396
BIOCHEMICAL SOCIETY TRANSACTIONS
be inferred both from the Ca' mobilization experiments
(Connolly et ul., 1984) and from some metabolic observations (Nedergaard, 1981), such an increased level has not
been directly demonstrated to date.
7. A Cu' -inducid opening oj K channels in the plusnia
nienibranr. This would be a feedback step in this cascade,
counteracting and stabilizing the membrane depolarization
and allowing the K + gradient to approach the Nernst
equilibrium. An 2-adrenergically stimulated release of
K ( " , R b + )has been observed (E. Nanberg, J. Nedergaard
& B . Cannon, unpublished work). Although the existence of
a potential-gated K channel cannot be excluded at present,
it is in agreement with the action of a Ca2+-dependent K +
channel that the CaZ ionophore A23187 can stimulate K
efflux from the cells (J. Nedergaard, E. Connolly, E.
Nanberg & N. Mohell, unpublished work). The ionophore
presumably acts by increasing cytosolic Ca2+,in parallel to
what happens when the N a + / C a ' + antiport is stimulated
physiologically.
+
+
+
+
+
+
+
Thp qicestiori of the iitiir~ersalitj~
of the phetionirnon
We have here described a possible active regulatory role
of the mitochondrial Na + / C a 2 antiport in brown adipose
tissue. I t is possible that the antiport in this tissue is
unusually important because of the very high abundance of
mitochondria in the tissue, and because of the limited
presence of other Ca' regulatory systems (endoplasmic
reticulum). We would however propose that the significance of the antiport and the cascade system sketched could
be more general. I t was, for example, initially believed that
the mitochondria from liver did not possess the Na' /Ca2
antiport. but a series of investigators now claim its presence
also here (Nedergaard & Cannon, 1980; Haworth et al.,
1980; Heffron & Harris, 1981; Harris & Heffron, 1982;
Goldstone & Crompton, 1982; J . Nedergaard, unpublished
work). It may be argued that the activity of the antiport in
liver is low, but functionally this is not the case, since also in
incubation of liver mitochondria an elevation of the extramitochondrial CaZ level of about 1 PM can be induced by
Na' (J. Nedergaard, unpublished work). I t may be added
that extracellular N a + has been demonstrated to be
essential for the mediation of r-adrenergic stimuli in liver
(Haylett & Jenkinson, 1972a,b).
Thus it is perhaps a general phenomenon that the mitochondrial Na /CaZ antiport is involved in the mediation
of r,-adrenergic (and/or other hormonal) stimuli.
+
+
+
+
+
+
This work was supported by the Swedish Natural Science
Research Council. The authors would like to thank Barbara
Cannon for valuable discussions.
Al-Shaikhaly, M. H. M., Nedergaard. J . &Cannon, B. (1979) Proc.
Nail. Acad. Sci. U . S . A . ,76. 2350 2353
Cannon. B. & Lindberg, 0. (1979) Methods Eiizrniol. 55. 65 78
Cannon, B., Nicholls, D. G. & Lindberg, 0. (1973) in Mc~chatii.sni.s
in Bioenergetics (Azzone, G . F., Ernster. L., Papa, S., Quagliariello. E. & Siliprandi, N., eds.), pp. 357 363. Academic Press.
New York
Cannon, B., Sundin, U., & Romert, L. (1977) FEBS Lett. 74, 4346
Carafoli, E. (1981) in Mitochondria and Micwsonics (Lee, C. P.,
Schatz, G.. & Dallner, G.. eds.), pp. 357 374. Addison-Wesley,
Reading, M A
Carafoli. E., Tiozzo, R., Lugli. G., Crovetti, F. & Kratzing, C.
(1974) J. Mol. Cell. Cardiol. 6. 3 6 1 ~371
Connolly, E., NAnberg, E. & Nedergaard, J . (1984) Eur. J.
Bk~cheni.in the press
Denton, R. M. & McCormack, J . G. (1981) FEBS Lcw. 119. I 8
Exton, J . H. (1980) Am. J. Phj~siol.238, E3 E l 2
Fain, J. N . & Garcia-Siinz, J . A. (1980) Lifi, Sci. 26. 1183- I194
Fink, S. A. & Williams, J . A. (1976) Am. J . Phwiol. 231, 700 706
Girardier, L. & Seydoux, J . (1971~)in Non.shirw"ng Thcwmgenc,.ris
(Jansky. L., ed.), pp. 255-270, Academia, Prague
Girardier, L. & Seydoux. J . (1971h) J. Physiol. (Paris)63, 147 186
Girardier, L.. Seydoux, J . & Clausen, T. (1968) J . Gcw. Physiol. 52.
925-940
Goldstone, T. P. & Crompton. M. (1982) Biochrni. J. 204. 3 6 9 ~371
Harris, E. J. & Heffron. J. J. A. (1982) Arch. Biochiwi. B i ~ ~ p h218,
y~.
531-539
Haworth, R. A., Hunter, D. R. & Berkoff, H. A. (1980) FEBS Lcw.
110, 216-218
Haylett, D. G. & Jenkinson. D. H. (1972a)J. Physiol. 225, 721 750
Haylett, D. G . & Jenkinson. D. H. (1972h)J. P h y s i d . 225, 751 772
Heffron. J . J . A. & Harris, E. J . (1981) Biochem. J. 194, 925 929
Horowitz, J . M., Horwitz, B. A. &Smith, R. E. (1971) E.\pricwtia
27, 1419-1421
Lilien, 0. M. (1976) J. Urol. 116, 277-281
Lilien, 0. M., Krauss, D. J., Manke, C. G., Euser, B. A . &
Subramanian, G . M. (1978) J . Urol. 119, 170-174
Locke, R., Rial, E., Scott, I. D. & Nicholls, D. G. (1982) Eur. J .
Biochem. 129, 373-380
Mohell, N., Svartengren, J . &Cannon, B. (1983) Eur. J . Pharniacol.
92, 15-25
Nedergaard, J . (1981) Eur. J. Biochem. 114. 159-167
Nedergaard, J. (1983) Eur. J . Biocheni. 133, 185 191
Nedergaard, J . & Cannon, B. (1980) Actu Chcm. Scand. B34, 149
151
Nedergaard. J . & Lindberg, 0.(1982) Inter. RCT.c.vlfJ/.74. 187 286
Nicholls, D. G. (1978) Biochem. J . 176, 463 474
Nicholls, D. G. (1979) Biochim. Biophp. Acta 549, I 29
Nicholls, D. G. & Akerman, K . (1982) Biochini. Bii~phy.~.
Acta 683.
57-88
Seydoux, J., Constantinidis, J., Tsacopoulos, M. & Girardier, L.
(1977) J. Physiol. (Paris) 73, 985-996
Strieleman, P. J.. Edson. C. E. & Shrago, E. (1983) Fed. Proc. 42,
I324
Electron flow in membranes
ROBERT J. P. WILLIAMS
Inorganic Chemistry Laboratory, Uniwrsit-v qj ' O.yjbrd,
O.yfiird OX1 3 Q R , U . K .
In 1961 two papers appeared describing the way external
energy could be transduced to give internal biochemical
energy, ATP (Mitchell, 1961; Williams, 1961). Both started
from electron transfer in membranes. Detailed analysis of
electron transfer in protein matrices (see Moore e t al., 1982)
now makes it clear that the electron can hop from site to site
covering about 1.5nm at each hop. The only problems that
remain in this area concern the disposition of and the
medium between sites. Given this hop distance it now
Abbreviation used: Q, coenzyme Q
seems very unlikely that electrons as such can travel across
membranes in some of the particles which transduce energy,
such as cytochrome oxidase. I say this since the construction
of this protein generates haem a and the associated Cu well
to the cytosolic side of the membrane so as to accept
electrons from cytochrome c and it has only one other
electron-transfer centre, the haem a , . Cu pair. The construction could hardly span a membrane and thus the idea of
an electron path across a membrane, a central part of
chemiosmosis (Mitchell, 1961) is likely to be incorrect in
this case. A more plausible scheme is that the electron
reactions are connected in the membrane to reactions, redox
or otherwise, of protons also in the membrane so that
energized protons (acids) are generated in the niemhrane
(Williams, 1961). These protons then diffuse by restricted
1984
397
606th MEETING, CORK
diffusion, i.e. in channels, firstly in the membrane and then
either directly (locally) to the ATP synthase (Williams,
1961) or establish of necessity chemiosmotic gradients
(Mitchell, 1961). Originally chemiosmosis had no proton
diffusion channels. I show elsewhere (Williams, 1984) that
very much evidence is now against the general equilibrations of chemiosmosis. This hypothesis appears now to
be at fault at all four steps; (a) the electron path, (b) the
proton path, (c) the direct field-driven synthesis of ATP, (d)
the need for equilibrated aqueous gradients. The alternative
of a more restricted electron flow, together with an
energized proton flow in membranes and a local flow of
protons through the ATP synthase accompanied by conformational change, is a more convincing description. It
should be noted that the energized proton flow in a
membrane (Williams, 1961), is of course a proton pump.
I wish now to turn to the experimental situation relating
to the recognized proton pumps, which are driven by
electron flow in the mitochondria1 and thylakoid chains.
(Note that the proton pump of bacteriorhodopsin, halorhodopsin and rhodopsin is driven instead by a conformational energy change used to generate the acidity in the
membrane.) What are the problems? They all rest in the
interaction between the electron and the proton flow. We
can dissect away the independent parts of this flow in the
membranes, that is of electrons alone (1-5nm hops) and of
protons alone (by rotational migration along - O H or H,O
chains; Williams, 1984). We are then left with the
proton/electron intercommunication which occurs at three
sites at least. They are in particles I, 111 and IV of mitochondria. In particle I the two-electron reaction is (see
Williams, 1970):
NADH (flavin H) + N A D (flavin) + 2e + 2H+
In particle 111 it is:
QH2-+Q+2e+2H+
In particle IV it is:
OH,
-+
10,+ 2e + 2H (FeOH, t FeO + 2e + 2H +)
+
(In all particles other, non-redox proton movements may
also be driven.) Notice that these are all two-electron
reactions of non-metals and not one-electron reactions of
metals as is used in simple electron flow (Williams, 1961). It
is generally true that non-metals react in two-electon steps
with proton involvement while metals in low oxidation
states, e.g. Fe(I1) and Cu(I), react by one-electron protonindependent steps in water. It is then the involvement of
non-metal couples which brings the proton and the electron
together in the membrane (Williams, 1961). One essential
feature of the non-metal reactions is that if they are part of a
catalytic transfer the non-metal group must cycle chemically. This applies to NAD (flavin) and Q but not to O,, which
is destroyed. Again if during the reactions energy is to be
conserved then the oxidation and reduction paths of the
cycling unit must not be directly connected physically.
Space must be used in the membrane to control the diffusion
from the reaction centres of both the products e and H so
that different e and H are used in oxidation and reduction
respectively. I described this as the dislocation of reaction
products (Williams, 1961), which has no membrane
counterpart in chemiosmosis and is quite separate from
subsequent translocation of either H + or e.
movement of the electron in the membrane other than by
transfer from metal site to metal site it is only through the
swinging-arm motions of the coenzymes (Williams, 1961) in
which H + and e move together as bound H atoms. When
the electron goes to a metal the proton must find its own
separate path. In particle IV the same situation arises since
oxygen does not cycle. In effect the two-electron reactions of
the high oxidation states of iron, e.g. FeO -+ Fe, are very
similar to the two-electron reaction N A D + -+ NADH.
Hydroxide leaves the first on reduction while protons leave
the second on oxidation due to the different sense of the
redox reaction, that is, reduction of oxygen (non-cycling)
and oxidation of substrate H (non-cycling). When we turn to
particle 111 there is a difficulty since the substrate of the
enzyme is now in a reversible chemical system. The
substrate, free QH,, first reacts with a bound Q in the
particle and the generated free 0'-must later react with a
second bound system to be re-reduced so as to complete the
cycle. The electrons and the protons are here travelling together
as bound H in the membrane. However, at some stage in the
reaction protons and electrons must be separated if energy
capture is to be achieved via energized protons. What
happens? Almost everybody has tried his hand at devising
schemes. Let us analyse them.
The problems of particle III
The first obvious point is that we need Q and QH, to
communicate between different major protein redox particles. This lateral diffusion in the membrane of Q/QH, is
essential if (1) redox equivalents once in the membrane are
to be kept there and since (2) redox equivalents in the
membrane must travel in a kinetically inert form and (3)
must be a source of H + on oxidation. Quinones are ideally
designed for this lateral two-dimensional diffirsion in the
membrane avoiding any communication with the aqueous
phases and only reacting in the membrane at chosen
centres. The significance of this lateral diffusion has
become strikingly obvious now that the lateral separation of
particles in thylakoids (Fig. 1) is established. We d o not
know the lateral separation in mitochondria. A major
problem now concerns the possible transverse diffusion of
Q/QH2, which is essential in chemiosmosis but is not
necessary if the membrane at particle 111 has proton and
electron transverse diffusion paths. [Note that if there is any
rate-limiting step for transverse movement of redox
W/A
Local pH high
\ 1 .
+
+
The cycles
In particle I the reactions of NADH or flavin H are those
of fixed sites. The electron leaves in one direction and the
proton in another. No problem arises since both these
entities are replaced by neutral hydrogen atoms from the
substrate which is consumed, e.g. malate. If there is any
Vol. 12
Local pH high
u,
0,
Fig. 1. The flow problem in thylakoids
Note the separation of the particles and the assumption that
chemiosmosis is not required. Local circuits are shown. PSI,
Photosystem I ; PS 11, photosystem 11.
BIOCHEMICAL SOCIETY TRANSACTIONS
398
equivalents at the Q/QHzreaction sites then the two sides of
the membrane are not at redox equilibrium and two Q/QH,
pools, one on each side of the membrane at different redox
potentials, are an energy store in and across the membrane,
i.e. not a store of free protons but of bound redox
equivalents in two pools of Q/QH,. In fact even if there
were aqueous pH gradients in redox equilibrium with
Q/QH, on both sides of the membrane then there would
always be a substrate gradient in the membrane. One
consequence is that Q/QH, gradients could be the essential
feature of energy transduction and proton gradients could
be secondary. Various observations on the relationships
(not chemiosmotic) between proton gradients and ATP
production could then be explained as properties of
such energized Q pools within membranes.]
Q cycles
Q and QH, are not reactive. We therefore need special
reaction sites not in particle I or IV. We now think that Qbound sites in particle 111 dislocate H + and e, e travels on
via cytochrome 6, the Rieske centre, cytochromes c, and c
(see Fig. 3). QH, must reform from different protons and
different electrons. Now there is a known redox-potential
drop in the particle which can be transduced to a proton
energy for ATP synthesis. What happens to e, H , Q and
QH, in the reactions of the particle? My first solution of this
problem (Williams, 1970), is shown in Fig. 2 where I have
+
Fig. 2. The form of Q cycle used by Williams (1970)
legitimately replaced X with Q for particle 111. I used a
mobile QH radical to help the cycle between two reaction
centres, nowadays written as coenzyme Qinand coenzyme
Q,,*. Before stating my later worries over this scheme I draw
attention to other types of scheme in which e and H + are
moved together transversely as either QH, or QH, or Q is
moved as Q or Q'-. The most elaborate are by Crofts et al.
(1982) and Slater (1983) and are very difficult to test. There
are some simpler ones, e.g. there are a variety by Mitchell
(1976) which are elaborated in Mitchell & Moyle (1982). My
problems with all the schemes are as follows.
(i) If Q'- and/or QH (the radicals) can migrate they can
carry e and H + in energized reactions forms and so break
the dislocation (or chemiosmotic gradient) since these forms
of Q do not need electron-transfer catalysts and have the
same pK, as uncouplers. I doubt very much that such
reactions are allowed to occur across membranes.
(ii) Q and QH, need catalysts so their migration is
apparently harmless. But can they migrate rapidly between
the reaction centres in vivo if these are on opposite sides of
highly rigid, organized and energized membranes (see
Hauska & Hurt, 1982)?
(iii) What is the construction of particle III? It has a wellestablished ability to pump protons and to move electrons
between many centres. Where are they in space, and what
are they for?
Taking point (ii) first, there is no proof yet that Q or QH,
can cross biological membranes at reasonable rates. The
work of Hauska & Hurt (1982) shows that in artificial
vesicle membranes the rate of Q/QH, transfer could be
adequate but this is a far cry from movement in the rigidly
structural convoluted membrane of Fig. 1. This problem
needs immediate study.
As far as the construction of particle I11 is concerned,
point (iii), it is generally taken today that cytochrome c , , the
Rieske protein, one cytochrome b and one bound Q site are
on one side of the membrane (Fig. 3). If this is true the
second cytochrome b is unlikely to be in direct electrontransfer contact since it is expected from sequence data to
be on the other side of the membrane. There could be a
second bound Q site towards this side of the membrane of
course. In this case we can understand why, on energization,
passing redox equivalents through the Q pool, we see the
differential energization of b cytochromes, and of the two
different bound Q sites. However, there is no need for Q to
migrate between the two sides of the membrane and it may
well be necessary to consider two Q/QH, pools and to
postulate electron and proton channels for their communication (Fig. 3). The model has no Q cycles (see Papa, 1976;
Wikstrom & Krebs, 1979).
This paper directs attention to the movement of electrons,
protons and bound H in membranes. It suggests that the
nature of the Q/QHz pool can be such that it acts as a
simultaneous system for energized e and H lateral transfer
as H. Protons and electrons are transferred transversely in
the membrane. The Q pools are energized and could drive
ATP synthesis without any essential chemiosmotic communication (see Fig. 1 and Fig. 3). We shall not solve the
problem without the structure of particle 111.
+
Fig. 3. A suggested form for the reactions of particle Iff in
mitochondria
N o Q cycle is used but movement of protons is shown in the
membrane and lateral flow of Q and QH, is required. There
is no flow of Q across the membrane.
Crofts, A. R., Meinhardt, S. W. & Bowyer, J. R. (1982) in Function
of Quinones in Energy Conseruing Systems (Trumpower, 9. L.,
ed.), pp. 477499, Academic Press, New York
Hauska, G . & Hurt, E. (1982) in Function of Quinones in Energy
Conserving Systems (Trumpower, 9. L. ed.), pp. 87-110,
Academic Press, New York
Mitchell, P. (1961) Nature (London) 191, 144-148
Mitchell, P. (1976) J . Theor. B i d . 62, 327-367
Mitchell, P. & Moyle, J. (1982) in Function of Quinones in Energy
Conseruing Systems (Trumpower, 9. L. ed.), pp. 553-573,
Academic Press, New York
1984
606th MEETING, CORK
Moore, G. R., Huang, Z.-X., Eley, C. G. S., Barker, H. A.,
Williams, G., Robinson, M. N. & Williams, R. J. P. (1982)
Furuduy Discuss. Chem. SOC.74, 31 1-329
Papa, S. (1976) Biochim. Biophys. Acta 456, 39-84
Slater, E. C. (1983) Trends Biochem. Sci. 8, 239-245
Wikstrom, M. K. F. & Krebs, K. (1979) Biochim. Biophys. Acta
549, 177-222
399
Williams, R. J. P. (1961) J. Theor. Biol. 1, 1-17
Williams, R. J. P. (1970) in Electron Trunsport and Energy Conservation (Tager, J. M., Papa, S., Quagliariello, E. Slater, E. C.,
eds.), pp. 7-23, Adriatica Editrice, Bari
Williams, R. J. P. (1984) in The Enzymes of the Bio/ogicu/
Membrunes (Martonosi, A., ed.), 2nd edn., Plenum Press, New
York, in the press
Some implications of fixed-charge formation during electron-transport-chainactivity
with the energized membrane they inhibit energy transduction and ATP synthesis and cause the release of protons into
the medium (Higuti et al., 1978); the last-named authors
comment that ‘the energy-dependent binding of ethidium
In 1969 Azzi and co-workers concluded that the fluores- cation to the membrane inhibits ATP synthesis by
cence changes of ANS which accompanied its reaction with neutralizing the negative charges created on the surface of
mitochondria ‘indicated structural changes of the mito- the C-side of the membrane’. A similar explanation had
chondrial membrane associated with energy conversion’ been given at much the same time for the action of Ca2+,
(Azzi et al., 1969). Azzi (1969) reported a 20% decrease in and for the same reasons: the release of protons into the
the amount of dye bound after adding succinate; from this, medium during Caz+uptake, and a reduction in synthesis
coupled with the fact that a positively charged dye, which, since Ca2 is a permeant ion and its association with
auramine-0, showed increased binding on energization, he a fixed-charge molecule must be supposed transient, was
deduced that ‘the interaction between dyes and the seen as a ‘lag’ period, delaying rather than inhibiting the
energized membrane is electrostatic’ and that ‘it is probably onset of synthesis (Archbold et al., 1979; McKay &
important in the mechanism of ATP synthesis that an Malpress, 1980). An involvement of fixed charges in the
asymmetrical charge distribution is associated with energy energizing process has also been assumed by Kell (1979),
conservation in the mitochondria1 membrane’. Norden- arguing from the general inadequacy of bulk-phase electrobrand & Ernster (1971) extended Azzi’s studies, differen- chemical forces to account for observed phosphate
tiating the non-energized and energized ANS binding and potentials.
fluorescence changes by several different kinetic indices;
It may seem surprising that the chemiosmotic hypothesis
they suggested that the energy-dependent reaction reflected in particular should have remained uninfluenced by these
‘a charge separation in a special locus ... distinct from non- varied observations. The reason seems to have been the
energy-dependent binding sites’, and that the energized delusive support which the hypothesis drew from a number
state was to be considered as a ‘heterogeneous concept .. . of experiments which were either irrelevant to the purpose
rather than a homogeneous entity such as that defined by a of identifying the nature of the protonmotive force, for
bulk chemical or electrical potential across the mito- which they were nonetheless used, or which were interpreted with a free inferential logic which left much to be
chondrial membrane’.
It was, however, a more homogeneous view of the mito- desired. Archbold et al. (1975, 1979) attempted to draw
chondrial energy transduction mechanism, in the form of attention to these remarkable lacunae in the arguments of a
the chemiosmotic hypothesis, which gained the authority of major hypothesis, and two examples will be mentioned here
a near consensus during the ensuing decade, a view that in general terms. (i) It is not correct to infer from the proton
held no explicit function for the fixed-charge changes movements in the cation-exchange reactions of an oxygendetected by the dye-binding studies. The chief alternative pulse experiment that bulk-phase transmembrane forces
hypothesis at this time, the ‘local energized proton’ will be operating in synthesis; nor, indeed, in the light of the
exposition (Williams, 1978), also lacked any essential role fixed-chargechanges during respiratory activity is it right to
for fixed-charge development, though the production of assume that transmembrane potentials are at any time, even
negatively charged groups on energization could be the during permeant cation exchange, the primary mediating
adventitious accompaniment of local proton release from force in energy transduction. (ii) To show that an artificially
apoproteins within the membrane.
imposed ApH+can lead to synthesis (e.g. Thayer & Hinkle,
In contrast to these prominent and contending hypoth- 1975) provides no proof of the in uiuo mechanism of
eses Archbold et al. (1974) regarded an increased negative synthesis; such experiments require the use of respiratoryfixed-charge presence in energized mitochondria as the inhibited particles and are therefore carried out in the
probable explanation for their observations on the absence absence of the fixed-charge development which is intrinsic
of proton movement into the bulk-phase during ATP to n o w 1 oxidative phosphorylation; they demonstrate no
synthesis. Following Azzi’s model (1969) they envisaged more than that the energy input for ATP synthesis may be
proton retention at the outer surface of the inner membrane presented successfully on an experimental basis in more
as the direct result of an increased surface negative than one form.
electrical field, an important factor not to be ignored by any
Two consequences may be expected to follow from an
energy transduction hypothesis (Archbold et al., 1975). increase in the negative surface charge of the coupling
Confidence in the development of these views (Archbold et membrane: an increase in the surface electrical field giving
al., 1979, 1 9 8 0 ~ ;Lappin & Malpress, 1980; Malpress, an enlargement of the diffuse double layer, and an increase
198la,b) owed much to the steadily accumulating evidence in the concentration of cations retained within the diffuse
for fixed-charge generation in the energized mitochondrion double layer. The retentive force will be electrostatic and
(Kamo etal., 1976; Quintanilha & Packer, 1977; Aiuchi and exerted chiefly on Ca2+ and H +.It is important to note that
Kobatake, 1979; Archbold et al., 19806; Malpress, 1981~). as soon as we cease to regard the protonmotive force as a
It was also noted that when positively charged dyes react generalized, dissipative bulk-phase property, and see it as
localized and non-dissipative, the surface area involved in
acid. energy transduction may become a mere fraction of the total
Abbreviation used : ANS, 1-anilino-8-naphthalene-sulphonic
FRANK H. MALPRESS
Department of Biochemistry. Queen’s University, Belfast
BT7 INN. N.Ireland, U.K.
+
VOl. 12