230
labelled human serum albumin ([12JI]d.HSA).To calculate the
pinocytic uptake of degradable substances, it is necessary to
sum both the tissue level of the substance and the release of
digestion products from the cells. The radioactivity associated
with the cells became constant after 3 h. This indicates that the
proteolytic capacity of the macrophage lysosomes is adequate to
degrade labelled albumin at the rate at which this substrate is
being pinocytosed; as a result, the acid-soluble radioactivity in
the media (digestion products) continued to rise throughout the
incubation period.
Uptake of [ V d . H S A was linear with time over 24 h and the
Endocytotic Index was 6pllmg of cell protein/h (corresponding
to 3ng/h per mg of cell protein), similar to the values reported
by Pratten et af. (1977) for uptake of bovine serum albumin by
rat peritoneal macrophages.
Uptake and degradation of 1Z51-I-labelled
Mannose-bovine serum
albumin (Man-BSA)
It has been shown (Stahl et al., 1980) that Man-BSA is
endocytosed by a receptor-mediated system in rat alveolar
macrophages. We confirmed this in our system and found that
uptake of [1251]Man-BSA at a concentration of 0.5 g/ml is linear
with time over 24 h. The radioactivity associated with the cells
became constant after 1 h. Allowing for non-specific binding and
uptake, the Endocytotic Index for specific uptake was found to
be 8.5ng/h per mg of cell protein (corresponding to a nominal
fluid intake of 17pl/h per mg of cell protein, which is
approximately three times greater than the rate of uptake for
[ IzJI]d.HSA. The rate of non-specific uptake was equal to that of
[ IZ5Ild.HSA.
Studies with inhibitors
It has frequently been suggested that microtubules and
microfilaments play an important part in the movement of
organelles in endocytic cells (Allison et al., 1971). Cytochalasin
B, which is believed to affect polymerization of microfilaments,
used at a concentration of l0pglml decreased uptake of
[3H]sucrose4 1% and [12sIld.HSA30%, which is consistent with
the report of Pratten et al. (1979), that this inhibitor could not
decrease uptake of colloidal [198Aulgold or 1251-labelledpolyvinylpyrolidone in rat peritoneal macrophages by more than
4096, and with previous work of one of us (Dean, 1979).
Temperature-dependency of uptake
As another means of distinguishing the membrane activities
involved in the endocytosis of the three substrates, we studied
their rate of uptake at various temperatures between 4 and 37OC
(Table 1). The effect of temperature on uptake of 13H1sucrose
(fluid-phase marker), [ lZ5I]d.HSA (adsorptive substrate) and the
receptor-mediated substrate [L2JIIMan-BSAare very similar, in
BIOCHEMICAL SOCIETY T R A N S A C n O N S
Table 1. Temperature-dependence of pinocytosis by mouse
macrophages
Each value represents data from three individual cultures from a
single population of cells. The tracers were used at the following
concentrations: [3H1sucrose, 3.5pg/ml; [ V d . H S A , 0.5pg/ml;
and [lZ5IIMan-BSA, OSpg/ml. The values for radioactivity
associated with the cells after lOmin at 4OC, a measure of
adsorption to the cell surface, corresponding to ng uptake/mg
of cell protein for sucrose, HSA and Man-BSA are respectively
0.01 kO.01; 0.15 kO.04; 0.4 1 kO.01.
Uptake (ng/h per mg of cell protein
Temperature
("(2)
4
10
15
20
25
32
37
I)H ISucrose
0.02 f 0.01
0.034 f 0.04
0.032 0.02
0.148f0.03
0.22 f0.02
0.3 f 0.003
0.4 f 0.02
['2511d.HSA
0.26 f0.1
0.26 f 0.0 1
0.3 f 0.04
1 fO.2
1.5 f 0.12
2f0.16
3 f 0.32
I"'IIMan-BSA
0.42 f 0.1
0.38f0.06
0.36 & 0.04
2.24 f 0.1
4.4 f 0.2
7.6 f 0.4
9.56f 1.1
that for all three, substrate capture was almost completely
abolished below 20°C.
In conclusion, our studies of two aspects (temperaturedependency and susceptibility to inhibitors) of fluid, adsorptive
and receptor-mediated pinocytosis, give no indication of
qualitative distinctions in the mechanism of membrane internalization per se. In spite of the association of HSA and
Man-BSA with the membrane, and the contrasting independence of sucrose from the membrane, the internalization of
all three markers is qualitatively similar. These results lend no
support to the concept (Allison et al., 1974) that macrophage
pinocytosis has two mechanistically distinct components.
macropinoc ytosis and micropinocytosis.
This work is supported by a grant from the Arthritis and
Rheumatism Council. We thank Dr. Philip Stahl, Washington
University School of Medicine, St. Louis, MO, U.S.A.. for providing
Man-BSA.
Allison, A. C., Davies, P. & de Petris, S . (1971) Narure (London) New
Biol. 232, 153-155
Allison, A. C. & Davies, P. (1974) Symp. Soc. Exp. Biol. 27.4 19-446
Dean, R. T. (1979) Biochem. SOC.Trans. 7,362-364
Jessup, W. & Dean, R. T. (1980) Biochem. J. 190,847-850
Pratten, M. K., Williams, K. E. & Lloyd, J. B. (1977) Biochem. J . 168.
365-372
Pratten, M. K. & Lloyd, J. B. (1979) Biochem. J. 180, 567-571
Stahl, P., Schlesinger, P. H., Sligandson, E., Rodman, J. S. & Lee. Y.C.
( 1980) Cell 19,207-2 15
Endogenous protein turnover during lysosomal storage in normal, Tay-Sachs' and Sandhops
fibroblasts
SHEENA M. COCKLE and ROGER T. DEAN
Cell Biology Research Group, Brunel University, Uxbridge,
Middx., U.K.
Genetics lesions leading to the synthesis of abnormal lysosomal
enzymes are now known to be responsible for the lipidoses in
which intracellular storage of glycolipids and glycosaminoglycans occurs; for example, Tay-Sachs' and Sandhoff s
diseases are characterized by a deficiency of hexosaminidase A
and hexosaminidase A + B respectively from fibroblasts which,
as a consequence, accumulate GM, ganglioside in the lysosomes
(Hers & Van Hoof, 1973). Research to date has been largely
concerned with the identification and characterization of the
abnormal enzymes, and little is yet known of the secondary
effects of the intralysosomal storage of non-degradable components on other cellular functions in which lysosomes participate.
A study of such secondary effects may be of considerable
importance in understanding the symptoms of the disease. in
many cases, severe mental retardation and blindness. Our
studies so far have been concerned with the effect of storage on
the degradation of endogenous proteins with a slow turnover in
fibroblasts in culture from patients with Tay-Sachs' and
Sandhoffs disease.
In order to standardize growth conditions for an investigation into the effect of lysosomal storage on protein degradation in cells in culture, it was necessary to monitor any
regulation of proteolysis with growth state. Some evidence
suggests that acceleration of protein degradation may occur as
1982
599th MEETING, BIRMINGHAM
23 I
Table 1. Comparison of protein degradation of intracellular proteins in normal and lipid-storage cells
Cells were seeded at lo4 (growth) and lo’ (confluent) cells per sterilin tissue-culture tube and grown in 1 ml of Eagle’s minimal
esential medium, 10% heat-inactivated foetal-calf serum, 100i.u. of penicillin and 1 OOpg of streptomycin/ml (growth medium)
for the times indicated below at 37OC in an incubator gassed with CO,/air ( I : 19). Cells were labelled for I6 h in labelling medium
(i.e. growth medium without leucine, except for 0.1pCi of [‘4Clleucine), washed, and the degradation allowed to proceed in
growth medium. Degradation (%)is expressed as:
Trichloroacetic acid-soluble (d.p.m. in medium and cells)
Total d.p.m. per culture
x
loo
Percentage degradation in 24 h (k s.D.:n = 4)
1
Growth state
( a ) Exponential growth
( a ) Confluent
(b) Confluent
(c) Confluent
Time after
seeding (h)
24
24
8
0
Normal
(line I )
18.2 (k1.2)
22.0 (k0.4)
23.7 (kO.2)
28.2 (k 1.9)
cells become quiescent at confluence in tissue culture (Hendil,
1977; Tanaka & Ichihara, 1976), although the effect may not be
universal (Baxter & Stanners, 1978; Bradley, 1977; Lee &
Engel, 1977). We have studied the effect of growth state on
protein degradation in a range of normal cell lines. Increases in
protein degradation at confluence of between 15 and 40% have
been observed in seven human fibroblast cell lines and also in
several continuous cell lines; namely, 3T3, BHK2 l c 13 and
BSC-I. This change in degradation cannot be explained by
depletion of serum components or certain amino acids which are
known to regulate protein degradation (Dean, 1980), because
the effect is still observed in the absence of serum or amino
acids.
Proteolysis appears to be regulated by growth state in a
similar manner in both normal and storage diseased cells,
because degradation is consistently higher in confluent cultures
compared with exponentially growing cultures in Tay-Sachs’
and SandhofPs fibroblasts (Table la). The time course of
degradation (up to 32h) is similar in normal and storagediseased fibroblasts (results not shown).
The diseased fibroblasts may progressively accumulate
storage material as they remain at confluence, making any
changes in protein degradation more apparent. For this reason,
degradation has been measured in normal and diseased
fibroblasts maintained for various times (0, 8, 24h) at
confluence before performing the experiment (Table la, l b and
Ic). A marked decrease in proteolysis was observed in normal
fibroblasts the longer they were maintained at confluence, such
that degradation after 24 h at confluence was 22% less than that
in control cells (Table lc) which were not maintained at
confluence before the experiment. In contrast, Sandhoff s
fibroblasts show no change in proteolysis after maintenance for
24h at confluence, although a decrease was observed at later
time points. Tay-Sachs’ fibroblasts show a decrease in proteolysis which is less marked (1 2%) after 24 h at confluence.
Comparison of degradation rates reveals no consistent
difference between lipid storage and normal fibroblasts (Table
1); for example, proteolysis after 24 h at confluence was higher
in Sandhoff’s but no different in Tay-Sachs’ compared with
normal fibroblasts. However, degradation rates in both lipidstorage-cell types are significantly lower than in normal
VOl.
10
Normal
(line 15)
18.3 (k0.3)
21.4 (k0.7)
22.4 (k1.0)
27.9 (k0.5)
Tay-Sach
21.3 (k0.7)
23.2 (k0.7)
23.8 (k0.5)
26.0 (20.4)
Sandhoff
19.6 (kl.0)
25.3( k0.6)
25.6 (k1.7)
25.8 (k0.9)
fibroblasts when cultures are freshly confluent. These
phenomena are a consequence of the complex nature of
proteolysis regulation as cells remain at confluence; for example,
the increase in proteolysis in Sandhoffs relative to normal
fibroblasts after 24h at confluence reflects a maintenance of
degradation in the Sandhoffs and a pronounced decrease in
degradation in the normal fibroblasts.
At present it is difficult to relate these complex changes
directly to intracellular storage. Previous work (Dean, 1979) has
shown that macrophages induced to store a number of
endocytosable molecules exhibit decreased degradation: for
example, 8Om~-sucroseinhibits degradation by 30%. We have
similarly found that 8Om~-sucrose inhibits degradation in
normal and lipid storage cells at confluence, although the extent
of inhibition is more pronounced in normal (20%) than in both
Tay-Sachs’ (6%) and Sandhoffs (13%). This difference may
indicate that lysosomal degradation is already partially inhibited
in these storage-disease cells, but we have as yet no clear
evidence for this. Indeed, dextran (200pglml) and gangliosides
(bovine brain, 200pglml) have no effect on proteolysis on either
normal or diseased fibroblasts when presented in the degradation medium, although this may be due to insufficient
internalization in 24 h.
Further studies with lysosomotropic agents are required to
determine the relevance of storage to these complex differences in
degradation in various conditions of growth and during storage.
We thank The Wellcome Trust for funding this work and The
Paediatric Research Unit, Guy’s Hospital. London, for their gift of
cells.
Baxter, G. C. & Stanners, C. P. (1978)J. Cell. Phjaiol. 96. 139-146
Bradley, M. 0. (1977) J . Biol. Chem. 252.53 10-53 15
Dean, R. T. (1979) Biochem. J . 180.339-345
Dean, R. T. (1980) in Degradation Processes in Heart and Skeletal
Muscle (Wildenthal, K.. ed.), pp. 3-30, Elsevier/North-Holland
Biomedical Press, Amsterdam
Hendil, K. B. (1977)J. Cell. Phvsiol. 92, 353-64
Hers, H. G. & Van Hoof, F. (1973) Lysosomes and Sforage Diseases.
Academic Press, New York and London
Lee, G. T. Y. & Engel, D. L. (1977) J. Cell. Phvsiol. 92, 293-302
Tanaka, K. & Ichihara, A. (1976) Exp. Cell. Res. 99. 1-6