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Interfacial regulation of bacterial sphingomyelinase activity
Biochimica et Biophysica Acta 1344 Ž1997. 230–240
Interfacial regulation of bacterial sphingomyelinase activity Marina Jungner, Henna Ohvo, J. Peter Slotte
)
˚ Akademi UniÕersity, P.O. Box 66, FIN-20521 Turku, Finland Department of Biochemistry and Pharmacy, Abo Received 15 July 1996; revised 7 October 1996; accepted 9 October 1996
Abstract The objective of this study was to define how the quality of the bufferrmembrane interface influences the activity of bacterial sphingomyelinase acting at the interface. The enzyme reaction was carried out in a zero-order trough using a surface barostat. This approach allowed for proper control of the physico-chemical properties of the substrate molecules. Since the molecular area of ceramide is smaller than that of sphingomyelin, the hydrolysis reaction could be followed ‘on-line’ from the monolayer area decrease at constant surface pressure. The hydrolysis reaction could be divided into two separate phases, the first being the lag-phase Žtime between enzyme addition and commencement of the monolayer area change., and the second phase being the actual hydrolysis reaction Žfrom which a maximal degradation rate could be determined.. The activity of sphingomyelinase Ž Staphylococcus aureus . toward bovine brain sphingomyelin Žbb-SM. was markedly enhanced by Mg 2q Žmaximal activation at 5 mM.. Mg 2q also influenced the lag-phase of the reaction Žthe lag-time increased markedly when the Mg 2q concentration decreased below 1 mM.. Saturated sphingomyelins Žbb-SM and N-palmitoyl sphingomyelin w N-P-SMx. were more slowly degraded than the mono-unsaturated N-oleoyl sphingomyelin Ž N-O-SM.. Both bb-SM and N-P-SM monolayers underwent a phase-transition at room temperature, whereas the N-O-SM monolayer did not. The phase-transition Žliquid-expanded to liquid-condensed. was observed to greatly increase the lag-time of the hydrolysis reaction. The activity of sphingomyelinase was also sensitive to the lateral surface pressure of the monolayer membrane. Maximal degradation rate was achieved at 20 mNrm Žwith bb-SM, 308C.; above this pressure the lag-time of the reaction increased sharply. The inclusion of 4 mol% of cholesterol into a w 3 Hxsphingomyelin monolayer markedly increased the extent of w 3 Hxsphingomyelin degradation, and shortened the lag-time of the reaction. The inclusion of 10 mol% of zwitterionic or negatively charged phospholipids to the w 3 Hxsphingomyelin monolayer did not affect the sphingomyelinase reaction significantly. In conclusion, this study has demonstrated that the physico-chemical properties of the substrate molecules have a dominating influence on the activity of a bacterial sphingomyelinase acting at the bufferrmembrane interface. Keywords: Sphingomyelinase; Sphingomyelin; Cholesterol; Monolayer membrane; Interfacial regulation; ŽBovine brain.
1. Introduction
Abbreviations: bb-SM, bovine brain sphingomyelin; N-P-SM, N-palmitoyl sphingomyelin; N-O-SM, N-oleoyl sphingomyelin. ) Corresponding author. Fax: q358 2 2654745; E-mail: [email protected]
Sphingomyelinases ŽEC 3.1.4.12. are enzymes which degrade sphingomyelin to ceramide and phosphocholine. Different sphingomyelinases have been found in various human tissues. The acid sphin-
0005-2760r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 6 . 0 0 1 4 7 - 6
M. Jungner et al.r Biochimica et Biophysica Acta 1344 (1997) 230–240
gomyelinase is present in lysosomes, and is a membrane-associated glycoprotein w1,2x. The acid sphingomyelinase has recently been purified to homogeneity, and its gene has been cloned w3,4x. Selective absence of the acid sphingomyelinase enzyme appears to be responsible for types A and B of Niemann–Pick disease w5x. In addition to the acid sphingomyelinase, many cells also contain a neutral sphingomyelinase, which is activated by Mg 2q. This enzyme is found with high specific activity in brain tissue w6x, but is also present in cells from many other tissues w7–10x. The neutral sphingomyelinase activity is mostly localized in the plasma membrane fraction w11,12x, and has been shown to be externally oriented in neuroblastoma cells w12x. Studies in recent years have led to the discovery of a sphingomyelin cycle, in which activation of a neutral sphingomyelinase results in selective degradation of sphingomyelin, yielding bioactive lipid intermediates like ceramide and its metabolites w13,14x. Activation of cellular sphingomyelin degradation can be accomplished by different receptor ligands Ž TNFa w15,16x, NGF w17x, g-interferon w15x, interleukin 1 b w18,19x, 1,25-dihydroxyvitamin D3 w20x.. The mechanism of sphingomyelinase activation has not been fully elucidated, although it has been shown that changes in membrane fluidity Žby volatile anesthetics. can have dramatic effects on the activity of neutral sphingomyelinase in brain synaptosomal membranes w21x. It is also known that some surfaceactive compounds Že.g., apolipoprotein C-III and bee venom melittin. can stimulate sphingomyelinase activity, presumably by interfering with the lateral distribution of sphingomyelin in the membrane w22,23x. Since sphingomyelinases function at the aqueousrmembrane interface, the quality of this interface must have profound effects on the activity of the enzymes. Although reports have been published in which the substrate specificity of different sphingomyelinases have been examined using micellar or liposomal substrate vehicles w22,24,25x, only one paper has been published in which the interfacial quality of the substrate membrane was controlled w26x. Yedgar and coworkers examined the activity of a bacterial neutral sphingom yelinase at the bufferrmonolayer interface w26x, using a surface barostat technique w27x. Their work revealed, among other things, that the optimal surface pressure for
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maximal enzyme activity was different with different sphingomyelin substrates. In the present work, we have extended the studies of Yedgar and coworkers, using the same enzyme Ž neutral sphingomyelinase from Staphylococcus aureus . and the surface barostat technique. Our emphasis has been to examine how the physical state of the substrate molecules Žas affected by temperature, surface pressure, and degree of sphingomyelin saturationrunsaturation. affected the activity of the soluble enzyme acting at the interface. Whereas Yedgar and coworkers w26x measured their enzyme activity from the reaction-induced decrease of monolayer surface pressure at constant monolayer area, our technique makes it possible to keep the lateral surface pressure constant, and measure the reaction-induced monolayer area decrease instead. This is a clear advantage, since the lateral surface pressure is known to affect rates of enzymes acting at interfaces, and needs to be controlled.
2. Materials and methods 2.1. Materials Sphingomyelinase Ž Staphylococcus aureus ., and the lipids used were obtained from Sigma Ž St. Louis, MO, USA.. Buffer salts used for preparation of subphase were the purest available Ž Merck, Germany. . The water was purified by reverse osmosis followed by passage through a Millipore UF Plus water purification system, to yield a product with a resistivity of 18.2 M Vrcm. w 3 HxSphingomyelin was synthesized from bovine brain sphingomyelin according to the procedure reported by Stoeffel w28x. The radioactive w 3 Hxsphingomyelin was purified using HPLC, and was diluted to a final specific activity of about 100 cpmrpmol. 2.2. Force-area isotherms Monolayers of the sphingomyelins used Žbovine brain sphingomyelin, bb-SM; N-palmitoyl-sphingomyelin, N-P-SM; N-oleoyl-sphingomyelin, N-O-SM. or their corresponding ceramide, were compressed on buffer Ž50 mM Tris, pH 7.4, 140 mM NaCl, 5 mM MgCl 2 . at 308C with a KSV 3000 surface barostat ŽKSV Instruments, Helsinki, Finland.. The barrier
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˚ 2rmolecule per min during speed did not exceed 10 A compression. Data were collected using proprietary KSV software. From these isotherms the mean molecular area values were obtained for a given surface pressure, and used for calculations of sphingomyelin degradation rates, as indicated below. 2.3. Sphingomyelin hydrolysis determined from the change in monolayer area at constant surface pressure Sphingomyelin monolayers were spread on the buffer surface of a zero-order trough w27x. The reaction compartment, which had a surface of 2827 mm2 and a volume of 30 ml, was connected to a lipid reservoir compartment with a glass bridge, which allowed monolayer continuity between the two compartments but kept subphases apart. The monolayer was compressed and maintained at a constant surface pressure Žusually 20 mNrm unless otherwise indicated.. When a stable monolayer was achieved, sphingomyelinase was injected into the stirred subphase without penetrating the surface monolayer, to yield a desired activity Ž6.7 mUrml unless otherwise indicated.. The degradation of monolayer sphingomyelin was detected from the change in monolayer area Žat constant surface pressure. . Since the area difference between the reaction substrate and product was known at a given surface pressure, the rate of monolayer area decrease could be converted to molar units.
3. Results The basis for the direct determination of sphingomyelin degradation in monolayers by sphingomyelinase lies in the fact that the reaction product Žceramide. has a smaller molecular area requirement compared to the parent molecule Žsphingomyelin. . Therefore, the hydrolysis reaction can be analyzed by measuring the monolayer area decrease as a function of time, provided that the monolayer surface pressure is kept constant. Fig. 1 shows the force-area isotherms of bovine brain sphingomyelin Ž bb-SM. and the corresponding ceramide, determined at 308C. Two important features can be seen from the isotherms. One is the phase-transition present in the isotherm of bb-SM. The transition onset point can be observed at a surface pressure of about 28 mNrm, corresponding ˚ 2. This bb-SM to a molecular area of about 68 A transition was abolished by the presence of bb-ceramide Žat 20 mol%, Fig. 1. . We will see later that the presence of a phase-transition has important consequences for the substrate properties of the sphingomyelin. The second important feature, from the point of view of this work, is the molecular area difference between bb-SM and ceramide at a given lateral surface pressure. At 20 mNrm Žat which pressure most degradation experiments were performed., the area difference between bb-SM and ceramide was about ˚ 2. Corresponding force–area isotherms were ob32 A
2.4. Sphingomyelin hydrolysis determined from the release of [ 3H]phosphocholine from [ 3H]sphingomyelin In some experiments radioactive sphingomyelin was used as the substrate, and the degradation thereof by sphingomyelinase was determined from the release of radiolabeled phosphocholine into the aqueous subphase. Monolayers of w 3 Hxsphingomyelin were spread as described in the previous section. In some experiments, the monolayer also contained cholesterol or various nonlabeled phospholipids. The enzyme was injected into the subphase Žfinal activity 6.7 mUrml., and aliquots of the subphase Ž 0.2 ml. were removed at time intervals for determination of the activity of the released w 3 Hxphosphocholine, using scintillation counting.
Fig. 1. Force–area isotherms at the airrbuffer interface. Isotherms of monolayers containing either bovine brain sphingomyelin, bovine brain ceramide, or a mixture of these lipids Ž20 mol% ceramide and 80 mol% sphingomyelin., were obtained at 308C.
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tained for N-P-SM, N-O-SM, and their corresponding ceramides Ž curves not shown. . The molecular area difference between the ceramide and the parent ˚ 2 for N-P-SM, and about 4 compound was about 28 A 2 ˚ for N-O-SM. The force–area isotherm of N-P-SM A also indicated the presence of a phase-transition, with the transition onset pressure being about 23 mNrm, ˚ 2 and 308C Žcurve not at an molecular area of 66 A shown.. N-O-SM had a fully expanded force–area isotherm Ždata not shown..
Fig. 3. Hydrolysis of bb-SM as a function of sphingomyelinase concentration in the subphase. Pure bb-SM monolayers were kept at 20 mNrm Žconstant surface pressure. on the buffer surface, and different amounts of sphingomyelinase were injected below the monolayer at 308C. Maximal hydrolysis rate as a time function was determined from several experiments. Each value is the mean from duplicate determinations Žrange"10%. from representative experiments.
Fig. 2. Hydrolysis of monolayer sphingomyelin by bacterial sphingomyelinase. A pure bb-SM monolayer was prepared at the airrbuffer interface, and compressed to 20 mNrm. The subphase buffer contained Tris-HCl Ž50 mM, pH 7.4. with 140 mM NaCl and 5 mM MgCl 2 . Sphingomyelinase was added to the stirred subphase, at 308C, to a final activity of 2.6 mUrml. Degradation of bb-SM by the enzyme led to a monolayer area decrease Žat constant surface pressure, 20 mNrm., as depicted in the upper panel. The definition of the lag-time is also shown. The lower panel shows the velocity of the monolayer area decrease per time unit as a time function, determined as the slope between two adjacent data points on the monolayer area decrease curve Župper panel.. The peak value indicates maximal monolayer area decrease which corresponds to maximal hydrolysis rate, and is converted to values of nmol bb-SM degraded per min during the course of the experiment. Most of the hydrolysis rates shown in subsequent figures are derived from similar data on monolayer area change as a time function.
The exposure at constant surface pressure of a pure sphingomyelin monolayer to sphingomyelinase in the subphase led to a time-dependent reduction of the monolayer area Ždetermined from the barrier movement which pushed new substrate into the reaction chamber., as shown in Fig. 2. The upper panel shows the area decrease Žgiven in square mm., and also shows the definition of the lag-time. The lower panel in Fig. 2 shows the slope between adjacent data points of the monolayer area Õersus time function Župper panel.. The slope gives the apparent reaction rate Žindicated as nmol sphingomyelin degraded per min., and also indicate the maximal rate achieved in the reaction. Both maximal hydrolysis rates, and lag-times were determined in subsequent experiments. The maximal rate of the degradation of bovine brain sphingomyelin by sphingomyelinase increased linearly as a function of added enzyme ŽFig. 3.. No saturation of the reaction rate increase could be seen with the sphingomyelinase activities tested. However, since the bacterial sphingomyelinase preparation contained 50% glycerol as a stabilizer, we did not use activities higher than about 6.7 mUrml to avoid adverse effects from the glycerol component.
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Fig. 4. Effect of Mg 2q-concentration on sphingomyelinase action. Monolayers of bb-SM were spread on the buffer surface to a surface pressure of 20 mNrm Žat 308C.. The subphase concentration of MgCl 2 was varied, and the hydrolysis induced by 6.7 mUrml sphingomyelinase in the subphase was determined from the monolayer area decrease Žpanel A.. Panel B shows the lag-time Žsee Fig. 2, upper panel. of the commencement of the hydrolysis reaction as a function of the subphase Mg 2q-concentration. Each value is the mean from duplicate determinations Žrange " 10%. from representative experiments.
The bacterial sphingomyelinase is known to be activated by Mg 2q w29x. As shown in Fig. 4, the addition of Mg 2q affected both the maximal reaction rate Ž panel A. as well as the lag-time for the commencement of the reaction Žpanel B.. It appeared, however, that whereas 1 mM Mg 2q was enough to maximally decrease the lag-time Ž panel B. , slightly more Mg 2q was needed to achieve maximal reaction rate Žpanel A; about 5 mM. . Consequently, 5 mM Mg 2q was included in all subsequent reactions.
The activity of the bacterial sphingomyelinase was markedly dependent on temperature. At room temperature, the rate of degradation of saturated sphingomyelins Žbb-SM and N-P-SM. was slow Ž Fig. 5. . When N-O-SM was used as substrate, the rate of degradation at room temperature was much higher than that observed for the saturated sphingomyelins. On a proportional basis, however, an increased temperature affected Ž increased. the rate of degradation of all tested sphingomyelins similarly ŽFig. 5..
Fig. 5. Rates of sphingomyelin hydrolysis at different temperatures. Monolayers containing either bb-SM, N-P-SM, or N-O-SM were prepared at the pure airrbuffer interface, and were kept at constant surface pressure Ž20 mNrm.. Sphingomyelinase Ž6.7 mUrml. was injected below the monolayer, and the maximal hydrolysis rate was determined at different temperatures. Each value is the mean from duplicate determinations Žrange " 10%. from representative experiments.
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Fig. 6. Lag-times of sphingomyelin hydrolysis at different temperatures. Monolayers containing either bb-SM, N-P-SM, or N-O-SM were prepared at the pure airrbuffer interface, and were kept at constant surface pressure Ž20 mNrm.. Sphingomyelinase Ž6.7 mUrml. was injected below the monolayer, and the lag-time for the commencement of the hydrolysis reaction was determined at different temperatures. Each value is the mean from duplicate determinations Žrange " 10%. from representative experiments.
The lag-time for the commencement of the hydrolysis reaction was also very sensitive to temperature. At room temperature, monolayers of both bb-SM and N-P-SM displayed long lag-times before hydrolysis by sphingomyelinase commenced ŽFig. 6.. However, when the temperature was increased, the lag-times decreased sharply, and stabilized on a steady level at temperatures above 258C. The degradation of a monolayer containing N-O-SM, which is altogether fluid at room temperature, and has no observable
phase-transition, did not show a dramatic change in lag-time over the temperature range of 20–378C ŽFig. 6.. Clearly, the observable lag-time for sphingomyelin degradation reaction was highly dependent on the physical state of the substrate molecules, as was the maximal reaction rate ŽFig. 5.. The lateral surface pressure of the substrate-containing monolayer membrane markedly influenced both reaction rates and lag-times, when the hydrolysis of bb-SM was determined at 308C. The reaction rate
Fig. 7. Effect of monolayer surface pressure on the hydrolysis of sphingomyelin. Pure monolayers of bb-SM were prepared and compressed to different lateral surface pressures. At constant surface pressure, the hydrolysis of bb-SM was initiated by injection of 6.7 mUrml of sphingomyelinase into the subphase, which was maintained at 308C. Panel A shows the maximal hydrolysis rate as a surface pressure function, whereas panel B gives the lag-time for the commencement of the reaction as a surface pressure function. Each value is the mean from duplicate determinations Žrange " 10%. from representative experiments.
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Fig. 8. Hydrolysis of w 3 Hxsphingomyelin by sphingomyelinase. Monolayers were prepared to contain w 3 Hxsphingomyelin from bovine brain, and these were maintained at a constant surface pressure of 20 mNrm Žat 308C.. Sphingomyelinase Ž6.7 mUrml. was injected into the subphase, and the degradation of monolayer w 3 Hxsphingomyelin Žopen circle. was determined from the release of w 3 Hxphosphocholine into the subphase, aliquots of which were removed at time intervals for radioactivity determination. Another set of monolayers were prepared to also contain 4 mol% cholesterol, in addition to bovine brain w 3 Hxsphingomyelin, and their degradation is indicated by the open squares. Each value is the mean"S.E.M. of three different monolayers at each temperature.
increased with increasing surface pressure up to about 20 mNrm Žmaximal reaction rate, cf. Fig. 7A., but thereafter the reaction rate decreased as the lateral surface pressure increased further Žlateral surface
pressures higher than about 28 mNrm could not be examined because of the monolayer instability of ceramide.. As the surface pressure increased above 20 mNrm, the lag-time of the reaction started to increase dramatically ŽFig. 7B.. A qualitatively similar surface pressure-dependence was observed when N-P-SM and N-O-SM monolayers were hydrolyzed by sphingomyelinase Ždata not shown.. To study the effects of colipids Že.g., cholesterol and other phospholipids. on reaction rates and lagtimes, we had to use radiolabeled sphingomyelin Žprepared from bb-SM. as substrate, and assay the hydrolysis reaction from the release of w 3 Hxphosphocholine into the subphase Žin binary and ternary monolayers all components may contribute to measurable area changes in a way which is not easily controllable.. Using this assay technique, we could see that when using a pure w 3 Hxsphingomyelin as substrate, about 5 nmol of w 3 Hxsphingomyelin was degraded by sphingomyelinase within 6–8 min at 308C ŽFig. 8.. Since the reaction chamber surface was 2827 mm2 , and the bb-SM molecular area at 308C ˚ 2 , one can calculate and 20 mNrm was about 75 A 3 that 3.8 nmol of w Hxsphingomyelin was initially exposed to sphingomyelinase Ž this number only includes the amount of w 3 Hxsphingomyelin on the surface of the reaction chamber. . Therefore, we can conclude that all of the initially present w 3 Hxsphingomyelin was degraded during the first 6–8 min. In addition, it appears that some sphingomyelin ‘leaked’ into the reaction chamber from the lipid reservoir compartment Ž by diffusion, and because unhydro-
Table 1 Effects of cophospholipids on hydrolysis of bovine brain w 3 Hxsphingomyelin Žw 3 Hxbb-SM. by sphingomyelinase Monolayer composition
w 3 Hxbb-SM degraded during first 8 min of reaction Žnmol.
100 mol% w 3 Hxbb-SM 90 mol% w 3 Hxbb-SMq 10 mol% dilauroyl phosphatidic acid 90 mol% w 3 Hxbb-SMq 10 mol% bovine brain phosphatidylserine 90 mol% w 3 Hxbb-SMq 10 mol% dipalmitoyl phosphatidylcholine 90 mol% w 3 Hxbb-SMq 10 mol% dipalmitoyl phosphatidylethanolamine 90 mol% w 3 Hxbb-SMq 10 mol% dipalmitoyl phosphatidylglycerol
4.5 " 0.5 4.0 " 0.1 4.0 " 0.2 5.0 " 0.4 4.5 " 0.3 4.6 " 0.3
Monolayers containing w 3 Hxbb-SM Ž90 mol%. and an unlabeled cophospholipid Žat 10 mol%. were prepared at the airrbuffer interface. Monolayers were maintained at 20 mNrm and 308C. Sphingomyelinase was injected into the subphase Žfinal concentration 6.7 mUrml. and the release of w 3 Hxphosphocholine into the aqueous subphase was determined by scintillation counting. Each value is the mean " S.E.M. of three different monolayers preparations of each composition.
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lyzed monolayer material was pushed into the reaction chamber, since the assay was performed in the constant surface pressure mode.. The lag-time for the commencement of the degradation of w 3 Hxsphingomyelin was about 2 min, which is similar to what was seen previously with nonlabeled substrate molecules. The inclusion of a trace amount of cholesterol in the sphingomyelin monolayer Žat 4 mol%. dramatically increased the amount of w 3 Hxsphingomyelin that was degradable by sphingomyelinase, and also shortened the lag-time for the reaction Ž Fig. 8.. An increased lateral diffusion probably supplied this additional w 3 Hxsphingomyelin onto the surface of the reaction chamber, where it was degraded. The addition of more cholesterol Žup to 25 mol%. to the sphingomyelin monolayer did not further enhance sphingomyelin degradation Ždata not shown.. The inclusion of 10 mol% of one of several different phospholipids Ž both zwitterionic and negatively charged. did not significantly affect rates of w 3 Hxsphingomyelin degradation ŽTable 1..
4. Discussion In this study we have examined how interfacial properties of the substrate membrane affect the activity of bacterial sphingomyelinase. The enzyme assay was performed using the surface barostat technique, the advantages of which include full control of the physical properties of the lipid substrate membrane. The trough used was of a zero-order type w27x, although the hydrolysis reaction measured did not obey zero-order kinetics Žbecause the reaction product remained in the membrane.. We could still, however, determine the lag-time for the commencement of the reaction, and the maximal rate of degradation. The actual reaction could be followed from the monolayer area decrease, since the removal of the phosphocholine group of sphingomyelin led to a smaller molecular area requirement of the resulting ceramide. The sphingomyelinase-induced degradation of sphingomyelin gave a sigmoidal monolayer area decrease as a time function. This is typical of enzyme reactions, in which both the substrate and the product reside in the monolayer membrane w30,31x. The latter part of the sigmoidal reaction curve was clearly a result of the fact that ceramide accumulates in the
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vicinity of the enzyme, and slows down the reaction due to substrate depletion. The initial part of the sigmoidal reaction curve is likely to depend on at least two different components, one being the association of enzyme with the monolayer, and the other being the actual commencement of the enzyme reaction. The association of sphingomyelinase with the monolayer membrane has not been determined directly in the present study. This is because the enzyme is not surface active Žw26x; data not shown. , and because of technical limitations. However, the results regarding the lag-time of the reactions can be interpreted to depend on enzyme association with the monolayers Žlonger lag-times indicate poorer association.. First, much less Mg 2q was required to lower the lag-time of the reaction, as compared to what was needed for full activation of the enzyme. This suggests that Mg 2q at low concentrations enhanced the association of enzyme with the substrate monolayer Žresulting in shorter lag times., whereas higher Mg 2q concentration were needed to fully activate the enzyme. Previous experiments with a Bacillus cereus sphingomyelinase have shown that adsorption of the enzyme to liposomal sphingomyelin membranes is enhanced by divalent cations, although a specific role for Mg 2q in adsorption was not demonstrated w32,33x. The Staphylococcus aureus enzyme Žused in this study. may differ from the Bacillus cereus enzyme with regard to the specificity of the metal ion required for adsorption. Secondly, the lag-times of the reactions were much longer when the substrate molecules were liquid-condensed as opposed to liquid-expanded. At room temperature Žabout 208C. both bb-SM and N-P-SM monolayers were dominantly liquid-condensed at 20 mNrm, and the lag-time for the commencement of the sphingomyelinase-reaction was long Ž40 and 15 min, for bb-SM and N-P-SM respectively. . However, the monolayer of N-O-SM at 208C and 20 mNrm was liquid-expanded, and the corresponding lag-time was only 2 min. Clearly, the physical state of the substrate markedly affected the lag-times of the reactions. Protein penetration into monolayers is known to be enhanced by a looser lateral packing Žhigher lateral compressibility. as opposed to a more condensed packing w34,35x. The observed effect of the physical state of the monolayer on the lag-times of
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the reactions is compatible with the view that a looser packing Že.g., N-O-SM monolayer. allows better or faster enzyme association, and consequently yields a shorter lag-time before the hydrolysis reaction commences. When monolayers of bb-SM or N-P-SM were hydrolyzed at a higher temperature, at which they were more liquid-expanded, the observable lagtimes were dramatically shortened, suggesting that enzyme association with the monolayers at higher temperatures was enhanced. It is interesting to note that the presence of small amounts of ceramide Ž20 mol%. in the bb-SM monolayer appeared to abolish the phase transition region at surface pressure below 40 mNrm. Consequently, as the sphingomyelinasecatalyzed reaction product Žceramide. is likely to affect the rate of its own initial formation. The effect of the lateral surface pressure on the lag-time of the reaction can be explained as follows. At higher surface pressures, the enzyme association with the monolayer is more difficult, and consequently longer lag-times are expected and were observed. The lag-times we determined for the sphingomyelinase reaction is clearly related to the ‘acceleration time’ described by Yedgar and coworkers w26x for the same monolayer reaction. Since we and they measured the sphingomyelinase reaction differently Žconstant pressure versus constant area., the lag-time or the acceleration-time variable is also defined differently. Despite the difference in the way the enzyme was assayed, our results regarding the lag-time of the reaction is in good general agreement with the acceleration time variable of the study of Yedgar and coworkers w26x. Assuming that the enzyme associates with the monolayers at sites where the local packing density is looser Žexpanded., one can envision that the sphingomyelin degradation reaction starts at these localizations and commences at the boundary line between expanded and condensed phases. Such a reaction sequence has actually been documented visually Žusing monolayer fluorescence microscopy. for two enzymes acting at the aqueousrmembrane interface. The enzyme phospholipase A 2 was shown to penetrate into the expanded domains of a dipalmitoylphosphatidylcholine monolayer, whereas the actual degradation of the phospholipid occurred along the liquidrcondensed domain interface w36,37x. Similarly, cholesterol oxidase, which oxidizes cholesterol
to cholestenone, also penetrated the substrate monolayers at loosely packed sites, from which the reaction subsequently commenced w38x. It is likely that the sequence of events with the sphingomyelinase system is similar to that of the phospholipase A 2 or cholesterol oxidase systems. However, the direct visualization of sphingomyelin degradation in monolayers by sphingomyelinase is hampered by the fact that sphingomyelin does not form clearly defined condensed domains, the fate of which could be visually followed Ž Slotte, unpublished observations.. Maximal reaction rates with the bacterial sphingomyelinase were observed at a lateral surface pressure of about 20 mNrm for all three sphingomyelin substrates tested. In the study of Yedgar and coworkers w26x, maximal reaction rate was observed at 19–22 mNrm for bb-SM. With N-P-SM, the optimal surface pressure was determined to lie within the range 16–20 mNrm w26x, whereas our study suggested that the optimal pressure was 20 mNrm. It is possible that the surface pressure-dependence of the sphingomyelinase reaction relates to the orientation of the phosphocholine head group of sphingomyelin in relation to the hydrophobic part of the molecule, a function which clearly depends on the degree of compression Ž and hence on lateral surface pressure.. However, the surface concentration of a substrate molecule in monolayers is also known to affect rates of enzyme-catalyzed conversions w31x, and may have contributed to the observed surface pressure-dependence of our enzyme assay. Another parameter which clearly affected maximal reaction rates was the degree of unsaturation of the substrate molecules. N-O-SM, which is more fluid than, e.g., N-P-SM, was degraded much faster at a given temperature. Clearly the general fluidity of the monolayer affected the rate by which enzyme and substrate molecules could diffuse toward each other Žand presumably also how efficiently the resulting ceramide could diffuse away from the enzyme.. The importance of monolayer fluidity in determining reaction rates was also seen when trace amounts Ž4 mol%. of cholesterol was included in a w 3 Hxsphingomyelin monolayer – both the lag-time Ženzyme association. decreased and the extent of w 3 Hxsphingomyelin degradation increased as a consequence of cholesterol inclusion. Whereas the feeding of free cholesterol or 7-dehydrocholesterol to cells have been
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shown to inhibit sphingomyelin degradation w39,40x, the effect of cholesterol on the action of sphingomyelinase in this model system was quite the opposite. The importance of membrane fluidity on affecting sphingomyelinase activity was also clearly demonstrated by Sandhoff and colleagues, who showed that halothane Žby increasing membrane fluidity. could dramatically enhance the degradation of sphingomyelin in isolated brain synaptosomal membranes w21x. Modest changes in monolayer surface Žnegative. charge, as effected by the inclusion of 10 mol% cophospholipids in the w 3 Hxsphingomyelin monolayer, did not however appear to affect the extent of w 3 Hxsphingomyelin degradation. The cophospholipids that were used were all saturated Ž save for bovine brain phosphatidyl serine. and were not expected to affect the fluidity of bovine brain w 3 Hxsphingomyelin. In conclusion, the results of this study have emphasized the importance of the physical state of the substrate molecules in determining rates of both enzymermembrane association and reaction rates for the bacterial sphingomyelinase. It is plausible to assume that the physical state of sphingomyelin present in biological membranes have similar effects on the activity of sphingomyelinases present in those membranes, or acting at the aqueousrmembrane interface. Future work with purified human sphingomyelinases, using this experimental approach, should settle this assumption one way or the other.
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Acknowledgements
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This study was supported by generous grants from the Sigrid Juselius Foundation, the Academy of Fin¨ ˚ land, the Oscar Oflund Foundation, and the Abo Akademi University.
w22x w24x w25x w26x
References w27x w1x Yamaka, T. and Suzuki, K. Ž1982. J. Neurochem. 38, 1753–1764. w2x Quintern, L.E., Weitz, G., Nehrkorn, H., Tager, J.M., Schram, A.W. and Sandhoff, K. Ž1987. Biochim. Biophys. Acta 922, 323–336. w3x Quintern, L.E., Schuchman, E.H., Levran, O., Suchi, M.,
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Ferlinz, K., Reinke, H. and Sandhoff, K. Ž1989. EMBO J. 8, 2469–2473. Schuchman, E.H., Suchi, M., Takahashi, T., Sandhoff, K. and Desnick, R.J. Ž1991. J. Biol. Chem. 266, 8531–8539. Brady, R.O. Ž1983. in The Metabolic Basis of Inherited Diseases ŽStanbury, J.B., Wyngaarden, J.B., Fredrickson, D.S., Goldstein, J.L. and Brown, M.S., eds.., 5th Ed., Chap. 41, McGraw-Hill, New York. Rao, B.G. and Spence, M.W. Ž1976. J. Lipid Res. 17, 506–515. Kolesnick, R.N. and Hemer, M.J. Ž1990. J. Biol. Chem. 265, 18803–18808. ˚ Ž1969. Biochim. Biophys. Acta 176, 339–347. Nilsson, A. Okazaki, T., Bielawaska, A., Bell, R.M. and Hannun, Y. Ž1990. J. Biol. Chem. 265, 15823–15831. Schneider, P.B. and Kennedy, E.P. Ž1967. J. Lipid Res. 8, 202–209. Hoestetler, K.Y. and Yakazaki, P.J. Ž1979. J. Lipid Res. 20, 456–463. Spence, M.W., Wakkary, J., Clarke, J.T.R. and Cook, H.W. Ž1982. Biochim. Biophys. Acta 719, 162–164. Hannun, Y. and Bell, R.M. Ž1993. Adv. Lipid Res. 25, 27–41. Hannun, Y. Ž1994. J. Biol. Chem. 269, 3125–3128. Kim, M.-Y., Linardic, C., Obeid, L. and Hannun, Y. Ž1991. J. Biol. Chem. 266, 484–489. Mathias, S., Dressler, K.A. and Kolesnick, R.N. Ž1992. Proc. Natl. Acad. Sci. USA 88, 10009–10013. Dobrowsky, R.T., Werner, M.H., Castellino, A.M., Chao, M.V. and Hannun, Y.A. Ž1994. Science 265, 1596–1599. Ballou, L.R., Chao, C.P., Holness, M.A., Barker, S.C. and Raghow, R. Ž1992. J. Biol. Chem. 267, 20044–20050. Mathias, S., Younes, A., Kan, C.-C., Orlow, I., Joseph, C. and Kolesnick, R.N. Ž1993. Science 259, 519–522. Okazaki, T., Bell, R.M. and Hannun, Y. Ž1989. J. Biol. Chem. 264, 19076–19080. Pellkofer, R. and Sandhoff, K. Ž1980. J. Neurochem. 34, 988–992. Pellkofer, R., Marsh, D., Hoffman-Bleihauer, P. and Sandhoff, K. Ž1982. J. Neurochem. 38, 1230–1235. Ahmad, T.Y., Beaudet, A.L., Sparrow, J.T and Morrisett, J.D. Ž1986. Biochem. 25, 4415–4420. Bartolf, M. and Franson, R.C. Ž1986. J. Lipid Res. 26, 57–63. Lister, M.D., Ruan, Z.-s. and Bittman, R. Ž1995. Biochim. Biophys. Acta 1256, 25–30. Yedgar, S., Cohen, R., Gatt, S. and Barenholz, Y. Ž1982. Biochem. J. 201, 597–603. Verger, R. and De Haas, G.H. Ž1973. Chem. Phys. Lipids 10, 127–136. Stoeffel, W. Ž1975. Methods Enzymol. 35, 533–541. Ikezawa, H., Matsushita, M., Tomita, M. and Taguchi, R. Ž1986. Arch. Biochem. Biophys. 249, 588–595. Slotte, J.P. Ž1992. Biochemistry 31, 5472–5477. Slotte, J.P. Ž1992. Biochim. Biophys. Acta 1123, 326–333.
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w32x Tomita, M., Taguchi, R. and Ikezawa, H. Ž1983. J. Biochem. 93, 1221–1230. w33x Ikezawa, H., Matshushita, M., Tomita, M. and Taguchi, R. Ž1986. Arch. Biochem. Biophys. 249, 588–595. w34x Op Den Kamp, J.A.F., De Gier, J. and Van Deenen, L.L.M. Ž1974. Biochim. Biophys. Acta 345, 253–256. w35x Phillips, M.C., Graham, D.E. and Hauser, H. Ž1975. Nature 254, 154–155. w36x Grainger, D.W., Reichert, A., Ringsdorf, H. and Salesse, C. Ž1989. FEBS Lett. 252, 74–83.
w37x Grainger, D.W., Reicher, A., Ringsdorf, H. and Salesse, C. Ž1990. Biochim. Biophys. Acta 1023, 365–379. w38x Slotte, J.P. Ž1995. Biochim. Biophys. Acta 1259, 180–186. w39x Maziere, G. ` J.C., Wolf, C., Maziere, ` C., Mora, L., Bereziat, ´´ and Polonovski, J. Ž1981. Biochem. Biophys. Res. Commun. 100, 1299–1304. w40x Maziere, J.C., Maziere, C., Mora, L., Gallie, F. and ` ` Polonovski, J. Ž1983. Biochem. Biophys. Res. Commun. 112, 860–865.
Interfacial regulation of bacterial sphingomyelinase activity Marina Jungner, Henna Ohvo, J. Peter Slotte
)
˚ Akademi UniÕersity, P.O. Box 66, FIN-20521 Turku, Finland Department of Biochemistry and Pharmacy, Abo Received 15 July 1996; revised 7 October 1996; accepted 9 October 1996
Abstract The objective of this study was to define how the quality of the bufferrmembrane interface influences the activity of bacterial sphingomyelinase acting at the interface. The enzyme reaction was carried out in a zero-order trough using a surface barostat. This approach allowed for proper control of the physico-chemical properties of the substrate molecules. Since the molecular area of ceramide is smaller than that of sphingomyelin, the hydrolysis reaction could be followed ‘on-line’ from the monolayer area decrease at constant surface pressure. The hydrolysis reaction could be divided into two separate phases, the first being the lag-phase Žtime between enzyme addition and commencement of the monolayer area change., and the second phase being the actual hydrolysis reaction Žfrom which a maximal degradation rate could be determined.. The activity of sphingomyelinase Ž Staphylococcus aureus . toward bovine brain sphingomyelin Žbb-SM. was markedly enhanced by Mg 2q Žmaximal activation at 5 mM.. Mg 2q also influenced the lag-phase of the reaction Žthe lag-time increased markedly when the Mg 2q concentration decreased below 1 mM.. Saturated sphingomyelins Žbb-SM and N-palmitoyl sphingomyelin w N-P-SMx. were more slowly degraded than the mono-unsaturated N-oleoyl sphingomyelin Ž N-O-SM.. Both bb-SM and N-P-SM monolayers underwent a phase-transition at room temperature, whereas the N-O-SM monolayer did not. The phase-transition Žliquid-expanded to liquid-condensed. was observed to greatly increase the lag-time of the hydrolysis reaction. The activity of sphingomyelinase was also sensitive to the lateral surface pressure of the monolayer membrane. Maximal degradation rate was achieved at 20 mNrm Žwith bb-SM, 308C.; above this pressure the lag-time of the reaction increased sharply. The inclusion of 4 mol% of cholesterol into a w 3 Hxsphingomyelin monolayer markedly increased the extent of w 3 Hxsphingomyelin degradation, and shortened the lag-time of the reaction. The inclusion of 10 mol% of zwitterionic or negatively charged phospholipids to the w 3 Hxsphingomyelin monolayer did not affect the sphingomyelinase reaction significantly. In conclusion, this study has demonstrated that the physico-chemical properties of the substrate molecules have a dominating influence on the activity of a bacterial sphingomyelinase acting at the bufferrmembrane interface. Keywords: Sphingomyelinase; Sphingomyelin; Cholesterol; Monolayer membrane; Interfacial regulation; ŽBovine brain.
1. Introduction
Abbreviations: bb-SM, bovine brain sphingomyelin; N-P-SM, N-palmitoyl sphingomyelin; N-O-SM, N-oleoyl sphingomyelin. ) Corresponding author. Fax: q358 2 2654745; E-mail: [email protected]
Sphingomyelinases ŽEC 3.1.4.12. are enzymes which degrade sphingomyelin to ceramide and phosphocholine. Different sphingomyelinases have been found in various human tissues. The acid sphin-
0005-2760r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 6 . 0 0 1 4 7 - 6
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gomyelinase is present in lysosomes, and is a membrane-associated glycoprotein w1,2x. The acid sphingomyelinase has recently been purified to homogeneity, and its gene has been cloned w3,4x. Selective absence of the acid sphingomyelinase enzyme appears to be responsible for types A and B of Niemann–Pick disease w5x. In addition to the acid sphingomyelinase, many cells also contain a neutral sphingomyelinase, which is activated by Mg 2q. This enzyme is found with high specific activity in brain tissue w6x, but is also present in cells from many other tissues w7–10x. The neutral sphingomyelinase activity is mostly localized in the plasma membrane fraction w11,12x, and has been shown to be externally oriented in neuroblastoma cells w12x. Studies in recent years have led to the discovery of a sphingomyelin cycle, in which activation of a neutral sphingomyelinase results in selective degradation of sphingomyelin, yielding bioactive lipid intermediates like ceramide and its metabolites w13,14x. Activation of cellular sphingomyelin degradation can be accomplished by different receptor ligands Ž TNFa w15,16x, NGF w17x, g-interferon w15x, interleukin 1 b w18,19x, 1,25-dihydroxyvitamin D3 w20x.. The mechanism of sphingomyelinase activation has not been fully elucidated, although it has been shown that changes in membrane fluidity Žby volatile anesthetics. can have dramatic effects on the activity of neutral sphingomyelinase in brain synaptosomal membranes w21x. It is also known that some surfaceactive compounds Že.g., apolipoprotein C-III and bee venom melittin. can stimulate sphingomyelinase activity, presumably by interfering with the lateral distribution of sphingomyelin in the membrane w22,23x. Since sphingomyelinases function at the aqueousrmembrane interface, the quality of this interface must have profound effects on the activity of the enzymes. Although reports have been published in which the substrate specificity of different sphingomyelinases have been examined using micellar or liposomal substrate vehicles w22,24,25x, only one paper has been published in which the interfacial quality of the substrate membrane was controlled w26x. Yedgar and coworkers examined the activity of a bacterial neutral sphingom yelinase at the bufferrmonolayer interface w26x, using a surface barostat technique w27x. Their work revealed, among other things, that the optimal surface pressure for
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maximal enzyme activity was different with different sphingomyelin substrates. In the present work, we have extended the studies of Yedgar and coworkers, using the same enzyme Ž neutral sphingomyelinase from Staphylococcus aureus . and the surface barostat technique. Our emphasis has been to examine how the physical state of the substrate molecules Žas affected by temperature, surface pressure, and degree of sphingomyelin saturationrunsaturation. affected the activity of the soluble enzyme acting at the interface. Whereas Yedgar and coworkers w26x measured their enzyme activity from the reaction-induced decrease of monolayer surface pressure at constant monolayer area, our technique makes it possible to keep the lateral surface pressure constant, and measure the reaction-induced monolayer area decrease instead. This is a clear advantage, since the lateral surface pressure is known to affect rates of enzymes acting at interfaces, and needs to be controlled.
2. Materials and methods 2.1. Materials Sphingomyelinase Ž Staphylococcus aureus ., and the lipids used were obtained from Sigma Ž St. Louis, MO, USA.. Buffer salts used for preparation of subphase were the purest available Ž Merck, Germany. . The water was purified by reverse osmosis followed by passage through a Millipore UF Plus water purification system, to yield a product with a resistivity of 18.2 M Vrcm. w 3 HxSphingomyelin was synthesized from bovine brain sphingomyelin according to the procedure reported by Stoeffel w28x. The radioactive w 3 Hxsphingomyelin was purified using HPLC, and was diluted to a final specific activity of about 100 cpmrpmol. 2.2. Force-area isotherms Monolayers of the sphingomyelins used Žbovine brain sphingomyelin, bb-SM; N-palmitoyl-sphingomyelin, N-P-SM; N-oleoyl-sphingomyelin, N-O-SM. or their corresponding ceramide, were compressed on buffer Ž50 mM Tris, pH 7.4, 140 mM NaCl, 5 mM MgCl 2 . at 308C with a KSV 3000 surface barostat ŽKSV Instruments, Helsinki, Finland.. The barrier
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˚ 2rmolecule per min during speed did not exceed 10 A compression. Data were collected using proprietary KSV software. From these isotherms the mean molecular area values were obtained for a given surface pressure, and used for calculations of sphingomyelin degradation rates, as indicated below. 2.3. Sphingomyelin hydrolysis determined from the change in monolayer area at constant surface pressure Sphingomyelin monolayers were spread on the buffer surface of a zero-order trough w27x. The reaction compartment, which had a surface of 2827 mm2 and a volume of 30 ml, was connected to a lipid reservoir compartment with a glass bridge, which allowed monolayer continuity between the two compartments but kept subphases apart. The monolayer was compressed and maintained at a constant surface pressure Žusually 20 mNrm unless otherwise indicated.. When a stable monolayer was achieved, sphingomyelinase was injected into the stirred subphase without penetrating the surface monolayer, to yield a desired activity Ž6.7 mUrml unless otherwise indicated.. The degradation of monolayer sphingomyelin was detected from the change in monolayer area Žat constant surface pressure. . Since the area difference between the reaction substrate and product was known at a given surface pressure, the rate of monolayer area decrease could be converted to molar units.
3. Results The basis for the direct determination of sphingomyelin degradation in monolayers by sphingomyelinase lies in the fact that the reaction product Žceramide. has a smaller molecular area requirement compared to the parent molecule Žsphingomyelin. . Therefore, the hydrolysis reaction can be analyzed by measuring the monolayer area decrease as a function of time, provided that the monolayer surface pressure is kept constant. Fig. 1 shows the force-area isotherms of bovine brain sphingomyelin Ž bb-SM. and the corresponding ceramide, determined at 308C. Two important features can be seen from the isotherms. One is the phase-transition present in the isotherm of bb-SM. The transition onset point can be observed at a surface pressure of about 28 mNrm, corresponding ˚ 2. This bb-SM to a molecular area of about 68 A transition was abolished by the presence of bb-ceramide Žat 20 mol%, Fig. 1. . We will see later that the presence of a phase-transition has important consequences for the substrate properties of the sphingomyelin. The second important feature, from the point of view of this work, is the molecular area difference between bb-SM and ceramide at a given lateral surface pressure. At 20 mNrm Žat which pressure most degradation experiments were performed., the area difference between bb-SM and ceramide was about ˚ 2. Corresponding force–area isotherms were ob32 A
2.4. Sphingomyelin hydrolysis determined from the release of [ 3H]phosphocholine from [ 3H]sphingomyelin In some experiments radioactive sphingomyelin was used as the substrate, and the degradation thereof by sphingomyelinase was determined from the release of radiolabeled phosphocholine into the aqueous subphase. Monolayers of w 3 Hxsphingomyelin were spread as described in the previous section. In some experiments, the monolayer also contained cholesterol or various nonlabeled phospholipids. The enzyme was injected into the subphase Žfinal activity 6.7 mUrml., and aliquots of the subphase Ž 0.2 ml. were removed at time intervals for determination of the activity of the released w 3 Hxphosphocholine, using scintillation counting.
Fig. 1. Force–area isotherms at the airrbuffer interface. Isotherms of monolayers containing either bovine brain sphingomyelin, bovine brain ceramide, or a mixture of these lipids Ž20 mol% ceramide and 80 mol% sphingomyelin., were obtained at 308C.
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tained for N-P-SM, N-O-SM, and their corresponding ceramides Ž curves not shown. . The molecular area difference between the ceramide and the parent ˚ 2 for N-P-SM, and about 4 compound was about 28 A 2 ˚ for N-O-SM. The force–area isotherm of N-P-SM A also indicated the presence of a phase-transition, with the transition onset pressure being about 23 mNrm, ˚ 2 and 308C Žcurve not at an molecular area of 66 A shown.. N-O-SM had a fully expanded force–area isotherm Ždata not shown..
Fig. 3. Hydrolysis of bb-SM as a function of sphingomyelinase concentration in the subphase. Pure bb-SM monolayers were kept at 20 mNrm Žconstant surface pressure. on the buffer surface, and different amounts of sphingomyelinase were injected below the monolayer at 308C. Maximal hydrolysis rate as a time function was determined from several experiments. Each value is the mean from duplicate determinations Žrange"10%. from representative experiments.
Fig. 2. Hydrolysis of monolayer sphingomyelin by bacterial sphingomyelinase. A pure bb-SM monolayer was prepared at the airrbuffer interface, and compressed to 20 mNrm. The subphase buffer contained Tris-HCl Ž50 mM, pH 7.4. with 140 mM NaCl and 5 mM MgCl 2 . Sphingomyelinase was added to the stirred subphase, at 308C, to a final activity of 2.6 mUrml. Degradation of bb-SM by the enzyme led to a monolayer area decrease Žat constant surface pressure, 20 mNrm., as depicted in the upper panel. The definition of the lag-time is also shown. The lower panel shows the velocity of the monolayer area decrease per time unit as a time function, determined as the slope between two adjacent data points on the monolayer area decrease curve Župper panel.. The peak value indicates maximal monolayer area decrease which corresponds to maximal hydrolysis rate, and is converted to values of nmol bb-SM degraded per min during the course of the experiment. Most of the hydrolysis rates shown in subsequent figures are derived from similar data on monolayer area change as a time function.
The exposure at constant surface pressure of a pure sphingomyelin monolayer to sphingomyelinase in the subphase led to a time-dependent reduction of the monolayer area Ždetermined from the barrier movement which pushed new substrate into the reaction chamber., as shown in Fig. 2. The upper panel shows the area decrease Žgiven in square mm., and also shows the definition of the lag-time. The lower panel in Fig. 2 shows the slope between adjacent data points of the monolayer area Õersus time function Župper panel.. The slope gives the apparent reaction rate Žindicated as nmol sphingomyelin degraded per min., and also indicate the maximal rate achieved in the reaction. Both maximal hydrolysis rates, and lag-times were determined in subsequent experiments. The maximal rate of the degradation of bovine brain sphingomyelin by sphingomyelinase increased linearly as a function of added enzyme ŽFig. 3.. No saturation of the reaction rate increase could be seen with the sphingomyelinase activities tested. However, since the bacterial sphingomyelinase preparation contained 50% glycerol as a stabilizer, we did not use activities higher than about 6.7 mUrml to avoid adverse effects from the glycerol component.
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Fig. 4. Effect of Mg 2q-concentration on sphingomyelinase action. Monolayers of bb-SM were spread on the buffer surface to a surface pressure of 20 mNrm Žat 308C.. The subphase concentration of MgCl 2 was varied, and the hydrolysis induced by 6.7 mUrml sphingomyelinase in the subphase was determined from the monolayer area decrease Žpanel A.. Panel B shows the lag-time Žsee Fig. 2, upper panel. of the commencement of the hydrolysis reaction as a function of the subphase Mg 2q-concentration. Each value is the mean from duplicate determinations Žrange " 10%. from representative experiments.
The bacterial sphingomyelinase is known to be activated by Mg 2q w29x. As shown in Fig. 4, the addition of Mg 2q affected both the maximal reaction rate Ž panel A. as well as the lag-time for the commencement of the reaction Žpanel B.. It appeared, however, that whereas 1 mM Mg 2q was enough to maximally decrease the lag-time Ž panel B. , slightly more Mg 2q was needed to achieve maximal reaction rate Žpanel A; about 5 mM. . Consequently, 5 mM Mg 2q was included in all subsequent reactions.
The activity of the bacterial sphingomyelinase was markedly dependent on temperature. At room temperature, the rate of degradation of saturated sphingomyelins Žbb-SM and N-P-SM. was slow Ž Fig. 5. . When N-O-SM was used as substrate, the rate of degradation at room temperature was much higher than that observed for the saturated sphingomyelins. On a proportional basis, however, an increased temperature affected Ž increased. the rate of degradation of all tested sphingomyelins similarly ŽFig. 5..
Fig. 5. Rates of sphingomyelin hydrolysis at different temperatures. Monolayers containing either bb-SM, N-P-SM, or N-O-SM were prepared at the pure airrbuffer interface, and were kept at constant surface pressure Ž20 mNrm.. Sphingomyelinase Ž6.7 mUrml. was injected below the monolayer, and the maximal hydrolysis rate was determined at different temperatures. Each value is the mean from duplicate determinations Žrange " 10%. from representative experiments.
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Fig. 6. Lag-times of sphingomyelin hydrolysis at different temperatures. Monolayers containing either bb-SM, N-P-SM, or N-O-SM were prepared at the pure airrbuffer interface, and were kept at constant surface pressure Ž20 mNrm.. Sphingomyelinase Ž6.7 mUrml. was injected below the monolayer, and the lag-time for the commencement of the hydrolysis reaction was determined at different temperatures. Each value is the mean from duplicate determinations Žrange " 10%. from representative experiments.
The lag-time for the commencement of the hydrolysis reaction was also very sensitive to temperature. At room temperature, monolayers of both bb-SM and N-P-SM displayed long lag-times before hydrolysis by sphingomyelinase commenced ŽFig. 6.. However, when the temperature was increased, the lag-times decreased sharply, and stabilized on a steady level at temperatures above 258C. The degradation of a monolayer containing N-O-SM, which is altogether fluid at room temperature, and has no observable
phase-transition, did not show a dramatic change in lag-time over the temperature range of 20–378C ŽFig. 6.. Clearly, the observable lag-time for sphingomyelin degradation reaction was highly dependent on the physical state of the substrate molecules, as was the maximal reaction rate ŽFig. 5.. The lateral surface pressure of the substrate-containing monolayer membrane markedly influenced both reaction rates and lag-times, when the hydrolysis of bb-SM was determined at 308C. The reaction rate
Fig. 7. Effect of monolayer surface pressure on the hydrolysis of sphingomyelin. Pure monolayers of bb-SM were prepared and compressed to different lateral surface pressures. At constant surface pressure, the hydrolysis of bb-SM was initiated by injection of 6.7 mUrml of sphingomyelinase into the subphase, which was maintained at 308C. Panel A shows the maximal hydrolysis rate as a surface pressure function, whereas panel B gives the lag-time for the commencement of the reaction as a surface pressure function. Each value is the mean from duplicate determinations Žrange " 10%. from representative experiments.
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Fig. 8. Hydrolysis of w 3 Hxsphingomyelin by sphingomyelinase. Monolayers were prepared to contain w 3 Hxsphingomyelin from bovine brain, and these were maintained at a constant surface pressure of 20 mNrm Žat 308C.. Sphingomyelinase Ž6.7 mUrml. was injected into the subphase, and the degradation of monolayer w 3 Hxsphingomyelin Žopen circle. was determined from the release of w 3 Hxphosphocholine into the subphase, aliquots of which were removed at time intervals for radioactivity determination. Another set of monolayers were prepared to also contain 4 mol% cholesterol, in addition to bovine brain w 3 Hxsphingomyelin, and their degradation is indicated by the open squares. Each value is the mean"S.E.M. of three different monolayers at each temperature.
increased with increasing surface pressure up to about 20 mNrm Žmaximal reaction rate, cf. Fig. 7A., but thereafter the reaction rate decreased as the lateral surface pressure increased further Žlateral surface
pressures higher than about 28 mNrm could not be examined because of the monolayer instability of ceramide.. As the surface pressure increased above 20 mNrm, the lag-time of the reaction started to increase dramatically ŽFig. 7B.. A qualitatively similar surface pressure-dependence was observed when N-P-SM and N-O-SM monolayers were hydrolyzed by sphingomyelinase Ždata not shown.. To study the effects of colipids Že.g., cholesterol and other phospholipids. on reaction rates and lagtimes, we had to use radiolabeled sphingomyelin Žprepared from bb-SM. as substrate, and assay the hydrolysis reaction from the release of w 3 Hxphosphocholine into the subphase Žin binary and ternary monolayers all components may contribute to measurable area changes in a way which is not easily controllable.. Using this assay technique, we could see that when using a pure w 3 Hxsphingomyelin as substrate, about 5 nmol of w 3 Hxsphingomyelin was degraded by sphingomyelinase within 6–8 min at 308C ŽFig. 8.. Since the reaction chamber surface was 2827 mm2 , and the bb-SM molecular area at 308C ˚ 2 , one can calculate and 20 mNrm was about 75 A 3 that 3.8 nmol of w Hxsphingomyelin was initially exposed to sphingomyelinase Ž this number only includes the amount of w 3 Hxsphingomyelin on the surface of the reaction chamber. . Therefore, we can conclude that all of the initially present w 3 Hxsphingomyelin was degraded during the first 6–8 min. In addition, it appears that some sphingomyelin ‘leaked’ into the reaction chamber from the lipid reservoir compartment Ž by diffusion, and because unhydro-
Table 1 Effects of cophospholipids on hydrolysis of bovine brain w 3 Hxsphingomyelin Žw 3 Hxbb-SM. by sphingomyelinase Monolayer composition
w 3 Hxbb-SM degraded during first 8 min of reaction Žnmol.
100 mol% w 3 Hxbb-SM 90 mol% w 3 Hxbb-SMq 10 mol% dilauroyl phosphatidic acid 90 mol% w 3 Hxbb-SMq 10 mol% bovine brain phosphatidylserine 90 mol% w 3 Hxbb-SMq 10 mol% dipalmitoyl phosphatidylcholine 90 mol% w 3 Hxbb-SMq 10 mol% dipalmitoyl phosphatidylethanolamine 90 mol% w 3 Hxbb-SMq 10 mol% dipalmitoyl phosphatidylglycerol
4.5 " 0.5 4.0 " 0.1 4.0 " 0.2 5.0 " 0.4 4.5 " 0.3 4.6 " 0.3
Monolayers containing w 3 Hxbb-SM Ž90 mol%. and an unlabeled cophospholipid Žat 10 mol%. were prepared at the airrbuffer interface. Monolayers were maintained at 20 mNrm and 308C. Sphingomyelinase was injected into the subphase Žfinal concentration 6.7 mUrml. and the release of w 3 Hxphosphocholine into the aqueous subphase was determined by scintillation counting. Each value is the mean " S.E.M. of three different monolayers preparations of each composition.
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lyzed monolayer material was pushed into the reaction chamber, since the assay was performed in the constant surface pressure mode.. The lag-time for the commencement of the degradation of w 3 Hxsphingomyelin was about 2 min, which is similar to what was seen previously with nonlabeled substrate molecules. The inclusion of a trace amount of cholesterol in the sphingomyelin monolayer Žat 4 mol%. dramatically increased the amount of w 3 Hxsphingomyelin that was degradable by sphingomyelinase, and also shortened the lag-time for the reaction Ž Fig. 8.. An increased lateral diffusion probably supplied this additional w 3 Hxsphingomyelin onto the surface of the reaction chamber, where it was degraded. The addition of more cholesterol Žup to 25 mol%. to the sphingomyelin monolayer did not further enhance sphingomyelin degradation Ždata not shown.. The inclusion of 10 mol% of one of several different phospholipids Ž both zwitterionic and negatively charged. did not significantly affect rates of w 3 Hxsphingomyelin degradation ŽTable 1..
4. Discussion In this study we have examined how interfacial properties of the substrate membrane affect the activity of bacterial sphingomyelinase. The enzyme assay was performed using the surface barostat technique, the advantages of which include full control of the physical properties of the lipid substrate membrane. The trough used was of a zero-order type w27x, although the hydrolysis reaction measured did not obey zero-order kinetics Žbecause the reaction product remained in the membrane.. We could still, however, determine the lag-time for the commencement of the reaction, and the maximal rate of degradation. The actual reaction could be followed from the monolayer area decrease, since the removal of the phosphocholine group of sphingomyelin led to a smaller molecular area requirement of the resulting ceramide. The sphingomyelinase-induced degradation of sphingomyelin gave a sigmoidal monolayer area decrease as a time function. This is typical of enzyme reactions, in which both the substrate and the product reside in the monolayer membrane w30,31x. The latter part of the sigmoidal reaction curve was clearly a result of the fact that ceramide accumulates in the
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vicinity of the enzyme, and slows down the reaction due to substrate depletion. The initial part of the sigmoidal reaction curve is likely to depend on at least two different components, one being the association of enzyme with the monolayer, and the other being the actual commencement of the enzyme reaction. The association of sphingomyelinase with the monolayer membrane has not been determined directly in the present study. This is because the enzyme is not surface active Žw26x; data not shown. , and because of technical limitations. However, the results regarding the lag-time of the reactions can be interpreted to depend on enzyme association with the monolayers Žlonger lag-times indicate poorer association.. First, much less Mg 2q was required to lower the lag-time of the reaction, as compared to what was needed for full activation of the enzyme. This suggests that Mg 2q at low concentrations enhanced the association of enzyme with the substrate monolayer Žresulting in shorter lag times., whereas higher Mg 2q concentration were needed to fully activate the enzyme. Previous experiments with a Bacillus cereus sphingomyelinase have shown that adsorption of the enzyme to liposomal sphingomyelin membranes is enhanced by divalent cations, although a specific role for Mg 2q in adsorption was not demonstrated w32,33x. The Staphylococcus aureus enzyme Žused in this study. may differ from the Bacillus cereus enzyme with regard to the specificity of the metal ion required for adsorption. Secondly, the lag-times of the reactions were much longer when the substrate molecules were liquid-condensed as opposed to liquid-expanded. At room temperature Žabout 208C. both bb-SM and N-P-SM monolayers were dominantly liquid-condensed at 20 mNrm, and the lag-time for the commencement of the sphingomyelinase-reaction was long Ž40 and 15 min, for bb-SM and N-P-SM respectively. . However, the monolayer of N-O-SM at 208C and 20 mNrm was liquid-expanded, and the corresponding lag-time was only 2 min. Clearly, the physical state of the substrate markedly affected the lag-times of the reactions. Protein penetration into monolayers is known to be enhanced by a looser lateral packing Žhigher lateral compressibility. as opposed to a more condensed packing w34,35x. The observed effect of the physical state of the monolayer on the lag-times of
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the reactions is compatible with the view that a looser packing Že.g., N-O-SM monolayer. allows better or faster enzyme association, and consequently yields a shorter lag-time before the hydrolysis reaction commences. When monolayers of bb-SM or N-P-SM were hydrolyzed at a higher temperature, at which they were more liquid-expanded, the observable lagtimes were dramatically shortened, suggesting that enzyme association with the monolayers at higher temperatures was enhanced. It is interesting to note that the presence of small amounts of ceramide Ž20 mol%. in the bb-SM monolayer appeared to abolish the phase transition region at surface pressure below 40 mNrm. Consequently, as the sphingomyelinasecatalyzed reaction product Žceramide. is likely to affect the rate of its own initial formation. The effect of the lateral surface pressure on the lag-time of the reaction can be explained as follows. At higher surface pressures, the enzyme association with the monolayer is more difficult, and consequently longer lag-times are expected and were observed. The lag-times we determined for the sphingomyelinase reaction is clearly related to the ‘acceleration time’ described by Yedgar and coworkers w26x for the same monolayer reaction. Since we and they measured the sphingomyelinase reaction differently Žconstant pressure versus constant area., the lag-time or the acceleration-time variable is also defined differently. Despite the difference in the way the enzyme was assayed, our results regarding the lag-time of the reaction is in good general agreement with the acceleration time variable of the study of Yedgar and coworkers w26x. Assuming that the enzyme associates with the monolayers at sites where the local packing density is looser Žexpanded., one can envision that the sphingomyelin degradation reaction starts at these localizations and commences at the boundary line between expanded and condensed phases. Such a reaction sequence has actually been documented visually Žusing monolayer fluorescence microscopy. for two enzymes acting at the aqueousrmembrane interface. The enzyme phospholipase A 2 was shown to penetrate into the expanded domains of a dipalmitoylphosphatidylcholine monolayer, whereas the actual degradation of the phospholipid occurred along the liquidrcondensed domain interface w36,37x. Similarly, cholesterol oxidase, which oxidizes cholesterol
to cholestenone, also penetrated the substrate monolayers at loosely packed sites, from which the reaction subsequently commenced w38x. It is likely that the sequence of events with the sphingomyelinase system is similar to that of the phospholipase A 2 or cholesterol oxidase systems. However, the direct visualization of sphingomyelin degradation in monolayers by sphingomyelinase is hampered by the fact that sphingomyelin does not form clearly defined condensed domains, the fate of which could be visually followed Ž Slotte, unpublished observations.. Maximal reaction rates with the bacterial sphingomyelinase were observed at a lateral surface pressure of about 20 mNrm for all three sphingomyelin substrates tested. In the study of Yedgar and coworkers w26x, maximal reaction rate was observed at 19–22 mNrm for bb-SM. With N-P-SM, the optimal surface pressure was determined to lie within the range 16–20 mNrm w26x, whereas our study suggested that the optimal pressure was 20 mNrm. It is possible that the surface pressure-dependence of the sphingomyelinase reaction relates to the orientation of the phosphocholine head group of sphingomyelin in relation to the hydrophobic part of the molecule, a function which clearly depends on the degree of compression Ž and hence on lateral surface pressure.. However, the surface concentration of a substrate molecule in monolayers is also known to affect rates of enzyme-catalyzed conversions w31x, and may have contributed to the observed surface pressure-dependence of our enzyme assay. Another parameter which clearly affected maximal reaction rates was the degree of unsaturation of the substrate molecules. N-O-SM, which is more fluid than, e.g., N-P-SM, was degraded much faster at a given temperature. Clearly the general fluidity of the monolayer affected the rate by which enzyme and substrate molecules could diffuse toward each other Žand presumably also how efficiently the resulting ceramide could diffuse away from the enzyme.. The importance of monolayer fluidity in determining reaction rates was also seen when trace amounts Ž4 mol%. of cholesterol was included in a w 3 Hxsphingomyelin monolayer – both the lag-time Ženzyme association. decreased and the extent of w 3 Hxsphingomyelin degradation increased as a consequence of cholesterol inclusion. Whereas the feeding of free cholesterol or 7-dehydrocholesterol to cells have been
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shown to inhibit sphingomyelin degradation w39,40x, the effect of cholesterol on the action of sphingomyelinase in this model system was quite the opposite. The importance of membrane fluidity on affecting sphingomyelinase activity was also clearly demonstrated by Sandhoff and colleagues, who showed that halothane Žby increasing membrane fluidity. could dramatically enhance the degradation of sphingomyelin in isolated brain synaptosomal membranes w21x. Modest changes in monolayer surface Žnegative. charge, as effected by the inclusion of 10 mol% cophospholipids in the w 3 Hxsphingomyelin monolayer, did not however appear to affect the extent of w 3 Hxsphingomyelin degradation. The cophospholipids that were used were all saturated Ž save for bovine brain phosphatidyl serine. and were not expected to affect the fluidity of bovine brain w 3 Hxsphingomyelin. In conclusion, the results of this study have emphasized the importance of the physical state of the substrate molecules in determining rates of both enzymermembrane association and reaction rates for the bacterial sphingomyelinase. It is plausible to assume that the physical state of sphingomyelin present in biological membranes have similar effects on the activity of sphingomyelinases present in those membranes, or acting at the aqueousrmembrane interface. Future work with purified human sphingomyelinases, using this experimental approach, should settle this assumption one way or the other.
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Acknowledgements
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This study was supported by generous grants from the Sigrid Juselius Foundation, the Academy of Fin¨ ˚ land, the Oscar Oflund Foundation, and the Abo Akademi University.
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