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We thank the Irish Dairy Industry for financial support.
Carroll, R. J., Thompson, M. P. & Farrell, H. M., Jr. (1970) Annu. Proc. Electron Microscope
SOC.Am. 28th, 150
Davies, D. T. & Law, A. J. R. (1977a)J. Dairy Res. 44, 213-221
Davies, D. T. & Law, A. J. R. (19776) J . Dairy Res. 44,447-454
Donnelly, W. J. (1977) J . Dairy Res. 44, 621-625
Grosclaude, F., Mercier, J. C. & Ribadeau-Dumas,B. (1973) Nefh. Milk Dairy J. 27, 328-340
Rose, D., Davies, D. T. & Yaguchi, M. (1969) J. Dairy Sci. 52,8-11
Human Erythrocyte Hereditary Spherocytosis: A Membrane Defect?
The Continuing Enigma
JOHN ROBINSON,* SHAUN McCANNt and MICHAEL N. McKILLEN*
*Department of’Biochemistry, University of Dublin, Trinity College, Dublin 2, Ireland,
and t Department of Haematology, Federated Dublin Voluntary Hospitals,
Meath Hospital, Heytesbury Street, Dublin 8, Ireland
Human hereditary spherocytosis, a disease affecting the shape and survival of the
erythrocyte, has been under intensive biochemical investigation over the past three
decades, but the nature of the molecular lesion caused by the disease still remains
elusive. It is widely assumed that the defect must involve the erythrocyte membrane
(Jacob, 1969), since no abnormality within a cytosol metabolic pathway has been
reported (Shafer, 1964). However, although a number of membrane parameters, such
as lipid (Van Deenen & de Gier, 1974) and protein composition (Boivin & Galand, 1977),
membrane-bound protein kinase activity (Zail, 1977), Na+ transport (Wiley, 1969) and
the determinants of membrane shape and deformability (Weed, 1975), have been
investigated in this disease, there is still no universal agreement as to the nature of the
primary defect, and the entire field is best described as being confused and in need of
direction (Zail, 1977).
We have investigated certain aspects of the peptide composition of both normal and
hereditary spherocytosis erythrocyte membranes on the assumption that the lesion
involves a defective membrane peptide. Our approach was to investigate (1) the SDS$/
polyacrylamide-gel electrophoresis profiles of SDS-dispersed erythrocyte membrane
peptides, (2) the isoelectric focusing/polyacrylamide-gelelectrophoresis profiles of nonionic detergent-dispersed erythrocyte membrane peptides, (3) the nearest-neighbour
membrane peptide composition using the cleavable cross-linking reagent, 3,3’-dimethyldithiobispropionimidate, followed by peptide resolution of SDS-dispersed peptides by
SDS/polyacrylamide-gel electrophoresis and (4) the effect of exogenous oxidative
stress (H2O2treatment) on the membrane peptide composition resolved by SDS/polyacrylamide-gel electrophoresis.
Blood from up to seven different female patients with hereditary spherocytosis was
used in this study. Blood samples were freshly drawn and used within 30min of sampling.
Erythrocyte membrane vesicles (‘ghosts’) were prepared according to standard procedures (Dodge et al., 1963).
SDS-dispersed membrane peptide profiles, resolved by SDS/polyacrylamide-gel
electrophoresis (in rods and vertical slabs) by standard procedures (Laemmli, 1970),
revealed no qualitative or quantitative differences between any of the patient or normal
control patterns. Peptides were detected by using both Coomassie Brilliant Blue R.250
staining (Fairbanks et al., 1971) and radioautography after radiolabelling with iodo[14C]acetamide. The gels were 7.5 or 10% with respect to acrylamide and were either
100 or 120mm long. The peptide profiles of both patient and normal control blood
samples were highly reproducible and were identical with the standard published profiles (Fairbanks et al., 1971; Steck, 1974). The 10% gels (120mm long slabs) gave the
best resolution of membrane peptides. These results are in contrast with those of Hayashi
1Abbreviation: SDS, sodium dodecyl sulphate.
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BlOCHEMICAL SOCIETY TRANSACTIONS
et al. (1974) and Nozawa et a/. (1974), who have reported an absence (or deficiency) of
band 4.2 from patients, but in agreement with those of Kitao e / a/. (1973), Kirkpatrick
et al. (1975) and Boivin & Galand (1977), who reported no alteration in membrane
peptide composition. We consider it naive to expect that this approach would reveal a
difference in peptide composition of the diseased membrane, when it is based on differences in molecular size. Presuniably the membrane defect will involve a point mutation
in a constituent peptide, which might possibly be revealed as a charge difference on
electrophoresis or in an altered conformation during topological or conforniational
probing.
Non-ionic-detergent (Nonidet P-40 plus 9 M-urea)-dispersed erythrocyte membrane
peptides from patients and normal controls were next resolved by isoelectric focusing in
polyacrylamide gel slabs (Anies & Nikaido, 1976). The carrier ampholytes covered the
pH range 4-6 and 3.5-10. The erythrocyte membrane peptides were detected as before.
The profiles contained at least 30 consistently resolved peptides and in contrast with
Boivin & Galand (1977), who reported no difference in peptide profiles, we found an
increased intensity of a band found in the patients at pH6.2. The material in this band
had an isoelectric point similar to that of human haemoglobin (HbA), and we suggest that
it is the result of increased haemoglobin binding to the hereditary spherocytosis erythrocyte membrane as a result of metabolic stress within the spherocyte, rather than representing the primary lesion. Increased binding of cytosol proteins to the membrane in
hereditary spherocytosis has been reported (Sears et al., 1975; Allen et al., 1977).
Probing the spherocyte membrane, by covalent cross-linking of membrane peptides,
to investigate the topology of the peptides should reveal subtle differences in peptide
relationships caused by the defect and the resultant shape change. Peptides of intact
erythrocyte suspensions, from blood from both patients and normal controls, were
subjected to cross-linking with the cleavable reagent, 3,3’-dimethyldithiobispropionimidate (Wang & Richards, 1975). The treated erythrocytes were then lysed and membrane vesicles prepared. SDS-dispersed membrane peptides were then resolved by
SDS/polyacrylamide-gel electrophoresis (3.2 % acrylamide). The results indicate that
there was no difference in the type or extent of cross-linking of membrane peptides in
erythrocytes from patients or controls, which could have been due to the fact that this
reagent only measures the accessibility of haemoglobin (and some other cytosol proteins)
to the spectrin complex on the cis-face of the erythrocyte membrane. Obviously it would
be worth extending this study to reagents that probe other membrane peptide interactions.
The ability of spherocytes to withstand an oxidative stress was next investigated.
Kahane et al. (1978) have demonstrated that erythrocyte membrane peptides from Bthalassaemia patients undergo increased cross-linking during exposure in uitro to an
exogenous oxidative stress induced by H,O, treatment. Intact isolated erythrocytes were
exposed to 8 m ~ - H , 0for
~ 2h at 37°C as described by Kahane et al. (1978). Membrane
vesicles were then isolated and the profiles of SDS-dispersed membrane peptides were
resolved by SDS/polyacrylamide-gel electrophoresis (10 % acrylamide). No difference
in the peptide composition of membranes from patients or normal controls was detected,
and it must be concluded that the capacity of the diseased spherocyte to resist oxidative
stress is identical with that of the normal erythrocyte.
Our failure, and that of other investigators, to detect the presence of a consistent and
identical defective membrane peptide in hereditary spherocytosis reflects either the
sensitivity of the analytical techniques used, or alternatively that the defect does not
involve a membrane peptide. If the defect involves a minor peptide present to the extent
of only a few copies per erythrocyte, then electrophoretic separation and proteinstaining techniques are not going to detect its presence.
We suggest that the best approach to the identification of the molecular lesion present
in this disease will lie in the direction of (1) probing the topology of the spherocyte,
(2) an analysis of the metabolic functions resident in spherocytes, (3) the application of
two-dimensional electrophoretic techniques, and (4) the separation of and experimentation with the grossly spherocytic population from patient’s blood.
1979
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Allen, D. W., Cadman, S., McCann, S. R. & Finkel, B. (1977)Blood49, 113-123
Arnes, G. F.-L. & Nikaido, K. (1976) Biochntiistry 15, 616-623
Boivin, P. & Galand, C. (1977) Noiru. Rev. Fr. Hertiotol. 18, 95-1 16
Dodge, J. T., Mitchell, C. & Hanahan, D. J. (1963) Arch. Bioc/te/rt.Biophys. 100, 119-130
Fairbanks, G., Steck, T. L. & Wallach, D. F. H. (1971) Biochemistry 10, 2606-2617
Hayashi, S., Koornoto, R., Yano, A,, Ishigarni, S . , Tsujino, G., Saeki, S. & Tanaka, T. (1974)
Biocheriz. Biophys. Res. Cottzniiur. 57, 1038-1044
Jacob, H. S. (1969) Annu. Rev. Med. 20,41-46
Kahane, I., Shifter, A. & Rachmileivitz, E. A. (1978) FEBS Lett. 85, 267-270
Kirkpatrick, F. H., Woods, G. M . & La Celle, P. C. (1975) Blood46, 945-954
Kitao, T., Kawamura, H., Hattori, K . & Takeshita, M. (1973) Clirt. Chinr. Acta 43, 319-321
Laemmli, U. K. (1970) Nature (London) 227, 681-684
Nozawa, Y., Noguchi, T., Iida, H., Fukushima, H., Sekiya, T. & Ito, Y. (1974) Clin. C h i m Acfa
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Sears, D. A., Friedman, J. M. &White, D. R. (1975) J. Lob. Clin. Med. 86, 722-732
Shafer, A. W. (1964) Blood23,417-526
Steck, T. L. (1974) J. Cell Biol. 62, 1-19
Van Deenen, L. L. M. & de Gier, J. (1974) in The Red Blood Cell (Surgenor, D. MacN., ed.),
pp. 147-21 1, Academic Press, London
Wang, K. & Richards, F. M. (1975) J. Biol. Cheni. 250, 6622-6626
Weed, R. I. (1975) Arch. Intern. Med. 135, 1316-1323
Wiley, J. S. (1969) Nature (London) 221, 1222-1224
Zail, S. S. (1977) Br. J. Huemutol. 37, 305-310
Some Effects of Rubidium Chloride on the Motor Activity and Brain
Serotonin Concentrations of Rats
BARTOLOME RIBAS, ROSA I. ACOBETTRO, CARLOS MATE and
ANGEL SANTOS RUIZ
Departamento de Bioquimica, Facultad de Farmaciu, Universidad Complutense,
Madrid-3, Spain
Previous work (Eichelman, 1974) has shown different effects for various monovalent
cations on the concentration of serotonin (5-hydroxytryptamine) in the brain, and
contrasting effects of lithium and rubidium on the behaviour of rats and mice after
treatment with RbCl and LiCl in the presence of monoamine oxidase inhibitors (Carroll
&Sharp, 1971). Without monoamine oxidase inhibitors, there is a n increase in serotonin
concentration in rat brain (Betes et al., 1975) as shown in the present work and also
enzymic changes in the y-aminobutyrate cycle after rubidium treatment (Ribas et al.,
1978).
The object of the present work was to confirm that treatment with RbCl in the absence
of monoamine oxidase inhibitors induces a n activation of rat motor activity, accompanied by changes in serotonin concentrations in the six main cerebral structures.
Serotonin and 5-hydroxyindol-3-ylaceticacid were determined in male Wistar rats
with a weight of 180-21Og. The group used as control was injected with 0.9% (w/v)
NaCl and the other rats were treated intraperitoneally during 14 days with 1 m-equiv.
of RbCl/kg body wt. per day. The animals were killed 24h after the last injection, which
were given 3, 6 and 14 days after the beginning of the experiment. Serotonin and 5hydroxyindol-3-ylacetic acid were determined by the method of Curzon &Green (1970).
The ‘rearing activity’was determined after 3 days of treatment with 3 and 6m-equiv. of
RbCl/kg body wt. per day. Control and test groups were intraperitoneally injected with
a solution of freshly prepared pargyline at a dose of 75mg/kg body wt. and the ‘rearing
activity’ was then measured. The injections of pargyline to the different groups were a t
30min intervals. Activity was registered during periods of 30 min by a Tedeschi-type
Actograph.
After the administration of 14 doses of 1 m-equiv. of RbCl/kg body wt. of rat, there
was a statistically significant increase in serotonin concentration in the hypothalamus
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