n-acetylglucosamine-6-phosphate deacetylase in hepatocytes, kupffer cells and sinusoidal endothelial...

N-Acetylglucosamine-6-phosphate Deacetylase in Hepatocytes, Kupffer Cells and Sinusoidal Endothelial

Cells from Rat Liver

PATRICK CAMPBELL,' JERRY N. THOMPSON,~~ 3, J . ROBERT E. FRASER,' TORVARD C. LAURENT,~ HAKAN PERTOFT7 AND LENNART RODEN', 3,

'School of Dentistry, 'Laboratory of Medical Genetics and the Departments of 3Biochemistry, 'Medicine and 5Pediatrics, Cniuersity of Alabama at Birmingham, Birmingham, Alabama 35294; 6Department of Medicine, University of

Melbourne, Melbourne, Australia, and the 'Department of Medical and Physiological Chemistry, University of Uppsala, Uppsala, Sweden

The activity of N-acetylglucosamine-6-phosphate deacetylase, a key enzyme in the pathway of N- acetylglucosamine catabolism, was measured in hepatocytes, Kupffer cells and sinusoidal endothelial cells from rat liver and cultured human skin fibroblasts. Kupffer cells and endothelial cells had similar high levels of deacetylase activity that were more than twice the level observed in fibroblasts. In contrast, hepatocytes had extremely low activity (several hundredfold less than Kupffer cells and endothelial cells). A major implication of deacetylase deficiency in hepatocytes is that N-acetylglucosamine generated as a result of the catabolism of complex carbohydrates in these cells cannot enter glycolysis and must be largely reused for the synthesis of plasma glycoproteins and other N-acetylglucosamine-containing macromolecules. (HEP- ATOLOGY 1990;11:199-204.)

Hepatocytes, Kupffer cells and sinusoidal endothelial cells are all active in the uptake and catabolism by the liver of circulating glycoproteins and glycosaminoglycans. Several specific receptors take up these compounds, and it is notable that the different receptors are not evenly distributed between the three cell types. Thus the Ashwell receptor, which recognizes glycoconjugates terminating in galactose, is found on hepatocytes (l), whereas the binding site for hyaluronan is restricted to sinusoidal endothelial cells (2-4). The endocytosed material is degraded in the lysosomes to monosaccharides, which are used in energy production or recycled in biosynthetic pathways.

Relatively little attention has been paid to the fate

Received August 4, 1988; accepted August 15, 1989. Supported by NIH grants AM 31101, DE 2670 and DE 8252; grants from

the Swedish Medical Research Council (project 03X-4) and the Gustaf V 80th Birthday fund; and the National Health and Medical Research Council and the Arthritis and Rheumatism Foundation of Australia.

Address reprint requests to: Lennart Rod&, P.O. Box 500, University of Alabama at. Birmingham, Birmingham, AL 35294.

31/1/17510

of monosaccharides released from the complex carbohydrates, but recent studies of hyaluronan metabolism prompted us to examine its catabolism and to search for differences in the metabolic capabilities of the major liver cell types.

It has recently been shown that a significant turn- over of hyaluronan exists in the blood circulation (5, 6) and that the polysaccharide is taken up almost entirely by the liver (7). After intravenous injection of [3Hlacetyl-labeled hyaluronan, the acetyl groups are removed and degraded largely to 3Hz0, which is de- tectable in plasma within 10 to 20 min (7). In this process the sinusoidal endothelial cells are responsible not only for uptake and degradation of the polysaccharide to monosaccharides (2,4) but also for liberation of the acetyl groups, as has been indicated by studies of isolated endothelial cells in culture (4).

The metabolic pathway by which the release of the acetyl groups occurs is shown in Figure 1. It is seen that the N-acetylglucosamine generated by the lyso- soma1 hydrolases is first phosphorylated and that the product, N-acetylglucosamine-6-phosphate, is deacet- ylated in what may be regarded as the first step in the catabolism of the monosaccharide. In our study, the activity of the deacetylase catalyzing this reaction was measured directly in extracts of isolated sinusoidal endothelial cells. Because N-acetylglucosamine is also produced in hepatocytes and Kupffer cells by the lysosomal degradation of many glycoproteins, similar analyses of these cell types have been carried out. This work has shown the presence of high deacetylase activity in endothelial cells and Kupffer cells but a virtual lack of the enzyme in hepatocytes, suggesting that the major part of theN-acetylglucosamine generated by the catabolism of glycoconjugates in the latter cells is reused in the synthesis of new complex carbohydrate molecules (8).

MATERIALS AND METHODS N-[3H]acetylglucosamine-6-phosphate was prepared as de-

scribed (9) and had a specific activity of 2.1 x 10' cpmlmg.

199

200 CAMPBELL ET AL.

H ya I urona n

HEPATOLOGY

U DP - N- Acetylglucosamine

E E F h o s p h o r y I ase)

I

(Hyaluronidase) N- Acetylglucosamine-1-P

(p- Glucuronidase) (Mutase)

(Deacety lase)

Acetate

Oligosaccharides

ADP 1 - Hexosaminidase

N- Acetylglucosamine U N - A c e t y l g l u c o s a r n i n e - 6 - P (Kinase)

Glucuronic acid

Glucosamine- 6-P

Dearninase)

Fructose-6- P

FIG. 1. Pathways of hyaluronan catabolism and N-acetylglucosamine usage in mammalian tissues.

ScintiVerse E (a universal liquid scintillation cocktail), ScintiLene (a xylene-based liquid scintillation cocktail nor- mally used for nonaqueous samples) and isoamyl alcohol were supplied by Fisher Scientific, Atlanta, GA. Materials for isolation of liver cells were obtained as described previously (10). Other chemicals were of reagent grade.

Isolation of Rat Liver Cells. Preparation of parenchymal cells (hepatocytes), Kupffer cells and sinusoidal endothelial cells from livers of Sprague-Dawley rats was carried out as described previously (10). Single-cell suspensions were obtained using collagenase perfusion (11) and were fractionated as described below. The parenchymal cells were pelleted by centrifugation at 50 g for 2 min, resuspended in physiological saline and sedimented through a Percoll cushion with a density of 1.08 gm/ml to remove cell debris and remaining nonparenchymal cells. The cells were then cultured for 2 to 4 hr in fibronectin-coated dishes. The nonparenchymal cells in the supernatant from the first centrifugation were banded in a discontinuous Percoll gradient at a density of 1.066 gm/ml, and the purified cells were seeded on plastic culture dishes (Costar, Cambridge, MA). Within 15 min, the Kupffer cells adhered to the plastic. The media with the unattached cells were transferred to fibronectin-coated culture dishes. After 2 hr, when the endothelial cells had adhered, the cultures were carefully washed to remove debris and unattached cells. The cells were scraped from the culture dishes and suspended in a final volume of 2 ml of physiological saline. The suspensions were stored at - 70" C for 2 to 4 wk before analysis. In some experiments, the cells were suspended in 2 ml of 0.01 moUL Tris-HC1, pH 8.0, and their deacetylase activity was determined immediately.

This procedure yielded Kupffer cells and endothelial cells of at least 90% to 95% purity and hepatocytes that were 99% pure (10, 12).

Fibroblast Cultures. Normal human skin fibroblasts were cultured as described previously (13) and were analyzed after 5 to 10 passages.

Assay of N-acetylglucosamine-6-phosphate Deacetylase.

Liver cell extracts used in the assay of the deacetylase were prepared as described below. A frozen suspension of each cell type was rapidly thawed to room temperature and, after brief vortexing, a 0.5-ml aliquot was mixed with an equal volume of 0.02 mol/L Tris-HC1, pH 8.0. The mixture was centrifuged at 10,000 g for 1 min; the supernatant was assayed for deacetylase activity aRer further dilution as needed in 0.01 mol/L Tris buffer. In one experiment, the 10,000 g pellet from each of the three cell types was resuspended in 0.5 ml of 0.01 mol/L Tris-HCI, pH 8.0, containing 50 mmol/L KCI and 0.5% Triton X-100 (Sigma Chemical Co., St. Louis, MO). The mixture was vortexed and allowed to stand at 4' C for 15 min and, after three rapid freeze/ thaw cycles, the extract was clarified by centrifugation. When fresh cells were analyzed, the suspension in 0.01 mol/L Tris-HC1, pH 8.0, was sonicated (MSE sonicator, Measuring and Scientific Equipment Ltd., Crawley, Sussex, England, at setting "High, #5") for 1 min, with cooling in ice. Insoluble material was removed by centrifugation for 1 min at 10,000 rpm.

Cultured fibroblasts were suspended in physiological saline; the suspension was sonicated and centrifuged as described previously (13), and the supernatant was mixed with an equal volume of 0.02 mol/L Tris-HC1, pH 8.0, and assayed.

Assay of deacetylase activity was carried out as described (9). Reaction mixtures had a total volume of 100 p1 and contained N-[3H]acetylglucosamine-6-phosphate (50,000 cpm, counted in ScintiVerse E; specific activity, 2.1 x 10' cpm/mg) in 25 p1 of 0.01 mol/L Tris-HC1, pH 8.0, cell extract prepared as described above (10 to 75 pl); and Tris buffer (0 to 65 pl). The protein concentration in the reaction mixtures was 5 to 975 pg/ml when extracts of Kupffer cells and endothelial cells were assayed, 165 to 6,570 pg/ml for assays of the hepatocyte extracts and 100 to 725 pg/ml when fibroblasts were analyzed. After incubation at 37" C for 30 min, reactions were terminated by addition of 100 pl of a mixture of 1 mol/L monochloroacetic acid, 0.5 mol/L NaOH and 2 mol/L NaC1. Released [3H]acetate was measured after addition of 3 ml of ScintiLene containing 10% isoamyl alcohol. Because solubil-

Vol. 11, No. 2,1990 N-ACE'I'YLGLUCOSAMINE-6-PHOSPHATE DEACETYLASE IN RAT LIVER CELLS 20 1

ity of water in this scintillation cocktail is low, a two-phase system is obtained; the unused substrate remains in the aqueous phase and does not generate scintillation. In contrast the released acetate (as acetic acid in the acidified reaction mixture) is soluble in the organic phase because of the presence of isoamyl alcohol in the latter. Its radioactivity may thus be measured without interference from the substrate. Specific activities for the deacetylase (cpm/hr/mg protein) were calculated from a linear portion of the enzyme concentration curves without correction for the efficiency of the ex- traction of labeled acetate into the organic phase.

Protein was determined by the method of Bradford (14) with BSA (Sigma Chemical Go., St. Louis, MO) or bovine gamma globulin (Bio-Rad Laboratories, Richmond, CA) as standard.

RESULTS Assay of N-acetylglucosamine-6-phosphate deacety-

lase activity in extracts of hepatocytes, Kupffer cells and endothelial cells, which had been prepared from a single rat liver, gave the results shown in Figure 2. Most striking was the great disparity between hepatocytes and the other two cell types. Whereas the activities were similar in the latter two cell types, the activity of the hepatocytes was several hundred times less (note that the scale for the protein content of the hepatocyte assay mixtures in Figure 2 is 10 times larger than that for nonparenchymal cells).

The low deacetylase activity in the hepatocytes raised the possibility that an inhibitor might be present in these cells, but mixing experiments in which hepatocyte extract was added to extracts of Kupffer cells or endothelial cells indicated that this was not the case. It was observed, however, that addition of hepatocyte protein at a final concentration of approximately 1 mg/ml in the assay mixtures, representing a 15-fold excess over the concentrations of Kupffer cell or endothelial cell protein, reduced the apparent deacetylase activity by approximately 30%. This effect was not specific for hepatocyte extracts but was also observed on addition of BSA or hemoglobin to partially purified preparations of rat liver deacetylase (9). The reasons for the apparent inhibition are not clear, but the possibility was ruled out that trapping of (3H)-acetic acid occurred on formation of the voluminous precipitates in the assay mixtures containing added protein.

The large difference in deacetylase activity between hepatocytes and sinusoidal cells leaves open the possibility that the activity in the hepatocyte fraction may be due to contamination by nonparenchymal cells. This possibility will be discussed later.

The N-acetylglucosamine-6-phosphate deacetylase activities in liver cell fractiQns from nine separate experiments are shown in Table 1. In all instances, the virtual absence of deacetylase activity in hepatocytes was confirmed. However, there was considerable variation in absolute activity from one experiment to another. Nevertheless, within each set of cells the activities of Kupffer cells and endothelial cells were always close, suggesting that the variation was due at least in part to differences between experimental animals. Be-

5 10 15 I I I

50 100 150 Protein (Pglassay)

FIG. 2. Assay of N-acetylglucosamine-6-phosphate deacetylase in liver cells. 0-0 = Kupffer cells, 0-0 = endothelial cells, A-A = hepatocytes. Upper scale for protein concentration (pg protein per assay) applies to Kupffer cell and endothelial cell extracts. Lower scale applies to hepatocyte extracts.

cause lability of the enzyme on storage was another possible reason for the variation, samples of fresh cells (preparations 5 to 9) were analyzed in addition to the cells examined initially, which had been frozen for at least 1 wk (preparations 1 to 4). With one exception, the fresh cells exhibited more activity than cells of the same type that had been frozen. On prolonged storage and reassay, a substantial decrease in activity was observed. Lability on storage therefore seemed to cause some of the observed variation, but differences in experimental animals apparently also played a role because the activities varied considerably even within the group of fresh cells.

A preparation of cultured human skin fibroblasts that had been stored frozen for several days had a specific activity of the same magnitude as that of the nonparenchymal cells (219,000 cpm/hr/mg compared with an average of 524,000 cpm/hr/mg and 454,000 cpml hr /mg, respectively, for similarly treated Kupffer cells and endothelial cells.

DISCUSSION This investigation has shown that the activity of N-

acetylglucosamine-6-phosphate deacetylase in rat he-

202 CAMPBELL ET AL. HEPATOLOGY

TABLE 1. N-acetylglucosamine-6-phosphate deacetylase activity in rat liver cells

Activity (cpmlhrlmg protein) Activity ratio

Cell Kupffer cells Endothelial cells

no. Hepatocytes cells cells Hepatocytes Hepatocytes preparation Kupffer Endothelial

1 4,000 469,000 400,000 117 100 2 660 500,000 586,000 758 888 3 2,450 971,000 704,000 396 287 4 1,340 157,000 125,000 117 93 5 3,380 1,069,000 1,054,000 316 312 6 1,232,000 1,801,000 7 600,000 817,000 8 1,912,000 9 4,490 1,245,000 1,194,000 277 266

Ir, one experiment, insoluble pellets were extracted with buffer containing detergent. Approximately 15% of total protein in initial cell extracts was found in detergent-solubilized material. Specific activity of deacetylase in detergent extracts was 113 to 112 of that observed for soluble supernatants. Total activity was 5% to 10% of the total activity in the initially soluble fractions. Values in this table have not been corrected for protein content and activity of the insoluble pellets.

Protein content was measured by the method of Bradford (141, with BSA (preparations 1 to 4) and bovine gammaglobulin (preparations 5 to 9) as standards. Protein values obtained with the gamma-globulin standard were divided by 1.58 to compensate for the lower color yield of t15s standard.

patocytes is extremely low compared with levels measured in extracts of Kupffer cells and sinusoidal endothelial cells. In view of the large difference, we must consider the possibility that all activity in the hepatocyte fraction was derived from contaminating Kupf- fer cells and endothelial cells.

To properly estimate the potential contribution of the nonparenchymal cells to the total enzyme activity, we must take into account not only the percentage of these cells in the hepatocyte preparations but also the large difference in size among the three cell types. According to Knook and Sleyster (151, the protein content of lo6 hepatocytes, endothelial cells and Kupffer cells is 1,280, 46 and 114 kg, respectively. Therefore contamination with 1% Kupffer cells would result in the presence of only 0.1% of extraneous protein in the hepatocyte fraction. Even so, the contribution of the contaminating cells to the total activity in the hepatocyte fraction cannot be ignored. Indeed, if the hepatocyte preparations contained 1% sinusoidal cells, almost all the activity in hepatocyte fraction 2 could be due to their presence. We cannot exclude the possibility that the hepatocyte preparations used for deacetylase assays may have been contaminated with more than 1% nonparenchymal cells, although other preparations used for assessment of purity always contained at least 99% hepatocytes (10, 12).

The low activity or absence of deacetylase in hepatocytes has important implications for the overall metabolism of N-acetylglucosamine in these cells. The deacetylase catalyzes the only reaction by which N-acetylglucosamine, after obligatory phosphorylation by a specific kinase (16-20), can enter the degradative pathway (Fig. 1). Absence of deacetylase implies that N-acetylglucosamine generated by the catabolism of complex carbohydrates (21) must be reused for synthesis of new glycoconjugates. This hypothesis is not un- reasonable despite the tremendous capacity for de nouo

synthesis exhibited by hepatocytes, which allows these cells to synthesize an amount of protein equal to their own protein content during a 24-hr period (22-24). Because much of the hepatocytes’ output consists of N-acetylglucosamine-containing serum proteins, it stands to reason that the cells draw on the substantial N-acetylglucosamine pool generated by the lysosomal degradation of glycoproteins taken up by receptor- mediated endocytosis (1, 21). It should also be pointed out that hepatocytes have a low rate of glycolysis under normal aerobic conditions and depend largely on fatty acids for oxidation and energy production (25). Little glycolysis occurs directly from glucose entering the cell, and the glucose is largely converted to glycogen, from which it is subsequently mobilized by glycogenolysis when needed. At physiological extracellular glucose concentrations, glycogenolysis will occur, but much of the glucose-6-phosphate so generated is converted to free glucose by glucose-6-phosphatase and enters the bloodstream (25). It is thus apparent that when the need for glycolysis arises, the pertinent metabolites are likely to be supplied by glycogenolysis rather than by use of monosaccharides derived from glycoprotein degradation.

In previous studies of glycoprotein and proteoglycan synthesis by the liver and other tissues, it has usually been assumed that the various monosaccharide com- ponents originate directly from glucose. However, as has been shown above, our data lead us to conclude that N-acetylglucosamine must be reused for synthesis of new glycoconjugate molecules. (Elimination of the monosaccharide from the cells before phosphorylation is another possibility, but seems to be a less likely fate than reuse.)

It remains to be determined what proportion of the glycoprotein-bound N-acetylglucosamine is derived from recycled monosaccharide rather than de nouo synthesis from glucose. It should be emphasized here that

Vol. 11, NO. 2,1990 N-ACETYLGLUCOSAMINE-6-PHOSPHATE DEACETYLASE IN RAT LIVER CELLS 203

the concept of recycling of glycoconjugate monosaccharides is by no means new. In 1959, Spiro (26) reported kinetic studies of the biosynthesis of protein-bound glucosamine in the living rat, which showed that the turn- over time of serum glucosamine (approximately 2 hr) was much shorter than the time estimated for total resynthesis from glucose (56 hr). It was suggested that breakdown and resynthesis of glycoproteins in the liver with reuse of most of the glucosamine was a possible explanation for this discrepancy. However, the design of these experiments was such that no conclu- sions could be drawn about the detailed pathways of recycling.

Some more recent studies support the notion that recycling is a major source of monosaccharide compo- nents in glycoconjugate synthesis. MacNicoll et al. (27) found that the galactosamine moiety of radiolabeled chondroitin sulfate could be used by perfused liver for synthesis of serum glycoproteins. Rome and Hill (28) reported that more than 50% of the hexosamines produced by lysosomal degradation in cultured human skin fibroblasts was reused for synthesis of new macromolecules. This high degree of reuse (which actually represented a minimal estimate because only products secreted into the medium were measured) is surprising in view of the comparatively high activity of N- acetylglucosamine-6-phosphate deacetylase found in this study. Finally, a mutant of mouse fibroblasts, de- ficient in the N-acetyltransferase, which catalyzes formation of N-acetylglucosamine-6-phosphate from glucosamine 6-phosphate and acetylcoenzyme A, was ca- pable of synthesizing glycoproteins at about 50% of the level observed for wild-type cells, suggesting that recycling of N-acetylglucosamine played a major role in the monosaccharide economy of these cells (29-31).

The high activity of N-acetylglucosamine-6- phosphate deacetylase in sinusoidal endothelial cells and Kupffer cells makes reuse of N-acetylglucosamine in these cell types less likely. In fact, the most radioactivity from [3Hlacetyl-labeled hyaluronan was found in 3H20 after degradation in uiuo (7) and in [3H]acetate after degradation in cultures of liver endothelial cells (4).

It should be noted that the high deacetylase activity of the nonparenchymal cells is in keeping with their recognized role as major tools for the degradation of complex carbohydrates and other macromolecules. This role is manifested structurally in the presence of a high content of lysosomes in these cells as compared with hepatocytes (32, 33). It is also reflected in their high content of glycosidases and other lysosomal hydrolases, which was demonstrated previously by Knook and Sleyster (15) and, more recently, in studies from our laboratory (34).

REFERENCES

1. Schwartz AL. The hepatic asialoglycoprotein receptor. CRC Crit Rev Biochem 1984;16:207-223.

2. Fraser JRE, Alcorn D, Laurent TC, Robinson AP, Ryan GB. Uptake of circulating hyaluronic acid by the rat liver: cellular localization in situ. Cell Tissue Res 1985;242:505-510.

3. Laurent TC, Fraser JRE, Pertoft H, Smedsrprd B. Binding of

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

hyaluronate and chondroitin sulphate to liver endothelial cells. Biochem J 1986;234:653-658. Smedsr@d B, Pertoft H, Eriksson S, Fraser JRE, Laurent TC. Studies in vitro on the uptake and degradation of sodium hyaluronate in rat liver endothelial cells. Biochem J 1984;223:

Laurent UBG, Laurent TC. On the origin ofhyaluronate in blood. Biochem Int 1981;2:195-199. Tengblad A, Laurent UBG, Lilja K, Cahill RNP, Engstrom- Laurent A, Fraser JRE, Hanson HK, et al. Concentration and relative molecular mass of hyaluronate in lymph and blood. Bio- chem J 1986;236:521-525. Fraser JRE, Laurent TC, Pertoft H, Baxter E. Plasma clearance, tissue distribution and metabolism of hyaluronic acid injected intravenously in the rabbit. Biochem J 1981;200:415-424. Thompson JN, Campbell P, Fraser JRE, Pertoft H, Laurent TC, Roden L. N-Acetylglucosamine 6-phosphate deacetylae and lysosomal hydrolases in hepatocytes, Kupffer cells, and sinusoidal endothelial cells from rat liver [Abstract]. Fed Proc 1987;46:2093. Campbell P, Laurent TC, Roden L. Assay and properties of N- acetylglucosamine 6-phosphate deacetylase in rat liver. Anal Biochem 1987;166:134-141. Smedsrprd B, Pertoft H. Preparation of pure hepatocyte and re- ticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence. J Leu- coc Biol 1985;38:213-230. Obrink B. Hepatocyte-collagen adhesion. Methods Enzymol

Smedsrprd B, Pertoft H, Eggertsen G, Sundstrom C. Functional and morphological characterization of cultures of Kupffer cells and liver endothelial cells prepared by means of density sepa- ration in Percoll, and selective substrate adherence. Cell Tissue Res 1985;241:639-649. Thompson JN, Roden L, Reynertson R. Oligosaccharide sub- strates for heparin sulfamidase. Anal Biochem 1986;152:412- 422. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-254. Knook DL, Sleyster EC. Isolated parenchymal, Kupffer and endothelial rat liver cells characterized by their lysosomal enzyme content. Biochem Biophys Res Commun 1980;96:250-257. Datta A. Studies on hog spleen N-acetylglucosamine kinase: I. Purification and properties of N-acetylglucosamine kinase. Biochim Biophys Acta 1970;220:51-60. Ghosh SI, Roseman S. Enzymatic phosphorylation of N-acetyl- D-mannosamine. Proc Natl Acad Sci USA 1961;47:955-958. Leloir LF, Cardini C9, Olavarria JM. Phosphorylation of ace- tylhexosamines. Arch Biochem Biophys 1958;74:84-91. Reyes AI, Cdrdenas ML. All hexokinase isoenzymes coexist in rat hepatocytes. Biochem J 1984;221:303-309. Saeki H, Suzuki R, Tsuiki S. Purification and properties of N - acetylglucosamine kinase from rat liver. Tohoku J Exp Med

Stockert RJ, Morel1 AG. Endocytosis of glycoproteins. In: Arias I, Popper H, Schachter D, Shafritz DA, eds. The liver: biology and pathobiology. New York: Raven Press, 1982:205-217. Grieninger G. Isolated hepatocytes in culture: a model for study- ing the control of plasma protein synthesis. In: Glaumann H, Peters T Jr, Redman C, eds. Plasma protein secretion by the liver. New York: Academic Press, 1983:155-193. Grieninger G, Granick S. Synthesis and secretion of plasma proteins by embryonic chick hepatocytes: changing patterns during the first three days of culture. J Exp Med 1978;147:1806-1823. Haschemeyer AEV. Kinetics of protein synthesis in higher or- ganisms in vivo. Trends Biochem Sci 1976;1:133-136. Seifter S, Englard S. Energy metabolism. In: Arias I, Popper H, Schachter D, Shafritz DA, eds. The liver: biology and pathobiology. New York: Raven Press, 1982:219-249. Spiro RG. Studies on the biosynthesis of glucosamine in the intact rat. J Biol Chem 1959;234:742-748. MacNicoll AD, Wusteman FS, Winterburn PJ, Powell GM, Cur- tis CG. Degradation of [3Hlchondroitin 4-sulphate and reutili- zation of the [3Hlhexosamine component by the isolated perfused rat liver. Biochem J 1980;186:279-286.

617-626.

1982;82:513-529.

1971;105:87-98.

204 CAMPBELL ET AL. HEPATOLOGY

28. Rome LH, Hill DF. Lysosomal degradation of glycoproteins and glycosaminoglycans: efflux and recycling of sulfate and N - acetylhexosamine. Biochem J 1986;235:707-713.

29. Neufeld EJ, Pastan I. A mutant fibroblast cell line defective in glycoprotein synthesis due to a deficiency of glucosamine phosphate acetyltransferase. Arch Biochem Biophys 1978;188:323- 327.

30. Pouyssegur J , Pastan I. Mutants of Balb/c 3T3 fibroblasts defective in adhesiveness to substratum. Evidence for alteration in cell surface proteins. Proc Natl Acad Sci USA 1976;73:544- 548.

31. Pouyssegur J , Pastan I. Mutants of mouse fibroblasts altered in the synthesis of cell surface glycoproteins. J Biol Chem 1977;252: 1639-1646.

32. Blouin A, Boldender RP, Weibel ER. Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma: a stereological study. J Cell Biol 1977;72:

33. Blouin A. Anatomy, ultrastructure and morphometry of the liver. In: Glaumann H, Peters T Jr , Redman C, eds. Plasma protein secretion by the liver. New York Academic Press, 1983;31-53.

34. Roden L, Campbell P, Fraser JRE, Laurent TC, Pertoft H, Thompson JN. Enzymic pathways of hyaluronan catabolism. In: Evered D, Whelan J, eds. The biology of hyaluronan. Chichester: John Wiley & Sons, 1989:60-86.

441-455.

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