583rd MEETING. CAMBRIDGE
1075
simple consequence of metabolic switching, since the predicted retardation of 3-hydroxylation for the ZH4-labelledanalogue did not occur. Indeed, 3-hydroxylation was actually more rapid for this analogue than for biphenyl when microsomal-fraction from noninduced animals was used. Moreover, the predicted increase in 4-hydroxylation for the ZH4-labelledanalogue did not occur; indeed, there was a trend towards a lower rate for 4-hydroxylation of this analogue compared with the ZH,-labelled and the non-deuterated forms. A possible explanation for the above anomalies may be that deuterium substitiuton might slightly alter, and to various extents, the affinity of the substrate for the different microsomal mono-oxygenases. The steric requirement of hydrogen appears to be significantly more than that of deuterium (Lee et al., 1978). Deuterium substitution can also change lipophilicity, as evidenced by the ability of high-pressure liquid chromatography to resolve deuterated compounds from their unlabelled counterparts (see, e.g., Farmer et al., 1978). Such an explanation could account for species differences in the isotope effect for 3-hydroxylation of biphenyl (Billings & McMahon, 1978) and the dependence on the method of induction (Table 1) of the relative rates of hydroxylation at the various sites of the biphenyl variants used in the present study. To test this second hypothesis, further studies on the effect of selective deuteration on hydroxylation of biphenyl need to be carried out with the separate components of the microsomal monooxygenase system. Akawie, R. I., Scarborough, J. M. & Burr, J. G. (1959) J. Org. Chem. 24,946-949 Billings, R. E. & McMahon, R. E. (1978) Mol. Pharmucol. 14,145-154 Burke, M . D. & Prough, R. A. (1977) Anal. Biochem. 83,46&473 Burke, M. D., Benford, D. J., Bridges, J. W. & Parke, D. V. (1977) Biochem. SOC. Trans. 5, 1370-1372 Farmer, P. B., Foster, A. B., Jarman, M., Oddy, M. R. & Reed, D. J. (1978)J. Med. Chem. 21, 514-520 Homing, M. G . , Haogele, K . D., Sommer, K. R., Nowlin, J., Stafford, M. & Thenot, J. P. (1975) Proc. Int. Symp. Stable Isoropes 2nd. (Klein, E. R. & Klein, P. D., eds.), pp. 41-54, National Technical Information Service, U S . Department of Commerce, Springfield Lee,S.-Y.,Rarth, G., Kieslich, K. & Djerassi, C. (1978)J. Am. Chem. SOC. 100,3965-3966 McPherson, F. J., Bridges, J. W. & Parke, D. V. (1976) Biochem. Pharmacol.25,1345-1350
Evidence for a Model of Regeneration of a Protonated Species, bR, from a Phototransient, M, in the Photochemical Cycle of Bacteriorhodopsin from Halobacterium halobium MARY E. EDGERTON and COLIN GREENWOOD School of Biological Sciences, Unfversity of East Anglia, Norwich NR4 7TJ, U.K.
The purple membrane of Halobacterium halobium contains a single protein bound via a Schiff base to a retinal (Oesterhelt et a!., 1973; Lewis et al., 1974). This proteinretinal complex, bacteriorhodopsin, undergoes a light-driven reaction cycle that results in a vectorial flow of protons across the membrane (Oesterhelt & Stoeckenius, 1973). The photocycle can be described by the following sheme, defined by Lozier et al. (1979, the superscripts denoting the approximate wavelength maxima (nm) :
Studies carried out on species M show that it has a deprotonated Schiff base (Lewis et al., 1974) and that, simultaneous with its decay, a proton is released from the membrane to the exterior medium (Lozier et al., 1975). Decay of species M is not always monophasic, as was once thought, but shows biphasicity under conditions of low temperature and
Vol. 7
1076
BIOCHEMICAL SOCIETY TRANSACTIONS
Table 1. Decay kinetics of species M in Halobacterium halobium purple membrane at 412nm in thepHrange 6.80-10.80 Flash-photolysis experiments were carried out at 22°C on purple membrane at 2 - 5 p ~ adjusted to a measured pH with 0.1 M NaOH. The results here give the initial absorbance changes due to the decay phase(s), A, and A,. The decay rate(s) is given as k , and k,. PH 6.80 9.00 9.40 10.30 10.80
A, 0.175 0.162f0.004 0.185f0.017 0.122f0.005 0.097 f0.007
ki (s-') 168 149f2 171 f 9 313f 13 303 k 18
A2
k2 (s-')
0.077k0.004 0.062 f0.001
47+3 1852
high ionic strength (Eisenbach et al., 1976; Korenstein et al., 1978). Hurley et al. (1978) have shown by static spectra that at very low temperatures species M is photolysed to M', a species with a very similar absorption spectrum. The photochemical cycle has been modified to accommodate this new information, and now appears as: bR-+K+L+M+
M'+bR
L O ,
Here 412nm decay is shown to have biphasic characteristics at high pH values, and one of the reactions appears to be associated with the reprotonation of the Schiff base necessary to regenerate species bR. Experiments were carried out with purple membrane at 2 - 5 p ~prepared as described by Oesterhelt & Stoeckenius (1974) adjusted to a measured pH with 0.1 M NaOH. An Applied Photophysics flash photolysis was used and the data were input to a Digital PDP 8/A Mini-Computer via a Data Lab 905 Transient Recorder. Results were found by linear regression for the monpohasic reactions and an iterative procedure adapted from Wilkins et al. (1974) for biphasic reactions. These results are given in Table 1 . Species M4I2 decays at room temperature with a single rate of 161 k 11s-', independent of pH in the range 6.80-9.40. Above pH9.4 the decay becomes biphasic, with 61 % decaying at a rate of 310k 13s-I and the remaining 39% at a rate that has a possible dependence on pH (see Table 1). Given that decay of species M is associated with a deprotonation at the membrane surface, it follows that there must be at least one intermediate between species M and the regenerated species bR that is involved in a reprotonation of the Schiff base. This hypothetical intermediate is termed x in the following model. The decay rate of intermediate x would then have a linear dependence on increasing H+. If it has an extinction coefficient comparable with, though not equal to, that of species M at 412nm, then at pH 6.80 species M would decay to intermediate x at a rate of 161s-,, whereupon intermediate x would apparently disappear at a rate lo4 times that of the decay at pH 10.80. If k z is taken to represent the decay rate of intermediate x, then it would not be resolvable in the pH range 6.80-9.40. The suggested photocycle then becomes : bR + K
+
L
-+
M
+ y,
-+
bR
L O ,
with L-tM representing a transfer of a proton away from the Schiff base, M-+x a release of the proton to the exterior medium, and x-bR a reprotonation of the Schiff base. The sudden change of the M-tx rate from 150s-' to 350s-I between pH9.4 and 10.3 may be interpreted as being due to the decay of two different species of M to intermediate x that have an equilibrium whose pK lies between 9.4 and 10.3. The high-pH species 1979
583rd MEETING. CAMBRIDGE
1077
could either be a deprotonated or hydroxylated form and is denoted as MoH-. The proposed model is now completed as: r ( O ) i bR+K+L+M+x+bR
11 /”
MOH-
Since no O species is observed at these high pH values, this pathway is left as a possible branch for return to species bR under conditions other than those reported here. This model supplies the reprotonation step necessary for regeneration of species bR in the photochemical cycle, and also explains why decay of species M is apparently monophasic at neutral pH in distilled water. High ionic strength or very low temperature may slow down the reprotonation step so that the decay of species M is apparently biphasic under these conditions. Intermediate x could represent the M species at a slightly higher energy level, which would accommodate the work of Hurley et al. (1978), with intermediate x identified as species M‘, formed by photolysis of species M. The proposed model can be seen to unify a number of models already extant, and to give further insight into the nature of the photochemical intermediates and their function in the proton pumping role of bacteriorhodopsin. M. E. E. is grateful for a postgraduate fellowship from the Marshall Aid Commemoration Commission. We thank Dr. Donald Barber, Dr. Peter Brimblecombe and Dr. Terry Moore for helpful discussions.
Eisenbach, M., Bakker, P., Korenstein, R. & Caplan, C. R. (1976) FEBS Lett. 71, 228-231 Hurley, J. B., Becher, B. & Ebrey, J. G. (1978) Nurure (Londun) 272,87-88 Korenstein, R., Hess, B. & Kuschmitz, D. (1978) FERSLetf. 93, 266-270 Lewis, A., Spoonhower, J., Bogomolni, R. A,, Lozier, R. H. & Stoeckenius, W. (1974) Proc Natl. Acud. Sci. U.S.A. 71,44624466 Lozier, R. H., Bogomolni, R. A. & Stoeckenius, W. (1975) Bioghys. J . 15,955-962 Oesterhelt, D. & Stoeckenius, W. (1973) Pruc. Natl. Acad. Sci. U.S.A.70,2853-2857 Oesterhelt, D. & Stoeckenius, W. (1974) Methods Enzynrol. 31A, 667-678 Oesterhelt, D., Meentzen, M & Schumann, L. (1973) Eur. J. Biochem. 40,453-463 Wilkins, C. L., Klopfenstein,C. E., Isenhour, T. L. & Jurs, P. C. (1974) Introduction ro Cotnputer Prugrammingfor Chemists: BASIC Version, .411yn and Bacon, Boston
Phosphatidylcholine Degradation in Mouse Plasma MICHAEL P. T. GILLETT, ELIZABETH M. COSTA and JAMES S. OWEN* Departmento de Bioquimica, Centro de Ciencias Bioldgicas, Universidade Federal de Pernambuco, Recije-PE, Brazil
We have previously shown differences between the phosphatidylcholine-cholesterol acyltransferase (EC 2.3.1.43, hereafter called ‘acyltransferase’) of mouse plasma and that of other species, including man and rat (Owen et al., 1979). The mouse acyltransferase was partially inhibited by mercaptoethanol (Owen, 1977) and could not be measured by the method of Stokke & Norum (1971), which depends on re-activating the acyltransferase with mercaptoethanol after equilibrating radiolabelled cholesterol with endogenous lipoprotein cholesterol, during which time the acyltransferase is inhibited with a disulphide. Instead mouse acyltransferase activity was measured either by incubation of plasma whose cholesterol had been labelled by prior injection in viuo of ~ ~ - [ 2 - ~ H ] m e v a l oacid n i c lactone (Owen et al., 19780) or by chemically determining the decrease in free cholesterol concentration (Owen et al., 1979). During this latter
* Present address: Department of Biochemistry and Chemistry, Royal Free Hospital School of Medicine, University of London, 8 Hunter Street, London WClN IBP, U.K. Vol. 7
1075
simple consequence of metabolic switching, since the predicted retardation of 3-hydroxylation for the ZH4-labelledanalogue did not occur. Indeed, 3-hydroxylation was actually more rapid for this analogue than for biphenyl when microsomal-fraction from noninduced animals was used. Moreover, the predicted increase in 4-hydroxylation for the ZH4-labelledanalogue did not occur; indeed, there was a trend towards a lower rate for 4-hydroxylation of this analogue compared with the ZH,-labelled and the non-deuterated forms. A possible explanation for the above anomalies may be that deuterium substitiuton might slightly alter, and to various extents, the affinity of the substrate for the different microsomal mono-oxygenases. The steric requirement of hydrogen appears to be significantly more than that of deuterium (Lee et al., 1978). Deuterium substitution can also change lipophilicity, as evidenced by the ability of high-pressure liquid chromatography to resolve deuterated compounds from their unlabelled counterparts (see, e.g., Farmer et al., 1978). Such an explanation could account for species differences in the isotope effect for 3-hydroxylation of biphenyl (Billings & McMahon, 1978) and the dependence on the method of induction (Table 1) of the relative rates of hydroxylation at the various sites of the biphenyl variants used in the present study. To test this second hypothesis, further studies on the effect of selective deuteration on hydroxylation of biphenyl need to be carried out with the separate components of the microsomal monooxygenase system. Akawie, R. I., Scarborough, J. M. & Burr, J. G. (1959) J. Org. Chem. 24,946-949 Billings, R. E. & McMahon, R. E. (1978) Mol. Pharmucol. 14,145-154 Burke, M . D. & Prough, R. A. (1977) Anal. Biochem. 83,46&473 Burke, M. D., Benford, D. J., Bridges, J. W. & Parke, D. V. (1977) Biochem. SOC. Trans. 5, 1370-1372 Farmer, P. B., Foster, A. B., Jarman, M., Oddy, M. R. & Reed, D. J. (1978)J. Med. Chem. 21, 514-520 Homing, M. G . , Haogele, K . D., Sommer, K. R., Nowlin, J., Stafford, M. & Thenot, J. P. (1975) Proc. Int. Symp. Stable Isoropes 2nd. (Klein, E. R. & Klein, P. D., eds.), pp. 41-54, National Technical Information Service, U S . Department of Commerce, Springfield Lee,S.-Y.,Rarth, G., Kieslich, K. & Djerassi, C. (1978)J. Am. Chem. SOC. 100,3965-3966 McPherson, F. J., Bridges, J. W. & Parke, D. V. (1976) Biochem. Pharmacol.25,1345-1350
Evidence for a Model of Regeneration of a Protonated Species, bR, from a Phototransient, M, in the Photochemical Cycle of Bacteriorhodopsin from Halobacterium halobium MARY E. EDGERTON and COLIN GREENWOOD School of Biological Sciences, Unfversity of East Anglia, Norwich NR4 7TJ, U.K.
The purple membrane of Halobacterium halobium contains a single protein bound via a Schiff base to a retinal (Oesterhelt et a!., 1973; Lewis et al., 1974). This proteinretinal complex, bacteriorhodopsin, undergoes a light-driven reaction cycle that results in a vectorial flow of protons across the membrane (Oesterhelt & Stoeckenius, 1973). The photocycle can be described by the following sheme, defined by Lozier et al. (1979, the superscripts denoting the approximate wavelength maxima (nm) :
Studies carried out on species M show that it has a deprotonated Schiff base (Lewis et al., 1974) and that, simultaneous with its decay, a proton is released from the membrane to the exterior medium (Lozier et al., 1975). Decay of species M is not always monophasic, as was once thought, but shows biphasicity under conditions of low temperature and
Vol. 7
1076
BIOCHEMICAL SOCIETY TRANSACTIONS
Table 1. Decay kinetics of species M in Halobacterium halobium purple membrane at 412nm in thepHrange 6.80-10.80 Flash-photolysis experiments were carried out at 22°C on purple membrane at 2 - 5 p ~ adjusted to a measured pH with 0.1 M NaOH. The results here give the initial absorbance changes due to the decay phase(s), A, and A,. The decay rate(s) is given as k , and k,. PH 6.80 9.00 9.40 10.30 10.80
A, 0.175 0.162f0.004 0.185f0.017 0.122f0.005 0.097 f0.007
ki (s-') 168 149f2 171 f 9 313f 13 303 k 18
A2
k2 (s-')
0.077k0.004 0.062 f0.001
47+3 1852
high ionic strength (Eisenbach et al., 1976; Korenstein et al., 1978). Hurley et al. (1978) have shown by static spectra that at very low temperatures species M is photolysed to M', a species with a very similar absorption spectrum. The photochemical cycle has been modified to accommodate this new information, and now appears as: bR-+K+L+M+
M'+bR
L O ,
Here 412nm decay is shown to have biphasic characteristics at high pH values, and one of the reactions appears to be associated with the reprotonation of the Schiff base necessary to regenerate species bR. Experiments were carried out with purple membrane at 2 - 5 p ~prepared as described by Oesterhelt & Stoeckenius (1974) adjusted to a measured pH with 0.1 M NaOH. An Applied Photophysics flash photolysis was used and the data were input to a Digital PDP 8/A Mini-Computer via a Data Lab 905 Transient Recorder. Results were found by linear regression for the monpohasic reactions and an iterative procedure adapted from Wilkins et al. (1974) for biphasic reactions. These results are given in Table 1 . Species M4I2 decays at room temperature with a single rate of 161 k 11s-', independent of pH in the range 6.80-9.40. Above pH9.4 the decay becomes biphasic, with 61 % decaying at a rate of 310k 13s-I and the remaining 39% at a rate that has a possible dependence on pH (see Table 1). Given that decay of species M is associated with a deprotonation at the membrane surface, it follows that there must be at least one intermediate between species M and the regenerated species bR that is involved in a reprotonation of the Schiff base. This hypothetical intermediate is termed x in the following model. The decay rate of intermediate x would then have a linear dependence on increasing H+. If it has an extinction coefficient comparable with, though not equal to, that of species M at 412nm, then at pH 6.80 species M would decay to intermediate x at a rate of 161s-,, whereupon intermediate x would apparently disappear at a rate lo4 times that of the decay at pH 10.80. If k z is taken to represent the decay rate of intermediate x, then it would not be resolvable in the pH range 6.80-9.40. The suggested photocycle then becomes : bR + K
+
L
-+
M
+ y,
-+
bR
L O ,
with L-tM representing a transfer of a proton away from the Schiff base, M-+x a release of the proton to the exterior medium, and x-bR a reprotonation of the Schiff base. The sudden change of the M-tx rate from 150s-' to 350s-I between pH9.4 and 10.3 may be interpreted as being due to the decay of two different species of M to intermediate x that have an equilibrium whose pK lies between 9.4 and 10.3. The high-pH species 1979
583rd MEETING. CAMBRIDGE
1077
could either be a deprotonated or hydroxylated form and is denoted as MoH-. The proposed model is now completed as: r ( O ) i bR+K+L+M+x+bR
11 /”
MOH-
Since no O species is observed at these high pH values, this pathway is left as a possible branch for return to species bR under conditions other than those reported here. This model supplies the reprotonation step necessary for regeneration of species bR in the photochemical cycle, and also explains why decay of species M is apparently monophasic at neutral pH in distilled water. High ionic strength or very low temperature may slow down the reprotonation step so that the decay of species M is apparently biphasic under these conditions. Intermediate x could represent the M species at a slightly higher energy level, which would accommodate the work of Hurley et al. (1978), with intermediate x identified as species M‘, formed by photolysis of species M. The proposed model can be seen to unify a number of models already extant, and to give further insight into the nature of the photochemical intermediates and their function in the proton pumping role of bacteriorhodopsin. M. E. E. is grateful for a postgraduate fellowship from the Marshall Aid Commemoration Commission. We thank Dr. Donald Barber, Dr. Peter Brimblecombe and Dr. Terry Moore for helpful discussions.
Eisenbach, M., Bakker, P., Korenstein, R. & Caplan, C. R. (1976) FEBS Lett. 71, 228-231 Hurley, J. B., Becher, B. & Ebrey, J. G. (1978) Nurure (Londun) 272,87-88 Korenstein, R., Hess, B. & Kuschmitz, D. (1978) FERSLetf. 93, 266-270 Lewis, A., Spoonhower, J., Bogomolni, R. A,, Lozier, R. H. & Stoeckenius, W. (1974) Proc Natl. Acud. Sci. U.S.A. 71,44624466 Lozier, R. H., Bogomolni, R. A. & Stoeckenius, W. (1975) Bioghys. J . 15,955-962 Oesterhelt, D. & Stoeckenius, W. (1973) Pruc. Natl. Acad. Sci. U.S.A.70,2853-2857 Oesterhelt, D. & Stoeckenius, W. (1974) Methods Enzynrol. 31A, 667-678 Oesterhelt, D., Meentzen, M & Schumann, L. (1973) Eur. J. Biochem. 40,453-463 Wilkins, C. L., Klopfenstein,C. E., Isenhour, T. L. & Jurs, P. C. (1974) Introduction ro Cotnputer Prugrammingfor Chemists: BASIC Version, .411yn and Bacon, Boston
Phosphatidylcholine Degradation in Mouse Plasma MICHAEL P. T. GILLETT, ELIZABETH M. COSTA and JAMES S. OWEN* Departmento de Bioquimica, Centro de Ciencias Bioldgicas, Universidade Federal de Pernambuco, Recije-PE, Brazil
We have previously shown differences between the phosphatidylcholine-cholesterol acyltransferase (EC 2.3.1.43, hereafter called ‘acyltransferase’) of mouse plasma and that of other species, including man and rat (Owen et al., 1979). The mouse acyltransferase was partially inhibited by mercaptoethanol (Owen, 1977) and could not be measured by the method of Stokke & Norum (1971), which depends on re-activating the acyltransferase with mercaptoethanol after equilibrating radiolabelled cholesterol with endogenous lipoprotein cholesterol, during which time the acyltransferase is inhibited with a disulphide. Instead mouse acyltransferase activity was measured either by incubation of plasma whose cholesterol had been labelled by prior injection in viuo of ~ ~ - [ 2 - ~ H ] m e v a l oacid n i c lactone (Owen et al., 19780) or by chemically determining the decrease in free cholesterol concentration (Owen et al., 1979). During this latter
* Present address: Department of Biochemistry and Chemistry, Royal Free Hospital School of Medicine, University of London, 8 Hunter Street, London WClN IBP, U.K. Vol. 7
