J. Embryol. exp. Morph., Vol. 15, 3, pp. 291-295, June 1966
Printed in Great Britain
291
Inhibition by cc-methyl-norvaline of the uptake
of valine and leucine into haemoglobin
of the chick embryo
By E. M. DEUCHAR 1 & A. M. L. DRYLAND 1
From the Department of Anatomy, University College London
We reported recently (Deuchar & Dryland, 1965) that a-methyl-norvaline, an
amino acid whose structure resembles both valine and leucine, inhibits the
uptake of these two normal amino- acids into protein in explanted chick embryos
and is also itself incorporated into embryonic proteins.
Since the first effect of a-methyl-norvaline that we had noted (Deuchar &
Dryland, 1964) was a reduction in the quantity of haemoglobin formed in the
cells of the area vasculosa, it was of special interest to see whether this inhibitor
blocked the uptake of valine and/or leucine into haemoglobin in particular and
whether it was at the same time taken up itself into haemoglobin. The results of
experiments in which haemoglobin has been extracted from area vasculosa
explants and the uptake of radioactively labelled amino acids into it has been
measured, are reported here.
MATERIALS AND METHODS
Area vasculosae from chick embryos of 48 h incubation were isolated as
described in our first paper (1964) and explanted in watch-glass cultures on
agar/saline/glucose medium (Spratt, 1948). 1 /*c/ml of 14C-labelled valine or
leucine was added to the medium, as in our previous experiments (1965). In the
experiments designed to measure the uptake of the analogue into haemoglobin,
2-5 mg of tritium-labelled analogue, containing 0-02/4C of activity, was added
per ml of culture medium. Labelling was by the TR1 procedure (Radiochemical
Centre, Amersham); radiochemical purity was 85%. Chromatographic analysis
showed only faint traces of impurities. Explants were incubated for 20 h at 37 °C,
then the red cells were isolated as follows:
After removal from the media, the explants were transferred to a Petri dish of
ice-cold Pannett-Compton saline and washed in this for a few minutes, followed
by a wash in ice-cold 0-9 % sodium chloride solution (buffered to pH 7 by the
addition of 0-2 g/1. of sodium bicarbonate). They were then placed in cold
1
Authors' address: Department of Anatomy, University College, Gower St, London,
W.C.I, England.
292
E. M. DEUCHAR & A. M. L. DRYLAND
magnesium- and calcium-free Hanks's saline1 at pH 8-5, then filtered through
Turtox nylon cloth no. D, 50 /i2 pore size, and transferred to a centrifuge tube,
in an ice bath. After 10 min the disaggregating tissues were squirted up and
down in a pipette several times, which reduced them mainly to a cell suspension.
After a further 10 min this suspension was centrifuged at 2000 rev/min for 10
min, the supernatant discarded, and the precipitate washed with cold 0-9 %
NaCl and recentrifuged. The final precipitate was taken up into a melting-point
tube whose end was then sealed in a flame, and centrifuged at low speed for
5 min, bringing the red cells to the lower end of the tube, which could then be
broken off. Its contents, as little contaminated as possible with other cells, were
transferred to a microtube in which they were haemolysed overnight at 6 °C
with distilled water. Next morning the cell debris was removed by centrifuging
and the supernatant applied to the origin line on an 'Oxoid' cellulose acetate
strip, 2-5 x 12 cm, for electrophoresis (cf. Deuchar & Dryland, 1964). After a
2\ h run at 0-4 mA/cm in 0-4 M phosphate buffer, pH 6-8, the strips were 'fixed'
in 3 % trichloroacetic acid, blotted, and stained by the naphthalene-black and
ortho-dianisidine methods (Owen, Silberman & Got, 1958). Two haemoglobin
bands were clearly distinguishable in extracts from control and explants and
also identifiable, though the colour was fainter, in extracts from analoguetreated explants. With each electrophoresis run, a sample of adult haemoglobin
was included as a check that the conditions had been normal.
The quantity of haemoglobin present in the bands was estimated by clearing
the acetate strips in 'Dekalin' (decahydronaphthalene) to render them transparent. They were then scanned with the 'Chromoscan' colour densitometer
(Joyce Loebl Ltd.), using filter no. 5-042. The Chromoscan integral values for
areas under the peaks on the trace graph were then converted into fig of haemoglobin by reference to values obtained with standard haemoglobin solutions.
Five [A aliquots of these were applied to buffer-wetted cellulose acetate strips
which were 1 cm wide, the width of the Chromoscan slit. After fixing and staining
as above, the relationship between the standard haemoglobin concentrations
and the integral Chromoscan reading was linear, over the range from 2 to 1 fig
of haemoglobin (Fig. 1).
The radioactivity in the haemoglobin bands was estimated after restoring the
cellulose acetate to opaqueness in ether, drying it, then cutting transverse strips,
approximately £ cm wide, to correspond with the peaks on the Chromoscan
trace and the maximal naphthalene-black staining. The part of the strips not
containing protein was cut into £cm strips for background determinations.
These strips were placed in bottles containing 4 ml of NE 220 scintillation-fluid
(Nuclear Enterprises, Ltd.) and counted on a liquid scintillation counter (Isotope
Developments Ltd.). The activity per fig of haemoglobin could then be estimated.
It had already been ascertained that there was no quenching due to the presence
1
Modified Hank's saline (calcium- and magnesium-free). In 1 1. distilled H 2 0: 1 g glucose,
8 g NaCl, 4g KC1, 006 g KH2PO4, 006 g Na2HPO4, 10g EDTA.
Inhibition by a-methyl-norvaline
293
of naphthalene-black stain. The counting efficiency, tested with standard
samples applied to the same size strips of electrophoresis paper, was 40 % for
14
C and 9 % for tritium.
300 r
200
s
100
I
6
/^g Hb/strip
Standard Hb
solutions
1
|2
!
V
3
14
!
T
5
• 6
!
3'
7 ;i
I
j
4'
8
9
10
Fig. 1. Graph showing the linear relationship between Chromoscan integrator
readings and haemoglobin concentration, using four standard solutions (referred
to as V, 2', 3', 4') applied to electrophoresis strips.
EXPERIMENTAL RESULTS
(a) Valine uptake. Table 1 a gives the results of two experiments in which
explants were incubated for 20 h on medium containing 1 ficjm\ of 14C-valine,
either with or without analogue (2-5 mg/ml) present. It is clear that the uptake
of valine into haemoglobin is markedly reduced in the presence of analogue.
(b) Leucine uptake. Table 1 b gives the results of experiments designed similarly to those of (a). The uptake of leucine into haemoglobin is very much
reduced in the presence of the analogue.
(c) Uptake of analogue. In the presence of tritium-labelled analogue alone, too
little haemoglobin was formed for reliable quantitative estimates to be made on
the Chromoscan, so these results cannot be expressed as specific activities.
Table 2 gives the radioactivity counts in absolute terms, however, and these
show clearly that tritium is taken up into both of the haemoglobin bands
separated by electrophoresis.
JEEM 15
294
E. M. DEUCHAR & A. M. L. DRYLAND
Table 1 a. Valine uptake into haemoglobin {countsjminjfig Hb)
Experiment
1
2
Treated explants
Control
explants
(1 /tc/ml. 14C14
( C-valine
valine + 2-5
1 /tc/ml) mg/ml analogue)
244
20
226
71
Table 1 b. Leucine uptake into haemoglobin {countsjminjfig Hb)
Experiment
1
2
Control
explants
(1 /<c/ml
14
C-leucine)
789
683
Treated explants
(1 /tc/ml. 14Cleucine + 2-5
mg/ml analogue)
135
230
Table 2. Uptake of tritium-labelled cc-methyl norvaline
into haemoglobin (counts/mini 100 sees)
Band 1 (nearest origin)
Band 2 (furthest from origin)
Controls
(no analogue)
111
100
Experimental
explants
(005/tc/ml. of
tritiated
analogue)
920
657
CONCLUSION
These results serve to supplement and confirm our previous work (1964,1965),
as they demonstrate clearly that a-methyl-norvaline, which we had already
shown to inhibit haemoglobin formation in the area vasculosa of the chick
embryo, inhibits the uptake of both leucine and valine into haemoglobin. The
present results also show that a-methyl-norvaline is itself incorporated into the
haemoglobin, so that we may reasonably conclude that it acts as a true molecular analogue of these amino acids, by competing with them in reactions that
lead to haemoglobin synthesis. From previous work (Deuchar & Dryland, 1965)
it had already appeared that the analogue was taken up into proteins. This must
be an active process, too, and not mere chance adsorption from the medium,
since in two early experiments where the explants were stunted and unhealthy
there was no uptake of the labelled analogue. It is for future work to discover at
which particular steps of synthesis the competition for uptake into the haemoglobin occurs. The analogue might, for instance, inhibit the activation of the
normal amino acids, as Sharon & Lipmann (1957) reported for a tryptophan
Inhibition by a-methyl-norvaline
295
analogue. Or it might become incorporated into some protein precursor of
haemoglobin in preference to the normal amino acids, like the valine analogue,
a-amino-/?-chlorobutyric acid, studied by Rabinovitz & McGrath (1959). One
of the points which emerged earlier in our work (Deuchar & Dryland, 1964)
was that treatment of explants with a-methylnorvaline had detectable histological results too—slowing down the rate of maturation of the red blood cells.
It would be most interesting, if possible, to discover the link between the biochemical and histological inhibitions here.
SUMMARY
a-methyl-norvaline inhibits the uptake of valine and, even more strongly, of
leucine into the haemoglobin formed in explanted areae vasculosae of the 48 h
chick embryo. There is also some uptake of this analogue itself into the haemoglobin.
RESUME
Inhibition, par la a-methyl-norvaline, de Vincorporation de la valine
et de la leucine dans l'hemoglobine de Vembryon de poulet
La a-methyl-norvaline inhibe l'incorporation de la valine et, meme plus
fortement, celle de la leucine dans l'hemoglobine formee dans des explants de
l'aire vasculaire d'embryons de poulet de 48 heures. II y a de plus une certaine
incorporation de cet analogue lui-meme dans l'hemoglobine.
We should again like to thank Miss Gillian Weedon and Miss Susan Bevan for technical
assistance. One of us (E.M.D.) is indebted to the British Empire Cancer Campaign for a
grant covering the cost of the Chromoscan apparatus. Fig. 1 was drawn by Mrs J. Astafiev,
whom we should also particularly like to thank for her help.
REFERENCES
E. M. & DRYLAND, A. M. L. (1964). Effects of a-methyl-norvaline on synthesis of
haemoglobin in the area vasculosa of the chick embryo. Nature, Lond., 201, 832-3.
DEUCHAR, E. M. & DRYLAND, A. M. L. (1965). Inhibition by a-methyl-norvaline of valine
and leucine uptake into protein in the chick embryo. J. Embryol. exp. Morph. 13, 275-83.
DEUCHAR,
OWEN, J. A., SILBERMAN, H. J. & GOT, C. (1958). Detection of haemoglobin, haemoglobin-
haptoglobin complexes and other substances with peroxidase activity after zone electrophoresis. Nature, Lond., 182, 1373.
RABINOVITZ, M. & MCGRATH, H. (1959). Protein synthesis by rabbit reticulocytes. II. Interruption of the pathway of haemoglobin synthesis by a valine analogue. /. biol. Chem. 234,
2091-5.
SHARON, N. & LIPMANN, F. (1957). Reactivity of analogues with pancreatic tryptophanactivating enzyme. Archs. Biochem. Biophys. 69, 219-27.
SPRATT, N. T. (1948). Development of the early chick blastoderm on synthetic media. /. exp.
Zool. 107, 39-64.
{Manuscript received 5 August 1965, revised 25 November 1965)
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