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On rat liver mitochondrial monoamine oxidase activity and lipids
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 190, No. 2, September, pp. 847-849, 1978
COMMUNICATIONS On Rat Liver Mitochondrial
Monoamine
Oxidase Activity and Lipids’
Following earlier observations on the retention of 5-hydroxytryptamine oxidizing activity by a purified preparation of monoamine oxidase from rat liver mitochondria, this fraction has been obtained in a water-soluble form by Triton X-109 gradient gel filtration and DEAE-Bio-Gel A chromatography. The soluble fraction appears to depend on Triton X100 and phospholipids for its activity. The results seem to implicate membrane lipid components in the expression of rat liver mitochondrial monoamine oxidase activity. Mitochondrial MAO* is a flavin-dependent enzyme that regulates intracellular levels of biogenic amines by a two-step oxidative sequence (1, 2). This enzyme is generally recognized as a marker for the rat liver mitochondrial outer membrane (2, 3). Although the literature is replete with reports on the diversity of mitochondrial MAO (4), the issue has not been unequivocally settled yet. The current postulate concerning the heterogeneity of MAO is that this enzyme exists in two forms, MAO-A and MAO-B, distinction between which is based on differing sensitivities to substrate-selective inhibitors and also on the basis of substrate specificity (5, 6). In general, the A form is considered to preferentially attack benzylamine (or P-phenylethylamine) and the B form is believed to predominantly oxidize 5-HT. Purified preparations of liver MAO appear to be highly active upon benzylamine or /3-phenylethylamine while displaying negligible or no activity towards 5-HT (7-14). In these instances, 5-HT deaminating activity seems to decline rapidly during the course of fractionation of the enzyme. This could be due to the binding of the solubilizing detergent to the functional site of the enzyme, thereby influencing the affinity of the binding site(s) of MAO for different monoamines. Alternatively, alteration of the lipid environment of mitochondrial MAO might affect its activity towards certain substrates. Such possibilities have been alluded to in a recent report (15) wherein we have shown a high 5HT deaminating activity is associated with an electrophoretically homogeneous mitochondrial MAO preparation under certain conditions of solubilization and fractionation. Our attention was subsequently directed to the question whether the expression of certain monoamine deaminating activities is a reflection of the lipid environment of the enzyme. Inasmuch as MAO is fiiy bound to the mitochondrial membrane (2, 3), it is very likely that membrane components influence the activity of the enzyme. In the present 1 Supported by grants from the Medical Research Council of Canada. ’ Abbreviations used: MAO, monoamine oxidase [amine:oxygen oxidoreductase (deaminating), EC 1.4.3.4.1; 5-HT, 5-hydroxytryptamine.
study, the mitochondrial MAO preparation described previously has been obtained in a water-soluble form and attempts have been made to investigate the involvement of lipotropic compounds in the activity of this fraction. Rat liver mitochondria were prepared as described earlier (15). MAO was assayed polarographically with a YSl oxygen monitor (Yellow Springs Instrument Co., Inc.) as reported previously (15). MAO activity in the fractions obtained from chromatographic columns was determined by a coupled assay system with beef liver aldehyde dehydrogenase (8). Protein was estimated by the method of Lowry et al. (16) using crystalline bovine serum albumin as the standard. Lipid phosphorus determinations were performed according to previously published procedures (17). Lipid samples were dispersed by sonication as described by Rydstrom et al. (18). DEAE-Bio-Gel A was procured from Bio-Rad Laboratories. The sources of other chemicals have been indicated previously (15). The mitochondrial fraction subjected to osmotic shock (about 450-480 mg of protein) was suspended in 30 ml of 0.01 M potassium phosphate buffer (pH 7.6) containing 1 II~M EDTA and 0.1 mu dithiothreitol (buffer A). A solution of Triton X-100 (20%, w/v) in the same buffer was added dropwise to the mitochondrial suspension (final concentration, l%), stirred gently for 30 min, and centrifuged at 100,OOOgfor 60 min in a Spinco preparative ultracentrifuge (model L). Three such extractions were performed and the extracts were pooled (87 ml). Finely powdered (NH&S04 was added to this extract (O-40% saturation) and, after 45 min, the mixture was centrifuged at 20,000g for 20 min. The floating, yellow precipitate was collected by aspirating the underlying fluid, suspended in buffer A containing 0.15% Triton X-100, and centrifuged at 40,000g for 30 min. The clear supernatant obtained was applied to a column of Sepharose 6B (2.5 X 100 cm) to which had been applied 200 ml of a O-0.15% Triton X-100 gradient in buffer A. Fractions of 5 ml volume were collected and assayed for MAO activity with tyramine or 5-HT as the substrate. The active fractions were pooled and fractionated with (NH&SO, (O-40% saturation) as described above, the floating, yellow precipitate obtained on centrifugation was suspended in 0.01 M 847 0003-9861/78/1902-0847$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
848
KANDASWAMI
Tris-HCl buffer (pH 7.6) containing 1 mM EDTA and 0.1 mru dithiothreitol (buffer B), and dialyzed for 12 hr against buffer B with several changes of the buffer. This fraction was applied to a column of DEAE-BioGel A (2 x 25 cm), equilibrated with buffer B. The column was washed extensively with buffer B and was developed with 50-ml portions of buffer B containing 0.01 to 0.05% sodium deoxycholate, and NaCl was then gradually introduced. MAO was found to elute in presence of 0.05 M NaCl and 0.05% sodium deoxycholate. The DEAE-Bio-Gel A eluate was subjected to (NH&S04 fractionation (O-40% saturation) and the jelly-like precipitate obtained was suspended in buffer B and dialyzed against the same buffer (for 12 hr. All the above operations were performed at 0-5”C, unless otherwise stated. When gel filtration of the Triton X-100 extract is conducted on a column of Sepharose 6B to which has been applied a gradient of Triton X-100, one broad peak of protein as well as one peak of MAO activity are noticed, whereas two peaks of MAO activity are found to elute when gel filtration is performed at a fixed concentration of Triton X-100 (0.5%). A considerable amount of aggregated material is found in the latter case and also there is a loss in activity. Incidentally, at higher concentrations, Triton X-100 has been found to be inhibitory for MAO activity. Perhaps the increase in detergent to protein ratio at the gel filtration step might have affected the lipid-protein interactions of MAO, thereby causing a reduction in enzyme activity noticed. Swanljung (19) has suggested that the problem of increasing detergent to protein ratio during purification of membrane-bound enzymes may be minimized by the application of the detergent as a gradient in the column. Besides, it may facilitate the removal of a portion of the detergent from the sample as the membrane-bound enzyme that is soluble in a lower detergent concentration moves much ahead of the detergent in the column (19). The eluate from the Sepharose 6B column is found to be more active on 5-HT than against benzylamine as also observed earlier (15). This is in accord with the report of Houslay and Tipton (6) on MAO activity of mitochondrial outer membrane. A gradual removal of Triton X-100 from the Sepharose 6B column eluate has been attempted by DEAE-Bio-Gel A chromatography. Repeated washing of the DEAE-Bio-Gel A column with the starting buffer seems to be effective in reducing Triton X-109 which can be conveniently monitored (20). Triton X-100 is exchanged with sodium deoxycholate in the column as the latter detergent appears less difficult to remove from solubilized protein than the nonionic detergent (21). For instance, sodium deoxycholate could be removed from solubilized preparations by gel filtration or dialysis (20, 21). Upon (NH&SO4 fractionation of the DEAE-Bio-Gel A column eluate, the enzyme sediments to the bottom of the centrifuge tube and is fully soluble in buffer contaming no detergent. On dialysis of the enzyme against
AND
D’IORIO
buffer without any detergent, no precipitation is found. This preparation appears to be similar to the beef liver amine oxidase (22) which exhibited these features and was regarded as soluble. After the DEAE-Bio-Gel A chromatography step, there is a considerable reduction in the specific activity of the enzyme. This may be attributed to the effect of sodium deoxycholate which, in general, is considered to be less mild in its effects than Triton X-100 (21). However, rechromatography of Sepharose 6B column eluate on a column of Sepharose 6B in presence of 0.05% sodium deoxycholate results in no loss of MAO activity. The enzyme activity elutes as a single peak and an increase in specific activity is discernible after this step. These column fractions are found to have a considerable amount of lipid phosphate and this may be a factor in the retention of activity. The DEAEBio-Gel A column fraction, on the other hand, displays only 10 to 20% of the activity of the eluate from the Sepharose 6B column. The enzyme activity appears to be partially restored in the presence of diphosphatidylglycerol and Triton X-100 (Table I). It is likely that the soluble mitochontial MAO fraction obtained is devoid of membrane components essential for the expression of the enzyme activity. Apparently, ionexchange chromatography would have resulted in the removal of bound Triton X-100. Sodium deoxycholate, though effective in dislodging the enzyme from the gel matrix by hydrophobic interactions, might not adequately substitute for the lipid environment of the enzyme. Besides, this detergent could also be disrupting lipid-protein interactions essential for activity. At TABLE
I
EFFECT OF TRITON X-100 AND DIPHOSPHATIDYLGLYCEROL ON MONOAMINE OXIDASE ACTIVITY~ Sample*
Addition
Specific activity (units/mg of protein) 5-HT
Column eluate Column eluate
Tyramine
-
14.1
a.2
(DPG’ + Triton X-WV+
25.3
19.7
a MAO activity was assayed by the polarographic method. One unit of enzyme activity is the amount of protein required for the consumption of 1 natom of oxygen/min under the standard experimental conditions. ‘The sample assayed was the eluate from the DEAE-Bio-Gel A column. ’ DPG, diphosphatidylglycerol. ‘The eluate from DEAE-Bio-Gel A column was incubated with DPG (200 ILL) and Triton X-106 (4 mg) for 10 min before the addition of the substrate. DPG was dispersed in buffer B (see text) by sonication.
MITOCHONDRIAL
MONOAMINE
this juncture, it is pertinent to recall the findings of Sierens and D’Iorio (23), showing that solubilization of mitochondrial MAO by ionic detergents like sodium deoxycholate, led to a considerable loss in MAO activity towards 5-HT, possibly due to lipid removal. Based on present results, Triton X-100 seems to be necessary to stabilize mitochondrial MAO. This could perhaps be explained on the basis of preservation of polarapolar orientation of the protein by micelle formation. However, it looks as though Triton X-166 as well as an anionic phospholipid are required for restoration of MAO activity. Perhaps the detergent may be needed to disperse the phospholipid. To our knowledge, this is the fast indication of a possible direct involvement of lipid components in the activity of mitochondrial MAO. The present observations are particularly interesting in the light of our recent results (C. Kandaswami and A. D’Iorio, manuscript submitted for publication) illustrating that MAO activity against various substrates was affected under conditions facilitating the extraction of acidic phospholipids from the mitochondria. It is tempting to state that preferential inactivation of certain monoamine deaminating activities during the course of purification of mitochondrial MAO may be due to a change in the lipid environment of the enzyme. It appears that in our case at the (NH&SO4 precipitation stage, we are preferring the lipid-replete fraction for further fractionation of the enzyme. This could be a contributory factor in preserving 5-HT oxidizing activity. Tret’yakov et al. (24) reported that extraction of lipids by acetone from rat liver mitochondria led to a substantial change in the substrate specificity of MAO. These authors contend that the active sites of MAO are embedded in two different environments in the mitochondrial outer membrane, 5-HT deaminating activity being oriented towards protein-lipid layers. White and Glassman (25) have visualized a similar picture with respect to A and B sites of human MAO. These authors have reported that loss of MAO activity in Triton X-160~solubilized mitochondrial extracts could be prevented by acidic phospholipids. Further studies are evidently required on the problem of modulation of mitochondrial MAO activity by membrane lipids. ACKNOWLEDGMENTS We express our appreciation to Mrs. B. Betz for her competent assistance in this work. Our thanks are due to Mrs. Helene Amyot for Secretarial help.
OXIDASE
AND
849
LIPIDS
4. DIAZ-BORGES, J. M., AND D’IORIO, A. (1972) Adv.
Biochem. Psychopharmacol. 5,70-89. 5. JOHNSTON, J.P. (1968) Biochem. Pharmacol. 17, 1285-1297. 6. HOUSLAY, M. D., AND TIPTON, K. F. (1974) Biothem. J. 139,645-652. 7. TIPTON, K. F. (1972) Adv. Biochem. Psychopharmacol. 5, 11-24. 8. HOUSLAY, M. D., AND TIPTON, K. F. (1973) Biothem. J. 135, 173-186. 9. NARA, S., GOMES, B., AND YASIJNOBU, K. T. (1966)
J. Biol. Chem. 241,2774-2780. 10. Hsu, M. (1973) Ph.D. dissertation, Northwestern University, Evanston, Ill. 11. AKOPIAN, 2. I., STESINA, L. N., AND GORKIN, V. Z. (1971) J. Biol. Chem. 246,4610-4618. 12. ORELAND, L. (1971) Arch. Biochem. Biophys. 146, 410-421. 13. SEVERINA, I. S. (1973) Eur. J. Biochem. 38,
239-246. 14. DENNICK, R. G., AND MAYER, R. J. (1977) Biothem. J. 161, 167-174. 15. KANDASWAMI, C., DIAZ-BORGES, J. M., AND D’IORIO, A. (1977) Arch. Biochem. Biophys. 183,273-280. 16. LOWRY, 0. H., ROSEBOROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 17. AMES, B. N. (1966) in Methods in Enzymology (Neufeld, E., and Ginsburg, V., eds.), Vol. 8, pp. 115-118, Academic Press, New York. 18. RYDSTRBM, J., HOEK, J. B., ERICSON, B. G., AND HUNDAL, T. (1976) Biochim. Biophys. Acta 430,
419-425. 19. SWANLJLJNG, P. (1971) Anal.
chopharmacol. 5, 207-226. 3. SOTTOCASA, G. L. (1976) in Biochemical Analysis of membranes (Maddy, A. H., ed.), pp. 55-78, Chapman and Hall, London.
43,
Chem. 248,793-799. 21. HELENIUS, A., AND SIMONS, K. (1975) Biochim. Biophys. Acta 415,29-79. 22. YASUNOBU, K. T., AND GOMES, B. (1971) in Methods in Enzymology (Tabor, H., and Tabor, C. W., eds.), Vol. 17, Part B, pp. 709-717, Academic Press, New York. 23. SIERENS, L., AND D’IORIO, A. (1970) Canad. J.
Biochem. 48,659-663. 24. TRET’YAKOV, A. V., MUKHLENOV, A. G., AND GURKALO, V. K. (1976) Dokl. Biochem. 228.231-234. 25. WHITE, H. L., AND GLASSMAN, A. T. (1977) J.
Neurochem. 29,987-997. C. KANDASWAM? A. D’IORIO
REFERENCES 1. GREENAWALT, J. W., AND DECKER, G. L. (1976) Fed. Proc. 35, 1435. 2. GREENAWALT, J. W. (1972) Adv. Biochem. Psy-
Biochem.
382-387. 20. SPATZ, L., AND STRITTMATTER, P. (1973) J. Biol.
Department of Biochemistry University of Ottawa Ottawa, Ontario KlN 6N5, Canada Received April 25, 1978 3 To whom correspondence
should be addressed.
COMMUNICATIONS On Rat Liver Mitochondrial
Monoamine
Oxidase Activity and Lipids’
Following earlier observations on the retention of 5-hydroxytryptamine oxidizing activity by a purified preparation of monoamine oxidase from rat liver mitochondria, this fraction has been obtained in a water-soluble form by Triton X-109 gradient gel filtration and DEAE-Bio-Gel A chromatography. The soluble fraction appears to depend on Triton X100 and phospholipids for its activity. The results seem to implicate membrane lipid components in the expression of rat liver mitochondrial monoamine oxidase activity. Mitochondrial MAO* is a flavin-dependent enzyme that regulates intracellular levels of biogenic amines by a two-step oxidative sequence (1, 2). This enzyme is generally recognized as a marker for the rat liver mitochondrial outer membrane (2, 3). Although the literature is replete with reports on the diversity of mitochondrial MAO (4), the issue has not been unequivocally settled yet. The current postulate concerning the heterogeneity of MAO is that this enzyme exists in two forms, MAO-A and MAO-B, distinction between which is based on differing sensitivities to substrate-selective inhibitors and also on the basis of substrate specificity (5, 6). In general, the A form is considered to preferentially attack benzylamine (or P-phenylethylamine) and the B form is believed to predominantly oxidize 5-HT. Purified preparations of liver MAO appear to be highly active upon benzylamine or /3-phenylethylamine while displaying negligible or no activity towards 5-HT (7-14). In these instances, 5-HT deaminating activity seems to decline rapidly during the course of fractionation of the enzyme. This could be due to the binding of the solubilizing detergent to the functional site of the enzyme, thereby influencing the affinity of the binding site(s) of MAO for different monoamines. Alternatively, alteration of the lipid environment of mitochondrial MAO might affect its activity towards certain substrates. Such possibilities have been alluded to in a recent report (15) wherein we have shown a high 5HT deaminating activity is associated with an electrophoretically homogeneous mitochondrial MAO preparation under certain conditions of solubilization and fractionation. Our attention was subsequently directed to the question whether the expression of certain monoamine deaminating activities is a reflection of the lipid environment of the enzyme. Inasmuch as MAO is fiiy bound to the mitochondrial membrane (2, 3), it is very likely that membrane components influence the activity of the enzyme. In the present 1 Supported by grants from the Medical Research Council of Canada. ’ Abbreviations used: MAO, monoamine oxidase [amine:oxygen oxidoreductase (deaminating), EC 1.4.3.4.1; 5-HT, 5-hydroxytryptamine.
study, the mitochondrial MAO preparation described previously has been obtained in a water-soluble form and attempts have been made to investigate the involvement of lipotropic compounds in the activity of this fraction. Rat liver mitochondria were prepared as described earlier (15). MAO was assayed polarographically with a YSl oxygen monitor (Yellow Springs Instrument Co., Inc.) as reported previously (15). MAO activity in the fractions obtained from chromatographic columns was determined by a coupled assay system with beef liver aldehyde dehydrogenase (8). Protein was estimated by the method of Lowry et al. (16) using crystalline bovine serum albumin as the standard. Lipid phosphorus determinations were performed according to previously published procedures (17). Lipid samples were dispersed by sonication as described by Rydstrom et al. (18). DEAE-Bio-Gel A was procured from Bio-Rad Laboratories. The sources of other chemicals have been indicated previously (15). The mitochondrial fraction subjected to osmotic shock (about 450-480 mg of protein) was suspended in 30 ml of 0.01 M potassium phosphate buffer (pH 7.6) containing 1 II~M EDTA and 0.1 mu dithiothreitol (buffer A). A solution of Triton X-100 (20%, w/v) in the same buffer was added dropwise to the mitochondrial suspension (final concentration, l%), stirred gently for 30 min, and centrifuged at 100,OOOgfor 60 min in a Spinco preparative ultracentrifuge (model L). Three such extractions were performed and the extracts were pooled (87 ml). Finely powdered (NH&S04 was added to this extract (O-40% saturation) and, after 45 min, the mixture was centrifuged at 20,000g for 20 min. The floating, yellow precipitate was collected by aspirating the underlying fluid, suspended in buffer A containing 0.15% Triton X-100, and centrifuged at 40,000g for 30 min. The clear supernatant obtained was applied to a column of Sepharose 6B (2.5 X 100 cm) to which had been applied 200 ml of a O-0.15% Triton X-100 gradient in buffer A. Fractions of 5 ml volume were collected and assayed for MAO activity with tyramine or 5-HT as the substrate. The active fractions were pooled and fractionated with (NH&SO, (O-40% saturation) as described above, the floating, yellow precipitate obtained on centrifugation was suspended in 0.01 M 847 0003-9861/78/1902-0847$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
848
KANDASWAMI
Tris-HCl buffer (pH 7.6) containing 1 mM EDTA and 0.1 mru dithiothreitol (buffer B), and dialyzed for 12 hr against buffer B with several changes of the buffer. This fraction was applied to a column of DEAE-BioGel A (2 x 25 cm), equilibrated with buffer B. The column was washed extensively with buffer B and was developed with 50-ml portions of buffer B containing 0.01 to 0.05% sodium deoxycholate, and NaCl was then gradually introduced. MAO was found to elute in presence of 0.05 M NaCl and 0.05% sodium deoxycholate. The DEAE-Bio-Gel A eluate was subjected to (NH&S04 fractionation (O-40% saturation) and the jelly-like precipitate obtained was suspended in buffer B and dialyzed against the same buffer (for 12 hr. All the above operations were performed at 0-5”C, unless otherwise stated. When gel filtration of the Triton X-100 extract is conducted on a column of Sepharose 6B to which has been applied a gradient of Triton X-100, one broad peak of protein as well as one peak of MAO activity are noticed, whereas two peaks of MAO activity are found to elute when gel filtration is performed at a fixed concentration of Triton X-100 (0.5%). A considerable amount of aggregated material is found in the latter case and also there is a loss in activity. Incidentally, at higher concentrations, Triton X-100 has been found to be inhibitory for MAO activity. Perhaps the increase in detergent to protein ratio at the gel filtration step might have affected the lipid-protein interactions of MAO, thereby causing a reduction in enzyme activity noticed. Swanljung (19) has suggested that the problem of increasing detergent to protein ratio during purification of membrane-bound enzymes may be minimized by the application of the detergent as a gradient in the column. Besides, it may facilitate the removal of a portion of the detergent from the sample as the membrane-bound enzyme that is soluble in a lower detergent concentration moves much ahead of the detergent in the column (19). The eluate from the Sepharose 6B column is found to be more active on 5-HT than against benzylamine as also observed earlier (15). This is in accord with the report of Houslay and Tipton (6) on MAO activity of mitochondrial outer membrane. A gradual removal of Triton X-100 from the Sepharose 6B column eluate has been attempted by DEAE-Bio-Gel A chromatography. Repeated washing of the DEAE-Bio-Gel A column with the starting buffer seems to be effective in reducing Triton X-109 which can be conveniently monitored (20). Triton X-100 is exchanged with sodium deoxycholate in the column as the latter detergent appears less difficult to remove from solubilized protein than the nonionic detergent (21). For instance, sodium deoxycholate could be removed from solubilized preparations by gel filtration or dialysis (20, 21). Upon (NH&SO4 fractionation of the DEAE-Bio-Gel A column eluate, the enzyme sediments to the bottom of the centrifuge tube and is fully soluble in buffer contaming no detergent. On dialysis of the enzyme against
AND
D’IORIO
buffer without any detergent, no precipitation is found. This preparation appears to be similar to the beef liver amine oxidase (22) which exhibited these features and was regarded as soluble. After the DEAE-Bio-Gel A chromatography step, there is a considerable reduction in the specific activity of the enzyme. This may be attributed to the effect of sodium deoxycholate which, in general, is considered to be less mild in its effects than Triton X-100 (21). However, rechromatography of Sepharose 6B column eluate on a column of Sepharose 6B in presence of 0.05% sodium deoxycholate results in no loss of MAO activity. The enzyme activity elutes as a single peak and an increase in specific activity is discernible after this step. These column fractions are found to have a considerable amount of lipid phosphate and this may be a factor in the retention of activity. The DEAEBio-Gel A column fraction, on the other hand, displays only 10 to 20% of the activity of the eluate from the Sepharose 6B column. The enzyme activity appears to be partially restored in the presence of diphosphatidylglycerol and Triton X-100 (Table I). It is likely that the soluble mitochontial MAO fraction obtained is devoid of membrane components essential for the expression of the enzyme activity. Apparently, ionexchange chromatography would have resulted in the removal of bound Triton X-100. Sodium deoxycholate, though effective in dislodging the enzyme from the gel matrix by hydrophobic interactions, might not adequately substitute for the lipid environment of the enzyme. Besides, this detergent could also be disrupting lipid-protein interactions essential for activity. At TABLE
I
EFFECT OF TRITON X-100 AND DIPHOSPHATIDYLGLYCEROL ON MONOAMINE OXIDASE ACTIVITY~ Sample*
Addition
Specific activity (units/mg of protein) 5-HT
Column eluate Column eluate
Tyramine
-
14.1
a.2
(DPG’ + Triton X-WV+
25.3
19.7
a MAO activity was assayed by the polarographic method. One unit of enzyme activity is the amount of protein required for the consumption of 1 natom of oxygen/min under the standard experimental conditions. ‘The sample assayed was the eluate from the DEAE-Bio-Gel A column. ’ DPG, diphosphatidylglycerol. ‘The eluate from DEAE-Bio-Gel A column was incubated with DPG (200 ILL) and Triton X-106 (4 mg) for 10 min before the addition of the substrate. DPG was dispersed in buffer B (see text) by sonication.
MITOCHONDRIAL
MONOAMINE
this juncture, it is pertinent to recall the findings of Sierens and D’Iorio (23), showing that solubilization of mitochondrial MAO by ionic detergents like sodium deoxycholate, led to a considerable loss in MAO activity towards 5-HT, possibly due to lipid removal. Based on present results, Triton X-100 seems to be necessary to stabilize mitochondrial MAO. This could perhaps be explained on the basis of preservation of polarapolar orientation of the protein by micelle formation. However, it looks as though Triton X-166 as well as an anionic phospholipid are required for restoration of MAO activity. Perhaps the detergent may be needed to disperse the phospholipid. To our knowledge, this is the fast indication of a possible direct involvement of lipid components in the activity of mitochondrial MAO. The present observations are particularly interesting in the light of our recent results (C. Kandaswami and A. D’Iorio, manuscript submitted for publication) illustrating that MAO activity against various substrates was affected under conditions facilitating the extraction of acidic phospholipids from the mitochondria. It is tempting to state that preferential inactivation of certain monoamine deaminating activities during the course of purification of mitochondrial MAO may be due to a change in the lipid environment of the enzyme. It appears that in our case at the (NH&SO4 precipitation stage, we are preferring the lipid-replete fraction for further fractionation of the enzyme. This could be a contributory factor in preserving 5-HT oxidizing activity. Tret’yakov et al. (24) reported that extraction of lipids by acetone from rat liver mitochondria led to a substantial change in the substrate specificity of MAO. These authors contend that the active sites of MAO are embedded in two different environments in the mitochondrial outer membrane, 5-HT deaminating activity being oriented towards protein-lipid layers. White and Glassman (25) have visualized a similar picture with respect to A and B sites of human MAO. These authors have reported that loss of MAO activity in Triton X-160~solubilized mitochondrial extracts could be prevented by acidic phospholipids. Further studies are evidently required on the problem of modulation of mitochondrial MAO activity by membrane lipids. ACKNOWLEDGMENTS We express our appreciation to Mrs. B. Betz for her competent assistance in this work. Our thanks are due to Mrs. Helene Amyot for Secretarial help.
OXIDASE
AND
849
LIPIDS
4. DIAZ-BORGES, J. M., AND D’IORIO, A. (1972) Adv.
Biochem. Psychopharmacol. 5,70-89. 5. JOHNSTON, J.P. (1968) Biochem. Pharmacol. 17, 1285-1297. 6. HOUSLAY, M. D., AND TIPTON, K. F. (1974) Biothem. J. 139,645-652. 7. TIPTON, K. F. (1972) Adv. Biochem. Psychopharmacol. 5, 11-24. 8. HOUSLAY, M. D., AND TIPTON, K. F. (1973) Biothem. J. 135, 173-186. 9. NARA, S., GOMES, B., AND YASIJNOBU, K. T. (1966)
J. Biol. Chem. 241,2774-2780. 10. Hsu, M. (1973) Ph.D. dissertation, Northwestern University, Evanston, Ill. 11. AKOPIAN, 2. I., STESINA, L. N., AND GORKIN, V. Z. (1971) J. Biol. Chem. 246,4610-4618. 12. ORELAND, L. (1971) Arch. Biochem. Biophys. 146, 410-421. 13. SEVERINA, I. S. (1973) Eur. J. Biochem. 38,
239-246. 14. DENNICK, R. G., AND MAYER, R. J. (1977) Biothem. J. 161, 167-174. 15. KANDASWAMI, C., DIAZ-BORGES, J. M., AND D’IORIO, A. (1977) Arch. Biochem. Biophys. 183,273-280. 16. LOWRY, 0. H., ROSEBOROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 17. AMES, B. N. (1966) in Methods in Enzymology (Neufeld, E., and Ginsburg, V., eds.), Vol. 8, pp. 115-118, Academic Press, New York. 18. RYDSTRBM, J., HOEK, J. B., ERICSON, B. G., AND HUNDAL, T. (1976) Biochim. Biophys. Acta 430,
419-425. 19. SWANLJLJNG, P. (1971) Anal.
chopharmacol. 5, 207-226. 3. SOTTOCASA, G. L. (1976) in Biochemical Analysis of membranes (Maddy, A. H., ed.), pp. 55-78, Chapman and Hall, London.
43,
Chem. 248,793-799. 21. HELENIUS, A., AND SIMONS, K. (1975) Biochim. Biophys. Acta 415,29-79. 22. YASUNOBU, K. T., AND GOMES, B. (1971) in Methods in Enzymology (Tabor, H., and Tabor, C. W., eds.), Vol. 17, Part B, pp. 709-717, Academic Press, New York. 23. SIERENS, L., AND D’IORIO, A. (1970) Canad. J.
Biochem. 48,659-663. 24. TRET’YAKOV, A. V., MUKHLENOV, A. G., AND GURKALO, V. K. (1976) Dokl. Biochem. 228.231-234. 25. WHITE, H. L., AND GLASSMAN, A. T. (1977) J.
Neurochem. 29,987-997. C. KANDASWAM? A. D’IORIO
REFERENCES 1. GREENAWALT, J. W., AND DECKER, G. L. (1976) Fed. Proc. 35, 1435. 2. GREENAWALT, J. W. (1972) Adv. Biochem. Psy-
Biochem.
382-387. 20. SPATZ, L., AND STRITTMATTER, P. (1973) J. Biol.
Department of Biochemistry University of Ottawa Ottawa, Ontario KlN 6N5, Canada Received April 25, 1978 3 To whom correspondence
should be addressed.