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BIOCHEMICAL SOCIETY TRANSACTIONS
The diacylglycerol concentration of the haemolymph therefore shows a significant, but
temporary, rise. After cessation of flight release of adipokinetic hormone (Mayer &
Candy, 19696) may no longer occur and further release of diacylglycerol eventually
ceases. Locusts reflown at this point show a rapid depletion of haemolymph diacylglycerol as it is taken up by flight muscles at a greater rate than it is released from the
fat-body.
During a 30min rest period after flight the diacylglycerol concentration of haemolymph rises by 5.9mg/ml. If the total haemolymph volume of a locust is about 0.4ml
(Walker et al., 1970) this corresponds to a value of 4.7mg/h as a minimum rate of release
of diacylglycerol from the fat-body of a locust during flight. Beenakkers (1965) has
calculated that locust flight muscles use 23mg of fatty acid/h per g duringflight. Since an
average male desert locust has 0.23g of flight muscle (Candy, 1970) this corresponds to
approx. 5.5mg/h for the rate of diacylglycerol oxidation by a locust. Diacylglycerol
release from the fat-body can thus quantitatively account for much of the lipid oxidized
by muscle during flight.
Further experiments were carried out to investigate whether diacylglycerols of differing fatty acid composition are used at differing rates during flight. Fig. 2(a) shows that
the relative proportions of diacylglycerols with different numbers of fatty acid carbons
is very similar in haemolymph collected from locusts at rest, after flight or after flight
followed by rest. Similar results were obtained when the individual fatty acids were
measured (Fig. 2b). These results indicate that the flight muscle shows no marked
preference for any particular diacylglycerol or any fatty acid component of the diacylglycerols, but rather uses them in proportion to their relative concentrations in the
haemolymph.
Beenakkers, A. M. Th. (1965) J . Insect Physiol. 11, 879-888
Candy, D. J. (1970) J. Insect Physiol. 16, 531-543
Christie, W. W. (1972) Analyst (London) 97, 221-223
Mayer, R. J. & Candy, D. J. (1969~)Comp. Biochem. Physiol. 31B, 409-418
Mayer, R. J. & Candy, D. J. (1969b) J. Insect Physiol. 15, 611-620
Walker, P. R., Hill, L. & Bailey, E. (1970) J. Insect Physiol. 16, 1001-1015
Weis-Fogh, T. (1952) Phil. Trans. Roy. SOC.London Ser. B 237, 1-36
Pinocytosis and Intracellular Proteolysis in Experimentally Induced
Lysosomal Storage
ANNE V. S. ROBERTS, SUSAN E. NICHOLLS, KENNETH E. WILLIAMS
and JOHN B. LLOYD
Biochemistry Research Unit, University of Keele, Keele, Staffs. ST5 5BG, U.K.
Lysosomal storage disease is now a well-authenticated phenomenon in human pathology
(Hers & Van Hoof, 1973). The term embraces many distinct diseases but in all cases
morphological examination of affected tissues reveals progressive accumulation within
the cell of abnormally large lysosomes (diam. 1-2pm) filled with indigestible polymeric
material. In several cases a single defective or missing enzyme has been identified, making
possible attempts to correlate this enzymic deficit with the nature of the accumulated
metabolites. What is not understood, however, is precisely why progressive lysosomal
engorgement causes a deterioration of cell function. More specifically it is not known
whether the lysosomes of affected cells, modified as they are both in size and content,
retain the ability to function normally. A number of functional abnormalities of the
vacuolar system seem possible. The accumulation of some metabolite that is normally
degraded by the missing enzyme may so alter the intralysosomal environment that the
activities of other lysosomal enzymes are depressed. This could lead to a secondary
accumulation of substrates, and heterogeneity of the storage product. Alternatively, the
gross distension of the vacuolar system seen in lysosomal storage disease could inhibit
1974
550th MEETING, ENGLEFIELD GREEN
1097
endocytosis and thus profoundly affect cells in which this function is important. A depressed rate of pinocytosis would also hinder attempts at therapy involving replacement
of the absent enzyme.
The typical features of lysosomal storage can be reproduced experimentally in certain
cells by administration, either in vivo or in tissueculture, of compounds that thelysosomal
enzymes cannot digest (Lloyd, 1973). Thus sucrose, dextran, Triton WR-1339 and many
other compounds have been shown to accumulate in the vacuolar system of several types
of mammalian cell causing an increase in the average size of the lysosomes. In the present
paper we report studies on the rate of pinocytosis of 1251-labelledpolyvinylpyrrolidone
and 1251-labelledbovine serum albumin by yolk-sac epithelial cells from rats that had
previously received injections of vacuolating agents.
The light and electron histology of the rat visceral yolk sac have been described by
Padykula et al. (1966). Its epithelial cells are active in micropinocytosis and they capture
many substances which congregate in the prominent vacuolar system of the cells (for
references see Williams et al., 1971). Schultz et al. (1966) demonstrated that administration in vivo of Triton WR-1339 led to a distension of the vacuolar system of the yolk sac
similar to that observed in liver (Wattiaux et al., 1963). We have examined the effect on
yolk-sac morphology of Triton WR-1339, sucrose and polyvinylpyrrolidone. Substances
were administered in vivo under different regimes and the tissue examined at 17.5 days of
pregnancy. With Triton WR-1339 the most profound changes were seen 48h after
500mg/kg had been administered intraperitoneally. The average diameter of the vacuole
profiles was larger (usual range 0.7-2.1 pm) than in tissue from untreated animals
(0.5-0.9pm). The vacuoles appeared highly electronlucent and were crowded together
in the apical cytoplasm so that the normal rounded outline became distorted. Similar
changes were seen 36 h after intraperitoneal injection of polyvinylpyrrolidone (mean
mol.wt. 40000; 1000mg/kg), with vacuole profiles usually 2.C2.5pm in diameter. At
24h after an intraperitoneal injection of sucrose (1000mg/kg) the vacuoles in the apical
cytoplasm again appeared crowded compared with untreated tissue, but in electron
density more closely resembled the vacuoles in control tissue.
Lloyd et al. (1972) described an organ-culture system with rat yolk sac in which the rate
of substrate uptake by pinocytosis is both constant and reproducible. The rate of uptake
can be expressed as an Endocytic Index, defined as the volume of medium whose substrate content has been processed by unit quantity of tissue in unit time. The value of this
parameter was determined for the non-digestible substrate 1251-labelledpolyvinylpyrrolidone with both normal tissue and tissue from rats pretreated with the vacuolating agents
under the regimes described above. The results (Table 1) indicate that rates of pinocytosis
are unaffected.
Although pinocytic uptake of the hydrolysable substrate 1251-labelledbovine serum
albumin is followed by intracellular digestion, an Endocytic Index can still be calculated
(Moore et al., 1974); its value was shown to vary from batch to batch depending on the
degree of denaturation of the protein. During the present experiments two batches of
1251-labelledbovine serum albumin were employed ;both had been exposed to acetic acid
at pH3.5 for 1h a t 37°C (cf. batch C of Moore et al., 1974). Table 2 shows the Endocytic
Table 1. Effect of vacuolating agents accumulated in viuo on the rate of uptake of lZ5Zlabelled polyvinylpyrrolidone by 17.5-day rat yolk sac cultured in vitro
Vacuolating agent
(injected intraperitoneally)
None (control)
Triton WR-1339 (500mg/kg; 48h before death)
Sucrose (1000mg/kg; 24h before death)
Polyvinylpyrrolidone
(1000mg/kg; 36h before death)
VOl. 2
No. of
Endocytic Index
experiments (,ul/h per mg of protein)
10
3
3
3
1.57f 0.25
1.49 f 0.26
1.47 f 0.26
1.59f0.25
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BIOCHEMICAL SOCIETY TRANSACTIONS
Table 2. Effect of vacuolating agents (see Table 1 for doses and route of administration)
on the rate of uptake of '251-labelledbovine serum albumin by 17.5-day rat yolk sac cultured
in vitro
Vacuolating agent
None (control)
None (control)
Triton WR-1339
Sucrose
Polyvinylpyrrolidone
Batch of 12sI-labelled
bovine serum albumin
No. of
expts.
Endocytic Index
b l / h per mg of protein)
I
I1
I
I1
3
4
3
3
3
15.4f 1.2
25.3 rf: 5.7
13.8 rf: 2.2
22.9k 2.8
28.5 f 6.3
I1
Index of 1251-labelledbovine serum albumin in tissue from animals treated with vacuolating agents; no difference from controls was seen. In all experiments the amount of substrate contained in the tissue became constant after 1-2h, indicating that the rate-determining step in the overall process of uptake and digestion is uptake. Thus there is no
evidence that lysosomal enzymes are inhibited.
These experiments show, first, that it is possible to simulate the morphological features
of lysosomal storage disease in rat yolk sac by administration of appropriate substances
to the mother in vivo and, secondly, that these pre-treated yolk sacs show no alteration in
either pinocytic or digestive capacity. The substances used so far have all been uncharged
molecules and thus unlikely to exert inhibitory effects on lysosomal enzymes, but it is
interesting that cells with a grossly distended vacuolar system may pinocytose as readily
as normal cells and that, for the vacuolating agents studied, the alteration of the intralysosomal milieu by large amounts of a foreign compound appears not to affect the efficiency of digestion. These results contrast with the finding of Wagner et al. (1971) that
pinocytosis of sucrose by Chang liver cells in culture led to a 'blockade' of uptake when
the cells had become highly vacuolated.
We thank the M.R.C. for their support of this work and Professor A. R. Gemmell of the
Department of Biology, Keele University, for the generous provision of electron-microscope
facilities.
Hers, H. G. &Van Hoof, F. (eds.) (1 973) Lysosomes and Storage Diseases, Academic Press, New
York and London
Lloyd, J. B. (1973) in Lysosomes and Storage Diseases (Hers, H. G . & Van Hoof, F., eds.), pp.
173-195, Academic Press, New York and London
Lloyd, J. B., Williams, K. E., Beck, F. & Kidston, M. E. (1972) Biochem.J. 1 2 8 , 1 4 4 ~ - 1 4 5 ~
Moore, A. T., Williams, K. E. & Lloyd, J. B. (1974) Biochem. SOC.Trans. 2, 648-650
Padykula, H. A., Deren, J. L. & Wilson, T. H. (1966) Develop. Biol. 13, 311-348
Schultz, P. W., Reger, J. F. & Schultz, R. L. (1966) Amer. J. Anat. 119, 199-233
Wagner, R., Rosenberg, M. & Estensen, R. (1971) J. Cell Biol. 50, 804-817
Wattiaux, R., Wibo, M. & Baudhuin, P. (1963) Lysosomes, Ciba Found. Symp. 176-196
Williams, K. E., Lloyd, J. B., Davies. M. & Beck, F. (1971) Biochem. J. 125,303-308
Analytical Subcellular Fractionation of Guinea-pig Myocardium
with Special Reference to the Localization of the Adenosine Triphosphatases
FREDERICK J. BLOOMFIELD and T. J. PETERS
Department of Medicine, Royal Postgraduate Midical School, London W12 OHS, U.K.
Previous subcellular fractionation studies on heart muscle have usually been preparative
in nature. With this approach, particular organelles are isolated to a high degree of
homogeneity, usually with little regard to yield, e.g. mitochondria (Green et al., 1955),
1974