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The activities of ribosome-degrading enzymes and of a proteinase inhibitor in Tetrahymena pyriformis during starvation
Int. J. Biochem. Vol. 17, No. 7, pp. 799 803, 1985
0020-711X/85 $3.00+0.00 Copyright ~C 1985 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
THE ACTIVITIES OF R I B O S O M E - D E G R A D I N G E N Z Y M E S A N D OF A P R O T E I N A S E INHIBITOR IN T E T R A H Y M E N A P Y R I F O R M I S D U R I N G S T A R V A T I O N H. G. KLEMPERER and D. J. PILLEY Department of Biochemistry, University of Birmingham, P.O. Box 363, Birmingham Bl5 2TT, U.K. [Tel. 021-472-1301]
(Received 27 September 1984) In starved Tetrahymena acid RNase decreased but acid proteinase and a heat-stable proteinase inhibitor both increased. 2. A greater proportion of proteinase than RNase was released from the lysosomes of growing cells by homogenization, but progressively less proteinase was released during starvation. 3. Inhibitors of protein synthesis enhanced the decrease in RNase and prevented the increase in proteinase and proteinase inhibitor. Chloroquine caused a large increase in both enzymes. 4. Proteinase and RNase may be located in different lysosomes which together participate in ribosome destruction, but this process is unlikely to be limited by the total amount of these lysosomal hydrolases. Abstract--1.
MATERIALS AND METHODS
INTRODUCTION
The destruction o f ribosomes in the ciliate Tetrahymena is a useful model of r i b o s o m e b r e a k d o w n in eukaryotic cells. B r e a k d o w n is initiated by starvation and is suppressed by chloroquine, an inhibitor of lysosome function (Klemperer a n d Pilley, 1982). R i b o s o m e s have also been observed inside the lysosome-derived a u t o p h a g i c vacuoles that form in starving Tetrahymena (Nilsson, 1970). Lysosomal R N a s e a n d proteinase readily digest ribosomes in vitro at acid (intralysosomal) pH, but at neutral pH the R N a s e is inactive a n d proteinase action can be suppressed by a heat-stable inhibitor present in the cytosol (Klemperer a n d Pilley, 1985). This inhibitor is similar to the " c y s t a t i n s " found in m a n y other cells a n d it may serve to protect cell c o m p o n e n t s against proteinase released from the lysosomes (Lenney, 1980; G r e e n et al., 1984). The rate of ribosome b r e a k d o w n in Tetrahymena during starvation soon diminishes (Kristiansen a n d Kriiger, 1979; Eckert a n d Kaffenberger, 1980). F o r example, total R N A (which is largely r i b o s o m a l R N A ) decreases 15~o in the first h o u r but only a p p r o x 5~0 per hr after 3 or 4 hr (Klemperer a n d Pilley, 1982). F u r t h e r m o r e , ribosome b r e a k d o w n is inhibited in the presence o f certain proteinase inhibitors a n d of inhibitors of protein synthesis. These facts suggest t h a t ribosome destruction is controlled by the activity of specific enzymes. Certain lysosomal hydrolase activities m a y increase while others decrease in Tetrahymena cells starved for approx one day (e.g. Levy and Elliott, 1968; Lloyd et al., 1971), but i n f o r m a t i o n on the early stages o f starvation is limited. Thus in the first few h o u r s proteinase m a y decrease slightly while R N a s e increases (Mfiller, 1972) or decreases (Rothstein a n d Blum, 1974) but the m e c h a n i s m s involved remain unclear. This p a p e r describes the actions of inhibitors of r i b o s o m e breakd o w n on these lysosomal hydrolases and on the cytosolic proteinase inhibitor.
Tetrahymena pyrijbrmis (strain T) was cultured at 28'C on an orbital shaker in a growth medium as described previously (Klemperer and Pilley, 1982). Cells in exponential phase (density 2 × l05 cells/ml) were harvested at 0°C and washed by resuspension in ice-cold starvation medium (consisting of 1 mM MgCI 2, 2 mM KCI, 2 mM potassium phosphate, pH 7.2, 50 mM NaC1) followed by centrifugation. The cells were finally suspended in starvation medium at 28~C to give a density of approx 2 × 105cells/ml, and incubated in the same way as cultured cells. Cell fractionation Cells in 100 ml of starvation medium were chilled to 0 C , washed with Medium A (1 mM MgC12, 5 mM 2-mercaptoethanol, 50mM Tris-HCI, pH7.4, 100mM KC1, 250mM sucrose) and resuspended in 2 ml of Medium A. Essentially 100°/o of the cells were then disintegrated by 20-30 strokes in a modified Dounce homogenizer with a clearance of 50/~m (Philpott and Stanier, 1956). The homogenate was centrifuged for 10 rain at 4'C and 33,000g to give a pellet that included the lysosomes remaining intact alter this homogenization procedure. The supernatant was centrifuged for 90min at 160,000g to give a cell supernatant (approx 2 ml) that contained the cytosol fraction (including the proteinase inhibitor) as well as lysosomal enzymes released by the homogenization procedure. To extract lysosomal enzymes from the 33,000g pellet, this pellet was suspended in 2 ml of 10 mM sodium acetate (pH 5.0) containing 5 mM 2-mercaptoethanol, and incubated for 5 min at 28=C. The suspension was then centrifuged for 90 min at 160,000 g to give a supernatant that contained essentially all of the RNase and proteinase activities in a soluble form.
Preparation of lysosomal extract Cells suspended in 0.25 M sucrose at 0 C were homogenized in the presence of Celite as described previously (Klemperer and Pilley, 1985) under conditions that minimize lysosomal breakage (but disrupt only 60'~J0of the cells). Celite and unbroken cells were removed by centrifugation at 200g, and a pellet that included the lysosomes was collected by centrifugation at 33,000 g. A lysosome extract containing the proteinase used to determine the proteinase inhibitor was prepared from this pellet as described above.
799
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H . G . KLEMPERER and D. ]. PILLEY digested in 60 rain was determined as above (proteinase determination). Inhibitor activity is expressed as units of proteinase inhibited per mg of total cell protein.
75
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RESULTS
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Lysosomal RNase and proteinase during starvation 25
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I 50 ;L:I of
~_ 100 heai'ed cell
I 150
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Fig. 1. Inhibition of lysosomal proteinase by proteinase inhibitor in heated cell supernatant. In the absence of inhibitor lysosome extract (0.2 ml) digested 135 #g of azocasein in 60min at pH 7.4. An inhibition of 35~o by 60#1 of supernatant corresponds to 135 × 0 . 3 5 - 4 7 units of inhibitor.
Determination of protein and qf enzyme actieities Cells suspended in starvation medium were collected by centrifugation for 3 min at 1000g and taken for determination of total cell protein by the method of Lowry et al. (1951). Enzyme activities in cell fractions (cell supernatant and extract from 38,000g pellet) were measured at 28C in the presence of 1 mM EDTA, 5 mM 2-mercaptoethanol and 50 mM sodium acetate pH 5.0. Under these conditions the amount of product was proportional to the time of incubation. For proteinase the reaction mixture (0.5 ml) contained 5 mg of azocasein. After 60 min of incubation 1 ml of 5% trichloroacetic acid was added, the mixture was centrifuged to remove insoluble material, and the absorption of the supernatant was measured at 340nm. At pH 5.0 the cytosolic proteinase inhibitor has little activity (Klemperer and Pilley, 1985) and its effect on determinations of proteinase in cell supernatant can be ignored. For RNase determinations the reaction mixture (0.15 ml) contained 0.5 mg of Torula RNA (M~ 20-30,000) and was incubated tbr 10 min. After the addition of 3ml of 0.4M HC104 and centrifugation, the supernatant was taken to measure the absorption at 260 nm. RNase in the medium from which the cells had been harvested was measured similarly except that the incubation time was 60 rain. Enzyme activity is given as #g of azocasein or RNA digested in 60 or 10rain respectively and is expressed relative to total protein (rather than to DNA, the cellular content of which varies in different growth states: Bolund and Ringertz, 1966). Determination of proteinase inhibitor Tetrahymena cytosol contains a heat-stable inhibitor of the sulphydryl cathepsins (also inhibited by leupeptin) which contribute approx. 70~ of the total proteinase activity in the lysosomal extract (Klemperer and Pilley, 1985). Like the cystatins in other cells this inhibitor has tightbinding, reversible kinetics such that inhibition is proportional to inhibitor concentration over a wide range (Lenney et al., 1979; Green et al., 1984). Determinations were made in this linear range and under the conditions (pH 7.4) where the inhibitor is most active (Fig. 1). Tetrahymena cell supernatant was heated for 2rain at 100°C to inactivate proteinase, and then centrifuged to remove denatured protein. Aliquots of the supernatant were added to a reaction mixture (0.5 ml) in Medium A containing 5 mg of azocasein and 0.2 ml of lysosome extract (sufficient to digest approx 0.14 mg of azocasein in 60 min). The amount of azocasein
These acid hydrolases are confined to the lysosome-containing fraction of the cells after very gentle homogenisation, but procedures that break 100% o f the cells release a p r o p o r t i o n o f the enzymes into the cell supernatant where they can be measured at pH 5.0. The cytosolic proteinase inhibitor has little activity at this pH and its effect can be ignored. Figure 2 shows the sum for each enzyme of activity released into the supernatant plus enzyme extracted from the remaining lysosomes at low ionic strength. After 1.5hr o f starvation, RNase decreased to approx 501',,, and after 4 hr to 10% o f the initial value. By contrast, total proteinase increased 1005~i in 1.5 hr and then returned to approx, the initial value. Total cell protein decreased only slowly during starvation (Fig. 2: inset). During starvation the cells released RNase but not proteinase into the medium. The RNase that appeared in the medium in 1.5 hr accounted for less than 501~i] of the decrease in RNase in the cells during this period (legend to Fig. 2). Growing cells released RNase at approximately the same rate as cells in starvation medium, The p r o p o r t i o n of the total lysosomal enzyme remaining in the lysosome fraction after homog-
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Fig. 2. Lysosomal RNase and proteinase in starved ceils. Units of enzyme activity are expressed as #g of substrate digested in 10 min (RNase) or 60 min (proteinase) at pH 5.0. Total activity (i.e. supernatant value plus value for extract from 33,000 g pellet) is given for each enzyme. Inset shows total cell protein (100~'o = initial value of approx 30mg/ 100 ml). Each point is the mean +_ SEM of data from four experiments. RNase in the medium (units/mg of cell protein) was 86_+ 10 units after 1.5 hr in starvation medium and 71 _+ 8 units after 1.5 hr in growth medium.
Lysosomal enzymes in starvation Table 1. Percentage of total enzyme remaining in lysosomesafter homogenization
801
imide (2/~g/ml). Puromycin (1 m M ) inhibited to the same extent as cycloheximide (result not shown).
Activity in lysosomes (% of total activity) Starvation (hr) 0 1.5
4 6
DISCUSSION
~ RNase 43-+2
Proteinase 21_+1 48 + 3 78_+2 75+2
49 + 4
49-+2 51 -+3
RNase and proteinasewere determinedas in Fig. 2. Enzymeactivity retained in the lysosomes (extracted from 33,000g pellet) expressed as % of total enzyme activity. Values are means+ SEM of data from four experiments. The % of proteinase retained increasessignificantlyfrom 0 to 1.5 hr and from 1.5 to 4hr (P < 0.01).
enization provides a measure o f lysosome stability. Table 1 shows that during starvation there was little change in the p r o p o r t i o n o f R N a s e retained in the lysosomes but a large increase in the p r o p o r t i o n o f retained proteinase. Initially more proteinase than R N a s e was released but after 4 hr o f starvation this situation was reversed.
The high lysosomal proteinase and R N a s e activities in Tetrahymena may reflect a role in the digestion o f phagocytosed food. Enzyme levels changed rapidly in starvation, when phagocytosis is inhibited and autophagic activity is stimulated (Levy and Elliott, 1968; Nilsson, 1970). Enzyme activity can be assumed to reflect total a m o u n t o f enzyme protein since these lysosomal hydrolases do not appear to exist as inactive forms. Tetrahymena lysosomes contain several distinct acid RNases (Lazarus and Scherbaum, 1967) and acid proteinases (Klemperer and Pilley, 1985), but for the purpose o f a preliminary discussion each class o f hydrolase can be treated as if its c o m p o n e n t enzymes all responded to starvation etc. in the same way. The decrease in R N a s e (50% in 1.5 hr) found in
E
Effect o f inhibitors on proteinase and RNase in starvation
~ so
The activities o f these enzymes were altered in the presence o f inhibitors o f protein synthesis and o f lysosome function. These inhibitors were added at concentrations that are k n o w n to inhibit ribosome b r e a k d o w n without causing visible changes in cell m o r p h o l o g y or motility during 1.5 hr o f starvation. Cycloheximide (2/~g/ml) and puromycin (1 m M ) at concentrations that inhibit protein synthesis by 70-80% (Klemperer and Pilley, 1982) prevented the increase in proteinase. The presence o f puromycin resulted in an even greater depression o f R N a s e than usual after 1.5 hr o f starvation (Table 2). By contrast, both proteinase and R N a s e were greatly increased by the presence o f chlorquine. This drug also increased the p r o p o r t i o n o f R N a s e released from the lysosomes during homogenization. However n o n e o f these inhibitors had a significant effect on the release o f R N a s e from the cells into the medium.
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Time of storvotion (hr)
Proteinase inhibitor during starvation Figure 3 shows that the inhibitor present in cell s u p e r n a t a n t increased t h r o u g h o u t 6 hr o f starvation. The increase at 1.5 hr was prevented by cyclohex-
Fig. 3. Proteinase inhibitor in cell supernatant. Inhibition by heated supernatant determined as in Fig. 1. Each point is the mean ___SEM of four results. Cycloheximide (2/~g/ml) inhibited the increase at 1.5 hr (P < 0.01).
Table 2. Effect of inhibitors on proteinase and RNase in starved cells Enzyme activity Proteinase in cells Starvation (hr) 0 1.5 1.5 1.5 1.5
Inhibitor
Cycloheximide Puromycin Chloroquine
Total 81 +3 172-+4 82 + 2* 76 -+4* 282 _+8*
% in lysosomes 21 ,+ 1 48-+3 52 + 3 45 ,+ 2 53 -+ 2
RNase in cells Total 363-+I-19 158-+11 147 -+ 15 86 + 4* 539 + 17"
% in lysosomes 43-+2 49-+4 56 -+ 2 51 _+3 32 + 3*
RNase in medium -86-+10 66 -+ 8 75 _+8 95 + 3
Total enzymeactivity (units/mg of cell protein) was determinedas in Fig. 2 in cells and medium after incubation without inhibitor or with cycloheximide(2 ,ug/ml), puromycin (1 mM) or chloroquine (0.3 mM). The % activity remaining in the lysosomesafter homogenisation is expressedas in Table 1. Values are means -+ SEM of data from four experiments.Data for cells without inhibitor (first two lines) are taken from Fig. 2 and Table 1. *These values are significantlydifferent from 1.5 hr values without inhibitor (P < 0.01).
802
H . G . KLEMPERERand D. J. PIt,LEY
these experiments occurs in some other strains of Tetrahymena (see Introduction) but the large transient increase in proteinase has not been observed previously. Since these enzymes may be turning over (v.i.) variations in the balance of synthesis and destruction, depending on the precise conditions, may account for conflicting data on enzyme levels in starvation. Acid RNase and proteinase seemed to be controlled independently of one another since they changed in opposite directions during the first 1.5 hr of starvation. Furthermore, RNase (but not proteinase) was released into the medium, as are some lysosomal hydrolases in certain mammalian cells (e.g. Hawkins, 1980; Stahl, 1983). This release occurred at the same rate as in growing cells and it was insufficient to account for the decrease in cell RNase which was therefore undergoing net breakdown. RNase and proteinase also behaved as if they were located in lysosomes that differed in mechanical fragility. Acid proteinase in growing cells was more readily released into the supernatant during homogenisation, and the release of RNase, but not of proteinase, was increased when the cells had previously been incubated with chloroquine. These two enzymes may therefore occur in different types of lysosome. Acid RNase and certain hydrolases (other than proteinase) in Tetrahvmena have previously been shown to differ in their responses to nutrition, and seem to occur in distinct lysosomes (Lloyd et al.. 1971: Rothstein and Blum, 1973, 1974). Heterogeneity has also been observed in mammalian lysosomes (summarized e.g. in Rothstein and Blum, 1974; Pertoft et al., 1978). The fact that acid proteinase m ]etrahymena became increasingly resistant to release during starvation suggests a change in function. Thus there may be an increasing proportion of this enzyme in primary lysosomes, which are known to be more resistant to lysis by shearing forces than the larger lysosomederived vacuoles (Deter and Duve, 1967). This could reflect a progressive decrease in proteinase digestion after the initial stages of starvation, which was achieved, not by a decrease in enzyme (as is the case with RNase) but by restricting the function of the lysosomes that contain proteinase. Substances that inhibit ribosome breakdown (Klemperer and Pilley, 1982) altered the levels of RNase and proteinase. In the presence of puromycin RNase decreased even further after 1.5 hr of starvation (25~i instead of 50'I; of the initial value), suggesting that RNase was turning over and that the enzyme activity reflected the relative rates of synthesis and destruction. Direct evidence for proteinase turnover is lacking since inhibitors of protein synthesis merely prevented the increase in this enzyme. However, chloroquine greatly increased both proteinase and RNase in starved cells. A possible explanation is that chloroquine inhibited breakdown but not synthesis of these enzymes, since this drug accumulates in lysosomes (de Duve et al., 1974) and interferes with the flux of enzymes from the Golgi apparatus through lysosomes to autophagic vacuoles (reviewed in Dean et al., 1984). These findings are relevant to the breakdown of ribosomes by lysosomes. Paradoxically RNase de-
creased most rapidly during the early rapid phase of ribosome breakdown. Proteinase did increase transiently during this period but then remained at approximately the same level as in growing cells. Ribosomes account for 90': o of the total cell R N A but not more than 20c); of the total protein (Hallberg and Bruns, 1976; Dreisig et al., 1984). Other cell components are therefore the major proteinase substrate even though ribosomes are initially degraded faster than total protein. Consequently lysosomal hydrolases by themselves are unlikely to determine the rate of ribosome destruction. Although these enzymes readily digest ribosomes under intralysosomal conditions, enzyme released into the cytoplasm would not attack ribosomes because of the higher pH and the proteinase inhibitor in the cytosol (Klemperer and Pilley, 1985). Some initiating reaction that favours entry of ribosomes into lysosomes is likely to be a rate-limiting factor. Similarly the digestion of endocytosed material by mammalian cells is limited by the rate of transfer into lysosome-derived vacuoles (e.g. Kielian and Cohn, 1980; Stockert, 1983: Tolleshaug et al., 1979). The apparent location of RNase and proteinase in different lysosomes implies that several lysosomes together participate in the formation of a digestive vacuole, as is also suggested by the appearance of autophagic vacuoles in Tetrahymena (Nilsson, 1970) and of phagocytic vacuoles in mammalian cells (e.g. Kielian and Cohn, 1980). The increase in proteinase inhibitor which occurs in starvation would not influence the digestion of ribosomes in such vacuoles, but it would provide additional protection of cytoplasmic constituents if there was a greater risk of enzyme leakage from lysosomes during the complex fusion events of vacuole formation. Acknowledgement This work was supported in part by a grant from the Wellcome Trust. REFERENCES
Bolund L. and Ringertz N. R. (1966) Intracellular distribution of histone-like proteins in Tetrahymena pyri/brmis. Exp. CeU Res. 44, 606 613. Dean R. T., Jessup W. and Roberts C. R. (19841 Effects of exogenous amines on mammalian cells, with particular reference to membrane flow. Biochem. J. 217, 27 40. de Duve C.. de Barsy T., Poole B., Trouet A., Tulkens P. and van Hoof F. (1974) Lysosomotropic agents. Biochem. Pharmac. 23, 2495 2531. Deter R. L. and de Duve C. (1967) Influence of glucagon, an inducer of cellular autophagy, on some physical properties of rat liver lysosomes. J. Cell Biol. 33, 437-449. Dreisig H.. Andreascn P. H. and Kristiansen K. (1984) Regulation of ribosome synthesis in Tetrahvmena pyri[ormis. Eur. d. Biochcm. 140, 469 475. Eckert W. A. and Kafl'enberger W. (1980) Regulation of rRNA metabolism in Tetrahymena pyri/brmis. Eur. J. Cell Biol. 21, 53 62. Green G. D. J.. Kembhavi A. A., Davies M. E. and Barrett A. J. (1984) Cystalin-like cysteine proteinase inhibitors fl'om human liver. Biochem. J. 218, 939-946. Hallberg R. L. and Bruns P. J. (1976) Ribosome biosynthesis in Tetrahymena pyr!'fi)rmis. J. ('ell Biol. 71, 383-.394. Hawkins tt. K. (I 980) Reactions of lysosomes to cell injury. In Pathohioh)gy o/ Cell Membranes (Edited by Trump B. F. and Arstila A. V.) Vol. 2, pp. 251 285. Academic Press, New York.
Lysosomal enzymes in starvation Kielian M. and Cohn Z. A. (1980) Phagosome-lysosome fusion in mouse macrophages. J. Cell Biol. 85, 754-765. Klemperer H. G. and Pilley D. J. (1982) The breakdown of Tetrahymena ribosomes in vivo. Biochem. J. 208, 831-837. Klemperer H. G. and Pilley D. J. (1985) The breakdown of Tetrahymena ribosomes by lysosomal enzymes: inhibition by cytosol. Int. J. Biochem. 17, 399-404. Kristiansen K. and Kriiger A. (1979) Phosphorylation and degradation of ribosomes in starved Tetrahymena pyriformis. Exp. Cell Res. 118, 159-169. Lazarus L. H. and Scherbaum O. H. (1967) Isolation and specificity of the intracellular ribonuclease from Tetrahymena pyri/brmis. Bioehim. biophys. Acta 142, 368 384. Lenney J. F. (1980) Inhibitors associated with the proteinases of mammalian cells and tissues. Current Topics in Cellular Regulation (Edited by Prescott D. M.) Vol. 17, pp. 25-57. Academic Press, New York. Lenney J. F., Tolan J. R., Sugai W. J. and Lee A. G. (1979) Thermostable endogenous inhibitors of cathepsins B and H. Eur. J. Biochem. 101, 153-161. Levy M. R. and Eliott A. M. (1968) Biochemical and ultrastructural changes in Tetrahymena pyriformis during starvation. J. Protozool. 15, 208-222. Lloyd D., Brightwell R., Venables S. E., Roach G. I. and Turner G. (1971) Subcellular fractionation of Tetrahymena pyriformis ST by zonal centrifugation: changes in activities and distribution of enzymes during the growth cycle and starvation. J. gen. Microbiol. 65, 209 223. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall
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R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Mfiller M. (1972) Secretion of acid hydrolases and its intracellular source in Tetrahymena pyriformis. J. Cell Biol. 52, 478 487. Nilsson J. R. (1970) Cytolysomes in Tetrahymena pyriformis. C. r. Tray. Lab. Carlsberg 38, 87-106. Pertoft H., Wfirmeg~,rd B. and H66k M. (1978) Heterogeneity of lysosomes originating from rat liver parenchymal cells. Biochem. J. 174, 309-317. Rothstein T. L. and Blum J. J. (1973) Lysosomal physiology in Tetrahymena I. Effect of nutrients on three acid hydrolases. J. Cell Biol. 57, 630,641. Rothstein T. L. and Blum J. J. (1974) Lysosomal physiology in Tetrahymena IV. Effect of dichloroisoproterenol on the intracellular source of released acid hydrolases. Exp. Cell Res. 87, 168 174. Stahl P. (1983) Lysosomes and mononuclear phagocytes. In Receptor-mediated endocytosis: Receptors and Recognition, Series B (Edited by Cuatrecasas P. and Roth T.) Vol. 15, pp. 139 165. Chapman & Hall, London. Stockert R. J. (1983) Asialoglycoproteins: hepatic clearance and degradation of serum proteins. In Receptor-mediated endocytosis: Receptors and Recognition, Series B (Edited by Cuatrecasas P. and Roth T.) Vol. 15, pp. 167 185. Chapman & Hall, London. Tolleshaug H., Berg T., Fr61ich W. and Norum K. R. (1979) lntracellular location and degradation of asialofetuin in isolated rat hepatocytes. Biochim. Biophys Acta 585, 71 84.
0020-711X/85 $3.00+0.00 Copyright ~C 1985 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
THE ACTIVITIES OF R I B O S O M E - D E G R A D I N G E N Z Y M E S A N D OF A P R O T E I N A S E INHIBITOR IN T E T R A H Y M E N A P Y R I F O R M I S D U R I N G S T A R V A T I O N H. G. KLEMPERER and D. J. PILLEY Department of Biochemistry, University of Birmingham, P.O. Box 363, Birmingham Bl5 2TT, U.K. [Tel. 021-472-1301]
(Received 27 September 1984) In starved Tetrahymena acid RNase decreased but acid proteinase and a heat-stable proteinase inhibitor both increased. 2. A greater proportion of proteinase than RNase was released from the lysosomes of growing cells by homogenization, but progressively less proteinase was released during starvation. 3. Inhibitors of protein synthesis enhanced the decrease in RNase and prevented the increase in proteinase and proteinase inhibitor. Chloroquine caused a large increase in both enzymes. 4. Proteinase and RNase may be located in different lysosomes which together participate in ribosome destruction, but this process is unlikely to be limited by the total amount of these lysosomal hydrolases. Abstract--1.
MATERIALS AND METHODS
INTRODUCTION
The destruction o f ribosomes in the ciliate Tetrahymena is a useful model of r i b o s o m e b r e a k d o w n in eukaryotic cells. B r e a k d o w n is initiated by starvation and is suppressed by chloroquine, an inhibitor of lysosome function (Klemperer a n d Pilley, 1982). R i b o s o m e s have also been observed inside the lysosome-derived a u t o p h a g i c vacuoles that form in starving Tetrahymena (Nilsson, 1970). Lysosomal R N a s e a n d proteinase readily digest ribosomes in vitro at acid (intralysosomal) pH, but at neutral pH the R N a s e is inactive a n d proteinase action can be suppressed by a heat-stable inhibitor present in the cytosol (Klemperer a n d Pilley, 1985). This inhibitor is similar to the " c y s t a t i n s " found in m a n y other cells a n d it may serve to protect cell c o m p o n e n t s against proteinase released from the lysosomes (Lenney, 1980; G r e e n et al., 1984). The rate of ribosome b r e a k d o w n in Tetrahymena during starvation soon diminishes (Kristiansen a n d Kriiger, 1979; Eckert a n d Kaffenberger, 1980). F o r example, total R N A (which is largely r i b o s o m a l R N A ) decreases 15~o in the first h o u r but only a p p r o x 5~0 per hr after 3 or 4 hr (Klemperer a n d Pilley, 1982). F u r t h e r m o r e , ribosome b r e a k d o w n is inhibited in the presence o f certain proteinase inhibitors a n d of inhibitors of protein synthesis. These facts suggest t h a t ribosome destruction is controlled by the activity of specific enzymes. Certain lysosomal hydrolase activities m a y increase while others decrease in Tetrahymena cells starved for approx one day (e.g. Levy and Elliott, 1968; Lloyd et al., 1971), but i n f o r m a t i o n on the early stages o f starvation is limited. Thus in the first few h o u r s proteinase m a y decrease slightly while R N a s e increases (Mfiller, 1972) or decreases (Rothstein a n d Blum, 1974) but the m e c h a n i s m s involved remain unclear. This p a p e r describes the actions of inhibitors of r i b o s o m e breakd o w n on these lysosomal hydrolases and on the cytosolic proteinase inhibitor.
Tetrahymena pyrijbrmis (strain T) was cultured at 28'C on an orbital shaker in a growth medium as described previously (Klemperer and Pilley, 1982). Cells in exponential phase (density 2 × l05 cells/ml) were harvested at 0°C and washed by resuspension in ice-cold starvation medium (consisting of 1 mM MgCI 2, 2 mM KCI, 2 mM potassium phosphate, pH 7.2, 50 mM NaC1) followed by centrifugation. The cells were finally suspended in starvation medium at 28~C to give a density of approx 2 × 105cells/ml, and incubated in the same way as cultured cells. Cell fractionation Cells in 100 ml of starvation medium were chilled to 0 C , washed with Medium A (1 mM MgC12, 5 mM 2-mercaptoethanol, 50mM Tris-HCI, pH7.4, 100mM KC1, 250mM sucrose) and resuspended in 2 ml of Medium A. Essentially 100°/o of the cells were then disintegrated by 20-30 strokes in a modified Dounce homogenizer with a clearance of 50/~m (Philpott and Stanier, 1956). The homogenate was centrifuged for 10 rain at 4'C and 33,000g to give a pellet that included the lysosomes remaining intact alter this homogenization procedure. The supernatant was centrifuged for 90min at 160,000g to give a cell supernatant (approx 2 ml) that contained the cytosol fraction (including the proteinase inhibitor) as well as lysosomal enzymes released by the homogenization procedure. To extract lysosomal enzymes from the 33,000g pellet, this pellet was suspended in 2 ml of 10 mM sodium acetate (pH 5.0) containing 5 mM 2-mercaptoethanol, and incubated for 5 min at 28=C. The suspension was then centrifuged for 90 min at 160,000 g to give a supernatant that contained essentially all of the RNase and proteinase activities in a soluble form.
Preparation of lysosomal extract Cells suspended in 0.25 M sucrose at 0 C were homogenized in the presence of Celite as described previously (Klemperer and Pilley, 1985) under conditions that minimize lysosomal breakage (but disrupt only 60'~J0of the cells). Celite and unbroken cells were removed by centrifugation at 200g, and a pellet that included the lysosomes was collected by centrifugation at 33,000 g. A lysosome extract containing the proteinase used to determine the proteinase inhibitor was prepared from this pellet as described above.
799
800
H . G . KLEMPERER and D. ]. PILLEY digested in 60 rain was determined as above (proteinase determination). Inhibitor activity is expressed as units of proteinase inhibited per mg of total cell protein.
75
5o
RESULTS
o
b_ 'B
Lysosomal RNase and proteinase during starvation 25
45 x-
o~ O
I 50 ;L:I of
~_ 100 heai'ed cell
I 150
I 200
supernatont
Fig. 1. Inhibition of lysosomal proteinase by proteinase inhibitor in heated cell supernatant. In the absence of inhibitor lysosome extract (0.2 ml) digested 135 #g of azocasein in 60min at pH 7.4. An inhibition of 35~o by 60#1 of supernatant corresponds to 135 × 0 . 3 5 - 4 7 units of inhibitor.
Determination of protein and qf enzyme actieities Cells suspended in starvation medium were collected by centrifugation for 3 min at 1000g and taken for determination of total cell protein by the method of Lowry et al. (1951). Enzyme activities in cell fractions (cell supernatant and extract from 38,000g pellet) were measured at 28C in the presence of 1 mM EDTA, 5 mM 2-mercaptoethanol and 50 mM sodium acetate pH 5.0. Under these conditions the amount of product was proportional to the time of incubation. For proteinase the reaction mixture (0.5 ml) contained 5 mg of azocasein. After 60 min of incubation 1 ml of 5% trichloroacetic acid was added, the mixture was centrifuged to remove insoluble material, and the absorption of the supernatant was measured at 340nm. At pH 5.0 the cytosolic proteinase inhibitor has little activity (Klemperer and Pilley, 1985) and its effect on determinations of proteinase in cell supernatant can be ignored. For RNase determinations the reaction mixture (0.15 ml) contained 0.5 mg of Torula RNA (M~ 20-30,000) and was incubated tbr 10 min. After the addition of 3ml of 0.4M HC104 and centrifugation, the supernatant was taken to measure the absorption at 260 nm. RNase in the medium from which the cells had been harvested was measured similarly except that the incubation time was 60 rain. Enzyme activity is given as #g of azocasein or RNA digested in 60 or 10rain respectively and is expressed relative to total protein (rather than to DNA, the cellular content of which varies in different growth states: Bolund and Ringertz, 1966). Determination of proteinase inhibitor Tetrahymena cytosol contains a heat-stable inhibitor of the sulphydryl cathepsins (also inhibited by leupeptin) which contribute approx. 70~ of the total proteinase activity in the lysosomal extract (Klemperer and Pilley, 1985). Like the cystatins in other cells this inhibitor has tightbinding, reversible kinetics such that inhibition is proportional to inhibitor concentration over a wide range (Lenney et al., 1979; Green et al., 1984). Determinations were made in this linear range and under the conditions (pH 7.4) where the inhibitor is most active (Fig. 1). Tetrahymena cell supernatant was heated for 2rain at 100°C to inactivate proteinase, and then centrifuged to remove denatured protein. Aliquots of the supernatant were added to a reaction mixture (0.5 ml) in Medium A containing 5 mg of azocasein and 0.2 ml of lysosome extract (sufficient to digest approx 0.14 mg of azocasein in 60 min). The amount of azocasein
These acid hydrolases are confined to the lysosome-containing fraction of the cells after very gentle homogenisation, but procedures that break 100% o f the cells release a p r o p o r t i o n o f the enzymes into the cell supernatant where they can be measured at pH 5.0. The cytosolic proteinase inhibitor has little activity at this pH and its effect can be ignored. Figure 2 shows the sum for each enzyme of activity released into the supernatant plus enzyme extracted from the remaining lysosomes at low ionic strength. After 1.5hr o f starvation, RNase decreased to approx 501',,, and after 4 hr to 10% o f the initial value. By contrast, total proteinase increased 1005~i in 1.5 hr and then returned to approx, the initial value. Total cell protein decreased only slowly during starvation (Fig. 2: inset). During starvation the cells released RNase but not proteinase into the medium. The RNase that appeared in the medium in 1.5 hr accounted for less than 501~i] of the decrease in RNase in the cells during this period (legend to Fig. 2). Growing cells released RNase at approximately the same rate as cells in starvation medium, The p r o p o r t i o n of the total lysosomal enzyme remaining in the lysosome fraction after homog-
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Fig. 2. Lysosomal RNase and proteinase in starved ceils. Units of enzyme activity are expressed as #g of substrate digested in 10 min (RNase) or 60 min (proteinase) at pH 5.0. Total activity (i.e. supernatant value plus value for extract from 33,000 g pellet) is given for each enzyme. Inset shows total cell protein (100~'o = initial value of approx 30mg/ 100 ml). Each point is the mean +_ SEM of data from four experiments. RNase in the medium (units/mg of cell protein) was 86_+ 10 units after 1.5 hr in starvation medium and 71 _+ 8 units after 1.5 hr in growth medium.
Lysosomal enzymes in starvation Table 1. Percentage of total enzyme remaining in lysosomesafter homogenization
801
imide (2/~g/ml). Puromycin (1 m M ) inhibited to the same extent as cycloheximide (result not shown).
Activity in lysosomes (% of total activity) Starvation (hr) 0 1.5
4 6
DISCUSSION
~ RNase 43-+2
Proteinase 21_+1 48 + 3 78_+2 75+2
49 + 4
49-+2 51 -+3
RNase and proteinasewere determinedas in Fig. 2. Enzymeactivity retained in the lysosomes (extracted from 33,000g pellet) expressed as % of total enzyme activity. Values are means+ SEM of data from four experiments. The % of proteinase retained increasessignificantlyfrom 0 to 1.5 hr and from 1.5 to 4hr (P < 0.01).
enization provides a measure o f lysosome stability. Table 1 shows that during starvation there was little change in the p r o p o r t i o n o f R N a s e retained in the lysosomes but a large increase in the p r o p o r t i o n o f retained proteinase. Initially more proteinase than R N a s e was released but after 4 hr o f starvation this situation was reversed.
The high lysosomal proteinase and R N a s e activities in Tetrahymena may reflect a role in the digestion o f phagocytosed food. Enzyme levels changed rapidly in starvation, when phagocytosis is inhibited and autophagic activity is stimulated (Levy and Elliott, 1968; Nilsson, 1970). Enzyme activity can be assumed to reflect total a m o u n t o f enzyme protein since these lysosomal hydrolases do not appear to exist as inactive forms. Tetrahymena lysosomes contain several distinct acid RNases (Lazarus and Scherbaum, 1967) and acid proteinases (Klemperer and Pilley, 1985), but for the purpose o f a preliminary discussion each class o f hydrolase can be treated as if its c o m p o n e n t enzymes all responded to starvation etc. in the same way. The decrease in R N a s e (50% in 1.5 hr) found in
E
Effect o f inhibitors on proteinase and RNase in starvation
~ so
The activities o f these enzymes were altered in the presence o f inhibitors o f protein synthesis and o f lysosome function. These inhibitors were added at concentrations that are k n o w n to inhibit ribosome b r e a k d o w n without causing visible changes in cell m o r p h o l o g y or motility during 1.5 hr o f starvation. Cycloheximide (2/~g/ml) and puromycin (1 m M ) at concentrations that inhibit protein synthesis by 70-80% (Klemperer and Pilley, 1982) prevented the increase in proteinase. The presence o f puromycin resulted in an even greater depression o f R N a s e than usual after 1.5 hr o f starvation (Table 2). By contrast, both proteinase and R N a s e were greatly increased by the presence o f chlorquine. This drug also increased the p r o p o r t i o n o f R N a s e released from the lysosomes during homogenization. However n o n e o f these inhibitors had a significant effect on the release o f R N a s e from the cells into the medium.
"6 ~" 60 " =;
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o
I
I
I
1
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4
6
Time of storvotion (hr)
Proteinase inhibitor during starvation Figure 3 shows that the inhibitor present in cell s u p e r n a t a n t increased t h r o u g h o u t 6 hr o f starvation. The increase at 1.5 hr was prevented by cyclohex-
Fig. 3. Proteinase inhibitor in cell supernatant. Inhibition by heated supernatant determined as in Fig. 1. Each point is the mean ___SEM of four results. Cycloheximide (2/~g/ml) inhibited the increase at 1.5 hr (P < 0.01).
Table 2. Effect of inhibitors on proteinase and RNase in starved cells Enzyme activity Proteinase in cells Starvation (hr) 0 1.5 1.5 1.5 1.5
Inhibitor
Cycloheximide Puromycin Chloroquine
Total 81 +3 172-+4 82 + 2* 76 -+4* 282 _+8*
% in lysosomes 21 ,+ 1 48-+3 52 + 3 45 ,+ 2 53 -+ 2
RNase in cells Total 363-+I-19 158-+11 147 -+ 15 86 + 4* 539 + 17"
% in lysosomes 43-+2 49-+4 56 -+ 2 51 _+3 32 + 3*
RNase in medium -86-+10 66 -+ 8 75 _+8 95 + 3
Total enzymeactivity (units/mg of cell protein) was determinedas in Fig. 2 in cells and medium after incubation without inhibitor or with cycloheximide(2 ,ug/ml), puromycin (1 mM) or chloroquine (0.3 mM). The % activity remaining in the lysosomesafter homogenisation is expressedas in Table 1. Values are means -+ SEM of data from four experiments.Data for cells without inhibitor (first two lines) are taken from Fig. 2 and Table 1. *These values are significantlydifferent from 1.5 hr values without inhibitor (P < 0.01).
802
H . G . KLEMPERERand D. J. PIt,LEY
these experiments occurs in some other strains of Tetrahymena (see Introduction) but the large transient increase in proteinase has not been observed previously. Since these enzymes may be turning over (v.i.) variations in the balance of synthesis and destruction, depending on the precise conditions, may account for conflicting data on enzyme levels in starvation. Acid RNase and proteinase seemed to be controlled independently of one another since they changed in opposite directions during the first 1.5 hr of starvation. Furthermore, RNase (but not proteinase) was released into the medium, as are some lysosomal hydrolases in certain mammalian cells (e.g. Hawkins, 1980; Stahl, 1983). This release occurred at the same rate as in growing cells and it was insufficient to account for the decrease in cell RNase which was therefore undergoing net breakdown. RNase and proteinase also behaved as if they were located in lysosomes that differed in mechanical fragility. Acid proteinase in growing cells was more readily released into the supernatant during homogenisation, and the release of RNase, but not of proteinase, was increased when the cells had previously been incubated with chloroquine. These two enzymes may therefore occur in different types of lysosome. Acid RNase and certain hydrolases (other than proteinase) in Tetrahvmena have previously been shown to differ in their responses to nutrition, and seem to occur in distinct lysosomes (Lloyd et al.. 1971: Rothstein and Blum, 1973, 1974). Heterogeneity has also been observed in mammalian lysosomes (summarized e.g. in Rothstein and Blum, 1974; Pertoft et al., 1978). The fact that acid proteinase m ]etrahymena became increasingly resistant to release during starvation suggests a change in function. Thus there may be an increasing proportion of this enzyme in primary lysosomes, which are known to be more resistant to lysis by shearing forces than the larger lysosomederived vacuoles (Deter and Duve, 1967). This could reflect a progressive decrease in proteinase digestion after the initial stages of starvation, which was achieved, not by a decrease in enzyme (as is the case with RNase) but by restricting the function of the lysosomes that contain proteinase. Substances that inhibit ribosome breakdown (Klemperer and Pilley, 1982) altered the levels of RNase and proteinase. In the presence of puromycin RNase decreased even further after 1.5 hr of starvation (25~i instead of 50'I; of the initial value), suggesting that RNase was turning over and that the enzyme activity reflected the relative rates of synthesis and destruction. Direct evidence for proteinase turnover is lacking since inhibitors of protein synthesis merely prevented the increase in this enzyme. However, chloroquine greatly increased both proteinase and RNase in starved cells. A possible explanation is that chloroquine inhibited breakdown but not synthesis of these enzymes, since this drug accumulates in lysosomes (de Duve et al., 1974) and interferes with the flux of enzymes from the Golgi apparatus through lysosomes to autophagic vacuoles (reviewed in Dean et al., 1984). These findings are relevant to the breakdown of ribosomes by lysosomes. Paradoxically RNase de-
creased most rapidly during the early rapid phase of ribosome breakdown. Proteinase did increase transiently during this period but then remained at approximately the same level as in growing cells. Ribosomes account for 90': o of the total cell R N A but not more than 20c); of the total protein (Hallberg and Bruns, 1976; Dreisig et al., 1984). Other cell components are therefore the major proteinase substrate even though ribosomes are initially degraded faster than total protein. Consequently lysosomal hydrolases by themselves are unlikely to determine the rate of ribosome destruction. Although these enzymes readily digest ribosomes under intralysosomal conditions, enzyme released into the cytoplasm would not attack ribosomes because of the higher pH and the proteinase inhibitor in the cytosol (Klemperer and Pilley, 1985). Some initiating reaction that favours entry of ribosomes into lysosomes is likely to be a rate-limiting factor. Similarly the digestion of endocytosed material by mammalian cells is limited by the rate of transfer into lysosome-derived vacuoles (e.g. Kielian and Cohn, 1980; Stockert, 1983: Tolleshaug et al., 1979). The apparent location of RNase and proteinase in different lysosomes implies that several lysosomes together participate in the formation of a digestive vacuole, as is also suggested by the appearance of autophagic vacuoles in Tetrahymena (Nilsson, 1970) and of phagocytic vacuoles in mammalian cells (e.g. Kielian and Cohn, 1980). The increase in proteinase inhibitor which occurs in starvation would not influence the digestion of ribosomes in such vacuoles, but it would provide additional protection of cytoplasmic constituents if there was a greater risk of enzyme leakage from lysosomes during the complex fusion events of vacuole formation. Acknowledgement This work was supported in part by a grant from the Wellcome Trust. REFERENCES
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R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Mfiller M. (1972) Secretion of acid hydrolases and its intracellular source in Tetrahymena pyriformis. J. Cell Biol. 52, 478 487. Nilsson J. R. (1970) Cytolysomes in Tetrahymena pyriformis. C. r. Tray. Lab. Carlsberg 38, 87-106. Pertoft H., Wfirmeg~,rd B. and H66k M. (1978) Heterogeneity of lysosomes originating from rat liver parenchymal cells. Biochem. J. 174, 309-317. Rothstein T. L. and Blum J. J. (1973) Lysosomal physiology in Tetrahymena I. Effect of nutrients on three acid hydrolases. J. Cell Biol. 57, 630,641. Rothstein T. L. and Blum J. J. (1974) Lysosomal physiology in Tetrahymena IV. Effect of dichloroisoproterenol on the intracellular source of released acid hydrolases. Exp. Cell Res. 87, 168 174. Stahl P. (1983) Lysosomes and mononuclear phagocytes. In Receptor-mediated endocytosis: Receptors and Recognition, Series B (Edited by Cuatrecasas P. and Roth T.) Vol. 15, pp. 139 165. Chapman & Hall, London. Stockert R. J. (1983) Asialoglycoproteins: hepatic clearance and degradation of serum proteins. In Receptor-mediated endocytosis: Receptors and Recognition, Series B (Edited by Cuatrecasas P. and Roth T.) Vol. 15, pp. 167 185. Chapman & Hall, London. Tolleshaug H., Berg T., Fr61ich W. and Norum K. R. (1979) lntracellular location and degradation of asialofetuin in isolated rat hepatocytes. Biochim. Biophys Acta 585, 71 84.