F1h
607th MEETING, LONDON
69 1
15r
0 I0
'I'
E
d
Bound
Free
(d.p.m. x 104)
Bound
Free
(d.p.m.x 104)
Fig. 1. Eadie-Hofsteeplots of [3Hlglutamate binding to rat brain synaptic membranes
in the presence of various concentrations of triJluoperazine (a) and calmidazolium (b)
Intercept on abscissa indicates BmaX./Kd,
and intercept on ordinate indicates B,,,. ( a )
Control ( 0 ) 9p~-trifluoperazine
;
(A);45pM-trifluoperazine(m); 91 pM-trifluoperazine (0).(b) Control ( 0 ) ;0.9p~-calmidazolium(A);9p~-calmidazolium(m).
glutamate from that bound in the presence of 0 . 0 5 - 2 p ~ [3H]glutamate over a lOmin incubation at 37°C (see Sharif
& Roberts, 1980). Trifluoperazine and calmidazolium were
added in the concentration range 10-4-10-6~.
Fig. 1 shows Eadie-Hofstee plots of [3H]glutamate
binding in the presence of varying concentrations of
trifluoperazine or calmidazolium. The effect of both
compounds was to decrease maximum binding at equilibrium (BmaX)
without affecting the equilibrium dissociation
constant (&). Calmidazolium was 50Ck1000 times more
potent than trifluoperazine.
Trifluoperazine and calmidazolium are both inhibitors of
Ca2 -calmodulin. Calmidazolium, unlike trifluoperazine,
does not interact with dopamine receptors, and has been
+
reported to be 500 times more effective at inhibiting Ca2
calmodulin-dependent phosphodiesterase (Gierzan et al.,
1981; Van Belle, 1981). Glutamate binding to synaptic
membranes is decreased by 60% on washing with EGTA,
some of which can be recovered on adding Ca2 back (M.
Bardsley, unpublished work).
These results suggest that the Ca2+ activation of
glutamate binding to cerebellar membranes is mediated by
calmodulin.
+
-
+
Gierzan, K., Wiithrich, A. & Bader, H. (1981) EERC 101,418-425
Sharif, N . A. & Roberts, P. J. (1980) J . Neurochem. 34, 779-784
Van Belle, H.,(1981) Biochem. SOC.Trans. 9, 133P
Low concentrations of glucose precipitate a rapid dephosphorylation of ATP in ram
spermatozoa exposed to a-chlorohydrin
W. CHRISTOPHER L. FORD and ANNE HARRISON
Department of Physiology and Biochemistry, University of
Reading, Whiteknights, Reading RG6 ZAJ, U.K.
a-Chlorohydrin and 6-chloro-6-deoxysugars have a rapid
and reversible contraceptive action in male animals. The
effect is brought about by the selective inhibition of
glyceraldehyde 3-phosphate dehydrogenase in spermatozoa
which blocks glycolysis, although these cells can still oxidize
substrates such as lactate to maintain motility (see Jones,
1978, 1983; Ford, 1982). Ram spermatozoa treated with
1 mM-RS-a-chlorohydrin remained motile for > 1 h when
incubated in the absence of glucose but rapidly became
immotile if 5mM-glucose was present. The loss of motility
was associated with a massive conversion of ATP to AMP
Vol. 12
(Ford & Harrison, 1983). The present experiments were
designed to define more precisely the conditions required
for glucose-induced ATP dissipation to occur and if possible
to discover other biochemical changes associated with it.
Ram cauda epididymal spermatozoa were washed in
phosphate-buffered physiological saline (Ford et al., 1981)
and suspended in the same buffer at a concentration of
about lo*cells/ml. Portions of the suspension were incubated in a shaking water bath at 34°C for lOmin with 0,0.05 or
1.OmM-RS-a-chlorohydrinbefore 0,O.1 or 1.Orn~-D-glucose
was added to aliquots from each portion. Incubation was
continued and samples were taken after 0,lO and 30min for
the assay of ATP and glycolytic intermediates as described
previously (Ford & Harrison, 1981; Ford et al., 1981).
There was no accumulation of lactate in spermatozoa
692
BIOCHEMICAL SOCIETY TRANSACTIONS
Table 1. The ej’ect
of a-chlorohydrin and glucose on the concentration of ATP and of fructose 1.6-bisphosphate in
ram spermatozoa
Spermatozoa were incubated at 34°C for lOmin with RS-a-chlorohydrin before D-glucose was added to the incubations.
Samples were taken 0, 10 and 30min after the addition of glucose. The concentration (nmol/108 spermatozoa) of ATP at
Omin was 28 f 1.6,28 f 3.0 or 24 k 4.2 with 0,0.05 or 1.OmM-a-chlorohydrinrespectively; the concentration of fructose 1,6bisphosphate was <0.1 in all cases. The data are the m e a n k s . ~ . of
~ .three experiments.
ATP concn.
(nmol/108 spermatozoa)
Fructose 1,6-bisphosphate concn.
(nmol/108 spermatozoa)
I
[a-Chlorohydrin] (mM)
0
0.05
1 .O
[Glucose] (mM)
{!:;
{
8.1
1 .O
1
lOmin
35k3.3
34 f2.4
32k5.2
30k7.8
32k4.3
29k6.5
29 f6.0
11k3.5
12k4.2
incubated with 0 or 0.1 mwglucose but control spermatozoa
produced about 0.5,umol of lactate/108 sperm per 30min
from 1.OmM-glucose.This was inhibited by 60% by 0 . 0 5 m ~ and completely by 1.OmM-a-chlorohydrin.The ability of the
spermatozoa to maintain a stable ATP concentration
depended on the concentration of both a-chlorohydrin and
glucose. With 0.05mwa-chlorohydrin the presence of
1.OmM-ghcose was required to decrease the ATP concentration and this did not occur until 30min. With 1.OmM-achlorohydrin the ATP concentration had declined by 30min
even in the absence of glucose but O.lm~-glucosewas
sufficient to produce a large loss of ATP after lOmin (Table
1). Whether or not a combination of a-chlorohydrin
concentration and glucose concentration produced a loss of
ATP was correlated with its ability to increase the
concentration of glycolytic intermediates. With 1.OmM-achlorohydrin both 0.1 mM- and 1.OmM-glucose produced a
very large increase in the concentration of fructose 1,6bisphosphate after lOmin and with 0.05m~-a-chlorohydrin
1.OmM-glucose caused fructose 1,6-bisphosphate to accumulate after 30min. There was no accumulation of
fructose 1,6-bisphosphate with 1.OmM-a-chlorohydrin and
no glucose (Table 1). The concentration of dihydroxyacetone phosphate and of glyceraldehyde 3-phosphate increased in a similar way to that of fructose 1,6-bisphosphate
but glucose 6-phosphate accumulated to a much smaller
extent.
The results were analysed by a multifactorial analysis of
variance: for fructose 1,6-bisphosphate the effect of
incubation time, glucose and a-chlorohydrin concentrations
and all interactions between them were highly significant
30min
28+ 10.4
33 f5.5
36 f8.9
26 k6.6
26 k 5.5
12k4.5
13k2.8
8 k 2.9
5k 1.8
lOmin
<0.1
<0.1
0.4k0.3
<0.1
<0.1
1.8k0.6
<0.1
21 k0.9
21 k0.9
30min
0.2f0.00
0.2 kO.11
0.4k0.2
0.4k0.00
0.1 k0.1
12k0.4
<0.1
15k2.1
28 k0.8
(P<O.OOl); for ATP the effect of incubation time, achlorohydrin concentration and the interaction between the
concentrations of glucose and of a-chlorohydrin were
significant at the P<O.OOl level and the effect of glucose
was significant at the P<O.Ol level.
The mechanism of the glucose-induced ATP loss in
spermatozoa treated with a-chlorohydrin is unknown but it
could result from a futile phosphorylation4ephosphorylation cycle; such a cycle can occur in spermatozoa incubated
with 2-deoxyglucose (Hiipakka & Hammerstedt, 1978), or
simply from the consumption of ATP in the production of
glycolytic intermediates. Whatever, it can explain the
spermicidal action of a-chlorohydrin because it can be
caused by low concentrations of the drug and spermatozoa
are exposed to high concentrations of fructose in semen and
to glucose in female reproductive tract fluids.
The work was financed by grant no. G822472 SB from the
M.R.C.
Ford, W. C. L. (1982) in Progress Towards a Male Contraceptive
(Jeffcoate, S . L. & Sandler, M. eds.), pp. 159-183, Wiley,
Chichester
Ford, W. C. L. & Harrison, A. (1981) J. Reprod. Fert. 63, 75-79
Ford, W. C. L. &Harrison, A. (1983) in The Sperm Cell(Andre, J.,
ed.), pp. 63-66, Martinus Nijhoff, The Hague
Ford, W. C. L., Harrison, A. & Waites, G. M. H. (1981) J. Reprod.
Fert. 63, 61-73
Hiipakka, R. A. & Hammerstedt, R. H. (1978) Biol. Reprod. 19,
368-379
Jones, A. R. (1978) Life Sci. 23, 1625-1646
Jones, A. R. (1983) Aust. J. Biol. Sci. 36, 333-350
Simian-virus-40 DNA transfection of synchronized CV-1 cells
ALASTAIR J. STRAIN, WILLIAM A. H. WALLACE
and ANDREW H. WYLLIE
Department of Pathology, University of Edinburgh Medical
School, Teviot Place. Edinburgh EH8 9AG. U.K.
Transfection of mammalian cells with calcium phosphateDNA co-precipitates (Graham & Van der Eb, 1973) is now
one of the most commonly used methods for gene-transfer
Abbreviations used: HU, hydroxyurea; SV40,Simian virus 40.
studies (Scangos & Ruddle, 1981). The relative efficiency,
however, of such transfers is low even when enhanced by
carrier DNA. DNA which is taken up by cells under these
conditions can become integrated into the host-cell genome
where it may be expressed. We have been investigating the
events following calcium phosphate transfection of SV40
DNA into cells permissive for the virus. As the SV40 DNA
remains episomal, this permits analysis of factors affecting
stability of the transfected DNA as distinct from those
processes involved with integration. From our previous
1984