1058
BIOCHEMICAL SOCIETY TRANSACTIONS
Dobrota, M., Hinton, R. H., El.-Aaser, A. A. A., Fitzsimons, J. T. R. &Reid,E. (1978)Biochem. SOC.Trans. 6,291-293
Dobrota, M., Burge, M. L. E. & Hinton, R. H. (1979) Eur. J. Cell. Biol. in the press Goldberg, A. L. & St John, A. C. (1976) Annu. Rev. Biochem. 45,747-803 Gregoriadis, G . (1975) in Lysosomes in Biology and Pathology (Dingle, J. T. & Dean, R. T., eds.), vol. 4, pp. 265-294, North Holland, Amsterdam Hinton, R. H. & Mullock, B. M. (1977) Clin. Chim. Acta78,159-162 Holtzman, E. (1976) Lysosomes: a Survey, Springer Verlag, Wien Mullock, B. M., Issa, F. S. & Hinton, R. H. (1977) Clin.Chim. Acra 79, 129-140 Mullock, B. M., Dobrota, M. & Hinton, R. H. (1978) Biochim. Biophys. Acra 543,497-507 Muto, M. & Fujita, T. (1977) in Kupffer Cells and other Liver Sinusoidal Cells (Wisse, E. & Knook, D. L., eds.), pp. 109-1 19, Elsevier, Amsterdam Orlans, E., Peppard, J., Reynolds, J. & Hall, J. (1978) J. Exp. Med. 147,588-592 Segal, H. L. (1976) Curr. Top. Cell. Regul. 11, 183-201 Spector, I. M. (1974) Nature (London) 249,66 Steer, C . (1978) Bull. Kupffer Cell Foundation 1,26-36 Tolleshaug, H., Ose, T., Berg, T., Wandel, M. & Norum, K. R. (1977) in Kupffer Cells andother Liver Sinusoidal Cells (Wisse, E. & Knook, D. L., eds.), pp. 333-341, Elsevier, Amsterdam
Functional Arginine Residues in Bovine Testicular Hyaluronidase PETER GACESA, KENNETH S. DODGSON and ANTHONY H. OLAVESEN Department of Biochemistry, University College, P.O. Box 78, Cardiff CFl 1XL, Wales U.K.
Arginine residues in a number of enzymes have been shown to act as positively charged binding sites for anionic substrates or coenzymes. These enzymes act on a wide range of substrates such as polyanions (Borders et al., 1975), phosphorylated glycolytic intermediates (Riordan et al., 1977) and compounds containing carboxyl groups (Riordan, 1973). As bovine testicular hyaluronidase degrades polyanionic substrates such as hyaluronic acid and chondroitin sutphates, it seems possible that one or more arginine residues on the enzyme may interact with the carboxy and/or sulphate groups on the substrate during the catalytic process. This possibility was investigated by chemical modification of the enzyme with butane-2,3-dione, a reagent specific for arginine residues (Yankeelov, 1972). Hyaluronic acid was used as substrate, as the absence of ester sulphate groups in this polymer allows the interaction between carboxy groups and arginine residues to be studied without complication. Bovine testicular hyaluronidase (EC 3.2.1.35) was purchased from Miles Laboratories (Slough, Berks., U.K.) with a specific activity of approx. 300units/mg and purified by the method of Gorham (1974) (see also Pope et al., 1976) to a specific activity of 41000units/mg. [The unit of activity is as defined by Humphrey (1957).] The stock solution of enzyme (5 mg/ml in 0.1 M-NaCI) was diluted to give a final concentration of 10,ug (approx. 400 units) in either 50m~-sodiumborate buffer, pH8.3, or 50m~-Hepes buffer, pH8.3 (total volume of incubation mixture 0.7ml). The enzyme was inactivated at 20°C with a final concentration of 16.3 m~-butane-2,3-dione.In certain experiments the incubation mixture also contained 20m~-sodiumD-glucuronate, an inhibitor of testicular hyaluronidase. At timed intervals after addition of the butane-2,3-dione 100,d samples were withdrawn and residual enzyme activity was measured in an assay system (total volume 0.51111) containing 0.5mg of potassium hyaluronate in 0.1 Msodium citrate buffer, pH4.0, and 0.15~-NaCI.After lOmin incubation at 37°C the reaction was stopped by the simultaneous addition of 1 0 0 ~ of 1 0.7 M-potassium tetraborate, pH9.2 and lop1 of 6~-Na,cO,. The liberated reducing N-acetyl-D-glucosamine was assayed by the method of Reissig et al. (1955). Fig. 1 shows that bovine testicular hyaluronidase was inactivated by 16.3mM-butane2,3-dione in the presence of either borate or Hepes buffer and that the reaction was Abbreviation used : Hepes, 4-(2-hydroxyethyl)-l-piperazine-ethanesulphonicacid. 1979
1059
5S3rd MEETING, CAMBRIDGE
Time (min)
Fig. 1. Inactivation of hyaluronidase by butane-2,3-dione Bovine testicular hyaluronidase was incubated with 16.3m~-butane-2,3-dionein the presence of 5Omhl-borate buffer (O), 5Omhl-borate buffer and sodium D-glucuronate (o), SOmhl-Hepes buffer (B) or 50mhl-Hepes buffer and sodium D-glucuronate (0). Control activities were measured in borate buffer (A) and Hepes buffer (A). All experiments were performed at pH8.3.
first-order with respect to time. Results were quantified by measuring the time taken for 50% inactivation of the enzyme to occur (t+). The relatively rapid inactivation rates in borate buffer (t+ 3min) and in Hepes buffer (t+ 11.5min) suggest that the butane2,3-dione reacted specifically with arginine residues. The t+ values obtained compared well with results for the inactivation of several glycolyticenzymes by a similar concentration of butane-2,3-dione (Riordan et al., 1977). The higher inactivation rate in the presence of borate buffer has been ascribed to the formation of an intermediate boratestablilized complex (Riordan, 1973). Addition of sodium D-glucuronate, an inhibitor of hyaluronidase (Highsmith et al.,1975), moderated the inactivation of the enzyme by butane-2,3-dione (Fig. 1). Sodium D-glucuronate was effective in increasing the t+ values to 9.0min and 27.0min in borate and Hepes buffer respectively. It could be argued that the borate buffer and the sodium D-glucuronate interact, diminish the borate enhancement effect and hence produce this result. However, there is no evidence that sugars can interact with Hepes buffer, so the observed results with sodium D-glucuronate appear to represent true protection of the enzyme against inactivation by butane-2,3dione. Moreover, the addition of D-glucose, which is not an inhibitor of hyaluronidase but interacts with borate, produced only a smaller increase in t t to 6.0min with borate buffer and had no effect on t+with Hepes buffer. Thus the protection afforded by sodium D-glucuronate may be ascribed to a direct effect on the enzyme, and is not the result of an interaction with the buffer. Collectively these studies suggest that one or more arginine residues close to the active site of testicular hyaluronidase bind the carboxy groups of hyaluronic acid. It is interesting to compare this enzyme with lysozyme, an enzyme with a similar mode of action. Modification of arginine residues of lysozyme removes activity towards the anionic substrates such as cell walls, but does not affect the activity with neutral substrates (Davies & Neuberger, 1969). The results described in this present communication with bovine testicular hyaluronidase support the hypothesis that at least one positively charged arginine residue interacts with a carboxy group on the substrate. Preliminary results from amino acid analysis suggest that at least one arginine residue is modified during the inactivation process.
Vol. 7
1060
BIOCHEMICAL SOCIETY TRANSACTIONS
This work was supported by the Science Research Council. Borders, C. L., Jr., Riordan, J. F. & Auld, D. S. (1975) Biochem. Biophys. Res. Commun. 66, 490-496 Davies, R. C . & Neuberger, A. (1969) Biochim. Biophys. Acta 178,306-317 Gorham, S. D. (1974) Ph.D. Thesis, University of Wales Highsmith, S.,Garvin, J. H., Jr. & Chipman, D. M. (1975)J . Biol. Chem. 250, 7473-7480 Humphrey, J. H. (1957) Bull. W.H.O. 16,291-294 Pope, D. J., Rhodes, C. & Gorham, S. D. (1976) Br. Purenr 1425918 Reissig, J. L., Strominger,J. L. & Leloir, L. F. (1955)J. Biol. Chem. 217, 959-966 Riordan, J. F. (1973) Biochemistry 12,3915-3923 Riordan, J. F., McElvary, K. D. & Borders, C. L., Jr. (1977) Science 195,884-886 Yankeelov, J. A., Jr. (1972) Methods Enzymol. 25, 566-579
A Rapid Sensitive Assay for Glutathione S-Epoxidetransferase Activity : Species Differences in the Activity of the Hepatic Enzyme GIAN MARIA PACIFICI, ALAN R. BOOBIS, MARTlN J. BRODIE and DONALD S . DAVIES Department of Clinical Pharmacology, Royal Postgraduate Medical School, London W 12 OHS, U.K. Epoxides are often products of xenobiotic metabolism by microsomal mixed-function oxidases. Some of these epoxides are highly reactive and may be teratogenic or carcinogenic (Sims & Grover, 1974). Epoxides may be metabolized further by hydration to the diol, catalysed by microsomal epoxide hydratase, or by conjugation with glutathione, catalysed by cytosolic glutathione S-epoxide transferases (EC 2.5.1.18). [3H]Styrene oxide has been widely used as a substrate to study the metabolic fate of epoxides because of its relative chemical stability as compared with most other substrates for these enzymes, and its commercial availability in a high specific radioactivity tritiated form that has permitted the development of sensitive assays (Oesch, 1973). A method for the measurement of glutathione S-[3H]styrene oxide transferase has been described (Hayakawa et al., 1974) and is in widespread use. This method involves absorption on to charcoal, two extractions, concentration and paper chromatography. The reported recovery for this method is 52 % but it is not possible to determine recovery in all samples. We now report a rapid, sensitive method for determining glutathione S-[3H]styrene oxide transferase activity that permits 100 % recovery. The standard incubation mixture comprised loop1 of 0.1 M-sodium pyrophosphate buffer, pH 8.0, containing 1pmol of reduced glutathione, hepatic cytosol, prepared by differential centrifugation (Atlas et al., 1977), containing 40-450pg of protein, 5,uI of acetonitrile containing 1-2pmol of 7-[3H]styrene oxide with a specific radioactivity of 350c.p.m./nmol. [3H]Styrene oxide (The Radioachemical Centre, Amersham, Bucks., U.K.) was purified by solvent extraction to remove water-soluble decomposition products before use. The reaction was started by addition of the [3H]styrene oxide. Samples were incubated for 3-4min at 22°C and the reaction was stopped by addition of 2Opl of 4~-aceticacid. Unchanged styrene oxide was removed by extraction with 0.5ml of chloroform. This removed 83.2 2.9 % of total radioactivity. A portion (30~1) of the aqueous phase (25% of the sample) was applied to the loading zone of a 19channel LK5DF silica-gel plate (Whatman). The loading zone was pretreated with 80pl of a solution containing 30% (v/v) styrene oxide and 10% (w/v) styrene glycol in acetonitrile to decrease non-specific binding of the labelled reactants. Plates were dried in a hot-air current and chromatographed in chloroform/ethyl acetate (8 : 2, v/v). RF values for authentic styrene oxide and styrene glycol were 0.64 and 0.21 respectively, and styrene oxide glutathione conjugate remained at the loading zone. This was scraped from the plate into scintillation vials containing 2ml of methanol to extract the conjugate.
1979
BIOCHEMICAL SOCIETY TRANSACTIONS
Dobrota, M., Hinton, R. H., El.-Aaser, A. A. A., Fitzsimons, J. T. R. &Reid,E. (1978)Biochem. SOC.Trans. 6,291-293
Dobrota, M., Burge, M. L. E. & Hinton, R. H. (1979) Eur. J. Cell. Biol. in the press Goldberg, A. L. & St John, A. C. (1976) Annu. Rev. Biochem. 45,747-803 Gregoriadis, G . (1975) in Lysosomes in Biology and Pathology (Dingle, J. T. & Dean, R. T., eds.), vol. 4, pp. 265-294, North Holland, Amsterdam Hinton, R. H. & Mullock, B. M. (1977) Clin. Chim. Acta78,159-162 Holtzman, E. (1976) Lysosomes: a Survey, Springer Verlag, Wien Mullock, B. M., Issa, F. S. & Hinton, R. H. (1977) Clin.Chim. Acra 79, 129-140 Mullock, B. M., Dobrota, M. & Hinton, R. H. (1978) Biochim. Biophys. Acra 543,497-507 Muto, M. & Fujita, T. (1977) in Kupffer Cells and other Liver Sinusoidal Cells (Wisse, E. & Knook, D. L., eds.), pp. 109-1 19, Elsevier, Amsterdam Orlans, E., Peppard, J., Reynolds, J. & Hall, J. (1978) J. Exp. Med. 147,588-592 Segal, H. L. (1976) Curr. Top. Cell. Regul. 11, 183-201 Spector, I. M. (1974) Nature (London) 249,66 Steer, C . (1978) Bull. Kupffer Cell Foundation 1,26-36 Tolleshaug, H., Ose, T., Berg, T., Wandel, M. & Norum, K. R. (1977) in Kupffer Cells andother Liver Sinusoidal Cells (Wisse, E. & Knook, D. L., eds.), pp. 333-341, Elsevier, Amsterdam
Functional Arginine Residues in Bovine Testicular Hyaluronidase PETER GACESA, KENNETH S. DODGSON and ANTHONY H. OLAVESEN Department of Biochemistry, University College, P.O. Box 78, Cardiff CFl 1XL, Wales U.K.
Arginine residues in a number of enzymes have been shown to act as positively charged binding sites for anionic substrates or coenzymes. These enzymes act on a wide range of substrates such as polyanions (Borders et al., 1975), phosphorylated glycolytic intermediates (Riordan et al., 1977) and compounds containing carboxyl groups (Riordan, 1973). As bovine testicular hyaluronidase degrades polyanionic substrates such as hyaluronic acid and chondroitin sutphates, it seems possible that one or more arginine residues on the enzyme may interact with the carboxy and/or sulphate groups on the substrate during the catalytic process. This possibility was investigated by chemical modification of the enzyme with butane-2,3-dione, a reagent specific for arginine residues (Yankeelov, 1972). Hyaluronic acid was used as substrate, as the absence of ester sulphate groups in this polymer allows the interaction between carboxy groups and arginine residues to be studied without complication. Bovine testicular hyaluronidase (EC 3.2.1.35) was purchased from Miles Laboratories (Slough, Berks., U.K.) with a specific activity of approx. 300units/mg and purified by the method of Gorham (1974) (see also Pope et al., 1976) to a specific activity of 41000units/mg. [The unit of activity is as defined by Humphrey (1957).] The stock solution of enzyme (5 mg/ml in 0.1 M-NaCI) was diluted to give a final concentration of 10,ug (approx. 400 units) in either 50m~-sodiumborate buffer, pH8.3, or 50m~-Hepes buffer, pH8.3 (total volume of incubation mixture 0.7ml). The enzyme was inactivated at 20°C with a final concentration of 16.3 m~-butane-2,3-dione.In certain experiments the incubation mixture also contained 20m~-sodiumD-glucuronate, an inhibitor of testicular hyaluronidase. At timed intervals after addition of the butane-2,3-dione 100,d samples were withdrawn and residual enzyme activity was measured in an assay system (total volume 0.51111) containing 0.5mg of potassium hyaluronate in 0.1 Msodium citrate buffer, pH4.0, and 0.15~-NaCI.After lOmin incubation at 37°C the reaction was stopped by the simultaneous addition of 1 0 0 ~ of 1 0.7 M-potassium tetraborate, pH9.2 and lop1 of 6~-Na,cO,. The liberated reducing N-acetyl-D-glucosamine was assayed by the method of Reissig et al. (1955). Fig. 1 shows that bovine testicular hyaluronidase was inactivated by 16.3mM-butane2,3-dione in the presence of either borate or Hepes buffer and that the reaction was Abbreviation used : Hepes, 4-(2-hydroxyethyl)-l-piperazine-ethanesulphonicacid. 1979
1059
5S3rd MEETING, CAMBRIDGE
Time (min)
Fig. 1. Inactivation of hyaluronidase by butane-2,3-dione Bovine testicular hyaluronidase was incubated with 16.3m~-butane-2,3-dionein the presence of 5Omhl-borate buffer (O), 5Omhl-borate buffer and sodium D-glucuronate (o), SOmhl-Hepes buffer (B) or 50mhl-Hepes buffer and sodium D-glucuronate (0). Control activities were measured in borate buffer (A) and Hepes buffer (A). All experiments were performed at pH8.3.
first-order with respect to time. Results were quantified by measuring the time taken for 50% inactivation of the enzyme to occur (t+). The relatively rapid inactivation rates in borate buffer (t+ 3min) and in Hepes buffer (t+ 11.5min) suggest that the butane2,3-dione reacted specifically with arginine residues. The t+ values obtained compared well with results for the inactivation of several glycolyticenzymes by a similar concentration of butane-2,3-dione (Riordan et al., 1977). The higher inactivation rate in the presence of borate buffer has been ascribed to the formation of an intermediate boratestablilized complex (Riordan, 1973). Addition of sodium D-glucuronate, an inhibitor of hyaluronidase (Highsmith et al.,1975), moderated the inactivation of the enzyme by butane-2,3-dione (Fig. 1). Sodium D-glucuronate was effective in increasing the t+ values to 9.0min and 27.0min in borate and Hepes buffer respectively. It could be argued that the borate buffer and the sodium D-glucuronate interact, diminish the borate enhancement effect and hence produce this result. However, there is no evidence that sugars can interact with Hepes buffer, so the observed results with sodium D-glucuronate appear to represent true protection of the enzyme against inactivation by butane-2,3dione. Moreover, the addition of D-glucose, which is not an inhibitor of hyaluronidase but interacts with borate, produced only a smaller increase in t t to 6.0min with borate buffer and had no effect on t+with Hepes buffer. Thus the protection afforded by sodium D-glucuronate may be ascribed to a direct effect on the enzyme, and is not the result of an interaction with the buffer. Collectively these studies suggest that one or more arginine residues close to the active site of testicular hyaluronidase bind the carboxy groups of hyaluronic acid. It is interesting to compare this enzyme with lysozyme, an enzyme with a similar mode of action. Modification of arginine residues of lysozyme removes activity towards the anionic substrates such as cell walls, but does not affect the activity with neutral substrates (Davies & Neuberger, 1969). The results described in this present communication with bovine testicular hyaluronidase support the hypothesis that at least one positively charged arginine residue interacts with a carboxy group on the substrate. Preliminary results from amino acid analysis suggest that at least one arginine residue is modified during the inactivation process.
Vol. 7
1060
BIOCHEMICAL SOCIETY TRANSACTIONS
This work was supported by the Science Research Council. Borders, C. L., Jr., Riordan, J. F. & Auld, D. S. (1975) Biochem. Biophys. Res. Commun. 66, 490-496 Davies, R. C . & Neuberger, A. (1969) Biochim. Biophys. Acta 178,306-317 Gorham, S. D. (1974) Ph.D. Thesis, University of Wales Highsmith, S.,Garvin, J. H., Jr. & Chipman, D. M. (1975)J . Biol. Chem. 250, 7473-7480 Humphrey, J. H. (1957) Bull. W.H.O. 16,291-294 Pope, D. J., Rhodes, C. & Gorham, S. D. (1976) Br. Purenr 1425918 Reissig, J. L., Strominger,J. L. & Leloir, L. F. (1955)J. Biol. Chem. 217, 959-966 Riordan, J. F. (1973) Biochemistry 12,3915-3923 Riordan, J. F., McElvary, K. D. & Borders, C. L., Jr. (1977) Science 195,884-886 Yankeelov, J. A., Jr. (1972) Methods Enzymol. 25, 566-579
A Rapid Sensitive Assay for Glutathione S-Epoxidetransferase Activity : Species Differences in the Activity of the Hepatic Enzyme GIAN MARIA PACIFICI, ALAN R. BOOBIS, MARTlN J. BRODIE and DONALD S . DAVIES Department of Clinical Pharmacology, Royal Postgraduate Medical School, London W 12 OHS, U.K. Epoxides are often products of xenobiotic metabolism by microsomal mixed-function oxidases. Some of these epoxides are highly reactive and may be teratogenic or carcinogenic (Sims & Grover, 1974). Epoxides may be metabolized further by hydration to the diol, catalysed by microsomal epoxide hydratase, or by conjugation with glutathione, catalysed by cytosolic glutathione S-epoxide transferases (EC 2.5.1.18). [3H]Styrene oxide has been widely used as a substrate to study the metabolic fate of epoxides because of its relative chemical stability as compared with most other substrates for these enzymes, and its commercial availability in a high specific radioactivity tritiated form that has permitted the development of sensitive assays (Oesch, 1973). A method for the measurement of glutathione S-[3H]styrene oxide transferase has been described (Hayakawa et al., 1974) and is in widespread use. This method involves absorption on to charcoal, two extractions, concentration and paper chromatography. The reported recovery for this method is 52 % but it is not possible to determine recovery in all samples. We now report a rapid, sensitive method for determining glutathione S-[3H]styrene oxide transferase activity that permits 100 % recovery. The standard incubation mixture comprised loop1 of 0.1 M-sodium pyrophosphate buffer, pH 8.0, containing 1pmol of reduced glutathione, hepatic cytosol, prepared by differential centrifugation (Atlas et al., 1977), containing 40-450pg of protein, 5,uI of acetonitrile containing 1-2pmol of 7-[3H]styrene oxide with a specific radioactivity of 350c.p.m./nmol. [3H]Styrene oxide (The Radioachemical Centre, Amersham, Bucks., U.K.) was purified by solvent extraction to remove water-soluble decomposition products before use. The reaction was started by addition of the [3H]styrene oxide. Samples were incubated for 3-4min at 22°C and the reaction was stopped by addition of 2Opl of 4~-aceticacid. Unchanged styrene oxide was removed by extraction with 0.5ml of chloroform. This removed 83.2 2.9 % of total radioactivity. A portion (30~1) of the aqueous phase (25% of the sample) was applied to the loading zone of a 19channel LK5DF silica-gel plate (Whatman). The loading zone was pretreated with 80pl of a solution containing 30% (v/v) styrene oxide and 10% (w/v) styrene glycol in acetonitrile to decrease non-specific binding of the labelled reactants. Plates were dried in a hot-air current and chromatographed in chloroform/ethyl acetate (8 : 2, v/v). RF values for authentic styrene oxide and styrene glycol were 0.64 and 0.21 respectively, and styrene oxide glutathione conjugate remained at the loading zone. This was scraped from the plate into scintillation vials containing 2ml of methanol to extract the conjugate.
1979
![Effect of ionic strength and serum on the activity profile of bovine testicular hyaluronidase [proceedings].](/assets/img/hold.png)
