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BIOCHEMICAL SOCIETY TRANSACTIONS
Biochemical and Immunological Studies of the Monomine-Oxidizing Activities of Cultured Human Cells JOHN F. POWELL and IAN W. CRAIG Genetics Laboratory, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, U.K.
The enzyme monoamine oxidase [monoamine+ oxidoreductase (deaminating), EC 1.4.3.41 is believed to exist in several isoenzymic forms (see Sandler & Youdhim, 1972). On the basis of differential inhibitor sensitivity, Johnston (1968) suggested the existence of two forms of monoamine oxidase, designated A and B. Form A was highly sensitive to the inhibitor clorgyline (M & B 9302), whereas the form B was much less sensitive. These two forms also differed in substrate affinities, the A form showing a greater affinity for 5-hydroxytryptamine. Multiple enzymically active forms can be resolved by electrophoresis in polyacrylamide gels (Sandler & Youdhim, 1972). These forms exhibited different substrate affinities and inhibitor sensitivities. However, they were found to be conterted into a single form by treatment with chaotropic agents, suggesting that they represent a single enzyme species whose behaviour is modulated by attached membrane fragments (Houslay & Tipton, 1973). Other studies have attempted to define different forms of monoamine oxidase by immunological procedures. One of these (McCaulay & Racker, 1973) indicated that two immunologically distinct forms of the enzyme exist in bovine brain, one of which cross-reacts with bovine Iiver monoamine oxidase and exhibits low activity with 5-hydroxytryptamine and noradrenaline.
a
7
6
5
4
-log [Concn. of clorgyline (M)] Fig. 1. Inhibition of monoamine oxidase activity in extracts of ( 0 )ZMR32 and(.) MC63 cells by clorgyline Cells were harvested by trypsin treatment and sonicated on ice in 0.18~-potassium phosphate, pH7.0. Cell extracts were incubated at 37°C for 15min in a total volume of 0.2ml and in the presence of various concentrations of clorgyline. They were then assayed for monoamine oxidase activity as described in the text. 1977
566th MEETING, CAMBRIDGE
181
Crude mitochondria1 fraction (human placenta)
I J I I I I
Centrifuge 3500Og, 30min; Washed, 0.1 ht-potassium phosphate (pH7.4)
Pellet Sonicated 1 0 m h on ice in 0.1 M-potassium phosphate (pH7.4) with 1.36% (w/v) Triton X-100 and 0.1 M-bemylamine
Sonicate Centrifuge lOOOOOg, 60min
Supernatant Dialysis against 5 litres of 0.01 +potassium phosphate (pH8.0) with 10% (v/v) glycerol
Dialysis residue Loaded on DEAE-cellulose column
Most active fractions Chromatography on Sepharosc 6 8 column
Monoamine oxidase preparation Scheme I . Purification of human placental monoamine oxidase All work was performed at 4°C. Chromatography on DEAEcellulose and Sepharose 6B was performed by the procedures described by McCaulay & Racker (1973) for bovine brain monoamine oxidase.
This form presumably represents a type-B enzyme by the nomenclature of Johnston (1968). Studies in vivo have also suggested the existence of distinct monoamine oxidase activities showing different inhibitor sensitivities and substrate specificities (Roth & Gillis, 1975). We have undertaken studies on the monoamine oxidases of human cell lines grown under standard conditions to establish whether cultured cells could provide a suitable system for further investigation into the biochemical and genetic interrelationship of the A and B forms. Monoamine oxidase activity was assayed by a modilkation of the method of Wu & Dyck (1976). Tryptamine was used as substrate at a final concentration of 8 . 3 in~ a~total reaction volume of 0.2ml. [G-3H]Tryptamine was obtained from The Radiochemical Centre, Amersham, Bucks., U.K., and diluted with unlabelled substrate to give a specific radioactivity of 602mCi/mmol. Radioactive products were counted in a toluene-based scintillation mixture at an efficiency of 40% in a LKB 81OOO liquidscintillation counter. The assay was linear up to 30min over the range of enzyme activity used. Cell lines studied include a human neuroblastoma line (IMR32) (Tumilowicz et al., 1970) and a HeLa derivative (MC63) (Siege1 et al., 1976). They were grown in RPMl 1640 medium (Flow Laboratories, Paisley, Scotland, U.K.) supplemented with 5 % foetal calf serum.Antisera were raised in Dutch rabbits by intramuscular injeo tion of I mg of purified antigen together with an equaI volume of Freund's complete
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BIOCHEMICAL SOCIETY TRANSACTIONS
adjuvant; a second intramuscular injection of an equal amount of antigen in Freund’s incomplete adjuvant was given 1 month later. Then 1 week later rabbits were bled, and a crude immunoglobulin preparation was made by the method of Kekwick (1940). The results shown in Fig. 1 suggest that the human neuroblastoma line contains monoamine oxidase activity of an A type, whereas that of MC63 cells is predominantly type B. At a concentration of 10m-clorgyline, monoamine oxidase from IMR32 cells was almost completely inhibited, whereas that from MC63 cells was only slightly so. To clarify further the possible differences between monoamine oxidase in the two cell lines, 0.5 % (w/v) Triton X-100 extracts of the two cell lines were made to react with antiserum raised against purified human placental monoamine oxidase. The purification of this placental enzyme is briefly described in Scheme 1. This antiserum reacted with crude human placental mitochondrial extracts in 0.5 % (w/v) Triton X-100 to form a single precipitin line in double-diffusion tests, but failed to react with 0.5 % (w/v) Triton X-100 extracts of human platelets, or partially purified human platelet monoamine oxidase. These preparations should contain only type B enzyme (see Donnelly et al., 1976). The antiserum also reacted with crude extracts of IMR32-cell mitochondria but not those prepared from MC63 cells, even when the latter extract was in fourfold excess with respect to monoamine oxidase activity. Further work is required to clarify the nature of the antigen recognized by this antiserum. However, it is apparent that the neuroblastoma line expresses a form of monoamine oxidase with properties similar to those described for type A enzyme and can be distinguished from the form expressed in the HeLa-cellline derivative MC63. Clorgyline was a kind gift of Dr. A. Crichton of May and Baker Ltd. Donnelly, C. H., Richelson, E. & Murphy, D. L. (1976) Biochem.Pharmacol. 25, 1639-1643 Houslay, M. D. & Tipton, K. F. (1973) Biochem. J . 135, 173-186 Johnston, J. P. (1968) Biochem. Pharmacol. 17, 1285-1297 Kekwick, R. A. (1940) Biochem. J. 34,1248-1256 McCaulay, R. & Racker, E. (1973) MoZ. CeN Biochem. 1, 73-81 Roth, J. A. & Gillis, 0. N. (1975) J. Pharmacol. Exp. Ther. 194,537-544 Sandier, M. & Youdim, M. B. H. (1972) Phurmacol. Rev. 24, 331-348 Siegel, L., Jeffreys, A. J., Sly, W. & Craig, I. W. (1976) Exp. Cell Res. 102,298-310 Tumilowicz, J., Nichols, W., Cholon, J. & Green, A. (1970) Cancer Res. 30, 2110-2118 Wu, P. H. & Dyck, L. E. (1976) Anal. Biochem. 72,637-642
The Binding of a Specific Muscarinic Antagonist to Subcellular Fractions of the Bovine Caudate Nucleus MARTYN R. GREGG, SHEILA SPANNER and G. BRIAN ANSELL Department of Pharmacology (Preclinical), University of Birmingham Medical School, Birmingham B15 2TJ, U.K.
Quinuclidinyl benzilate is a potent, specific muscarinic antagonist (Albanus, 1970; Meyerhoffer, 1972) and the 3H-labelled compound has been used in the study of the muscarinic receptors in the brains of rat and monkey (Yamamura & Snyder, 1974; Yamamura et al., 1974a,b). Yamamura & Snyder (1974) found that whencrude subcellular fractions of rat brain were incubated with [3H]quinuclidinyl benzilate, 57 % of the specific binding was found in the crude mitochondria1 fraction (PJ and 35% in the microsomal fraction (P3). The specific binding of [3H]quinuclidinyl benzilate in the regions of rat brain was greatest in the striatum. In both the regional and the subcellular studies, the high specific binding of [3H]quinuclidinyl benzilate appeared to parallel choline acetyltransferase (EC 2.3.1.6) activity. The caudate nucleus, part of the striatum, is rich in cholinergic activity and is reported to have a high concentration of muscarinic receptors (Hiley & Burgen, 1974). In the present study [3H]quinuclidinyl benzilate was used as a marker for muscarinic receptors in subcellular fractions of the bovine caudate nucleus, b e f ~ r ethese enriched fractions were used for studies of choline metabolism. 1977
BIOCHEMICAL SOCIETY TRANSACTIONS
Biochemical and Immunological Studies of the Monomine-Oxidizing Activities of Cultured Human Cells JOHN F. POWELL and IAN W. CRAIG Genetics Laboratory, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, U.K.
The enzyme monoamine oxidase [monoamine+ oxidoreductase (deaminating), EC 1.4.3.41 is believed to exist in several isoenzymic forms (see Sandler & Youdhim, 1972). On the basis of differential inhibitor sensitivity, Johnston (1968) suggested the existence of two forms of monoamine oxidase, designated A and B. Form A was highly sensitive to the inhibitor clorgyline (M & B 9302), whereas the form B was much less sensitive. These two forms also differed in substrate affinities, the A form showing a greater affinity for 5-hydroxytryptamine. Multiple enzymically active forms can be resolved by electrophoresis in polyacrylamide gels (Sandler & Youdhim, 1972). These forms exhibited different substrate affinities and inhibitor sensitivities. However, they were found to be conterted into a single form by treatment with chaotropic agents, suggesting that they represent a single enzyme species whose behaviour is modulated by attached membrane fragments (Houslay & Tipton, 1973). Other studies have attempted to define different forms of monoamine oxidase by immunological procedures. One of these (McCaulay & Racker, 1973) indicated that two immunologically distinct forms of the enzyme exist in bovine brain, one of which cross-reacts with bovine Iiver monoamine oxidase and exhibits low activity with 5-hydroxytryptamine and noradrenaline.
a
7
6
5
4
-log [Concn. of clorgyline (M)] Fig. 1. Inhibition of monoamine oxidase activity in extracts of ( 0 )ZMR32 and(.) MC63 cells by clorgyline Cells were harvested by trypsin treatment and sonicated on ice in 0.18~-potassium phosphate, pH7.0. Cell extracts were incubated at 37°C for 15min in a total volume of 0.2ml and in the presence of various concentrations of clorgyline. They were then assayed for monoamine oxidase activity as described in the text. 1977
566th MEETING, CAMBRIDGE
181
Crude mitochondria1 fraction (human placenta)
I J I I I I
Centrifuge 3500Og, 30min; Washed, 0.1 ht-potassium phosphate (pH7.4)
Pellet Sonicated 1 0 m h on ice in 0.1 M-potassium phosphate (pH7.4) with 1.36% (w/v) Triton X-100 and 0.1 M-bemylamine
Sonicate Centrifuge lOOOOOg, 60min
Supernatant Dialysis against 5 litres of 0.01 +potassium phosphate (pH8.0) with 10% (v/v) glycerol
Dialysis residue Loaded on DEAE-cellulose column
Most active fractions Chromatography on Sepharosc 6 8 column
Monoamine oxidase preparation Scheme I . Purification of human placental monoamine oxidase All work was performed at 4°C. Chromatography on DEAEcellulose and Sepharose 6B was performed by the procedures described by McCaulay & Racker (1973) for bovine brain monoamine oxidase.
This form presumably represents a type-B enzyme by the nomenclature of Johnston (1968). Studies in vivo have also suggested the existence of distinct monoamine oxidase activities showing different inhibitor sensitivities and substrate specificities (Roth & Gillis, 1975). We have undertaken studies on the monoamine oxidases of human cell lines grown under standard conditions to establish whether cultured cells could provide a suitable system for further investigation into the biochemical and genetic interrelationship of the A and B forms. Monoamine oxidase activity was assayed by a modilkation of the method of Wu & Dyck (1976). Tryptamine was used as substrate at a final concentration of 8 . 3 in~ a~total reaction volume of 0.2ml. [G-3H]Tryptamine was obtained from The Radiochemical Centre, Amersham, Bucks., U.K., and diluted with unlabelled substrate to give a specific radioactivity of 602mCi/mmol. Radioactive products were counted in a toluene-based scintillation mixture at an efficiency of 40% in a LKB 81OOO liquidscintillation counter. The assay was linear up to 30min over the range of enzyme activity used. Cell lines studied include a human neuroblastoma line (IMR32) (Tumilowicz et al., 1970) and a HeLa derivative (MC63) (Siege1 et al., 1976). They were grown in RPMl 1640 medium (Flow Laboratories, Paisley, Scotland, U.K.) supplemented with 5 % foetal calf serum.Antisera were raised in Dutch rabbits by intramuscular injeo tion of I mg of purified antigen together with an equaI volume of Freund's complete
Vol. 5
182
BIOCHEMICAL SOCIETY TRANSACTIONS
adjuvant; a second intramuscular injection of an equal amount of antigen in Freund’s incomplete adjuvant was given 1 month later. Then 1 week later rabbits were bled, and a crude immunoglobulin preparation was made by the method of Kekwick (1940). The results shown in Fig. 1 suggest that the human neuroblastoma line contains monoamine oxidase activity of an A type, whereas that of MC63 cells is predominantly type B. At a concentration of 10m-clorgyline, monoamine oxidase from IMR32 cells was almost completely inhibited, whereas that from MC63 cells was only slightly so. To clarify further the possible differences between monoamine oxidase in the two cell lines, 0.5 % (w/v) Triton X-100 extracts of the two cell lines were made to react with antiserum raised against purified human placental monoamine oxidase. The purification of this placental enzyme is briefly described in Scheme 1. This antiserum reacted with crude human placental mitochondrial extracts in 0.5 % (w/v) Triton X-100 to form a single precipitin line in double-diffusion tests, but failed to react with 0.5 % (w/v) Triton X-100 extracts of human platelets, or partially purified human platelet monoamine oxidase. These preparations should contain only type B enzyme (see Donnelly et al., 1976). The antiserum also reacted with crude extracts of IMR32-cell mitochondria but not those prepared from MC63 cells, even when the latter extract was in fourfold excess with respect to monoamine oxidase activity. Further work is required to clarify the nature of the antigen recognized by this antiserum. However, it is apparent that the neuroblastoma line expresses a form of monoamine oxidase with properties similar to those described for type A enzyme and can be distinguished from the form expressed in the HeLa-cellline derivative MC63. Clorgyline was a kind gift of Dr. A. Crichton of May and Baker Ltd. Donnelly, C. H., Richelson, E. & Murphy, D. L. (1976) Biochem.Pharmacol. 25, 1639-1643 Houslay, M. D. & Tipton, K. F. (1973) Biochem. J . 135, 173-186 Johnston, J. P. (1968) Biochem. Pharmacol. 17, 1285-1297 Kekwick, R. A. (1940) Biochem. J. 34,1248-1256 McCaulay, R. & Racker, E. (1973) MoZ. CeN Biochem. 1, 73-81 Roth, J. A. & Gillis, 0. N. (1975) J. Pharmacol. Exp. Ther. 194,537-544 Sandier, M. & Youdim, M. B. H. (1972) Phurmacol. Rev. 24, 331-348 Siegel, L., Jeffreys, A. J., Sly, W. & Craig, I. W. (1976) Exp. Cell Res. 102,298-310 Tumilowicz, J., Nichols, W., Cholon, J. & Green, A. (1970) Cancer Res. 30, 2110-2118 Wu, P. H. & Dyck, L. E. (1976) Anal. Biochem. 72,637-642
The Binding of a Specific Muscarinic Antagonist to Subcellular Fractions of the Bovine Caudate Nucleus MARTYN R. GREGG, SHEILA SPANNER and G. BRIAN ANSELL Department of Pharmacology (Preclinical), University of Birmingham Medical School, Birmingham B15 2TJ, U.K.
Quinuclidinyl benzilate is a potent, specific muscarinic antagonist (Albanus, 1970; Meyerhoffer, 1972) and the 3H-labelled compound has been used in the study of the muscarinic receptors in the brains of rat and monkey (Yamamura & Snyder, 1974; Yamamura et al., 1974a,b). Yamamura & Snyder (1974) found that whencrude subcellular fractions of rat brain were incubated with [3H]quinuclidinyl benzilate, 57 % of the specific binding was found in the crude mitochondria1 fraction (PJ and 35% in the microsomal fraction (P3). The specific binding of [3H]quinuclidinyl benzilate in the regions of rat brain was greatest in the striatum. In both the regional and the subcellular studies, the high specific binding of [3H]quinuclidinyl benzilate appeared to parallel choline acetyltransferase (EC 2.3.1.6) activity. The caudate nucleus, part of the striatum, is rich in cholinergic activity and is reported to have a high concentration of muscarinic receptors (Hiley & Burgen, 1974). In the present study [3H]quinuclidinyl benzilate was used as a marker for muscarinic receptors in subcellular fractions of the bovine caudate nucleus, b e f ~ r ethese enriched fractions were used for studies of choline metabolism. 1977
