BIOCHEMICAL SOCIETY TRANSACTIONS
96
(b)
-LSP
-
-
H1A-
-HlB-
-HlO/ H3\
H2A H2B
-H 4 -
Fig. 1. Sodium dodecyl sulphatel polyacrylamide gel electrophoresis pattern of mouse
histones
(a) 1 and 2: two successive extracts (30min each) from mouse liver chromatin; 3 and 4:
the same from spleen chromatin; 5 and 6: the same from mouse kidney chromatin.
Abbreviation used: LSP, liver specific protein. (b) 1-4: four successive 0.25 M-HCl
extracts from mouse liver chromatin. Proteins were strained with Coomassie Brilliant
Blue, and gel electrophoresis was performed by the method of Laemmli (1970).
Chromatin was isolated from 30 mouse livers (35 g wet wt.). The first extraction yielded
40mg of total histones, the second extraction 4mg, the third extraction about 1mg and the
fourth extraction 0.61 mg of histones.
change the pattern of electrophoretic bands. Bio-Gel P-60
chromatography of nucleosomal histones from mouse liver
clearly separates a small peak that represents about 3 4 % of
total histones. Proteins from this peak during electrophoresis
give the same double band in the H3 dimer position as those
shown in Fig. 1. Similar results were obtained in our analysis of
rat liver histones, and the double band in the H3 dimer position
was present in both total histones and nucleosomal histones
isolated after preliminary extraction of H 1 histone with 5%
perchloric acid.
The nature of the proteins in the position of H 3 dimers is not
yet clear. The preliminary amino-acid analysis of the sample
isolated by Bio-Gel P-60 chromatography showed a rather
unique composition - a high content of aspartic acid
(9.2mol%), glutamic acid (14.7mol%), serine (10.1 mol%) and
lysine and arginine (6.8 and 7.6mol%). The proteins are
cysteine-free. The content of methionine is 1.9 mol%.
We thank Dr. I. H. Buchanan for his assistance in performing the
amino acid analysis of the protein sample and for his advice.
Bohm, E. L., Strickland, W. N., Strickland, M., Thwaits. B. H., Van Der
Westhuizen, D. R. & Von Holt, C. (1973) FEBS Lett. 34, 2 17-22 1
Chanda, S. K., Ickowicz, R. & Dounce, A. L. (1973) J. Biochem.
(Tokyo) 135,115-123
Cleland, W. W. (1964) Biochemistry 3,480-482
Goodwin, G. H., Sanders, C. &Johns, E. W. (1973) Eur. J . Biochem.
38, 14-19
Kornberg, R. D. (1977) Annu. Rev. Biochem. 46,93 1-954
Laemmli, U. K. (1970) Nature (London) 227,680-685
McGhee, J. D. & Felsenfeld, G. (1980) Annu. Rev. Biochem. 49,
1 1 15-1 156
Maizel, J. V. (1971) Methods Virol. 5, 179-246
Panyim, S., Bilek, D. & Chalkley, R. (1971) J. Biol. Chem. 246,
4206-42 15
Panyim, S . & Chalkley, R. (1969) Biochemistry 8, 3972-3979
Tarnowka, M. A., Baglioni, C. & Basilico, C. (1978) Cell 15, 163-171
ATP-stimulated breakdown of polylysine in rabbit reticulocyte cell-free extracts: differential
effects according to substrate molecular weight
V. C. WORTHINGTON and A. R. HIPKISS
Department of Biochemistry, King’s College London, Strand,
London WC2R 2 L S , U.K.
The rabbit reticulocyte possesses one or more proteolytic
activities which can be stimulated by ATP (Etlinger &
Goldberg, 1977; Daniels & Hipkiss, 1978; Hershko et al.,
1980). The role of the ATP during proteolysis is certainly not
clear. ATP may possibly stimulate the attachment of the
low-molecular-weight protein called ubiquitin to the substrate
via isopeptide bonds to lysine residues (Wilkinson et al., 1980);
alternatively ATP could directly activate the proteinase with no
involvement of ubiquitin or any other short protein (Etlinger &
Goldberg, 1977; Goldberg et al., 1978). Our studies have shown
that after the synthesis of rapidly degradable abnormal protein
in rabbit reticulocytes, inhibitors of ATP synthesis were less
effective in preventing the proteolysis of puromycin-peptides
than of normal-length aberrant proteins containing the lysine
analogue aminoethylcysteine (Hipkiss et al., 1982). This
observation may suggest that, analogous to the case in
Escherichia coli (Kemshead & Hipkiss, 1974, 1976), proteolysis
of large abnormal proteins requires ATP, whereas catabolism of
aberrant proteins of shortened chain length can proceed in the
absence of ATP. We have therefore tested this proposal, using
polylysine of various molecular weights as substrate, and a
reticulocyte cell-free extract as source of proteolytic activity.
Rabbit reticulocytes were obtained and cell-free extracts
prepared as previously described (Daniels et al., 1980).
1982
97
597th MEETING, LONDON
Polylysine (Sigma Chemical Co.) of various molecular weights
was incubated at 37OC (see legend to Table 1 for experimental
details). Lysine released into the trichloroacetic acid-soluble
fraction was measured in a Locarte automatic amino acid
analyser.
Table I(a) shows that polylysine is degradable in reticulocyte
cell-free extracts and that the proteolysis is stirnulatable by
ATP. The rate of proteolysis (which remained approximately
linear for at least 2h) decreased with increasing polylysine
molecular weight, whereas the relative effect of ATP increased
with substrate size. Similar differences in rates of lysine release
into the acid-soluble fraction were observed when the degradation of different-molecular-weight polylysines were compared at equimolar concentrations (Table Ib). Hence the
differences in proteolysis shown in Table l(a) are not a
consequence of variation in substrate molar concentration, but
are instead related to differences in polylysine molecular weight.
The present observations suggest therefore that cell-free
preparations of rabbit reticulocytes contain two types of
proteolytic activity: one an ATP-stimulatable proteinase which
can degrade polylysine of high and low molecular weights, and
the other an ATP-independent proteinase of higher activity than
the ATP-dependent enzyme, which is limited to degrading the
low-molecular-weight substrates. The mechanism of how such
size-discrimination occurs in proteinases is a matter for
speculation at present, however. The proposal for the existence
of size-discriminating proteinases in reticulocytes is consistent
with our repeated observation that inhibitors of ATP synthesis
frequently have little or no effect on the catabolism of
pulse-labelled abnormal proteins synthesized in the presence of
high concentrations of puromycin (Hipkiss et al., 1982).
Evidence for size-discriminating proteinases in E. coli has also
been obtained by ourselves (Kemshead & Hipkiss, 1974, 1976)
and by others (Cheng & Zipser, 1979; Manley, 1978).
The rapid, ATP-independent, proteolysis of shortened globin
chains (i.e. certain CNBr peptides) may involve at least
recognition of lysine residues, since blocking amino groups with
maleic anhydride or succinic anhydride results in a marked
inhibition of degradation (Hipkiss et al., 1982). Similarly,
treatment of E. coli CNBr peptides by succinic anhydride also
results in their decreased degradability in E. coli cell-free
extracts (A. R. Hipkiss, unpublished work). Finally, we suggest
that proteolysis of polylysine and puromycin-peptides may
proceed via a common process in reticulocyte cell-free preparations, since we have observed that the degradation of
puromycin-peptides is progressively inhibited upon addition of
increasing quantities of polylysine (V. C. Worthington & A. R.
Hipkiss, unpublished work). Whether ubiquitin is involved in
either the ATP-dependent or -independent proteolysis of
polylysine, we do not at present know.
Table 1. Effect of ATP on the proteolysis of polylvsine of
various molecular weights in reticulocyte cell free extracts
Each incubation contained 0.1 M-Tris/HCI, pH 7.75, 5 mMMgCI,, IOmM-KCI, 0.5 mwdithiothreitol, 2 ~ M - A T Pwhere
appropriate, and polylysine. Cell-free extract was added to 25%
(v/v) of the final incubation mixture. After incubation for 2 h at
37OC, 0.5 ml of 5% trichloroacetic acid was added. Lysine in the
acid-soluble fraction was determined in a Locarte automatic
amino acid analyser. Cell-free extracts were prepared from
reticulocytes depleted of ATP by 2 h incubation at 37OC in a
standard medium (Lingrel & Borsook, 1963). which also
contained 0.2m~-2,4-dinitrophenoland 20m~-2-deoxyglucose.
After lysis of washed, packed cells in 1.6~01.of water containing
1 mwdithiothreitol, the preparation was centrifuged first for
10min at 6 0 0 g and then for 2 h at lOOOOOg, and the supernatant fraction was finally dialysed against Borsook saline
(Lingrel & Borsook, 1963) for 20h at 4OC.
Lysine released
(nmol/2 h)
Stimulation
by ATP
Average mol. wt.
(%,)
+ATP
-ATP
of polylysine
17.0
49 1.4
420.0
(a) 3000 (10 mg/ml)
52.4
63.4
41.6
13000 (IOmg/ml)
127.2
28.4
12.5
40000 (IOmg/ml)
(b) 3000 (1.5mM)
13000 (1.5mM)
184.1
34.9
171.8
25.4
7.2
37.4
We thank Mr. C. James for the lysine determinations on the amino
acid analyzer and the University of London Central Research Fund for
financial support.
Cheng, Y.-S.E. & Zipser, D. (1979)J. Biol. Chem. 254,4698-4706
Daniels, R. S. & Hipkiss, A. R. (1978) Biochem. SOC. Trans. 6,
623-625
Daniels, R. S.,Worthington, V. C., Atkinson, E. M. & Hipkiss, A. R.
(1980) FEBS Lett. 113,245-248
Etlinger, J. D. & Goldberg, A. L. (1977) Proc. Null. Acad. Sci. U.S.A.
74,54-58
Goldberg, A. L., Kowit, J., Etlinger, J. & Klemes. Y. (1978) in Protein
Turnover and Lysosomal Function (Segal, H. & Doyle, D.. eds.), pp.
171-196, Academic Press, New York
Hershko, A., Ciechanover, A., Heller, H., Haas, A. L. & Rose, 1. A.
(1980)Proc. Narl. Acad. Sci. U.S.A.77, 1783-1786
Hipkiss, A. R., McKay, M. J., Daniels, R. S., Worthington, V. C. &
Atkinson, E. M. (1982)Acra Biol. Med. Ger., in the press
Kemshead, J. T.& Hipkiss, A. R. (1974) Eur. J . Biochem. 45.535-540
Kemshead, J. T.& Hipkiss, A. R. (1976)Eur.J. Biochem. 71. 185-192
Lingrel, J. B. & Borsook, M. (1963) Biochemistry 2. 309-3 14
Manley, J. L. (1978) J. Mol. Bid. 125,407432
Wilkinson, K. D., Urban, M. K. & Hass. A. L. (1980) J. Bid. Chem.
255,7529-7532
Activity and heat-stability of peptidase activity in cell-free extracts of rabbit reticulocytes and
erythrocytes: effects of bivalent metal ions
A. CROWTHER, E. M. ATKINSON and A. R. HIPKISS
Department of Biochemistry, King's College London, Strand,
London WC2R 2 L S , U.K.
Many cellular peptidases require or are activated by bivalent
metal ions, although the exact role of these cations remains
unclear (Armstrong et al., 1974; Patterson et al., 1975; Stern &
Mark, 1979). Peptidase activity has been demonstrated in
human erythrocytes (Scott & Kee, 1979; Pontremoli et al.,
1980) and the presence of such enzymes has been inferred in
mouse reticulocytes from inhibitor studies (Botbol & Scornik,
1979). Previous observations have shown that during
VOl. 10
maturation of the reticulocyte to the erythrocyte in the rabbit,
there is a marked decrease in peptidase activity (Atkinson &
Hipkiss, 1981). We have therefore compared the effect of a
number of divalent metal ions on the activity and heat-stability
of peptidase activity in cell-free extracts of rabbit reticulocytes
and erythrocytes.
Rabbit reticulocytes and erythrocytes were obtained and
cell-free extracts (600g-supernatant fractions) prepared as
previously described (Atkinson & Hipkiss, 198 1). Peptidase
activity with glycylglycine as substrate in 0.1 M-Tris/HCI,
pH7.5, was measured by the method of Binkley et al. (1968).
Heat-stability of the lysate peptidase activity was investigated