[49]
BEEF LIVER MONOAMINE OXIDASE
495
of molecular conformation around iron-sulfur centers S-2 and S-3 in the inactive preparations have been revealed as follows: 1. As shown in Fig. 4, EPR signals from center S-3 can be detected only in the reconstitutively active SDH preparations, but not in inactive ones. This indicates that a certain conformational change around this iron-sulfur cluster gives rise to a loss of the reconstitutive activity, namely, capability to transfer electrons to the respiratory chain. A close correlation between these two parameters can be further demonstrated by the parallel decrease of center S-3 signal and reconstitutive activity, upon exposure of SDH to the air. 2° 2. Spin relaxation of center S-2 is slower in reconstitutively inactive preparations than in active ones. This is reflected in the two following observations: (a) Quantitative detection of center S-2 spins at higher temperature range (>30°K) in reconstitutively inactive preparations, but not in active ones. (b) In all SDH preparations, centers S-1 and S-2 are closely located (< 10 ]k) and show spin-spin interactions, namely, release of power saturation of center S-I spins (longer T, component) by cross relaxation with S-2 spins (shorter T1 component). Conversely, center S2 spectra are affected by S-I spins in a low temperature range (4.2°-6°K), seen most clearly as broadening (active preparation) or splitting (inactive preparation) of the principal resonance signal, depending on the spin relaxation times of center S-24 (see Fig. 5). EPR characteristics of iron-sulfur centers in reconstitutively active and inactive succinate dehydrogenase preparations are summarized in Table II.
[49] P r e p a r a t i o n
of Monoamine Oxidase from Beef Liver Mitochondria B y JAMES I. SALACH, JR.
Two preparative procedures for monoamine oxidase have been described that yield an enzyme of high specific activity and purity. One utilizes beef kidney, 1 the other beef liver. 2 These enzyme preparations, of high specific activity, 3 are obtained in yields of 10% and 5-6%, rei H. Y. K. Chuang, D. R. Patek, and L. Hellerman, J. Biol. Chem. 249, 2381 (1974). 2 K. T. Yasunobu, I. Igaue, and B. Gomes, Adv. Pharmacol. 6, 43 (1%8); B. Gomes, I. lgaue, H. G. Kloepfer, and K. T. Yasunobu, Arch. Biochem. Biophys. 132, 16 (1%9). 3 Direct comparison of specific activities in these two preparations with the present preparation cannot be made. While the units of activity employed by these investigators may
496
FLAVOPROTEINS
[49]
spectively, o f the total in the mitochondria after c o m p l e x multiple chromatographic procedures. This m e t h o d presented is simple, rapid, and requires no chromatography. The yield is 3- to 5-fold higher. P r i n c i p l e . The method is b a s e d on modification of the lipids of the mitochondrial outer m e m b r a n e by treatment with phospholipases so that low concentrations of Triton X-100 will extract the e n z y m e nearly quantitatively. The detergent and phospholipids are r e m o v e d b y partition in a three-phase p o l y m e r system. 4 The e n z y m e , present in the interface b e t w e e n the middle and lower phases, is separated in good yield f r o m insoluble material and residual p o l y m e r s by centrifugation.
Assay Method The e n z y m e is assayed by a modification of the m e t h o d of T a b o r et ol. 5 Reagents
1. Potassium p h o s p h a t e , 0.2 M , p H 7.2 at 30 °, containing 0.6% (w/ v) Triton X-1006 2. Benzylamine, 0.1 M, neutralized to p H 7 with 6 M HCI P r o c e d u r e . Phosphate buffer, 1.0 ml, and glass-distilled water, 1.85 ml, are placed in a 4-ml quartz cuvette, brought to 30 ° by incubation for 3-5 min in a w a t e r bath, and placed in the s p e c t r o p h o t o m e t e r . A suitable aliquot of e n z y m e (10-50 ~1) is added and mixed, and the blank rate is m e a s u r e d at 250 nm. The reaction is initiated by addition of 0.1 ml of benzylamine. The reverse order of addition of e n z y m e and b e n z y l a m i n e can be used if desired. The value taken for the molar extinction of b e n z a l d e h y d e at 250 nm is 12,080. 7 One unit is defined as formation of 1.0 ttmol of product per minute. 8
be interconverted with the unit employed in this method, one or more of the conditions of the assay: volume, substrate concentration, pH, and temperature are also varied. A typical preparation of specific activity 2.28 units/mg in the present assay, when assayed by the method used for the beef kidney enzymel had an activity of 3.96 units/mg or 21,719 units/mg of the units used by Chuang et al. 1 4 P.-A. Albertsson, Biochemistry 12, 2525 (1973). C. W. Tabor, H. Tabor, and S. M. Rosenthal, J. Biol. Chem. 193, 265 (1951). 6 Distill off acetaldehyde from commercial Triton in a rotary evaporator at 80° and vacuum from a water aspirator for 3-4 hr before use. 7 "Organic Electronic Spectral Data," Vol. IV. Wiley (Interscience), New York, 1959. s At the substrate concentration used (3.3 mM) the reaction rates obtained are 90-95% of the rates at infinite substrate concentration measured by extrapolation of double reciprocal plots.
[49]
BEEF LIVER MONOAMINE OXIDASE
497
Measurement of Protein
Protein is determined by the biuret method 9 with the addition of 0.05 ml of 10% (w/v) sodium deoxycholate to solubilize lipids. One milligram of bovine serum albumin standard in 3.0 ml final volume is taken to give an absorbance at 540 nm of 0.095. Preparation of Mitochondria
Mitochondria are prepared essentially by the method of Kearney et al. lO Reagents
1. Sucrose, 0.25 M, potassium phosphate, 10 mM, EDTA, 0.5 mM, pH 7.0-7.2 at 0 ° 2. Sucrose, 0.25 M, neutralized to pH 7.0-7.2 at 0 ° with KOH 3. KC1, 0.15 M, Tris-phosphate, 10 mM, pH 7.0-7.2 at 0 ° 4. TEA 11 buffer, 0.1 M, pH 7.2, at 30 ° 5. CaC12, 1.0 M 6. Triton X-100, 10% (w/v) in water 6 7. Phospholipase A, partially purified, specific activity 15,000 units/mg 12 8. Phospholipase C, from Clostridium welchii, 13 type I, specific activity 5/zmol of water-soluble phosphorus released from egg 9 A. G. Gornall, C. J. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949). Protein may also be measured by the Lowry method (O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951)). This is recommended for the final step because of the smaller amounts of protein required. 10 E. B. Kearney, J. I. Salach, W. H. Walker, R. L. Seng, W. Kenney, E. Zeszotek, and T. P. Singer, Eur. J. Biochem. 24, 321 (1971). 1, TEA: triethanolamine (2,2',2"-nitrilotriethanol hydrochloride), molecular weight 185.65. 12 Phospholipase A is prepared from Naja naja venom by chromatography on Sephadex G75 by the method of T. C. Cremona and E. B. Kearney, J. Biol. Chem. 239, 2328 (1964). Fractions having a specific activity of 15,000-17,000 units per milligram of protein, or better, in the assay specified below are combined and used for the digestion. Note that the same amount of protein is used even if the phospholipase A sample has as much as 35,000 units of activity per milligram because excess phospholipase A does not interfere. Phospholipase activity is estimated with a recording pH meter by the method of J. 1. Salach, P. Turini, R. Seng, and T. P. Singer, J. Biol. Chem. 246, 331 (1971). This assay is less involved and more rapid than the manometric procedure used by Cremona and Kearney. One unit of phospholipase A activity is defined as that enzyme activity which releases sufficient fatty acid from purified egg lecithin to cause a decrease of 0.01 pH in 30 rain at pH 8.0 and 25 °. 13 Phospholipase C from C. welchii is used since it is reported to hydrolyze lysolecithin and phosphatidylethanolamine as well as lecithin [T. Takahashi and H. H. O. Schmid, Chem. Phys. Lipids 2, 220 (1968)].
498
FLAVOPROTEINS
9. 10. 11. 12.
[49]
lecithin per minute per milligram at pH 7.3 and 37 ° (Sigma Chemical Co.), is not further purified. Dextran, 250,000 average molecular weight, Sigma Chemical Co. Ficoll, Pharmacia Fine Chemicals, Inc. Polyethylene glycol 6000, Union Carbide Corp., Chemicals Div., or Sigma Chemical Co. Potassium phosphate buffer, 50 mM, pH 7.2 at 0 °
Procedure. Nine portions of liver, 250 g each, free of connective tissues, are chopped fine with sharp knives and passed through a coarse strainer at 0o-4 °. The tissue is then suspended to approximately 4 liters in sucrose-phosphate-EDTA and homogenized by hand with two passes in a loose-fitting glass/Teflon homogenizer. After homogenization the suspension is further diluted to 7 liters and centrifuged at 400 g14 for 15 min. Decant the supernatant solution through several layers of cheesecloth and dilute the solution with an equal volume of sucrose-phosphateEDTA. Centrifuge at 10,400 g for 15 min. Discard the supernatant solution. Homogenize the pellets in 0.25 M neutralized sucrose and dilute to 1.4 liters. Centrifuge at 7500 g for 15 min. Discard the supernatant solution. Homogenize the pellets in 0.15 M KCI-Tris-phosphate, dilute to 500-600 ml and centrifuge as in the preceding step. The mitochondrial pellets may be kept in ice if they are to be used the following day or in the freezer if they are to be used later. Extraction of Monoamine Oxidase
Washing of Particles and Phospholipase Digestion. Any convenient quantity of mitochondria may be extracted. A close-fitting glass/Teflon homogenizer 15 is used to homogenize the mitochondria in 10 volumes of cold distilled water. Centrifuge at 105,000 g for 15 min. From this point in the preparation all operations are performed at room temperature, 20 °25 °, including centrifugations, unless otherwise specified. Discard the supernatant solution and suspend the pellets in 0.1 M TEA buffer. Determine protein and activity. The suspended particles should be diluted with TEA buffer to a final protein concentration of 20-30 mg/ml. Add sufficient 1.0 M CaCI2 to make the final concentration 25 mM CaCI2. Add 1 mg of the partially purified phospholipase A for each 300 mg of protein and 1 mg of phospholipase C for each 500 mg of protein. Digest the 14All values for centrifugal force refer to the maximum. l~ Glenco Scientific, Inc., Cat. No. 8108-80, with 0.008 inch clearance.
[49]
BEEF LIVER MONOAMINE OXIDASE
499
particles for 2 hr, with stirring, at 30 °. Maintain the pH at 7.2 with 2 M NH4OH, as required. Centrifuge the suspended particles at 105,000 g for 15 min. Discard the supernatant solution and homogenize the pellets in 0.1 M TEA buffer. Dilute the suspension to 10-15 mg/ml. Triton Extraction. Add 10% Triton X-100 to give 1 mg of Triton per 3 mg of protein; homogenize briefly and stir for 25 min. Centrifuge at 105,000 g for 15 min as before. Retain the supernatant solution. Discard the pellets if less than 10% of the total activity remains in them. If in excess of 10% remains, wash the pellets in one-fourth the original volume of 0.1 M TEA buffer and centrifuge as before. Add the supernatant solution to the Triton extract. Polymer Partition. For each 4 ml of combined Triton extract and washes add: 0.440 g of dextran, 0.480 g of Ficoll, and 0.320 g of polyethylene glycol; mix the solids together, add the Triton extract, and then add 0.76 ml of distilled water for each 4 ml of combined extract. Stir until the solids are dissolved, approximately 30 min. Centrifuge the resulting emulsion at 60,000 g for 20 min. The emulsion separates into three phases, with a substantial interface between the bottom dextran phase and the middle Ficoll phase. The interface is generally sufficiently compact so that fixed-angle centrifuge rotors may be used without remixing when the rotor is stopped. A two-step centrifugation method l~a may be employed when large volumes are being centrifuged. This consists of centrifugation at 1500 g for 20 min in any large swinging-bucket rotor. The lower dextran phase and the upper polyethylene glycol phases will be relatively clear and can be discarded by aspirating away the clear portions. The middle Ficoll phase, which will be quite turbid, is retained together with loose interfacial material at the bottom of the Ficoll phase and can then be transferred to suitable tubes for the higher speed centrifugation. Carefully aspirate all the liquid phases from the semisolid interface. Suspend the interfacial material in 0.1 M TEA buffer diluting to 7-10 mg of protein per milliliter. Centrifuge at 41,000 g for 20 min. Discard the largely inactive pellets and transfer the supernatant solution to suitable centrifuge tubes. Centrifuge at 252,000 g for 60 min at 0o-4 °. The enzyme is obtained as a very small bright yellow pellet. Discard the supernatant solution and carefully drain the tubes, but do not allow the pellet to dry. Quantitatively transfer the pellet to a small glass/Teflon homogenizer and suspend to a small volume (1-4 ml, depending on the amount of mitochondria taken) with 50 mM sodium phosphate buffer. 15~ The two-step centrifugation method produces smaller yields because a single, tight interface is not obtained.
500
FLAVOPROTEINS
[49]
The solution of enzyme should be bright yellow and slightly opalescent. It may be clarified by centrifuging at 41,000 g for 5-10 min at 4 °. Storage. The enzyme will keep at least 1 month in buffer at 00-4 ° with minimum loss of activity when protected from light. It may be stored for much longer periods at - 1 0 ° to - 2 0 ° if the pellet is suspended in 50% (v/v) glycerol in 50 mM sodium phosphate, pH 7.2. This permits storage for several months without freezing, which inactivates the enzyme. Glycerol may be removed by diluting with phosphate buffer and centrifuging for 5 rain at 41,000 g at 0°-4 °, followed by centrifugation at 252,000 g for 60 min at 4 °. Comments on the Procedure. The specific activity of the enzyme in the last step may vary from 1.5 to 3.0 units per milligram of protein. This variability seems to depend on the quality of the mitochondria. The course of the preparation may be interrupted at any point after a centrifugation step. Pellets may be stored in ice overnight or over a weekend. The Triton extract may also be stored on ice overnight, however, addition of the polymers should not be delayed more than overnight since the enzyme is slowly inactivated in Triton solutions, even at 0 °. The extract should be quickly warmed in a 30 ° bath to room temperature before the polymers are added. Prolonged standing of the Triton extracts at room temperature should also be avoided. While there is no inactivation of the enzyme, the properties of the lipids in the extract change, possibly as a result of residual phospholipases, and the polymer partition step will no longer give a single compact interface.
Properties of the E n z y m e The enzyme obtained by this procedure is at least 85% pure, as judged by disc electrophoresis in sodium dodecyl sulfate-mercaptoethanol gels. The single polypeptide band seen in these gels has a molecular weight of 62,000. Flavin content, determined by absorbance at 455 nm of the flavin peptides in tryptic-chymotryptic digests and by a fluorometric procedure specific for the cysteinyl-8a-flavin of monoamine oxidase, TMwas 6.8 nmol of flavin per milligram of protein, by both methods, in the preparation given in the table. This corresponds to 1 mol of flavin per 146,000 g of protein. The enzyme is excluded from Sephadex G-200 in gel exclusion 16 A. L. Maycock, R. H. Abeles, J. I. Salach, and T. P. Singer,
(1976).
Biochemistry 15, 114
[49]
BEEF LIVER MONOAMINE OXIDASE
501
MONOAMINE OXIDASE PREPARATION FROM M1TOCHONDRIA OF 1. I kg BEEF LIVER
Step I. Water suspension of
mitochondria 2. Washed particles in T E A 3. Phospholipase-digested particles in Triton 4. Triton extract 5. Interface from polymers 6. 41,000 g supernatant 7. 252,000 g pellet
Volume
Total protein (rag)
Total activity (units)
Specific activity (units/mg)
(ml) 210
6216
132
0.021
100
150 207
4305 2752
112 108
0.026 0.039
85 82
193 64 95 2
1727 1105 700 22
96 80 62 57
0,056 0.072 0.089 2.54
73 61 47 43
Yield (%)
chromatography, indicating a higher apparent molecular weight, perhaps due to formation of polymers as described for the enzyme of beef liver.2 The purified enzyme from beef liver has a broad pH optimum between pH 7.2 and pH 11 and is active toward a number of primary aromatic amines, z The substrate and inhibitor specificity patterns of the enzyme are characteristic of the "B" form. 2' 17 Immunoprecipitation techniques, applied to the enzyme from beef liver, 18 have also indicated that the enzyme from this source contains but a single molecular form of monoamine oxidase. The enzyme has been shown to have covalently linked FAD as a coenzyme.~,19 This covalent linkage and the adjacent peptide sequence have been shown to be Ser-Gly-Gly-Cys-Tyr
I
flavin
with the flavin attached to the cysteinyl residue in a thioether link at the 8or- position of the flavin nucleus.19 Irreversible inhibition of the enzyme by pharmacologic agents of the acetylene type, such as propargylamine (N,N-dimethylpropynylamine), has been shown to occur by formation of an adduct with the N(5) position of the flavin nucleus and rearrangement of the inhibitor moiety to a flavocyanine compound. 16 17 j. p. J o h n s t o n , Biochem. Pharmacol. 17, 1285 (1968); and R. F. Squires, Biochem. Pharmacol. 17, 1401 (1968). 18 B. K. Hartman and S. Udenfriend, Adv. Biochem. Psychopharmacol. 5, 119 (1972). ~a W. H. Walker, E. B. K e a r n e y , R. L. Seng, and T. P. Singer, Fur. J. Biochem. 24, 328 (1971).
BEEF LIVER MONOAMINE OXIDASE
495
of molecular conformation around iron-sulfur centers S-2 and S-3 in the inactive preparations have been revealed as follows: 1. As shown in Fig. 4, EPR signals from center S-3 can be detected only in the reconstitutively active SDH preparations, but not in inactive ones. This indicates that a certain conformational change around this iron-sulfur cluster gives rise to a loss of the reconstitutive activity, namely, capability to transfer electrons to the respiratory chain. A close correlation between these two parameters can be further demonstrated by the parallel decrease of center S-3 signal and reconstitutive activity, upon exposure of SDH to the air. 2° 2. Spin relaxation of center S-2 is slower in reconstitutively inactive preparations than in active ones. This is reflected in the two following observations: (a) Quantitative detection of center S-2 spins at higher temperature range (>30°K) in reconstitutively inactive preparations, but not in active ones. (b) In all SDH preparations, centers S-1 and S-2 are closely located (< 10 ]k) and show spin-spin interactions, namely, release of power saturation of center S-I spins (longer T, component) by cross relaxation with S-2 spins (shorter T1 component). Conversely, center S2 spectra are affected by S-I spins in a low temperature range (4.2°-6°K), seen most clearly as broadening (active preparation) or splitting (inactive preparation) of the principal resonance signal, depending on the spin relaxation times of center S-24 (see Fig. 5). EPR characteristics of iron-sulfur centers in reconstitutively active and inactive succinate dehydrogenase preparations are summarized in Table II.
[49] P r e p a r a t i o n
of Monoamine Oxidase from Beef Liver Mitochondria B y JAMES I. SALACH, JR.
Two preparative procedures for monoamine oxidase have been described that yield an enzyme of high specific activity and purity. One utilizes beef kidney, 1 the other beef liver. 2 These enzyme preparations, of high specific activity, 3 are obtained in yields of 10% and 5-6%, rei H. Y. K. Chuang, D. R. Patek, and L. Hellerman, J. Biol. Chem. 249, 2381 (1974). 2 K. T. Yasunobu, I. Igaue, and B. Gomes, Adv. Pharmacol. 6, 43 (1%8); B. Gomes, I. lgaue, H. G. Kloepfer, and K. T. Yasunobu, Arch. Biochem. Biophys. 132, 16 (1%9). 3 Direct comparison of specific activities in these two preparations with the present preparation cannot be made. While the units of activity employed by these investigators may
496
FLAVOPROTEINS
[49]
spectively, o f the total in the mitochondria after c o m p l e x multiple chromatographic procedures. This m e t h o d presented is simple, rapid, and requires no chromatography. The yield is 3- to 5-fold higher. P r i n c i p l e . The method is b a s e d on modification of the lipids of the mitochondrial outer m e m b r a n e by treatment with phospholipases so that low concentrations of Triton X-100 will extract the e n z y m e nearly quantitatively. The detergent and phospholipids are r e m o v e d b y partition in a three-phase p o l y m e r system. 4 The e n z y m e , present in the interface b e t w e e n the middle and lower phases, is separated in good yield f r o m insoluble material and residual p o l y m e r s by centrifugation.
Assay Method The e n z y m e is assayed by a modification of the m e t h o d of T a b o r et ol. 5 Reagents
1. Potassium p h o s p h a t e , 0.2 M , p H 7.2 at 30 °, containing 0.6% (w/ v) Triton X-1006 2. Benzylamine, 0.1 M, neutralized to p H 7 with 6 M HCI P r o c e d u r e . Phosphate buffer, 1.0 ml, and glass-distilled water, 1.85 ml, are placed in a 4-ml quartz cuvette, brought to 30 ° by incubation for 3-5 min in a w a t e r bath, and placed in the s p e c t r o p h o t o m e t e r . A suitable aliquot of e n z y m e (10-50 ~1) is added and mixed, and the blank rate is m e a s u r e d at 250 nm. The reaction is initiated by addition of 0.1 ml of benzylamine. The reverse order of addition of e n z y m e and b e n z y l a m i n e can be used if desired. The value taken for the molar extinction of b e n z a l d e h y d e at 250 nm is 12,080. 7 One unit is defined as formation of 1.0 ttmol of product per minute. 8
be interconverted with the unit employed in this method, one or more of the conditions of the assay: volume, substrate concentration, pH, and temperature are also varied. A typical preparation of specific activity 2.28 units/mg in the present assay, when assayed by the method used for the beef kidney enzymel had an activity of 3.96 units/mg or 21,719 units/mg of the units used by Chuang et al. 1 4 P.-A. Albertsson, Biochemistry 12, 2525 (1973). C. W. Tabor, H. Tabor, and S. M. Rosenthal, J. Biol. Chem. 193, 265 (1951). 6 Distill off acetaldehyde from commercial Triton in a rotary evaporator at 80° and vacuum from a water aspirator for 3-4 hr before use. 7 "Organic Electronic Spectral Data," Vol. IV. Wiley (Interscience), New York, 1959. s At the substrate concentration used (3.3 mM) the reaction rates obtained are 90-95% of the rates at infinite substrate concentration measured by extrapolation of double reciprocal plots.
[49]
BEEF LIVER MONOAMINE OXIDASE
497
Measurement of Protein
Protein is determined by the biuret method 9 with the addition of 0.05 ml of 10% (w/v) sodium deoxycholate to solubilize lipids. One milligram of bovine serum albumin standard in 3.0 ml final volume is taken to give an absorbance at 540 nm of 0.095. Preparation of Mitochondria
Mitochondria are prepared essentially by the method of Kearney et al. lO Reagents
1. Sucrose, 0.25 M, potassium phosphate, 10 mM, EDTA, 0.5 mM, pH 7.0-7.2 at 0 ° 2. Sucrose, 0.25 M, neutralized to pH 7.0-7.2 at 0 ° with KOH 3. KC1, 0.15 M, Tris-phosphate, 10 mM, pH 7.0-7.2 at 0 ° 4. TEA 11 buffer, 0.1 M, pH 7.2, at 30 ° 5. CaC12, 1.0 M 6. Triton X-100, 10% (w/v) in water 6 7. Phospholipase A, partially purified, specific activity 15,000 units/mg 12 8. Phospholipase C, from Clostridium welchii, 13 type I, specific activity 5/zmol of water-soluble phosphorus released from egg 9 A. G. Gornall, C. J. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949). Protein may also be measured by the Lowry method (O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951)). This is recommended for the final step because of the smaller amounts of protein required. 10 E. B. Kearney, J. I. Salach, W. H. Walker, R. L. Seng, W. Kenney, E. Zeszotek, and T. P. Singer, Eur. J. Biochem. 24, 321 (1971). 1, TEA: triethanolamine (2,2',2"-nitrilotriethanol hydrochloride), molecular weight 185.65. 12 Phospholipase A is prepared from Naja naja venom by chromatography on Sephadex G75 by the method of T. C. Cremona and E. B. Kearney, J. Biol. Chem. 239, 2328 (1964). Fractions having a specific activity of 15,000-17,000 units per milligram of protein, or better, in the assay specified below are combined and used for the digestion. Note that the same amount of protein is used even if the phospholipase A sample has as much as 35,000 units of activity per milligram because excess phospholipase A does not interfere. Phospholipase activity is estimated with a recording pH meter by the method of J. 1. Salach, P. Turini, R. Seng, and T. P. Singer, J. Biol. Chem. 246, 331 (1971). This assay is less involved and more rapid than the manometric procedure used by Cremona and Kearney. One unit of phospholipase A activity is defined as that enzyme activity which releases sufficient fatty acid from purified egg lecithin to cause a decrease of 0.01 pH in 30 rain at pH 8.0 and 25 °. 13 Phospholipase C from C. welchii is used since it is reported to hydrolyze lysolecithin and phosphatidylethanolamine as well as lecithin [T. Takahashi and H. H. O. Schmid, Chem. Phys. Lipids 2, 220 (1968)].
498
FLAVOPROTEINS
9. 10. 11. 12.
[49]
lecithin per minute per milligram at pH 7.3 and 37 ° (Sigma Chemical Co.), is not further purified. Dextran, 250,000 average molecular weight, Sigma Chemical Co. Ficoll, Pharmacia Fine Chemicals, Inc. Polyethylene glycol 6000, Union Carbide Corp., Chemicals Div., or Sigma Chemical Co. Potassium phosphate buffer, 50 mM, pH 7.2 at 0 °
Procedure. Nine portions of liver, 250 g each, free of connective tissues, are chopped fine with sharp knives and passed through a coarse strainer at 0o-4 °. The tissue is then suspended to approximately 4 liters in sucrose-phosphate-EDTA and homogenized by hand with two passes in a loose-fitting glass/Teflon homogenizer. After homogenization the suspension is further diluted to 7 liters and centrifuged at 400 g14 for 15 min. Decant the supernatant solution through several layers of cheesecloth and dilute the solution with an equal volume of sucrose-phosphateEDTA. Centrifuge at 10,400 g for 15 min. Discard the supernatant solution. Homogenize the pellets in 0.25 M neutralized sucrose and dilute to 1.4 liters. Centrifuge at 7500 g for 15 min. Discard the supernatant solution. Homogenize the pellets in 0.15 M KCI-Tris-phosphate, dilute to 500-600 ml and centrifuge as in the preceding step. The mitochondrial pellets may be kept in ice if they are to be used the following day or in the freezer if they are to be used later. Extraction of Monoamine Oxidase
Washing of Particles and Phospholipase Digestion. Any convenient quantity of mitochondria may be extracted. A close-fitting glass/Teflon homogenizer 15 is used to homogenize the mitochondria in 10 volumes of cold distilled water. Centrifuge at 105,000 g for 15 min. From this point in the preparation all operations are performed at room temperature, 20 °25 °, including centrifugations, unless otherwise specified. Discard the supernatant solution and suspend the pellets in 0.1 M TEA buffer. Determine protein and activity. The suspended particles should be diluted with TEA buffer to a final protein concentration of 20-30 mg/ml. Add sufficient 1.0 M CaCI2 to make the final concentration 25 mM CaCI2. Add 1 mg of the partially purified phospholipase A for each 300 mg of protein and 1 mg of phospholipase C for each 500 mg of protein. Digest the 14All values for centrifugal force refer to the maximum. l~ Glenco Scientific, Inc., Cat. No. 8108-80, with 0.008 inch clearance.
[49]
BEEF LIVER MONOAMINE OXIDASE
499
particles for 2 hr, with stirring, at 30 °. Maintain the pH at 7.2 with 2 M NH4OH, as required. Centrifuge the suspended particles at 105,000 g for 15 min. Discard the supernatant solution and homogenize the pellets in 0.1 M TEA buffer. Dilute the suspension to 10-15 mg/ml. Triton Extraction. Add 10% Triton X-100 to give 1 mg of Triton per 3 mg of protein; homogenize briefly and stir for 25 min. Centrifuge at 105,000 g for 15 min as before. Retain the supernatant solution. Discard the pellets if less than 10% of the total activity remains in them. If in excess of 10% remains, wash the pellets in one-fourth the original volume of 0.1 M TEA buffer and centrifuge as before. Add the supernatant solution to the Triton extract. Polymer Partition. For each 4 ml of combined Triton extract and washes add: 0.440 g of dextran, 0.480 g of Ficoll, and 0.320 g of polyethylene glycol; mix the solids together, add the Triton extract, and then add 0.76 ml of distilled water for each 4 ml of combined extract. Stir until the solids are dissolved, approximately 30 min. Centrifuge the resulting emulsion at 60,000 g for 20 min. The emulsion separates into three phases, with a substantial interface between the bottom dextran phase and the middle Ficoll phase. The interface is generally sufficiently compact so that fixed-angle centrifuge rotors may be used without remixing when the rotor is stopped. A two-step centrifugation method l~a may be employed when large volumes are being centrifuged. This consists of centrifugation at 1500 g for 20 min in any large swinging-bucket rotor. The lower dextran phase and the upper polyethylene glycol phases will be relatively clear and can be discarded by aspirating away the clear portions. The middle Ficoll phase, which will be quite turbid, is retained together with loose interfacial material at the bottom of the Ficoll phase and can then be transferred to suitable tubes for the higher speed centrifugation. Carefully aspirate all the liquid phases from the semisolid interface. Suspend the interfacial material in 0.1 M TEA buffer diluting to 7-10 mg of protein per milliliter. Centrifuge at 41,000 g for 20 min. Discard the largely inactive pellets and transfer the supernatant solution to suitable centrifuge tubes. Centrifuge at 252,000 g for 60 min at 0o-4 °. The enzyme is obtained as a very small bright yellow pellet. Discard the supernatant solution and carefully drain the tubes, but do not allow the pellet to dry. Quantitatively transfer the pellet to a small glass/Teflon homogenizer and suspend to a small volume (1-4 ml, depending on the amount of mitochondria taken) with 50 mM sodium phosphate buffer. 15~ The two-step centrifugation method produces smaller yields because a single, tight interface is not obtained.
500
FLAVOPROTEINS
[49]
The solution of enzyme should be bright yellow and slightly opalescent. It may be clarified by centrifuging at 41,000 g for 5-10 min at 4 °. Storage. The enzyme will keep at least 1 month in buffer at 00-4 ° with minimum loss of activity when protected from light. It may be stored for much longer periods at - 1 0 ° to - 2 0 ° if the pellet is suspended in 50% (v/v) glycerol in 50 mM sodium phosphate, pH 7.2. This permits storage for several months without freezing, which inactivates the enzyme. Glycerol may be removed by diluting with phosphate buffer and centrifuging for 5 rain at 41,000 g at 0°-4 °, followed by centrifugation at 252,000 g for 60 min at 4 °. Comments on the Procedure. The specific activity of the enzyme in the last step may vary from 1.5 to 3.0 units per milligram of protein. This variability seems to depend on the quality of the mitochondria. The course of the preparation may be interrupted at any point after a centrifugation step. Pellets may be stored in ice overnight or over a weekend. The Triton extract may also be stored on ice overnight, however, addition of the polymers should not be delayed more than overnight since the enzyme is slowly inactivated in Triton solutions, even at 0 °. The extract should be quickly warmed in a 30 ° bath to room temperature before the polymers are added. Prolonged standing of the Triton extracts at room temperature should also be avoided. While there is no inactivation of the enzyme, the properties of the lipids in the extract change, possibly as a result of residual phospholipases, and the polymer partition step will no longer give a single compact interface.
Properties of the E n z y m e The enzyme obtained by this procedure is at least 85% pure, as judged by disc electrophoresis in sodium dodecyl sulfate-mercaptoethanol gels. The single polypeptide band seen in these gels has a molecular weight of 62,000. Flavin content, determined by absorbance at 455 nm of the flavin peptides in tryptic-chymotryptic digests and by a fluorometric procedure specific for the cysteinyl-8a-flavin of monoamine oxidase, TMwas 6.8 nmol of flavin per milligram of protein, by both methods, in the preparation given in the table. This corresponds to 1 mol of flavin per 146,000 g of protein. The enzyme is excluded from Sephadex G-200 in gel exclusion 16 A. L. Maycock, R. H. Abeles, J. I. Salach, and T. P. Singer,
(1976).
Biochemistry 15, 114
[49]
BEEF LIVER MONOAMINE OXIDASE
501
MONOAMINE OXIDASE PREPARATION FROM M1TOCHONDRIA OF 1. I kg BEEF LIVER
Step I. Water suspension of
mitochondria 2. Washed particles in T E A 3. Phospholipase-digested particles in Triton 4. Triton extract 5. Interface from polymers 6. 41,000 g supernatant 7. 252,000 g pellet
Volume
Total protein (rag)
Total activity (units)
Specific activity (units/mg)
(ml) 210
6216
132
0.021
100
150 207
4305 2752
112 108
0.026 0.039
85 82
193 64 95 2
1727 1105 700 22
96 80 62 57
0,056 0.072 0.089 2.54
73 61 47 43
Yield (%)
chromatography, indicating a higher apparent molecular weight, perhaps due to formation of polymers as described for the enzyme of beef liver.2 The purified enzyme from beef liver has a broad pH optimum between pH 7.2 and pH 11 and is active toward a number of primary aromatic amines, z The substrate and inhibitor specificity patterns of the enzyme are characteristic of the "B" form. 2' 17 Immunoprecipitation techniques, applied to the enzyme from beef liver, 18 have also indicated that the enzyme from this source contains but a single molecular form of monoamine oxidase. The enzyme has been shown to have covalently linked FAD as a coenzyme.~,19 This covalent linkage and the adjacent peptide sequence have been shown to be Ser-Gly-Gly-Cys-Tyr
I
flavin
with the flavin attached to the cysteinyl residue in a thioether link at the 8or- position of the flavin nucleus.19 Irreversible inhibition of the enzyme by pharmacologic agents of the acetylene type, such as propargylamine (N,N-dimethylpropynylamine), has been shown to occur by formation of an adduct with the N(5) position of the flavin nucleus and rearrangement of the inhibitor moiety to a flavocyanine compound. 16 17 j. p. J o h n s t o n , Biochem. Pharmacol. 17, 1285 (1968); and R. F. Squires, Biochem. Pharmacol. 17, 1401 (1968). 18 B. K. Hartman and S. Udenfriend, Adv. Biochem. Psychopharmacol. 5, 119 (1972). ~a W. H. Walker, E. B. K e a r n e y , R. L. Seng, and T. P. Singer, Fur. J. Biochem. 24, 328 (1971).
