BIOCHEMICAL SOCIETY TRANSACTIONS
690
Table 1. Recovery of hexoses in milk after injection into the lumen of the
mammary gland o f the lactating goat
Hexoses were determined spectrophotornetrically by enzymic techniques. Data are
means f S. E. for six animals.
Hexose recovered in milk (%)
Time after injection into the lumen . . .
Glucose
Galactose
Fructose
Na’, K’ and C1- concentrations in milk were normal
during the entire period of the experiment, indicating that
the tight junctions between the mammary epithelial cells
had not been affected by the treatment and hence
paracellular transport could not be taking place (Peaker,
1978).
The rapid loss of glucose from the lumen of the
mammary gland is evidence that this hexose rapidly equilibrates across the apical membrane and that, at equilibrium,
the concentration of glucose in milk equates with cytosolic
concentrations (Kuhn & White, 1975; Faulkner et al.,
1981). Transport of glucose appears t o be specific, as
galactose is lost at a much slower rate and loss of fructose
is negligible. The characteristics of the hexose transport
(glucose > galactose > fructose) are similar t o those
obtained with rat mammary epithelial cells (Threadgold &
Kuhn, 1984) and completely different from those of rat
2h
18 + 4
83t10
96+8
6h
7.8 + 3.1
63r9
102+9
16h
2.2 t 2.7
8.2 ? 5 . 2
7 8 + 10
Golgi vesicles (White et al., 1981), which demonstrate little
specificity in their hexose transport. If the properties of
the Colgi vesicles of the goat are similar to those reported
for the rat, the apical membrane has properties distinct
from those of the Golgi membrane, even though this
membrane is in part formed by fusion of Golgi vesicular
membrane during exocytosis.
f:aulkner, A., Chaiyabutr, N., Peaker, M., Carrick, D. T. & Kuhn,
N. J . (1981)J. Dairy Res. 48, 51-56
Kuhn, N. J. &White, A. (1975) Biochern J. 152, 153-155
Linzell, J. L. & Peaker, M. (1971) Physiol Rev. 51, 564-597
Peaker, M. (1978) in Lactation: A Comprehensive Treatise (Larson,
B. L., ed.), vol. l V , pp. 437-462, Academic Press, London and
New York
Threadgold, L. C. & Kuhn, N. J. (1984) Biochern. J. 218, 213-219
White, M. D., Kuhn, N. J. & Ward, S. (1981) Biochern. J. 194,
174-177
Studies on the location of the membrane-bound glyceraldehyde-3-phosphate
dehydrogenase
ROGER J. MOORE and R. B. BEECHEY
Department of Biochemistry and Agricultural Biochemistry,
University College of Wales, Penglais, Aberystwyth, Dyfed
SY23 300, U K .
The tetrameric glyceraldehyde-3-phosphate dehydrogenase
molecule contained within the human red blood cell ‘ghost’
is associated with the inner face of the plasma membrane
where it is known t o bind to the band Ill protein (Yu &
Steck, 1975). The association with the membrane is
regarded as superficial since it is relatively easy to disocciate
the enzyme-membrane complex by washing with salt
solutions (Kliman & Steck, 1980). The enzyme has a
thiolate ion at the active centre. We have studied the interaction of this group with a number of N-polymethylenecarboxymaleimides. These are molecules designed to
penetrate the membrane t o different depths. The results
suggest that the glyceraldehyde-3-phosphatedehydrogenase
complex is embedded within the membrane to the extent
that access t o the thiolate groups at the active centre is
impeded by the structure of the lipid bilayer within the
membrane.
N-Polymethylenecarboxymaleimideswere synthesized as
described by Griffiths et al., (1980).
0
Red blood cell ‘ghosts’ were prepared by the method of
Steck & Kant (1974). They showed a high degree of
intactness in that they would transport glucose and the
latency of the glyceraldehyde-3-phosphatedehydrogenase
activity was 20. These were rendered ‘leaky’ either by
treatment of the intact ghosts with 0.2% (w/v) Triton
X-100 or by omitting MgS04 from the medium in which
the cells were haemolysed.
The effects of N-octylmaleimide and the N-polyniethylenecarboxymaleimides on the glyceraldehyde-3-phosphate
dehydrogenase activity of the intact red cell ‘ghosts’ were
investigated by incubating the ‘ghosts’ with 8 5 nmol of
maleimide/mg of protein for 10 min. The excess maleimide
was quenched by the addition of 1 PI of mercaptoethanol.
Triton X-100 was added t o 0.2% and the activity of the
enzyme was assayed. None of the polymethylenecarboxymaleimides inhibited, but N-octylmaleimide completely
inhibited the activity. Thus the presence of a charged group
in the molecule prevented the movement across the
membrane of the maleimide moiety to the thiolate groups
at the active centre.
The activity of the soluble form of the enzyme was
completely inhibited at similar rates and by similar concentrations of N-octylmaleimide and all the N-polymethylene
carboxymaleimides used in this study (see Fig. 1 .) Thus
the presence of a charged group in the maleimide molecule
is neither essential nor detrimental t o inhibition.
The effects of the maleimides on the glyceraldehyde-3phosphate dehydrogenase activity bound t o the membranes
of leaky cell ‘ghosts’ was investigated. The results are shown
in Fig. 1 . It can be seen that there is a marked variation of
the inhibition with the length o f the N-polymethylene
1985
69 1
6 12th MEETING. LONDON
I
1
2
3
4
5
6
8
7
1.ciigfIi o l polyniefliylene chain
9
10
(11)
Fig. 1 . Structure-inhibition profile for the inhibition of
Rlyceraldeh.,.dc-3-ptiosphatc dehydrogenase actiiiity b y a
series o j N-~~o!,~tnetii~~letiecarbox~~tnaleimides
in (0)
unscaled 'ghosts' ( 0 ) Triton X - 1 00-treated 'ghosts', (m)
the soluble e n z y m e
chain. Thus when n = 1 the inhibition is only 25% but
when n = 2 the inhibition is complete. There is a progressive
diminution of inhibition up t o n = 7, followed by a small
but reproducible increase when n = 10. Glyceraldehyde-3phosphate gave almost complete protection against these
inhibitions, suggesting that the site of action of the
inhibitors is at the catalytic site on the enzyme. Thus the
presence of a membrane structure limits the ability of the
N-polymethylenecarboxymaleimidest o inhibit.
We interpret these results in terms of a model in which
the polymethylenecarboxymaleimidesfirst enter the bilayer
portion of the membrane, with the maleimide portion in
the membrane and the carboxylate ion remaining on the
surface of the membrane. Thus the depth of penetration of
the maleimide moiety is limited by the length of the polymethylene chain. The differential abilities of the N-polymethylenecarboxymaleimides to interact then depends on
the time that the thiolate group of the glyceraldehyde-3phosphate dehydrogenase molecule spends at that depth
within the membrane.
This model would imply that the active centres of the
enzyme are more deeply located within the membrane than
has been suspected previously.
Griffiths, D. G., Partis, M. D., Sharp, R. N. & Beechey, R. B. (1981)
FEBS Lett. 134.261-263
Klirnan, H. J. & Steck, 1'.L. ( 1 9 8 0 ) J .B i d . Chern. 255,6314-6321
Steck, T. L. & Kant. J . A. (1974)Methods Enzymol. 31, 172-180
Yu, J . & Steck, T. L. ( 1 9 7 5 ) J .Bid. Chern. 250,9176-9184
Ca2+fluxes and cytosolic Ca2+concentrations in normal and in cholesteroldepleted
erythrocytes measured with permeant Ca2+chelators
A, the cells were loaded with chelator (200-500 pmol/litre
of cells) in buffer A (75min, 37"C, haematocrit 30%) or
incubated without chelator under the same conditions.
Cells were washed again three times in buffer A. They were
then incubated in buffer A (37"C, haematocrit 10%) with
Cholesterol, a major component of plasma membranes in 45 Ca (5pCi/pmol of CaC12) (influx). The 45Ca content of
animal cells, is known t o modulate bilayer fluidity (Olfield the cells was measured in aliquots of the suspension after
& Chapman, 1972), to alter ionic permeabilities (Grunze three washes of the cells in ice-cold isotonic phosphate
& Deuticke, 1974) and to affect the activities of membrane- buffer without CaCI2 (buffer B) and lysis with tribound enzymes (Sabine, 1983). Its possible role in the chloracetic acid. After 120min at 37"C, aliquots of the
transport of Ca2+has never been studied. In erythrocytes, suspension were washed four times and re-incubated in
Ca2' exchange is the result of an inward leak and an buffer A (37"C, haematocrit 10%) (efflux). 45Ca content
outward ATP-dependent Caz+ pump flux (Schatzmann, was measured as above. Cell cholesterol and phospholipids
1973) which maintains a very low level of cytosolic Ca2+ were assayed as previously (Chailley et al., 1981).
CaZ+ content during influx and efflux is presented in
(1OnM) (Lew ct al.. 1981). The use of permeant Ca2'
Fig. l(a). As the Ca2+ exchange system was in steady-state,
chelators (Tsien, 1980, 1981) has made possible Ca2'
flux and cytosolic Ca2+ concentration measurements since all the steps including chelator loading were carried
without altering membrane properties (Lew el al., 1981). out in the presence of 1 mM-CaC12, the fluxes could be
These molecules in their free form have a high affinity for calculated from all the experimental values assuming a one
Ca2+. In their esterified form (Quin:! AM and MAPTAM), open-compartment system. This was done by plotting the
they can cross the cell membrane, are hydrolysed by log of the difference between Ca,, (45Ca uptake at radiointracellular esterases and thus create an internal measurable active equilibrium) and Cat (45Ca upthke at each time) for
the influx or the log of Cat for the efflux (Fig. Ib). Straight
pool of exchangeable Ca2+.
Human erythrocytes were prepared from fresh blood by lines were obtained for both fluxes in control cells. The
three washes in isotonic phosphate buffer containing 1 mM- slopes of these lines are the rate constants k (h-') and the
CaC12 (buffer A). Cholesterol depletion was carried out as product k . Ca,, gives the fluxes (pmol/litre of cells per h).
previously described (Chailley et al., 1981) by 15 h In cholesterol-depleted cells for both fluxes semi-linear
incubation with phosphatidylcholine vesicles, followed by curves were obtained, showing that cell exchangeable-Ca2'
1 h with adenine and inosine. Controls were incubated in behaved as a two-compartment system. In the experiment
the same way without vesicles. After three washes in buffer shown in Fig. 1 the size of the rapidly exchangeable
compartment was 4 pmol/litre of cells (determined from
the extrapolation at time 0 of the linear part of the curve).
*To whom correspondence should be addressed.
FLORENCE ROSIER and FRANCOISE GIRAUD*
Plij~siologiedc la Nutrition, Bat. 447, UA-CNRS 646,
lJ alliec I N S E M , Universitk Orsajl Paris XI, 91 405 Orsay
Cedcx, France
Vol. 13