Bound Monoamine Oxidase: Ph and Temperature Dependences

Inhibitory Effect of NaCI on Hog Kidney Mitochondrial Membrane- Bound Monoamine Oxidase: pH and Temperature Dependences

Yoko Omura

Department of Chemistry, Kanagawa Prefectural College of Nursing and Medical Technology, 50-1 Nakao-Cho, Yokohama 241, Japan

Received April 12, 1995 Accepted September 5, 1995

ABSTRACT-For a further understanding of the inhibitory effect of NaCI on hog kidney mitochondrial monoamine oxidase (MAO), the activity for benzylamine as substrate was assayed spectrophotometrically in the absence and presence of NaCI for mitochondrial outer membrane preparations as well as whole mitochondria. The effect of CaC12 was also examined for comparison. The inhibition by NaCI but not CaC12 was strongly pH dependent. The pH dependence of the inhibitory effect of NaCI in phosphate buffer was parallel to the pH dependence of the MAO activity itself. The point at which the slope of the Arrhenius plot in the absence of NaCl decreases with increasing temperature was to be 32.31C at pH 7.0 and 30.41C at pH 7.5 in phosphate buffer, while the Arrhenius plot in the presence of NaCI exhibited discontinuities without change in the slope in small temperature ranges, 39.21C -40.01C and 33.0'C -34.21C. It was estimated that the inhibitory effect of NaCl was due to a pH and temperature sensitive cooperative state change involving MAO protein and boundary lipids, while the effect of CaC12 could be induced by specific Ca 21 binding to acidic phospholipids.

Keywords: Hog kidney monoamine oxidase, Whole mitochondria, Mitochondrial outer membrane, Inhibitory effect of NaCl, Arrhenius plot

Monoamine oxidase (MAO, EC 1. 4. 3. 4) is known as ney mitochondrial MAO to determine if membranous a mitochondrial outer membrane enzyme, which oxida- MAO is present in vivo, we found that (NH4)2SO4 and tively deaminates neurotransmitters such as catechol- NaCl markedly inhibited the MAO activity at fairly low amines and also exogeneous biogenic amines. At least two salt concentrations. We have reported the inhibitory types of MAO (MAO-A and MAO-B) have been iden- effect of NaCI on MAO in hog kidney whole mitochon- tified based on the differences in their substrate specific- dria (23). The inhibition was reversible and noncompeti- ities and inhibitor sensitivities (1, 2) (for a more recent tive. The decrease in the inhibition of the Triton X-100 review, see Ref. 3). While there was convincing ex- solubilized MAO suggested that the mitochondrial mem- perimental support for the hypothesis that the two types brane was at least in part responsible for the inhibition: of MAO were distinct enzyme proteins (4-7), it was sug- 70% inhibition at a NaCI concentration of 1070(0.17 M), gested by several workers that they might be the same 85070 at 2070, 92010 at 3070 and 95070 at 5010 for whole protein in different membrane lipid environments (8-10). mitochondria in phosphate buffer, pH 7.0 at 38C;°53010 Recently, cloning of the cDNAs for MAO-A and MAO-B at 1010,67010 at 2070,72% at 3% and 73010at 5070under the demonstrated that the two forms of the enzyme share same conditions for Triton X-100 solubilized MAO (23). about 70% sequence identity and were encoded by differ- Therefore, preceding the purification and reconstitu- ent genes derived from the same ancestral gene. Full tion of MAO for the study on the artificial model system, length cDNA clones for human (11-13), bovine (14) and we investigated the mitochondrial MAO from both en- rat (15) MAO-A and human (11, 13) and rat (16) MAO-B zymatic and spectroscopic aspects by using mitochondrial have been characterized. However, the in vivo presence of outer membrane preparations to which MAO is tightly membranous MAO-A and MAO-B and the nature of bound. These investigations are expected to provide their interaction with lipids are still unsolved biochemical information about the functional state of hog kidney questions (3, 17-22). Further investigations are needed. mitochondrial membrane-bound MAO. In the process of purifying and reconstituting hog kid- To investigate the effect of NaCI on the spectral characteristics of the mitochondria) outer membranes, we described below. studied the effect of NaCI on the near infrared excited The sub fractionation of mitochondria by digitonin Fourier transform Raman spectra of hog kidney treatment was carried out essentially according to the mitochondrial outer membrane preparations. We ob- method of Schnaitman et al. and Okamoto et al. (27 - served a spectral change in the phospholipid acyl chain 29). A 2% digitonin solution was prepared just prior to CC stretching region, indicating that the presence of use by adding digitonin (biochemical use; Wako Chemi- NaCI decreases the gauche character of the phospholipid cals Ltd., Osaka) to the warm 0.25 M sucrose solution in acyl chains (24). The observed spectral change was differ- a boiling water bath. The cold 2% digitonin solution was ent from that in an aqueous dispersion of phosphatidyl- added to the mitochondrial fraction in the ice bath with choline, for which it has been reported that monovalent continuous stirring for 15 min so that the relative weight ions such as Na+ do not appreciably affect the I,,,,/ of digitonin to 10 mg mitochondrial protein was 1.2-1.5 'gaucheratio (25, 26). It was suggested that the presence of mg. The suspension was diluted by 34 ml of 0.25 M su- membrane proteins was required for this membrane con- crose and was further stirred for 1-2 min at 0'C. The densation effect by NaCI (24). It was also estimated that diluted suspension was centrifuged at 9,500 x g for 10 min the presence of NaCI might increase the amount of im- at 4C. The first 9,500 x g pellet (inner membrane and mobilized boundary lipid (24). matrix fractions) was suspended in 34 ml of 0.25 M su- In the present study, to elucidate the cause of the crose, and the suspension was recentrifuged at 9,500 x g inhibitory effect of NaCI on hog kidney mitochondrial for 10 min. The resulting pale yellow supernatant was MAO, measurements of the inhibitory effect of NaCI on combined with the first 9,500 x g supernatant. The com- mitochondrial outer membrane preparations as well as bined supernatant was centrifuged at 105,000 x g for 90 whole mitochondria were performed at various concen- min at 4 C by a Beckman L5-65 ultracentrifuge with a trations of NaCl and pH values. Furthermore, the inhibi- Type 65 rotor (Beckman, Palo Alto, CA, USA). tory effects of 0.1 M NaCI were measured in phosphate The 105,000 x g pellet is the outer membrane fraction. buffer, pH 6.0, 6.5, 7.0 and 7.5 at 29°C and 8.0 at 38C, The activity of MAO, which is a marker enzyme of the for mitochondrial outer membrane preparations and mitochondrial outer membrane, was assayed for the whole mitochondria. To evaluate the thermal properties 105,000 x g pellet at 29C in phosphate buffer, pH 7.5. of the effect of NaCI, I measured the MAO activity in hog The pellet was stored at -201C just until used. kidney mitochondrial outer membrane preparations for oxidative deamination of benzylamine as a substrate at Determination of protein concentration various temperatures in the absence and presence of The concentration of mitochondrial protein was rapid- NaCI, and Arrhenius plots were made from the data. The ly determined spectrophotometrically by the Coomasie results are reported and discussed in this article in terms blue G dye-binding assay (30) preceding the procedure of of a cooperative change of state involving membrane digitonin treatment. Bovine serum albumin was used for proteins as well as phospholipid acyl chains. the standard protein.

MATERIALS AND METHODS Standard assay of MAO activity The MAO activity was assayed spectrophotometrically Preparation of mitochondrial outer membranes on the basis of the method of Tabor et al. (31) by record- Hog kidney mitochondrial outer membranes were ing the absorbance at 250 nm due to benzaldehyde at 29C prepared as described in our previous paper (24). Briefly, in a standard assay system containing 0.1 ml of 0.1 M 50 g of hog kidney cortex was cut into small pieces and benzylamine hydrochloride as substrate, 1.0 ml of 0.2 M homogenized with a teflon homogenizer in 450 ml of 10 Na-K phosphate buffer adjusted to the appropriate pH by mM Na-K phosphate buffer, pH 7.5 containing 0.25 M mixing 0.2 M Na2HPO4 and KH2PO4 solution at the same sucrose (1 : 9 w/v). The homogenate was centrifuged at concentration and 0.5 ml of enzyme solution for the 3.0- 600 x g for 10 min at 4 °C, and the supernatant was recen- ml final volume of reaction mixture in the quartz cell of trifuged at 5,000 x g for 10 min. The resulting precipitate 10-mm path length. The final concentrations of phos- (crude mitochondrial fraction) was suspended in 200 ml phate buffer and substrate were 0.067 M and 3.33 x 10-3 of the same buffer (1 : 4 w/v) and centrifuged at 5,000 x g M, respectively. A blank cell was set up similarly in the for 10 min (mitochondrial fraction, 9-10 ml). One por- reference side except for omission of substrate, i.e., 0.1 tion of the mitochondrial fraction was diluted to 1/100 in ml of H20 was added instead of the same volume of sub- concentration by 0.25 M sucrose, and this was used to strate solution. The initial velocity 4A250/min was regard- determine the protein concentration. The mitochondrial ed as the MAO activity. preparation was immediately used for the procedure Measurements of inhibitory effects of salts on MAO RESULTS activity Concentration and pH dependences: The MAO activ- Concentration dependence of inhibitory effects of NaCI ity measurements were made by the procedure described and CaC12 on MAO above in the absence and the presence of NaCI in phos- Figure 1 shows the concentration dependence of the phate buffer at pH 6.5, 7.0 and 7.5 and in Tris-HC1 buffer inhibitory effect of NaC1 on the activity of hog kidney at pH 7.5 and 8.0. Similar experiments were performed mitochondrial MAO for whole mitochondria and with CaC12 for comparison, except that only Tris-HC1 mitochondrial outer membranes. Figure 2 shows the buffer was used for the calcium salt. The effect of MgC12 results of the similar experiments for CaC12 in Tris-HC1 on whole mitochondria was also investigated for com- buffer, pH 7.5 and pH 8.0. Stronger inhibition than in the parison to that of CaC12. case of NaCI was found. Since similar features of the in- The percent relative MAO activity at each pH in the hibitions by salts were found for both whole mitochon- presence of salt to that in the absence of salt was deter- dria and outer membranes, it is estimated that incomplete mined at each concentration of 0-0.6 M salt at 29C. In vesicles are formed in the outer membrane preparations the assay system, the same concentration of salt as in the sample cell was contained in the reference cell. pH dependence of inhibitory effect of NaCl at 291C and 38'C: The percent relative MAO activity in phosphate buffer in the presence of 0.1 M NaC1 to that in the absence of NaC1 was determined at pH 6.0, 6.5, 7.0, 7.5 and 8.0 at 29'C and 38C.

Temperature dependence of inhibitory effect of NaCl on MAO activity The inhibitory effect of 0.1 M NaCI on hog kidney mitochondrial MAO activity was measured at various temperatures by the procedure described above. The percent relative value of the MAO activity in the presence of NaC1 to that in the absence of NaC1 was determined at each temperature from 28C -441C in phosphate buffer, pH 7.0. Here, the percent inhibition means 100% minus the percent relative activity. The temperature of the reaction mixture in the reaction cuvette was controlled to be constant during recording of the absorbance at 250 nm. The temperature of the sample solution was monitored by a Chromel-Alumel thermocouple inserted into the cuvette in the cell holder thermostatically controlled by circulat- ing water at a constant temperature.

Temperature dependence of MAO activity in the absence and presence of NaCI The MAO activities in phosphate buffer at pH 7.0 and pH 7.5, respectively, were measured for mitochondrial outer membranes in the absence of NaCI at various temperatures; and Arrhenius plots were made by plotting the Fig. 1. Concentration dependence of inhibitory effect of NaC1 on value of - logiA250/min against ]IT. Here, T is the ab- hog kidney MAO activity at 291C for whole mitochondria (A) and outer membranes (B). All curves are from independent experiments. solute temperature. The same enzyme solution was used Each point was obtained from one set of measurement. a: Tris-HCI for one set of Arrhenius plots. The same kind of meas- buffer, pH 8.0; b: Tris-HCl buffer, pH 7.5; c: phosphate buffer, pH urements were performed for the reaction mixture in 7.5; d: phoshate buffer, pH 7.0; e: phosphate buffer, pH 6.5. The phosphate buffer, pH 7.0 in the presence of 0.1 M NaC1, enzyme solution was prepared by diluting whole mitochondria to 1150 or 1/100 in concentration (v/v) with 0.25 M sucrose for panel and an Arrhenius plot was made. A and by vigorously agitating the ultracentrifuge pellet with 10 ml of 0.25 M sucrose in the ultracentrifugation tube using a vortex mixer for panel B. Fig. 3. Concentration dependence of inhibitory effect of MgC12on hog kidney MAO activity for whole mitochondria at 291C. The enzyme solution was prepared by diluting whole mitochondria to 1/50 concentration (v/v) by 0.25 M sucrose. a: Tris-HCI buffer, pH 8.0; b: Tris-HC1 buffer, pH 7.5. Each point was obtained from one set of measurement.

Fig. 2. Concentration dependence of inhibitory effect of CaC12 on hog kidney MAO activity at 291C for whole mitochondria (A) and outer membranes (B). All curves are from independent experiments. Each point was obtained from one set of measurement. a: Tris-HC1 buffer, pH 8.0; b: Tris-HCl buffer, pH 7.5. The enzyme solution was prepared by diluting whole mitochondria to 1/100 in concentration (v/v) with 0.25 M sucrose for panel A and by vigorously agitating the ultracentrifuge pellet with 10 ml of 0.25 M sucrose in the ultracentrifugation tube using a vortex mixer for panel B.

as shown in the electron microscopic photograph (Fig. 9 in Ref. 29). However, in the case of NaCI, the inhibitions are rather stronger for outer membranes than for whole mitochondria (Fig. 1), while such a difference between whole mitochondria and outer membranes is not observed in the case of CaC12 (Fig. 2). The inhibition by NaC1 is strongly pH dependent over the entire concentration range. Increasing the pH decreases the inhibition in both phosphate and Tris-HCI buffer (Fig. 1), while that by CaC12 is almost pH independent (Fig. 2). The inhibitory effect of MgC12 showed an unexpected pH dependency as shown in Fig. 3. The inhibitory effect Fig. 4. Influence of the kind of phosphate buffer on inhibitory of MgC12 is rather more similar to that of NaCI in Tris- effects of NaCI (A) and KCI (B) on hog kidney mitochondrial MAO. HCl buffer, pH 7.5 and pH 8.0, than that of CaC12. The 0: Na-K phosphate buffer, 0: Na phosphate buffer, x : K phos- inhibition by MgC12 appears to be about twice as strong phate buffer. Each point was obtained from one set of measurement. as that by NaCI at the same concentration. As shown in buffer. Figure 4B shows the results of the similar experi- Fig.1, the inhibition by NaCI at pH 7.5 in phosphate ments for the inhibitory effects of KCI. The inhibitory buffer is stronger than that at the same pH in Tris-HC1 effects of KCI were observed to be identical to those of buffer. NaCl in all types of phosphate buffer. Figure 4A shows the concentration dependence of the inhibitory effects of NaCI on the MAO activity for the pH dependence of inhibition by NaCI at 291C and 38C whole mitochondrial preparation in Na-K (identical to Figure 5A shows the pH dependence of the inhibition the curve d in Fig. IA), K and Na phosphate buffers, pH of mitochondrial MAO activity by 0.1 M NaCI in phos- 7.0. The results are independent of the kind of phosphate phate buffer for both whole mitochondria and outer membranes at 29C and 38C. These curves (a, b, c and d) are each the sigmoid type, and their midpoints are between 7.0 and 7.5. There is no difference in inhibition between whole mitochondria and outer membranes at 29C.° However, the decrease in the inhibition with increasing temperature exhibits the largest value, 20%, at pH 7.0 for the outer membranes. At pH 8.0, the relative activity increase by temperature elevation is negligible in both whole mitochondria and outer membranes. Figure 5B shows the pH dependence of the MAO activity for mitochondrial outer membrane preparations in the absence and presence of NaCI at 29C. Figure 5 (A and B) shows that the pH dependence of the MAO activity in the presence of 0.1 M NaCl is parallel to that in the absence of NaCl and that the dependence of the inhibitory effect of 0.1 M NaCI on MAO activity is also parallel to that of the MAO activity. Similar results were also obtained at 38C and for whole mitochondria (data are not shown).

Fig. 5. pH dependence of hog kidney MAO activity in the absence and presence of NaCl. A: pH dependence of the inhibitory effect of 0.1 M NaCI on hog kidney mitochondrial MAO activity in phosphate buffer for whole mitochondria and outer membranes. a (O) and b (0): whole mitochondria at 291C and 381C, respectively; c (U`) and d (A): mitochondrial outer membranes at 291C and 38C, respectively. Curves a, b, c and d come from independent experi- Fig. 6. Temperature dependence of inhibitory effect of 0.1 M NaC1 ments. Each point was obtained from one set of measurement. En- on the MAO activity for hog kidney mitochondrial outer membranes zyme solution was prepared by diluting whole mitochondria to in phosphate buffer, pH 7.0. The relative activity in 076of the MAO 1/100 concentration (v/v) by 0.25 M sucrose (a, b) and by vigorously activity in the presence of NaCI to that in the absence of NaCI is agitating the ultracentrifuged pellet with 11 ml of 0.25 M sucrose plotted as a function of temperature in C . Points were obtained using a vortex mixer (c, d). B: pH dependence of MAO activity in the with two separate membrane preparations. Enzyme solution was absence (e) and presence (f) of 0.1 M NaCI at 291C. They are the prepared in a similar way as described in the captions for Figs. 1, original data points of curve c (A). 2 and 5 (8 ml of 0.25 M sucrose for the pellet). Fig. 7. Arrhenius plots of the MAO activity for mitochondrial outer membranes in the absence of NaCI in phosphate buffer, pH 7.0 and pH 7.5. 0: Enzyme solution was prepared in a similar way to that described in the captions for Figs. 1, 2 and 5 (8 ml of 0.25 M sucrose for the pellet). 0: Separate set of experiments from that expressed by 0. The membrane pellet used was the preparation ultracentrifuged at the same time as that for the data points 0. Each point was obtained from one measurement of activity.

Temperature dependence of inhibitory effect of NaCI on in hog kidney mitochondrial outer membranes; i.e., two MA O independent straight lines of the Arrhenius plot do not Figure 6 shows the temperature dependence of the necessarily intersect at the phase transition temperature. inhibitory effect of 0.1 M NaCI on the MAO activity for In fact, the usual phase transition temperature of mam- hog kidney mitochondrial outer membranes in phosphate malian membranes is much lower than the physiological buffer, pH 7.0. The inhibition does not change from 281C temperature and membranes have a fluid nature (32, 33). to 321C. However, between 321C to 381C, the inhibition It appears that the distribution of lipids in the membranes decreases by about 20%, and it increases again slightly in is heterogeneous. the range from 381C to 441C. Arrhenius plot in the presence of NaCI Arrhenius plot in the absence of NaCI Figure 8 shows the Arrhenius plot in the presence of Figure 7 shows the Arrhenius plots in the absence of 0.1 M NaCl in phosphate buffer, pH 7.0. The presence NaCI in phosphate buffer, pH 7.0 and pH 7.5. In both of NaC1 not only inhibited the MAO activity but also cases, as temperature is increased, the point at which the produced large changes in the features of the Arrhenius slope of the Arrhenius plot decreases is found at 32.31C plot. As temperature is increased, a discontinuous and 30.41C at pH 7.0 and 7.5, respectively. decrease of MAO activity without any change in the slope The breaks in the Arrhenius plots at 32.31C (pH 7.0) is detected between 39.2 *Cand 40.01C. The point at which and 30.41C (pH 7.5) (Fig. 7) do not necessarily corre- the slope decreases as temperature increases is found at spond to the thermotropic phase transition temperature 43.6'C. The discontinuous decrease at 40'C with increas- Fig. 8. Arrhenius plots of the MAO activity for mitochondrial outer membranes in the presence of 0.1 M NaCl in phosphate buffer, pH 7.0. 0: Enzyme solution was prepared in a similar way to that described in the captions for Figs. 1, 2 and 5 (8 ml of 0.25 M sucrose for the pellet). •: Separate set of experiments from that expressed by 0. The membrane pellet used was the preparation ultracentrifuged at the same time as that for the data points 0. Each point was obtained from one measurement of activity. ing temperature in the Arrhenius plot in the presence of surface potential concentrates counter ions and/or H+ NaCI appears to be an abrupt decrease in the frequency on the membrane surface. The electrostatic surface factor in the equation for the rate constant. This feature phenomenon would play a role in the effects of NaCI and appears to be similar to that observed in the sugar trans- temperature. port in Echerichia coli (34). The other discontinuity is However, the experimental result shown in Fig. 5 can detected between 33.0'C and 34.2C. This discrepacy be- not be interpreted in terms of electrostatic membrane tween the two parallel straight lines is somewhat small. surface properties such as membrane surface potential, surface charges and surface pK or pH shifts from bulk pK DISCUSSION or pH values or induced membrane fluidity change. Since the pK value of natural acidic phospholipids is lower than Inhibitory effects of salts on hog kidney mitochondrial 2 and it is lowered by the presence of NaCl, dissociable MAO phosphate groups on the membrane surface are fully It has been reported that the presence of negatively deprotonated and negatively charged even at pH 6.0 (35). charged acidic phospholipids such as cardiolipin, phos- Therefore, the observed effect of NaCI in Fig. 5 may be phatidylinositol and phosphatidylserine is an important due to such a cooperative pH sensitive state transition, factor in the rebinding of solubilized MAO to the mito- involving the MAO protein as well as membrane phos- chondrial residues and for determining the properties pholipids as reported in the Raman spectroscopic study of the enzyme (8, 19-22). Most biomembranes are nega- of erythrocyte membranes (36) rather than due to a pH tively charged under physiological conditions due to the dependence of membrane fluidity in the dissociation presence of acidic lipids. The resultant electrostatic process of acidic phospholipid with increases in pH; i.e., charge neutralization process with decreasing pH (35). no experimental data on the benzylamine concentration As shown in Fig. 5B, the overall behavior in the pH dependence of substrate specificity. While we leave this dependence of the MAO activity appears to be independ- subject for hog kidney mitochondrial MAO open to fur- ent of the presence of NaCl, although NaCl exhibits ther investigation, it would be adequate in this article to MAO inhibition. The pH dependence of the inhibitory discuss the experimental results on the basis of the hypo- effect of NaCl itself (Fig. 5A) and that of the absolute thesis that benzylamine at 3.33 X 10-3 M concentration MAO activity in the absence of NaC1 (curve e) may be used in the present experiments acts as a MAO-B sub- attributable to the same mechanism. The effect of NaCl strate. Considering the previous experimental results may be explained by the following proposal: First, NaCl reported in Refs. 39 and 41 and the fairly high km value directly influenced the MAO conformation itself includ- of benzylamine in comparison to that of 2-phenylethyl- ing the active site and then the conformational change in amine (in the case of hog kidney mitochondrial MAO, the the MAO protein induced a concerted conformational km value for benzylamine is estimated to be 290 pM at change of the phospholipid acyl chains from gauche to pH 7.4 (42); in the case of rat kidney homogenate, the km trans (24) and in turn, the membrane fluidity change value is 10 uM for 2-phenylethylamine and 260 pM for affected the MAO conformation and/or substrate diffu- benzylamine at pH 8.0 (43)). sion into the membranes. Taken together, the data from the present and previous The cause of the inhibitory effect of CaC12 is apparently studies (23, 24), suggest that the functional state of different from that of NaC1. It has been reported that MAO-B in hog kidney mitochondria which metabolizes divalent cations decreased the membrane fluidity by benzylamine as substrate is constituted by the MAO specific interactions with membrane acidic phospholipid protein that binds tightly to the specific acidic phospho- (37, 38). However, the distinct difference in the features lipids such as cardiolipine, phosphatidylserine and phos- of the inhibitory effect on MAO between CaC12 and phatidylinositol (19, 20) to form a separate phase and that MgC12 can not be interpreted in terms of the difference in the discontinuity in the Arrhenius plot in the absence of a simple membrane fluidity change. The effect specific to NaCl may reflect a change of state in this phase rather CaC12 observed in this study should be interpreted differ- than a phase transition in the bulk lipid bilayer. Present- ently from the effect of MgC12i for example, in terms of ly, it is not clear whether the break in the Arrhenius plot an isothermal phase separation induced by specific bind- is attributable to a cooperative conformation change of ing of Ca 2+ to acidic phospholipid, because it is known MAO-B protein through a boundary lipid-protein inter- that Mg2+ does not induce an isothermal phase separa- action or an intrinsic conformational change of the en- tion (38). The direct influence of pH and Ca 21 ions on the zyme protein. MAO conformation might play a role in the inhibitory The present conclusion is in agreement with that effect. However, different from the case of NaCl inhibi- derived from the investigation of the rebinding effect of tion, the specific interaction of Ca 21 with membrane the purified phospholipid to the lipid-depleted rat brain phospholipid may contribute more predominantly to the mitochondrial MAO (20). Huang and Faulkner estimated inhibitory effect. in their study (20) that phosphatidylcholine, a zwitteri- onic phospholipid, was responsible for the fluidity of the Functional state of hog kidney mitochondrial mem- bulk membrane bilayer which in turn modulated the brane-bound MAO which metabolizes benzylamine as activity of MAO-A, not that of MAO-B. It has been substrate reported that Triton X-100 - sodium cholate solubilization It is now clarified that the substrate specificity of MAO of hog kidney mitochondrial MAO did not decrease ap- isozymes depends on the concentration, the affinity and preciably the MAO activity (only a 9% decrease) (42). turnover rate of the substrate. It has been reported that This result also suggests that the detergent treatment 2-phenylethylamine, which is classified as a substrate of depleted little boundary lipid and that the benzylamine MAO-B, loses its substrate specificity for MAO-B in rat oxidation by MAO-B in hog kidney mitochondria was brain mitochondria at 125 and 1250 pM concentrations, regulated only by boundary lipids and not by the bulk while benzylamine (substrate of MAO-B) and serotonin bilayer. (substrate of MAO-A) do not (39). Similar results for rat The experimental result of the Arrhenius plot in the brain have been reported (40, 41). presence of NaCl (Fig. 8) does not give us any direct in- The ratio of the content of MAO-A to that of MAO-B formation about the cause of the inhibitory effect of in hog kidney mitochondria is roughly estimated to be NaCl itself but it gives information about the cause of the 2 : 3 from the height of the plateau in the double-sig- temperature dependence of the inhibitory effect. Such a moidal inhibition curve for deprenyl (MAO-B inhibitor) decrease in the activation energy at the transition temper- using tyramine as a substrate (42). At this time, we have ature as observed in the absence of NaCl is not detected in the presence of NaC1. This observation is consistent with ically separable multiple forms of rat liver monoamine oxi- the decrease in the inhibition with increasing temperature dase. Biochem J 135, 173 -186 (1973) in the range of 321C - 381C (Fig. 6). 9 Houslay MD and Tipton KF: A kinetic evaluation of monoamine oxidase in rat liver mitochondrial outer membranes. It appears that the present membrane system in the Biochem J 139, 645-652 (1974) presence of NaCI probably underwent a temperature- 10 Houslay MD: Lipid substitution of mitochondrial monoamine induced phase separation characterized by two tempera- oxidase can lead the abolition of clorgyline selective inhibition tures, which correspond to the observed discontinuities without alteration in the A/B ratio assessed by substrate utili- in the Arrhenius plot at 39.2C -40.0'C and at sation. Biochem Pharmacol 29, 3211-3213 (1980) 33.0'C -34.21C (Fig. 8). The state of thermotropic phase 11 Bach AWJ, Lan NC, Johnson DL, Abell CW, Bembenek ME, separations, where lipids are partially in fluid and partial- Kwan S-W, Seeburg PH and Shih AJ: cDNA cloning of human liver monoamine oxidase A and B: Molecular basis of differ- ly in solid states, may enhance the lateral compressibility ences in enzymatic properties. Proc Natl Acad Sci USA 85, of membrane lipids and facilitate the perpendicular 4934-4938 (1988) penetration into membranes of the unprotonated form of 12 Hsu Y-PP, Weyler W, Chen S, Sims KB, Rinehart WB, benzylamine, which has been considered to be the main Utterback MC, Powell JF and Breakefield XO: Structural fea- species that binds to the enzyme (44). tures of human monoamine oxidase A elucidated from cDNA Further investigations will be needed using purified and peptide sequences. J Neurochem 51, 1321 - 1324 (1988) MAO-A and MAO-B reconstituted into the liposomes of 13 Grimsby J, Chen K, Wang L-J, Lan NC and Shih JC: Human specific phospholipids from both the enzymatic and spec- monoamine oxidases A and B genes exhibit identical exonintron organization. Proc Natl Sci USA 88, 3637-3641 (1991) troscopic points of view. 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