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In situ determination of the molecular weight of hepatic γ-glutamyl transferase and γ-glutamyl hydrolase activities
Biochimica et Biophysica A cta, 707 (1982) 164-166
164
Elsevier Biomedical Press
BBA Report BBA 30030
IN SlTU DETERMINATION OF THE MOLECULAR WEIGHT O17 HEPATIC y-GLUTAMYL TRANSFERASE AND 7-GLUTAMYL HYDROLASE ACTIVITIES J.L. D I N G a, G.D. SMITH a,, A. SEARLE b and T.J. PETERS a
a Division of Clinical Cell Biology, M R C Clinical Research Centre, Harrow and b Department of Biochemistry, Brunel University, Uxbridge, Middlesex (U. K.)
Key words: Radiation inactivation," y-Glutamyl transferase," Molecular weight
The molecular weight of T-glutamyl transferase from normal rat liver and hepatoma tissue was determined by radiation-inactivation and found to be approx. 100000 in each case. The molecular weight previously reported for the subunit containing the y-glutamyl binding site (22000) is considerably less than that of the holoenzyme, suggesting that in situ the large subunit is implicated in both transferase and hydrolase activities.
y-Glutamyl transferase is a membrane-bound glycoprotein of considerable current interest which catalyses the transfer of v-glutamyl groups from y-glutamyl donors to amino acid and dipeptide acceptors or to water [1]. The molecular weight of ~,-glutamyl transferase, isolated by detergent extraction or with papain treatment, has been determined by a number of different techniques including gel filtration chromatography [2], polyacrylamide gel electrophoresis [3] and sedimentation equilibrium analysis [4]. A large range of molecular weights has been obtained, even with the enzyme purified from a similar source [5,6]. Bound detergent micelles or differential proteolysis of the hydrophobic peptide [7] could both result in this variation. We report here the determination in situ of the molecular weight of the enzyme, both its transferase and hydrolase activities, by irradiation inactivation. Although the technique of irradiation inactivation has been known for many years [8], it has not been widely used, since the results obtained apparently did not agree with those from other procedures. However, increased knowledge of protein
* To whom correspondence should be addressed. 0167-4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
structure has allowed a re-evaluation of the technique, and shown that previous discrepancies could be accounted for by the presence of subunits [9]. The molecular mass and mobility of hormone-receptor complexes have also been investigated with this technique [10,11]. Microsomal fractions were prepared from the livers of Sprague-Dawley rats (male, 200 g) and from rat hepatoma cells (JBl-line) [12] cultured to confluency. The tissues were homogenized with 5 vol. 10 mM Tris-HC1 buffer, pH 7.4, [13] in a Dounce homogenizer (Kontes Glass Co., Vineland, NJ, U.S.A.) with ten strokes each of the loose-fitting (type A) and the tight-fitting (type B) pestle. The homogenate was centrifuged at 40000 X g for 2 h at 4°C and the pellet was re-homogenized and passed through a 50-60/~m mesh filter. Aliquots of 0.5 ml were dispensed into 1-ml thinwalled ampoules (FBG-Trident Ltd., Temple Cloud, Avon, U.K.), and lyophilized. The glass vials were then sealed under vacuum (60-70 mmHg), and stored at 4°C. Irradiation of the samples was carried out within 24 h of lyophilization. Irradiation inactivation was carried out at room temperature with a linear accelerator (Vickers Ltd., U.K.) delivering 4 MeV electrons. The radiation
165
20,
10
TABLE I Molecular weights were obtained for ,/-glutamyl transferase and y-glutamyl hydrolase activities in normal rat liver and rat hepatoma cell. The values are the mean of three separate experiments together with the S.E.
O
~- 1-6 <
y-Glutamyl hydrolase y-Ghitamyl transferase
i
•E
1'4
E
~
Normal liver
Hepatoma cell
91800 -+ 18 000
107 000 ~ 3000
99400-*- 5000
102000--- 1100
1.2
1-0
0Io
L
6
8
Io
Dose (Mrad) Fig. 1. Graph of log percentage residual y-glutamyl transferase against irradiation dose for (O) hepatoma cell and (0) control liver membrane fraction. The line was fitted to the points by linear regression analysis. Molecular weights of 97000 (control) and 110000 (hepatoma) for the transferase activity were calculated [13] from the empirical formula M r =6.4.105/D37 where D37 is the dose (Mrad) of radiation at which 37% of the original activity remains.
dose was measured by Fricke dosimetry [14]. The vials containing the lyophilized samples were placed directly in the path of the electron beam. Increasing doses, at 0.115 Mrad/s, were applied for 0, 10, 20, 30, 40, 60 and 80 s to individual vials. The irradiated and control samples were rehydrated with 1.0 ml ice-cold distilled water and the enzyme activities were determined as described in Ref. 15. Fig. 1. shows a typical plot of log percent remaining activity against the dose of electrons used [13]. The values obtained for the molecular weights in control and hepatoma tissue are shown in Table I. There is no significant difference in the mean molecular weight for each activity from the two enzyme sources. By gel filtration, the Tritonsolubilised purified normal liver and hepatoma enzyme had molecular weights of 250000 and 235 000, respectively [ 12], considerably larger than the respective molecular weight data obtained by irradiation inactivation. This difference probably
reflects the presence of Triton micelles associated with the solubilised ~,-glutamyl transferase and illustrates one of the advantages of this technique in the determination of molecular weights of intact hydrophobic membrane proteins. The molecular weight of the rat liver ~,-glutamyl transferase obtained by the present technique is in close agreement with that obtained by gel filtration studies on papain/trypsin-solubilised human liver enzyme [4,16]. For isolated rat kidney enzyme, the 7-glutamyl binding site of the enzyme was found to be on the small subunit [17], which has a molecular weight of approx. 22000 [2]. However, the molecular mass of the hepatic enzyme determined by irradiation appears to have included the large subunit. It is implicit in this analysis that both subunits decay as a single target with energy transfer across their interface. This suggests that in the plasma membrane the large subunit has an important structural inter-relationship with the small subunit. We are grateful to Dr. R. Willson for his helpful comments, Mr. B. Wolfenden for expert technical assistance, Ms. Rosamund Greensted for secretarial assistance and to the Cancer Research Campaign (J.L.D.) and Medical Research Council (A.S.) for financial support. References 1 Meister, A. and Tate, S.S. (1976) Annu. Rev. Biochem. 45, 559-604 2 Tate, S.S. and Meister, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2599-2603 3 Tsuji, A., Matsuda, Y. and Katunoma, N. (1980) Clin. Chim. Acta 104, 361-366
166 4 Hughey, R.P. and Curthoys, N.P. (1976) J. Biol. Chem. 251, 7863-7870 5 Miller, S.P., Awasthi, Y.C. and Srivastava, S.K. (1976) J. Biol. Chem. 251, 2271-2278 6 Tate, S.S. and Ross, M.E. (1977) J. Biol. Chem. 252, 60426045 7 Horiuchi, S., Inoue, M. and Morino, Y. (1978) Eur. J. Biochem. 87, 429-437 8 Lea, D.E. (1955) Action of Radiations on Living Cells, 2nd Ed., Cambridge University Press, Cambridge 9 Kempner, E.S. and Schlegel, W. (1979) Anal. Biochem. 93, 2-10 10 Houslay, M.D., Ellory, J.C., Smith, G.A., Hesketh, T.R., Stein, J.M., Warren, G.B. and Metcalfe, J.C. (1977) Biochim. Biophys. Acta 467, 208-219
11 Harmon, J.T., Kempner, E.C. and Kahn, C.R. (1981) J. Biol. Chem. 256, 7719-7722 12 Ding, J.L., Smith, G.D. and Peters, T.J. (1981) Biochim. Biophys. Acta 661, 191-198 13 Kepner, G.R. and Macey, R.I. (1968) Biochim. Biophys. Acta 163, 188-203 14 Fricke, H. and Hart, E.J. (1966) in Radiation Dosimetry (Attix, F.H. and Roesch, W.C., eds.), Ch. 12, Academic Press, New York 15 Smith, G.D., Ding, J.L. and Peters, T.J. (1979) Anal. Biochem. 100, 136-139 16 Shaw, L.M., London, J.W. and Peterson, L.E. (1978) Clin. Chem. 24, 905-915 17 Tate, S.S. and Meister, A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 931-935
164
Elsevier Biomedical Press
BBA Report BBA 30030
IN SlTU DETERMINATION OF THE MOLECULAR WEIGHT O17 HEPATIC y-GLUTAMYL TRANSFERASE AND 7-GLUTAMYL HYDROLASE ACTIVITIES J.L. D I N G a, G.D. SMITH a,, A. SEARLE b and T.J. PETERS a
a Division of Clinical Cell Biology, M R C Clinical Research Centre, Harrow and b Department of Biochemistry, Brunel University, Uxbridge, Middlesex (U. K.)
Key words: Radiation inactivation," y-Glutamyl transferase," Molecular weight
The molecular weight of T-glutamyl transferase from normal rat liver and hepatoma tissue was determined by radiation-inactivation and found to be approx. 100000 in each case. The molecular weight previously reported for the subunit containing the y-glutamyl binding site (22000) is considerably less than that of the holoenzyme, suggesting that in situ the large subunit is implicated in both transferase and hydrolase activities.
y-Glutamyl transferase is a membrane-bound glycoprotein of considerable current interest which catalyses the transfer of v-glutamyl groups from y-glutamyl donors to amino acid and dipeptide acceptors or to water [1]. The molecular weight of ~,-glutamyl transferase, isolated by detergent extraction or with papain treatment, has been determined by a number of different techniques including gel filtration chromatography [2], polyacrylamide gel electrophoresis [3] and sedimentation equilibrium analysis [4]. A large range of molecular weights has been obtained, even with the enzyme purified from a similar source [5,6]. Bound detergent micelles or differential proteolysis of the hydrophobic peptide [7] could both result in this variation. We report here the determination in situ of the molecular weight of the enzyme, both its transferase and hydrolase activities, by irradiation inactivation. Although the technique of irradiation inactivation has been known for many years [8], it has not been widely used, since the results obtained apparently did not agree with those from other procedures. However, increased knowledge of protein
* To whom correspondence should be addressed. 0167-4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
structure has allowed a re-evaluation of the technique, and shown that previous discrepancies could be accounted for by the presence of subunits [9]. The molecular mass and mobility of hormone-receptor complexes have also been investigated with this technique [10,11]. Microsomal fractions were prepared from the livers of Sprague-Dawley rats (male, 200 g) and from rat hepatoma cells (JBl-line) [12] cultured to confluency. The tissues were homogenized with 5 vol. 10 mM Tris-HC1 buffer, pH 7.4, [13] in a Dounce homogenizer (Kontes Glass Co., Vineland, NJ, U.S.A.) with ten strokes each of the loose-fitting (type A) and the tight-fitting (type B) pestle. The homogenate was centrifuged at 40000 X g for 2 h at 4°C and the pellet was re-homogenized and passed through a 50-60/~m mesh filter. Aliquots of 0.5 ml were dispensed into 1-ml thinwalled ampoules (FBG-Trident Ltd., Temple Cloud, Avon, U.K.), and lyophilized. The glass vials were then sealed under vacuum (60-70 mmHg), and stored at 4°C. Irradiation of the samples was carried out within 24 h of lyophilization. Irradiation inactivation was carried out at room temperature with a linear accelerator (Vickers Ltd., U.K.) delivering 4 MeV electrons. The radiation
165
20,
10
TABLE I Molecular weights were obtained for ,/-glutamyl transferase and y-glutamyl hydrolase activities in normal rat liver and rat hepatoma cell. The values are the mean of three separate experiments together with the S.E.
O
~- 1-6 <
y-Glutamyl hydrolase y-Ghitamyl transferase
i
•E
1'4
E
~
Normal liver
Hepatoma cell
91800 -+ 18 000
107 000 ~ 3000
99400-*- 5000
102000--- 1100
1.2
1-0
0Io
L
6
8
Io
Dose (Mrad) Fig. 1. Graph of log percentage residual y-glutamyl transferase against irradiation dose for (O) hepatoma cell and (0) control liver membrane fraction. The line was fitted to the points by linear regression analysis. Molecular weights of 97000 (control) and 110000 (hepatoma) for the transferase activity were calculated [13] from the empirical formula M r =6.4.105/D37 where D37 is the dose (Mrad) of radiation at which 37% of the original activity remains.
dose was measured by Fricke dosimetry [14]. The vials containing the lyophilized samples were placed directly in the path of the electron beam. Increasing doses, at 0.115 Mrad/s, were applied for 0, 10, 20, 30, 40, 60 and 80 s to individual vials. The irradiated and control samples were rehydrated with 1.0 ml ice-cold distilled water and the enzyme activities were determined as described in Ref. 15. Fig. 1. shows a typical plot of log percent remaining activity against the dose of electrons used [13]. The values obtained for the molecular weights in control and hepatoma tissue are shown in Table I. There is no significant difference in the mean molecular weight for each activity from the two enzyme sources. By gel filtration, the Tritonsolubilised purified normal liver and hepatoma enzyme had molecular weights of 250000 and 235 000, respectively [ 12], considerably larger than the respective molecular weight data obtained by irradiation inactivation. This difference probably
reflects the presence of Triton micelles associated with the solubilised ~,-glutamyl transferase and illustrates one of the advantages of this technique in the determination of molecular weights of intact hydrophobic membrane proteins. The molecular weight of the rat liver ~,-glutamyl transferase obtained by the present technique is in close agreement with that obtained by gel filtration studies on papain/trypsin-solubilised human liver enzyme [4,16]. For isolated rat kidney enzyme, the 7-glutamyl binding site of the enzyme was found to be on the small subunit [17], which has a molecular weight of approx. 22000 [2]. However, the molecular mass of the hepatic enzyme determined by irradiation appears to have included the large subunit. It is implicit in this analysis that both subunits decay as a single target with energy transfer across their interface. This suggests that in the plasma membrane the large subunit has an important structural inter-relationship with the small subunit. We are grateful to Dr. R. Willson for his helpful comments, Mr. B. Wolfenden for expert technical assistance, Ms. Rosamund Greensted for secretarial assistance and to the Cancer Research Campaign (J.L.D.) and Medical Research Council (A.S.) for financial support. References 1 Meister, A. and Tate, S.S. (1976) Annu. Rev. Biochem. 45, 559-604 2 Tate, S.S. and Meister, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2599-2603 3 Tsuji, A., Matsuda, Y. and Katunoma, N. (1980) Clin. Chim. Acta 104, 361-366
166 4 Hughey, R.P. and Curthoys, N.P. (1976) J. Biol. Chem. 251, 7863-7870 5 Miller, S.P., Awasthi, Y.C. and Srivastava, S.K. (1976) J. Biol. Chem. 251, 2271-2278 6 Tate, S.S. and Ross, M.E. (1977) J. Biol. Chem. 252, 60426045 7 Horiuchi, S., Inoue, M. and Morino, Y. (1978) Eur. J. Biochem. 87, 429-437 8 Lea, D.E. (1955) Action of Radiations on Living Cells, 2nd Ed., Cambridge University Press, Cambridge 9 Kempner, E.S. and Schlegel, W. (1979) Anal. Biochem. 93, 2-10 10 Houslay, M.D., Ellory, J.C., Smith, G.A., Hesketh, T.R., Stein, J.M., Warren, G.B. and Metcalfe, J.C. (1977) Biochim. Biophys. Acta 467, 208-219
11 Harmon, J.T., Kempner, E.C. and Kahn, C.R. (1981) J. Biol. Chem. 256, 7719-7722 12 Ding, J.L., Smith, G.D. and Peters, T.J. (1981) Biochim. Biophys. Acta 661, 191-198 13 Kepner, G.R. and Macey, R.I. (1968) Biochim. Biophys. Acta 163, 188-203 14 Fricke, H. and Hart, E.J. (1966) in Radiation Dosimetry (Attix, F.H. and Roesch, W.C., eds.), Ch. 12, Academic Press, New York 15 Smith, G.D., Ding, J.L. and Peters, T.J. (1979) Anal. Biochem. 100, 136-139 16 Shaw, L.M., London, J.W. and Peterson, L.E. (1978) Clin. Chem. 24, 905-915 17 Tate, S.S. and Meister, A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 931-935