Download Protein degradation in mouse brain slices

BIOCHEMICAL SOCIETY TRANSACTIONS
214
Hanson. S. W. F.
10 1 102
We thank the Grampian Health Board for financial assistance.
Analysis of methyl esters of fatty acids was performed by Dr C.
Moffat at the Torry Research Station, Aberdeen, U.K.
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Ollcy, J. ( I 963) I ~ i o ~ l t c ~ rSOC.
u . Trwrs. 89.
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Besson, J. A . 0..Corrigan, F. M.. Forcman. E. I.. Eastwood, C. M..
Smith, F. W. & Ashcroft, G. W. (1985) nr. J. PJychicirry 146.
3 1-36
Received 2 0 June I 9 X X
Protein degradation in mouse brain slices
U T E ROMLONG,* D E B O R A H M. REECE,
PETER B. NUNN and A L A N R. HIPKISS
Department of Biochemistry, King's College, University of
London, Strand, London WC2R ZLS, U.K.
A number of neurological and neurodegenerative disorders
are associated with the accumulation of altered proteins
(fragmented, aggregated or ubiquitinatcd) in neuronal tissues
(Carrell, 1988). These observations suggest a role for protcolytic degradation, or possibly a defect in this process, in the
generation and accumulation of aberrant polypeptides in
some neurodegenerative conditions. Recent findings suggest
a role for neurotoxic and unusual neuroexitatory amino acids
in the aetiology of certain neurodegenerative disordcrs
(Spencer er ul., 1987). This has led us to speculate whether
those amino acids that are implicated as possible causativc o r
contributory agents in these diseases, might also be involved
in the modulation or perturbation of neuronal protein metabolism, thereby effecting aberrant protein accumulation.
Here we present results of preliminary studies in which we
have examined the effects of four unusual amino acids on
protein catabolism in mouse brain slices.
Mouse brain slices (around 30 mg), prepared immediately
after killing, were incubated in 2.5 ml of Krebs-Henscleit/
bicarbonate buffer (Krebs & Henseleit, 1932) at 37°C with
5.5 mM-glucose and gassed with 0 , / C 0 2 ( 1 9 1 ). ["SIMethionine (50 pCi at 1000 Ci/mmol) or [3H]leucine(10 pCi at 155
Ci/mmol were added as required. Radioactivity was incorporated into the trichloroacetic-acid-soluble fraction for up
to 2 h. Because incorporation was inhibited by cyclohexiniide ( 100 &mi), this observation was taken as prima
facie evidence for protein synthesis. To study protein breakdown, we employed a pulse-chase technique whereby slices
were labelled for 30 min with ["Slmethionine, washed frcc
from the radioisotope and reincubated with excess (100 pg/
ml) unlabelled methionine together with the amino acid to bc
investigated. Samples were then removed at intervals. Protein
was precipitated with trichloroacetic acid to 5% (w/v), rcsuspended and homogenized in I M-NaOH, and the
radioactivity determined after extraction with chloroform/
methanol (2: I m v/v). Protein concentrations were determincd
by the B C A method (Pearce) according to manufacturers
instructions.
Table 1 shows that the radioactivity (%) remaining in the
trichloroaeetic-acid-insoluble faction rapidly declined, with
u p to 90% being lost within 60 min. This result was not
unanticipated because the slices were not supplied with any
other amino acids (other than the radioactive one) and thus
substantial protein breakdown must be occurring to support
synthesis, and because of cleavage of the initiating mcthionine residues from the growing peptide chains. Experiments
employing ['H]leucine gave estimates of proteolysis of the
order of 30% in 1 h.
Both canavanine sulphate ( 100 pg/ml) and L-a-amino-p
methylaminopropionic acid ( 1 mM) stimulated degradation
of the 35S-labelled proteins. Kainic acid ( I mM) appeared to
*Present address: lnstitut fur Physiologischc Chemie, KonstanyGutschow- Str. 8,3000 Hannover 6 I , F.R.G.
Table 1. E8ect.s of conavonine sirlphare (I(X)pcg/ml), a$diominopropionic acid ( I mM), i~-a-omino-/3-rnerhylaminopropionic acid (lm,w) nnd kainic acid (I n i ~ )on prorein
breakdown in mouse brain slices
For experimental details see the text.
Protein breakdown
Conditions
(Oh)
7 min
30 min
Control
Plus canavanine
Plus a$-diaminopropionic acid
Plus L-a-amino-B-methylaminopropionic
acid
64
91
69
80
84
89
84
94
Control
Plus kainic acid
81
73
92
84
~
~
be inhibitory, while a,/3-diaminopropionic acid ( 1 mM),
an apparently non-toxic compound, had little effect (Table
1).
These studies, although very preliminary, demonstrate.
that mouse brain slices are amenable for the study of shortterm investigation of protein metabolism. Our results also
indicatq that the known neurotoxic amino acid L-a-amino-/%
methylaminopropionic acid, which causes a motor neuron
disease in macaques (Spencer et id, 1987), may enhance protein degradation in neuronal tissue. T h e mechanisms
invo!ved in this effect will be investigated. Canavaninc, which
also had stimulatory effect on proteolysis, is known to promote synthesis of aberrant polypeptides and to induce synthesis of stress proteins (some of which are proteinases) in
many cell types. Future studies will include an investigation
of the catabolism o f altered proteins (analogue-containing
and fragments) in the rodent brain, and the extent to which
neurotoxic amino acids modulate their catabolism. Of equal
importance will be the search for age-related changes in the
catabolism of both normal and aberrant proteins in thcse
preparations. T h e ability to degrade certain abnormal proteins has been found t o decline with age during reticulocyte
maturation (McKay et al., 1980), during senescence in cultured human fibroblasts it1 vitro (Wharton & Hipkiss, 1985)
and during ageing of the bovine lens core (Carmichael &
Hipkiss, 1988).
This work was supported in part by a grant from the Motor Neurone Disease Association to P.B.N.
Carmichacl, 1'. L. C! Hipkiss, A. R. ( 19x8) I~ioclrerii.SOC. '/kc/trs.17,
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Krcbs. H. A. I!! Henseleit. K. ( I 932) /-/opi)i~-SL.yler~s
Z. Plrysiol.
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McKay, M. J., Daniels, R. S. 6( Hipkiss, A. R. (1980) fjioclrcwi. J .
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Roy. D. N. SC liobertson. R. C. ( 1 9 8 7 ) .Scie,rw 237. 5 17-522
Wharton, S. A. 61 Hipkiss, A. R. ( 1 985) FEHS Lerr. 184, 249-253
Received 2 0 June 1988
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