based on the detection of newly formed thioester bonds at 232 nm (see Fig. 1, peak 2)
419
(6). The enzymes were isolated from normal rat liver peroxisomes (11). 3-Oxoacyl-CoAs were synthesized enzymatically from the corresponding fatty acylCoAs and purified as described (11). Thiolase activity was determined at 25°C by following the formation of new thioester bonds (measured at 232 nm, e 4500 M 21 cm 21 ) (6) after the addition of CoA (60 mM) to a reaction mixture consisting of 50 mM Tris–Cl, pH 8.0, 15 or 25 mM oxoacyl-CoA, and the purified enzyme preparation. In some experiments the enzyme activity was also measured by the commonly used Mg 21 – enolate assay at 303 nm. The reaction mixture (25°C) contained 50 mM Tris–HCl, pH 8.0, 5 mM MgCl 2 , 60 mM CoA, and 15 or 25 mM 3-oxoacyl-CoAs. The molar extinction coefficients for the Mg 21 – enolate complexes were determined as described (11). Acetyl-CoA production was estimated by following the reduction of NAD 1 in a coupled assay with citrate synthetase and malate dehydrogenase (6). The data of Fig. 2 demonstrate that there is a dose-dependent suppression of the thiolase activities by Mg 21 . Moreover, the degree of inhibition depends on the chain length of the 3-oxoacyl-CoAs and is more pronounced for the short-chain oxocompounds. However, at 5 mM, the minimal concentration used in the Mg 21 – enolate assay (4, 10), Mg 21 had only a very weak effect, except perhaps for thiolase A when catalyzing the cleavage of acetoacetyl-CoA [SCP-2/ thiolase has only very poor activity with acetoacetyl-
FIG. 2. Effect of Mg 21 on the cleavage activity of thiolase A and SCP-2/thiolase. (A) Thiolase A activity was measured with 60 mM CoA and 25 mM acetoacetyl-CoA (F), 15 mM 3-oxohexanoyl-CoA (E), and 15 mM 3-oxooctanoyl-CoA (■). In (B), SCP-2/thiolase activity with 15 mM 3-oxohexanoyl-CoA (E), 3-oxooctanoyl-CoA (■), and 3-oxodecanoyl-CoA (F) is shown. Thiolase activities were measured at 232 nm (new thioester bond formation). Results are expressed as percentages of thiolase activity measured without Mg 21.
420
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CoA (11) and was not tested with acetoacetyl-CoA in this experiment]. On the other hand, when the thiolase activities were measured in parallel by the formation of new ester bonds and by the disappearance of the Mg 21 – enolate complex, rates of the latter reaction were still seriously lower than the rates of new ester bond formation, when the short- to medium-chain substrates were used (C 6 and C 7 for thiolase A; C 6 , C 7 , and C 8 for SCP-2/thiolase; data not shown). Similarly, the stoichiometry of acetoacetylCoA cleavage (48 mmol acetyl-CoA formed/min z mg protein) by thiolase A in the presence of 5 mM Mg 21 closely agreed with the formation of new ester bonds (26 mmol/min z mg protein), but the rate of disappearance of the Mg 21 – enolate complex was drastically lower (11 mmol/min z mg protein). When 3-oxooctanoyl-CoA was used, the results of all three assays for thiolase A approximately coincided (98, 87, and 115 mmol/min z mg protein for thioester bond formation, acetyl-CoA production, and Mg 21 – enolate complex disappearance, respectively). These data are difficult to explain in terms only of an inhibitory effect of Mg 21 on the thiolytic cleavage reaction. They imply that the cleavage of 3-oxoacyl-CoAs (C 4 –C 7 ) by thiolase A as well as that of medium-chain 3-oxoacylCoAs by SCP-2/thiolase may not be the rate-limiting step in the case of the Mg 21 – enolate assay. Our data clarify the contradictory results related to the effect of Mg 21 on thiolase activity obtained by different authors (3, 5–10) and also indicate that the commonly used Mg 21– enolate assay leads to an underestimation of the thiolase activity, especially toward short- and medium-chain 3-oxoacyl-CoAs.
A Method for the Determination of the pH Optima of Proteases Using Unexposed Photographic Film Victor E. Buckwold, Milton Alvarado, Raquel Mesa Carraso, and Ricardo Amils 1 Departamento de Biologı´a Molecular, Centro de Biologı´a Molecular “Severo Ochoa,” Universidad Auto´noma de Madrid, Cantoblanco, 28049 Madrid, Spain Received August 21, 1998
The determination of the pH optima of proteases is usually time-consuming and tedious, as is, in general, the determination of the activity of proteases. One exception to this rule involves the determination of protease activity using unprocessed photographic film (1). In this method, a protease-containing solution is placed on an unexposed film which is then incubated at 42°C for 1 h. Following this, the film is washed with running water for 1 min while gently rubbing the film with the ball of the thumb. Samples containing protease activity show a clear zone where the protease has digested away the gelatin coating on the film. By successively diluting the sample 1:1 on the film with
1 To whom correspondence should be addressed. Fax: (3491) 3978087.
REFERENCES 1. Middleton, B. (1973) Biochem. J. 132, 717–730. 2. Clinkenbeard, K. D., Sugiyama, T., Moss, J., Reed, D., and Lane, M. D. (1973) J. Biol. Chem. 248, 2275–2284. 3. Middleton, B. (1974) Biochem. J. 139, 109 –121. 4. Huth, W., Jonas, R., Wunderlich, I., and Seubert, W. (1975) Eur. J. Biochem. 59, 475– 489. 5. Staack, H., Binstock, J. F., and Schulz, H. (1978) J. Biol. Chem. 253, 1827–1831. 6. Lazarow, P. B. (1978) J. Biol. Chem. 253, 1522–1528. 7. Krahling, J. B., and Tolbert, N. E. (1981) Arch. Biochem. Biophys. 209, 100 –110. 8. Miyazawa, S., Furuta, S., Osumi, T., Hashimoto, T., and Ui, N. (1981) J. Biochem. 90, 511–519. 9. Thompson, S. L., and Krisans, S. K. (1990) J. Biol. Chem. 265, 5731–5735. 10. Seedorf, U., Brysch, P., Engel, T., Schrage, K., and Assmann, G. (1994) J. Biol. Chem. 269, 21277–21283. 11. Antonenkov, V. D., Van Veldhoven, P. P., Waelkens, E., and Mannaerts, G. P. (1997) J. Biol. Chem. 272, 26023–26031.
FIG. 1. Determination of the pH optimum of a fungal protease. 50 ml of culture supernatant from the fungus Verano13 isolated from the Rı´o Tinto (Spain) was successively diluted in 50 ml of 50mM sodium phosphate buffer having a pH of between 6.5 and 8.5, as indicated, on unexposed X-Omat XAR-5 X-ray film (Kodak). The film was incubated for 1 h at 42°C and then was washed with running water. Clear zones indicate protease activity. This sample has a pH optimum of between 7.5 and 8.0, shown as clear zones in this pH range at the highest dilutions in which clear zones were observed (32X). Analytical Biochemistry 267, 420 – 421 (1999) Article ID abio.1998.2994 0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
NOTES & TIPS
tation method (7) and a silica matrix method (8) by testing respective yields and qualities of nucleic acids isolated from clinical samples (Fig. 3). We made a nucleic acid extract from DBS and added heparin to one half of the extract to a concentration that was twice as much as required for antiagglutination of equivalent amounts of whole blood absorbed in the DBS according to Sigma product specification (No. 210-6). Each half of the extract was further divided equally into several parts that were then subjected to nucleic acid isolation. As with the phenol extraction method, the cellulose matrix method was able to isolate nucleic acids as small as 50 nt as well as large genomic DNA which appeared to be less sheared than that isolated by the silica method. Furthermore, only the cellulose matrix method (using PEGcellulose as a precipitant) was able to separate heparin from DNA efficiently so that the isolated DNA could be completely digested by AluI restriction enzyme and amplified in the polymerase chain reaction (PCR). We have used the cellulose matrix method to purify nucleic acids from thousands of millimeter punches of DBS for molecular diagnostic studies. Additionally, we have isolated genomic DNA from blood and sputum, plasmid DNA from bacteria, and RNA from plants. We were also able to isolate HIV RNA from DBS for viral load studies. The functionality of the cellulose matrix can be attributed to the secondary fibrils. The presence of the secondary fibrils on the cellulose fiber surfaces suggests that free-moving colloidal cellulosic chains may be present (9). Thus nucleic acid molecules can coaggregate with or be adsorbed to the hydrophilic cellulosic chains under precipitating conditions and, therefore, reversibly bind to the cellulose matrix. On the other hand, the physical structures of the secondary fibrils may function as microfilters to retain precipitated nucleic acids. Acknowledgments. We thank D. Pan for technical assistance and G. F. Grady for critical review of the manuscript. This project was supported by NICHD Contract NO1-HD-5-3228.
REFERENCES 1. Yang, R. C. A., Lis, J., and Wu, B. (1979) Methods Enzymol. 65, 176 –182. 2. Vogelstein, B., and Gillespie, D. (1979) Proc. Natl. Acad. Sci. USA 76, 615– 619. 3. Semancik, J. S. (1986) Virology 155, 39 – 45. 4. Comeau, A., Hsu, H. W., Schwerzlar, M., Mushinsky, G., Walter, E., Hofman, L., and Grady, G. F. (1993) J. Pediatr. 123, 252–258. 5. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156 –159. 6. Chomczynski, P. (1989) United States Patent 4,843,155. 7. Strauss, W. M. (1989) in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seid-
man, J. G., Smith, J. A., and Struhl, K., Eds.),Vol. I, pp. 2.2.1– 2.2.3, Wiley, New York. 8. Boom, R., Sol, C. J. A., Salimans, M. M. M., Jansen, C. L., Wertheim-van Dillen, P. M. E., and van der Noordaa. J. (1990) J. Clin. Microbiol. 28, 495–503. 9. Kremer, R. D., and Tabb, D. (1990) Am. Lab. February, 136 – 143.
The Commonly Used Mg 21–Enolate Assay Can Lead to Underestimation of Thiolase Activity Vasily D. Antonenkov, Paul P. Van Veldhoven, and Guy P. Mannaerts Katholieke Universiteit Leuven, Departement Moleculaire Celbiologie, Afdeling, Farmacologie, Campus Gasthuisberg (O & N), Herestraat 49, B-3000 Leuven, Belgium Received August 18, 1998
Thiolases are ubiquitous enzymes that catalyze the thiolytic cleavage of different 3-oxoacyl-CoAs. 1 Acetoacetyl-CoA-specific thiolases use acetoacetyl-CoA as the sole substrate (1– 4), whereas 3-oxoacyl-CoA thiolases possess a broad substrate specificity and act on short-, medium-, and long-straight-chain 3-oxoacylCoAs (1, 5–11). A common method for the measurement of thiolase activity is UV registration (in the range of 303 to 313 nm) of the disappearance of the Mg 21 – enolate complex (Fig. 1, peak 1) during the thiolytic cleavage of 3-oxocompounds in the presence of Mg 21 (1, 3–5, 7–10). However, the true substrate for the thiolases is believed to be the oxo-tautomer and not the chelated or free enol form of 3-oxoacyl-CoAs (3). Thus, thiolase activity measurements in the presence of Mg 21 are based on the assumption that (i) Mg 21 has no direct inhibitory effect on the thiolases; (ii) Mg 21 does not suppress thiolase activity as a result of oxo-tautomer depletion; and (iii) the Mg 21 – enolate complex dissociation and enol to oxo-form conversion are not rate limiting in the cleavage assay. Although most authors did not find indications that Mg 21 would adversely affect thiolase activity with substrates of different chain length (5, 7–10), Middleton (3) and Lazarow (6) found that Mg 21 strongly inhibits the cleavage of acetoacetyl-CoA in rat liver cytosol and peroxisomes, respectively. This discrepancy prompted us to investigate the effect of Mg 21 on the cleavage activities of purified peroxisomal 3-oxoacylCoA thiolase A (thiolase A) and sterol carrier protein 1
Abbreviations used: CoA, coenzyme A; SCP, sterol carrier protein. Analytical Biochemistry 267, 418 – 420 (1999) Article ID abio.1998.3016 0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
NOTES & TIPS
FIG. 1. Absorption spectrum of 3-oxooctanoyl-CoA before and after incubation with purified peroxisomes. 3-Oxooctanoyl-CoA (25 mM) was incubated for 10 min at 25°C with a purified peroxisomal fraction (25 mg protein) in the presence of 25 mM MgCl 2 and 70 mM CoA in 50 mM Tris–HCl buffer, pH 8.0. The blank cuvette did not contain peroxisomes and Mg 21. Absorption spectrum before (—) and after (- - -) incubation.
2/3-oxoacyl-CoA thiolase (SCP-2/thiolase), using an alternative method based on the detection of newly formed thioester bonds at 232 nm (see Fig. 1, peak 2)
419
(6). The enzymes were isolated from normal rat liver peroxisomes (11). 3-Oxoacyl-CoAs were synthesized enzymatically from the corresponding fatty acylCoAs and purified as described (11). Thiolase activity was determined at 25°C by following the formation of new thioester bonds (measured at 232 nm, e 4500 M 21 cm 21 ) (6) after the addition of CoA (60 mM) to a reaction mixture consisting of 50 mM Tris–Cl, pH 8.0, 15 or 25 mM oxoacyl-CoA, and the purified enzyme preparation. In some experiments the enzyme activity was also measured by the commonly used Mg 21 – enolate assay at 303 nm. The reaction mixture (25°C) contained 50 mM Tris–HCl, pH 8.0, 5 mM MgCl 2 , 60 mM CoA, and 15 or 25 mM 3-oxoacyl-CoAs. The molar extinction coefficients for the Mg 21 – enolate complexes were determined as described (11). Acetyl-CoA production was estimated by following the reduction of NAD 1 in a coupled assay with citrate synthetase and malate dehydrogenase (6). The data of Fig. 2 demonstrate that there is a dose-dependent suppression of the thiolase activities by Mg 21 . Moreover, the degree of inhibition depends on the chain length of the 3-oxoacyl-CoAs and is more pronounced for the short-chain oxocompounds. However, at 5 mM, the minimal concentration used in the Mg 21 – enolate assay (4, 10), Mg 21 had only a very weak effect, except perhaps for thiolase A when catalyzing the cleavage of acetoacetyl-CoA [SCP-2/ thiolase has only very poor activity with acetoacetyl-
FIG. 2. Effect of Mg 21 on the cleavage activity of thiolase A and SCP-2/thiolase. (A) Thiolase A activity was measured with 60 mM CoA and 25 mM acetoacetyl-CoA (F), 15 mM 3-oxohexanoyl-CoA (E), and 15 mM 3-oxooctanoyl-CoA (■). In (B), SCP-2/thiolase activity with 15 mM 3-oxohexanoyl-CoA (E), 3-oxooctanoyl-CoA (■), and 3-oxodecanoyl-CoA (F) is shown. Thiolase activities were measured at 232 nm (new thioester bond formation). Results are expressed as percentages of thiolase activity measured without Mg 21.
420
NOTES & TIPS
CoA (11) and was not tested with acetoacetyl-CoA in this experiment]. On the other hand, when the thiolase activities were measured in parallel by the formation of new ester bonds and by the disappearance of the Mg 21 – enolate complex, rates of the latter reaction were still seriously lower than the rates of new ester bond formation, when the short- to medium-chain substrates were used (C 6 and C 7 for thiolase A; C 6 , C 7 , and C 8 for SCP-2/thiolase; data not shown). Similarly, the stoichiometry of acetoacetylCoA cleavage (48 mmol acetyl-CoA formed/min z mg protein) by thiolase A in the presence of 5 mM Mg 21 closely agreed with the formation of new ester bonds (26 mmol/min z mg protein), but the rate of disappearance of the Mg 21 – enolate complex was drastically lower (11 mmol/min z mg protein). When 3-oxooctanoyl-CoA was used, the results of all three assays for thiolase A approximately coincided (98, 87, and 115 mmol/min z mg protein for thioester bond formation, acetyl-CoA production, and Mg 21 – enolate complex disappearance, respectively). These data are difficult to explain in terms only of an inhibitory effect of Mg 21 on the thiolytic cleavage reaction. They imply that the cleavage of 3-oxoacyl-CoAs (C 4 –C 7 ) by thiolase A as well as that of medium-chain 3-oxoacylCoAs by SCP-2/thiolase may not be the rate-limiting step in the case of the Mg 21 – enolate assay. Our data clarify the contradictory results related to the effect of Mg 21 on thiolase activity obtained by different authors (3, 5–10) and also indicate that the commonly used Mg 21– enolate assay leads to an underestimation of the thiolase activity, especially toward short- and medium-chain 3-oxoacyl-CoAs.
A Method for the Determination of the pH Optima of Proteases Using Unexposed Photographic Film Victor E. Buckwold, Milton Alvarado, Raquel Mesa Carraso, and Ricardo Amils 1 Departamento de Biologı´a Molecular, Centro de Biologı´a Molecular “Severo Ochoa,” Universidad Auto´noma de Madrid, Cantoblanco, 28049 Madrid, Spain Received August 21, 1998
The determination of the pH optima of proteases is usually time-consuming and tedious, as is, in general, the determination of the activity of proteases. One exception to this rule involves the determination of protease activity using unprocessed photographic film (1). In this method, a protease-containing solution is placed on an unexposed film which is then incubated at 42°C for 1 h. Following this, the film is washed with running water for 1 min while gently rubbing the film with the ball of the thumb. Samples containing protease activity show a clear zone where the protease has digested away the gelatin coating on the film. By successively diluting the sample 1:1 on the film with
1 To whom correspondence should be addressed. Fax: (3491) 3978087.
REFERENCES 1. Middleton, B. (1973) Biochem. J. 132, 717–730. 2. Clinkenbeard, K. D., Sugiyama, T., Moss, J., Reed, D., and Lane, M. D. (1973) J. Biol. Chem. 248, 2275–2284. 3. Middleton, B. (1974) Biochem. J. 139, 109 –121. 4. Huth, W., Jonas, R., Wunderlich, I., and Seubert, W. (1975) Eur. J. Biochem. 59, 475– 489. 5. Staack, H., Binstock, J. F., and Schulz, H. (1978) J. Biol. Chem. 253, 1827–1831. 6. Lazarow, P. B. (1978) J. Biol. Chem. 253, 1522–1528. 7. Krahling, J. B., and Tolbert, N. E. (1981) Arch. Biochem. Biophys. 209, 100 –110. 8. Miyazawa, S., Furuta, S., Osumi, T., Hashimoto, T., and Ui, N. (1981) J. Biochem. 90, 511–519. 9. Thompson, S. L., and Krisans, S. K. (1990) J. Biol. Chem. 265, 5731–5735. 10. Seedorf, U., Brysch, P., Engel, T., Schrage, K., and Assmann, G. (1994) J. Biol. Chem. 269, 21277–21283. 11. Antonenkov, V. D., Van Veldhoven, P. P., Waelkens, E., and Mannaerts, G. P. (1997) J. Biol. Chem. 272, 26023–26031.
FIG. 1. Determination of the pH optimum of a fungal protease. 50 ml of culture supernatant from the fungus Verano13 isolated from the Rı´o Tinto (Spain) was successively diluted in 50 ml of 50mM sodium phosphate buffer having a pH of between 6.5 and 8.5, as indicated, on unexposed X-Omat XAR-5 X-ray film (Kodak). The film was incubated for 1 h at 42°C and then was washed with running water. Clear zones indicate protease activity. This sample has a pH optimum of between 7.5 and 8.0, shown as clear zones in this pH range at the highest dilutions in which clear zones were observed (32X). Analytical Biochemistry 267, 420 – 421 (1999) Article ID abio.1998.2994 0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.