Absence of keratan sulphate from skeletal tissues of mouse and rat.

The absence of keratan sulphate synthesis from skeletal tissues of young and mature mice and rats has been confirmed by (1) analysis of specific enzyme degradation products of newly synthesized glycosaminoglycans, and (2) immunohistochemistry and radioimmunoassay using a monoclonal antibody directed against keratan sulphate. Approx. 98% of the [35S]glycosaminoglycans synthesized in vivo by mouse and rat costal cartilage, and all of those of lumbar disc, are chondroitin sulphate. The remainder in costal cartilage were identified as heparan sulphate in mature rats. In contrast, [35S]glycosaminoglycans synthesized by cornea of both species comprised both chondroitin sulphate and keratan sulphate. In mice keratan sulphate accounted for 12-25% and in rats 40-50% of the total [35S]glycosaminoglycans, depending on the age of the animal. Experiments in vitro with organ culture of cartilage and cornea confirm these results. Absence of keratan sulphate from mouse costal cartilage and lumbar disc D1-proteoglycans was corroborated by inhibition radioimmunoassay with the monoclonal antibody MZ15 and by lack of staining for keratan sulphate in indirect immunofluorescence studies using the same antibody.     

Composition and distribution of glycosaminoglycans in cultures of human normal and malignant glial cells.

The glycosaminoglycans of human cultured normal glial and malignant glioma cells were studied. [35S]Sulphate or [3H]glucosamine added to the culture medium was incorporated into glycosaminoglycans; labelled glycosaminoglycans were isolated by DEAE-cellulose chromatography or gel chromatography. A simple procedure was developed for measurement of individual sulphated glycosaminoglycans in cell-culture fluids. In normal cultures the glycosaminoglycans of the pericellular pool (trypsin-susceptible material), the membrane fraction (trypsin-susceptible material of EDTA-detached cells) and the substrate-attached material consisted mainly of heparan sulphate. The intra- and extra-cellular pools showed a predominance of dermatan sulphate. The net production of hyaluronic acid was low. The accumulation of 35S-labelled glycosaminoglycans in the extracellular pool was essentially linear with time up to 72h. The malignant glioma cells differed in most aspects tested. The total production of glycosaminoglycans was much greater owing to a high production of hyaluronic acid and hyaluronic acid was the major cell-surface-associated glycosaminoglycan in these cultures. Among the sulphated glycosaminoglycans chondroitin sulphate, rather than heparan sulphate, was the predominant species of the pericellular pool. This was also true for the membrane fraction and substrate-attached material. Furthermore, the accumulation of extracellular 35S-labelled glycosaminoglycans was initially delayed for several hours and did not become linear with time until after 24 h of incubation. The glioma cells produced little dermatan sulphate and the dermatan sulphate chains differed from those of normal cultures with respect to the distribution of iduronic acid residues. The observed differences between normal glial and malignant glioma cells were not dependent on cell density; rather they were due to the malignant transformation itself.     

Renal glomerular proteoglycans. An investigation of their synthesis in vivo using a technique for fixation in situ.

Newly synthesized rat glomerular [35S]proteoglycans were labelled in vivo after injecting Na2[35S]SO4 intraperitoneally. At the end of the labelling period (7 h) the kidneys were perfused in situ with 0.01% (w/v) cetylpyridinium chloride. This fixed proteoglycans in the tissue and increased their recovery 2-3-fold during subsequent isolation of glomeruli from the renal cortex. The glomeruli were fractionated by a modified osmotic lysis and detergent extraction procedure [Meezan, Brendel, Hjelle & Carlson (1978) in The Biology and Chemistry of Basement Membranes (Kefalides, N.A., ed.), Academic Press, New York; Kanwar & Farquhar (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4493-4497] to obtain a basement membrane preparation. The proteoglycans released at each stage of the procedure were characterized using DEAE-Sephacel ion-exchange chromatography, chondroitinase ABC and HNO2 digestion and Sepharose CL-4B gel-permeation chromatography. About 85% of the [35S]proteoglycans synthesized were of the heparan sulphate variety, the remainder being chondroitin sulphate proteoglycans. Three sizes of heparan sulphate proteoglycans were identified. The largest (HS1, Kav. 0.47) accounts for 44% of the total extractable heparan sulphates. About one third of HS1 were extracted from the glomerular basement-membrane fraction with 8 M-urea and 4 M-guanidine hydrochloride but the remainder were released from the glomerulus during preparation of the fraction. The two smaller molecules (HS2, Kav. 0.56 and HS3, Kav. 0.68) accounted for 27% and 28% of the extractable heparan sulphate respectively and were not associated with the basement membrane fraction. HS1, HS2 and HS3 were also isolated from non-fixed glomeruli labelled in vivo but with much lower recovery. In glomeruli labelled in vitro, heparan sulphate accounted for only 35% of the proteoglycans, the remainder being of the chondroitin sulphate type. Proteoglycans similar to HS1, HS2 and HS3 were present in glomeruli labelled in vitro but, in addition, a large, highly charged heparan sulphate (HS1a) was extracted from the glomerular basement-membrane fraction of these glomeruli. It accounted for 6% of the total heparan sulphate.     

Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels.

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.     

Lysosomal degradation of glycoproteins and glycosaminoglycans. Efflux and recycling of sulphate and N-acetylhexosamines.

Lysosomal degradation of the carbohydrate portion of glycoproteins and glycosaminoglycans produces monosaccharides and sulphate, which must efflux from the lysosomes before re-entering biosynthetic pathways. We examined the degradation of glycoproteins and glycosaminoglycans by lysosomes isolated from cultured human diploid fibroblasts. Cells were grown for 24 h in medium containing [3H]glucosamine and [35S]sulphate. When lysosomes are isolated from these cells, they contain label primarily in macromolecules (glycoproteins and glycosaminoglycans). Glycoprotein degradation by isolated lysosomes was followed by measuring the release of tritiated sugars from macromolecules and efflux of these sugars from the organelles. Glycosaminoglycan degradation was monitored by the release of both tritiated sugars and [35S]sulphate. During macromolecule degradation, the total amounts of free [35S]sulphate, N-acetyl[3H]glucosamine and N-acetyl[3H]galactosamine found outside the lysosome parallels the amounts of these products released by degradation. The total degradation of glycoproteins and glycosaminoglycans by intact cultured cells was also examined. The lysosomal contribution to degradation was assessed by measuring inhibition by the lysosomotropic amine NH4Cl. After 48 h incubation, inhibition by NH4Cl exceeded 55% of glycoprotein and 72% of glycosaminoglycan degradation. Recycling of [3H]hexosamines and [35S]sulphate by intact cells was estimated by measuring the appearance of 'newly synthesized' radioactively labelled macromolecules in the medium. Sulphate does not appear to be appreciably recycled. N-Acetylglucosamine and N-acetylgalactosamine, on the other hand, are reutilized to a significant extent.     

Metabolism of glycosaminoglycans in CCl4-induced liver regeneration

The incorporation of [₃₅S]-SO₄ into glycosaminoglycans of liverin vivo and in in liver slices and into the glycosaminoglycans associated with the hepatic plasma membrane of rats at different periods after a heavy dose of CC1₄ have been studied. The incorporation of [35S]-SO4 into total glycosaminoglycans decreased to as low as 40% of the control at 24 h after the administration of CC14 and later on increased reaching a maximum on the 4th day. The amount of [35S]-SO4 incorporation into heparan sulphate was also reduced to about 40% of control at 12–24 h after the onset of injury and increased thereafter reaching a maximum on the 4th day. There was only a partial reduction in the synthesis of chondroitin sulphate in the early stage of injury and then it steadily increased reaching about 3 times the control level on 4–6 days. The [³⁵S]-SO4-incorporation into dermatan sulphate, after a slight initial decrease remained at the control levels. On the 8th day after the CCl₄-induced liver injury, the rate of [³⁵S]-SO₄-incorporation was almost equal to that in normal controls. The incorporation of [³⁵S]-SO₄ into hepatic plasma membrane glycosaminoglycans showed a similar change decreasing to about 35% of control at 24h followed by an increase, reaching normal levels on the 4th day after the administration of CC1₄. About 90% of the plasma membrane glycosaminoglycans was found to be heparan sulphate. The yield of plasma membrane from normal and CCl₄-induced regenerating liver was found to be similar and therefore the results obtained were not due to difference in the yield of the membrane preparation. The data also indicate that there was no difference in the degree of sulphation. The significance of these changes in the metabolism of sulphated glycosaminoglycans particularly plasma membrane heparan sulphate in tissue regeneration has been discussed.     

Metabolism of sulfated glycosaminoglycans in cultivated bovine arterial cells. I. Characterization of different pools of sulfated glycosaminoglycans.

"Fibroblast-like" cells from the intimal layer of bovine aorta were grown in culture. The formation, composition, molecular weight and turnover rate of different pools of glycosaminoglycans were investigated in cultures incubated in the presence [35S]sulfate or [14C]glucosamine. The newly synthesized glycosaminoglycans are distributed into an extracellular pool (37 - 58%), a cell-membrane associated or pericellular pool (23 - 33%), and an intracellular pool (19 - 30%), each pool exhibiting a characteristic distribution pattern of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronate. The distribution pattern of the extracellular glycosaminoglycans resembles closely that found in bovine aorta. A small subfraction of the pericellular pool - tentatively named "undercellular" pool--has been characterized by its high heparan sulfate content. The intracellular and pericellular [35S]glycosaminoglycan pools reach a constant radioactivity after 8-12 h and 24 h, respectively, whereas the extracellular [35S]glycosaminoglycans are secreted into the medium at a linear rate over a period of at least 6 days. The intracellular glycosaminoglycans are mainly in the process of degradation, as indicated by their low molecular weight and by their half-life of 7 h, but intracellular dermatan sulfate is degraded more rapidly (half-life 4-5 h) than intracellular chondroitin sulfate and heparan sulfate (half-life 7-8 h). Glycosaminoglycans leave the pericellular pool with a half-life of 12-14 h by 2 different routes: about 60% disappear as macromolecules into the culture medium, and the remainder is pinocytosed and degraded to a large extent. Extracellular and at least a part of the pericellular glycosaminoglycans are proteoglycans. Even under dissociative conditions (4M guanidinium chloride) their hydrodynamic volume is sufficient for partial exclusion from Sepharose 4B gel. The existence of topographically distinct glycosaminoglycan pools with varying metabolic characteristics and differing accessibility for degradation requiresa reconsideration and a more reserved interpretation of results concerning the turnover rates of glycosaminoglycans as determined in arterial tissue.     

Glycosaminoglycans of arterial basement membrane-like material from cultured rabbit aortic myomedial cells

Arterial basement membrane-like material was prepared by a sonication-differential centrifugation technique from cultures of rabbit aortic myomedial cells after metabolic labelling with [35S]sulphate and [3H]glucosamine. Labelled glycosaminoglycans were obtained from isolated basement membrane-like material by proteinase digestion and gel filtration. Glycosaminoglycans were identified by a combination of Sephadex G-50 chromatography and sequential degradation with nitrous acid, Streptomyces hyaluronidase, testicular hyaluronidase and chondroitinase ABC. The data showed that heparan sulphate and chondroitin sulphate were the predominant glycosaminoglycans of myomedial basement membrane-like material. Heparan sulphate accounted for about 55% of [3H]glucosamine-labelled glycosaminoglycans. In addition small amounts of hyaluronic acid was present. Only trace amounts of dermatan sulphate was found. The glycosaminoglycans were analysed by DEAE-cellulose chromatography. Two major peaks were found in the chromatogram consistent with the predominance of heparan sulphate and chondroitin sulphate.     

Structure and metabolism of rat liver heparan sulphate

Rat liver cells grown in primary cultures in the presence of [³⁵S]sulphate synthesize a labelled heparan sulphate-like glycosaminoglycan. The characterization of the polysaccharide as heparan sulphate is based on its resistance to digestion with chondroitinase ABC or hyaluronidase and its susceptibility to HNO₂ treatment. The sulphate groups (including sulphamino and ester sulphate groups) are distributed along the polymer in the characteristic block fashion. In ³H-labelled heparan sulphate, isolated after incubation of the cells with [³H]galactose, 40% of the radioactive uronic acid units are l-iduronic acid, the remainder being d-glucuronic acid. The location of heparan sulphate at the rat liver cell surface is demonstrated; part of the labelled polysaccharide can be removed from the cells by mild treatment with trypsin or heparitinase. Further, a purified plasma-membrane fraction isolated from rats previously injected with [³⁵S]sulphate contains radioactively labelled heparan sulphate. A proteoglycan macromolecule composed of heparan sulphate chains attached to a protein core can be solubilized from the membrane fraction by extraction with 6m-guanidinium chloride. The proteoglycan structure is degraded by treatment with papain, Pronase or alkali. The production of heparan [³⁵S]sulphate by rat liver cells incubated in the presence of [³⁵S]sulphate was followed. Initially the amount of labelled polysaccharide increased with increasing incubation time. However, after 10h of incubation a steady state was reached where biosynthetic and degradative processes were in balance.     

Studies on the in vitro incubation procedure for [35S]sulphate labelling of articular cartilage glycosaminoglycans

Articular cartilage from cow and calf femoral condyles was incubated in Tyrodes solution containing [35S]sulphate for different periods up to 80 min. Glycosaminoglycans from the cartilage tissue and incubation medium were fractionated on Cetylpyridinium chloride and ECTEOLA cellulose microcolumns. The incorporation of [35S]sulphate into all individual fractions of chondroitin sulphate and keratan sulphate was found to be linear from 20 to 80 min incubation time. As a rule the total specific activities of keratan sulphate and chondroitin sulphate were similar for both calves and cows. The proteoglycan material recovered from the medium amounted to about 1% of the tissue dry weight and was found to have a higher chondroitin sulphate: keratan sulphate ratio than the corresponding cartilage tissue for both calf and cow. The solubility profiles for the newly synthesised glycosaminoglycans, obtained from determination of the radioactivity in the individual fractions, were compared with those of glycosaminoglycans already present. These curves indicated that newly synthesised chondroitin sulphate had a higher average molecular size than that present in the tissue whereas the newly synthesised keratan sulphate had a smaller average molecular size. These newly synthesised components were also detected in the proteoglycans recovered from the incubation medium.     

Studies on the in vitro incubation procedure for [35]sulphate labelling of articular cartilage glycosaminoglycans

Articular cartilage from cow and calf femoral condyles was incubated in Tyrodes solution containing [³⁵S]sulphate for different periods up to 80 min. Glycosaminoglycans from the cartilage tissue and incubation medium were fractionated on Cetylpyridinium chloride and ECTEOLA cellulose microcolumns.The incorporation of [³⁵S]sulphate into all individual fractions of chondroitin sulphate and keratan sulphate was found to be linear from 20 to 80 min incubation time. As a rule the total specific activities of keratan sulphate and chondroitin sulphate were similar for both calves and cows.The proteoglycan material recovered from the medium amounted to about 1% of the tissue dry weight and was found to have a higher chondroitin sulphate: keratan sulphate ratio than the corresponding cartilage tissue for both calf and cow.The solubility profiles for the newly synthesised glycosaminoglycans, obtained from determination of the radioactivity in the individual fractions, were compared with those of glycosaminoglycans already present. These curves indicated that newly synthesised chondroitin sulphate had a higher average molecular size than that present in the tissue whereas the newly synthesised keratan sulphate had a smaller average molecular size. These newly synthesised components were also detected in the proteoglycans recovered from the incubation medium.     

Comparative study on glycosaminoglycans synthesized in peripheral and peritoneal polymorphonuclear leucocytes from guinea pigs.

Glycosaminoglycans synthesized in polymorphonuclear (PMN) leucocytes isolated from blood (peripheral PMN leucocytes) and in those induced intraperitoneally by the injection of caseinate (peritoneal PMN leucocytes) were compared. Both peripheral and peritoneal PMN leucocytes were incubated in medium containing [35S]sulphate and [3H]glucosamine. Each sample obtained after incubation was separated into cell, cell-surface and medium fractions by trypsin digestion and centrifugation. The glycosaminoglycans secreted from peripheral and peritoneal PMN leucocytes were decreased in size by alkali treatment, indicating that they existed in the form of proteoglycans. Descending paper chromatography of the unsaturated disaccharides obtained by the digestion of glycosaminoglycans with chondroitinase AC and chondroitinase ABC identified the labelled glycosaminoglycans of both the cell and the medium fractions in peripheral PMN leucocytes as 55-58% chondroitin 4-sulphate, 16-19% chondroitin 6-sulphate, 16-19% dermatan sulphate and 6-8% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found only in the medium fraction. In peritoneal PMN leucocytes there is a difference in the composition of glycosaminoglycans between the cell and the medium fractions; the cell fraction was composed of 60% chondroitin 4-sulphate, 5.5% chondroitin 6-sulphate, 16.8% dermatan sulphate and 13.9% heparan sulphate, whereas the medium fraction consisted of 24.5% chondroitin 4-sulphate, 28.2% chondroitin 6-sulphate, 33.7% dermatan sulphate and 10% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found in the cell, cell-surface and medium fractions. On the basis of enzymic assays with chondro-4-sulphatase and chondro-6-sulphatase, the positions of sulphation in the disulphated disaccharides were identified as 4- and 6-positions of N-acetylgalactosamine. Most of the 35S-labelled glycosaminoglycans synthesized in peripheral PMN leucocytes were retained within cells, whereas those in peritoneal PMN leucocytes were secreted into the culture medium. Moreover, the amount of glycosaminoglycans in peritoneal PMN leucocytes was significantly less than that in peripheral PMN leucocytes. Assay of lysosomal enzymes showed that these activities in peritoneal PMN leucocytes were 2-fold higher than those in peripheral PMN leucocytes.     

Heparan sulphate sulphotransferase. Properties of an enzyme from ox lung

A heparan sulphate sulphotransferase was partially purified from an ox lung homogenate by (NH(4))(2)SO(4) precipitation. Various glycosaminoglycans were assayed as sulphate acceptors with this enzyme. The highest acceptor activity was obtained with desulphated heparin and heparan sulphate, which indicates that sulphate transfer may be to free amino groups of the substrate. Some heparan sulphate was (35)S-labelled by incubation with the enzyme and re-isolated. On treatment of this heparan [(35)S]sulphate with nitrous acid and separation of the degradation products on Sephadex G-15, a major peak of radioactivity was obtained, and identified as [(35)S]sulphate by high-voltage electrophoresis at pH5.3. The [(35)S]sulphate is believed to be derived from N-[(35)S]sulphated groups of heparan [(35)S]-sulphate. That the ox lung preparation contained an N-sulphotransferase was confirmed by the isolation of 2-deoxy-2-[(35)S]sulphoamino-d-glucose as the major product from the flavobacterial degradation of heparan [(35)S]sulphate.     

Metabolism of sulfated glycosaminoglycans in rat hepatocytes. Synthesis of heparan sulfate and distribution into cellular and extracellular pools

Primary cultures of rat hepatocytes grown in a serum-free medium supplemented with [35S]sulfate synthesize 35S-labelled glycosaminoglycans at an almost constant rate for 58 h. Approx. 57% of the newly synthesized 35S-labelled glycosaminoglycans remain within the hepatocytes, approx. 30% become associated with the cell surface and only 13% are secreted into the medium. The amount of cell-surface-associated 35S-labelled glycosaminoglycans became constant within 36 h, whereas no equilibrium was reached in the intra- and extracellular pool. During a 24 h chase more than 50% of the intracellular and cell-surface-associated 35S-labelled glycosaminoglycans disappears, more than 80% of this material is degraded and radioactivity is recovered as inorganic sulfate. A minor part is released into the medium in a macromolecular form. Heparan sulfate accounts for more than 95% of the 35S-labelled glycosaminoglycans in each of the three pools. It is distinguished from heparan sulfates from other sources by the presence of unsubstituted glucosamine residues. In all three pools, heparan sulfate chains of mean molecular weights between 24 000 and 30 000 are part of an alkali labile proteoglycan. Intra- and extracellularly, however, part of the heparan sulfate appears to have little, if any, protein attached. Hepatocytes contain heparan sulfate-degrading endoglycosidase activity, which may contribute to the variation of molecular weights observed for the heparan sulfate.     

The effects of cycloheximide on the biosynthesis and secretion of proteoglycans by chondrocytes in culture.

Proteoglycans synthesized by rat chondrosarcoma cells in culture are secreted into the culture medium through a pericellular matrix. The appearance of [35S]sulphate in secreted proteoglycan after a 5 min pulse was rapid (half-time, t 1/2 less than 10 min), but that of [3H]serine into proteoglycan measured after a 15 min pulse was much slower (t 1/2 120 min). The incorporation of [3H]serine into secreted protein was immediately inhibited by 1 mM-cycloheximide, but the incorporation of [35S]sulphate into proteoglycans was only inhibited gradually(t 1/2 79 min), suggesting the presence of a large intracellular pool of proteoglycan that did not carry sulphated glycosaminoglycans. Cultures were pulsed with [3H]serine and [35S]sulphate and chased for up to 6 h in the presence of 1 mM-cycloheximide. Analysis showed that cycloheximide-chased cells secreted less than 50% of the [3H]serine in proteoglycan of control cultures and the rate of incorporation into secreted proteoglycan was decreased (from t 1/2 120 min to t 1/2 80 min). Under these conditions cycloheximide interfered with the flow of proteoglycan protein core along the route of intracellular synthesis leading to secretion, as well as inhibiting further protein core synthesis. The results suggested that the newly synthesized protein core of proteoglycan passes through an intracellular pool for about 70-90 min before the chondroitin sulphate chains are synthesized on it, and it is then rapidly secreted from the cell. Proteoglycan produced by cultures incubated in the presence of cycloheximide and labelled with [35S]sulphate showed an increase with time of both the average proteoglycan size and the length of the constituent chondroitin sulphate chain. However, the proportion of synthesized proteoglycans able to form stable aggregates did not alter.     

Nature of sulphated macromolecules in mouse Reichert''s membrane. Evidence for tyrosine O-sulphate in basement-membrane proteins.

Seven different sulphated macromolecules were detected in 6 M-guanidinium chloride extracts of metabolically [35S]sulphate-labelled mouse Reichert's membrane and were partially separated. Polypeptide bands of apparent Mr 50 000, 150 000 (tentatively identified as entactin) and 170 000 contained essentially tyrosine O-sulphate as the labelled component. Most of the radioactive sulphate was incorporated into three different proteoglycans, which could be separated by chromatography and density-gradient centrifugation before and after enzymic degradation. Enzymic analysis of glycosaminoglycans and of protein cores by immunoassays identified these components as low-density and high-density forms of heparan sulphate proteoglycan and a high-density form of chondroitin sulphate or dermatan sulphate proteoglycan.     

Proteoglycans synthesized in vitro by nude and normal mouse peritoneal macrophages

Peritoneal macrophages from nude mice were found to be functionally similar to 'activated' macrophages from normal mice. The objective of the present study was to characterize the proteoglycans synthesized and secreted in vitro by peritoneal macrophages isolated from nude and normal Balb/c mice and to investigate the relationship between macrophage 'activation' and changes in the proteoglycan patterns. Macrophages obtained by peritoneal lavage were seeded in Petri dishes. After 2 h incubation at 37 degrees C, the adherent cells (macrophages) were exposed to [35S]sulphate for the biosynthetic labelling of proteoglycans. After incubation, the cell and medium fractions were collected and analysed for proteoglycans and glycosaminoglycans. The glycosaminoglycans were identified and characterized by a combination of agarose gel electrophoresis and enzymatic degradation with specific mucopolysaccharidases. It was shown that 3/4 of the total 35S-labelled glycosaminoglycans were in the extracellular compartment after 24-48 h. The macrophages synthesized dermatan sulphate (68%), chondroitin sulphate (7%) and heparan sulphate (25%). Both cell and medium fractions of normal and nude mouse macrophages contained glycosaminoglycans with the same ratios, although the nude mouse macrophages synthesized 2-fold less glycosaminoglycans than the normal mouse macrophages. Lower levels of 35S-proteoglycans were also obtained from in vitro 'activated' macrophages, but the ratios of dermatan sulphate:chondroitin sulphate: heparan sulphate were altered in these cells as compared to the control. Furthermore, all the 35S-macromolecules found in the extracellular compartment of nude and normal control cells were of proteoglycan nature, in contrast to the medium fractions of 'activated' macrophages, which contain both intact proteoglycans and 'free' glycosaminoglycan chains. These results indicate that, at least as regards the proteoglycans and glycosaminoglycans, the nude mouse macrophages are not identical to the 'activated' macrophages from normal mice.     

Metabolism of macromolecular heparin in mouse neoplastic mast cells.

1. Polysaccharide in a heparin-producing mouse mastocytoma was pulse-labelled in vivo with [35S] sulphate, and after various periods of time was isolated from subcellular fractions. Such fractions were recovered from tissue homogenates by consecutive centrifugations at 1000g for 10min, 20000g for 20min and 100000g for 1h. Initially the 35S-labelled polysaccharide formed occurred principally in the second centrifugal fraction (20000g precipitate), with small amounts in the first (granular) and third (microsomal) fractions. Analysis for glycosyltransferase activity confirmed that glycosaminoglycans were formed chiefly in particles sedimenting at 20000g. Molecules of this newly synthesized polysaccharide were considerably larger than those of commercially available heparin, as judged from gel chromatography. 2. Within the first hour after injection of [35S]sulphate, most of the labelled polysaccharide was redistributed from the second to the first centrifugal fraction. During, and possibly also after, this shift, the macromolecular polysaccharide was degraded, ultimately to the size of commercial heparin. The degradation process appeared complete 6h after injection of [35S]sulphate. 3. Particulate subcellular fractions were incubated with macromolecular [35S]heparin and the products were analysed by gel chromatography. Significant degradation of the substrate occurred only with the second centrifugal fraction. Further characterization of this fraction, by density-gradient centrifugation in iso-osmotic colloidal silica, revealed a single visible band of particles, at approximately the same density at lysosomes. This band contained all the beta-glucuronidase, 35S-labelled endogenous polysacchride and heparin-degrading enzyme present in the second fraction.     

Synthesis of heparan sulfate proteoglycans by the isolated glomerulus

Incorporation of [35S]sulfate into newly synthesized macromolecules was studied in the isolated rat glomerulus and found to be linear between 6 and 24 h. When whole glomeruli were treated under conditions that dissociate proteoglycan aggregates, greater than 90% of incorporated label was extracted. Of this, 80-90% was found to be the heparan sulfate proteoglycan. Similarly, a linear incorporation of [35S]sulfate into a glomerular basement membrane-enriched fraction was due almost entirely to proteoheparan sulfate. This predominance of heparan sulfate among the newly sulfated glycosaminoglycans has previously been observed in vivo and in the perfused kidney, but different patterns have hitherto been described in vitro. The present results suggest that under certain conditions, the isolated glomerulus is a suitable in vitro model for the study of proteoglycan synthesis. The pattern of incorporation of proteoglycans into the glomerular basement membrane reflects the time course and distribution of their synthesis by the whole glomerulus.     

Regulation of heparan sulphate metabolism by adenosine 3′:5′-cyclic monophosphate in hepatocytes in culture

Freshly isolated rat hepatocytes maintained as monolayers in a serum-free medium synthesize sulphated glycosaminoglycans, most of which behave as heparan sulphate and are mainly distributed into intracellular compartments. Cyclic AMP, dibutyryl cyclic AMP, glucagon, noradrenaline, prostaglandin E(1), and theophylline, all drugs and hormones known to increase intracellular cyclic AMP concentrations, decreased the incorporation of (35)SO(4) (2-) into heparan sulphate of intra-, extra- and peri-cellular pools. The inhibition mediated by dibutyryl cyclic AMP was dose-dependent and observed as early as 2h after exposure to the drug. In the presence of 1mm-dibutyryl cyclic AMP, incorporation of (35)SO(4) (2-) or [(14)C]glucosamine into heparan sulphate was decreased to 40-50%, suggesting that dibutyryl cyclic AMP interfered with the synthesis of heparan sulphate. This was further supported by pulse-chase experiments, where dibutyryl cyclic AMP had no effect on the degradation of sulphated glycosaminoglycans. Heparan sulphates synthesized and secreted into the extracellular pool in the presence of dibutyryl cyclic AMP were smaller in size, whereas the degree of sulphation and molecular size of the heparan sulphate chains released by beta-elimination from these proteoglycans were not different from control values. In the presence of 1mm-cycloheximide, (35)SO(4) (2-) incorporation was decreased to 5%. Addition of p-nitrophenyl beta-d-xyloside, an artificial acceptor of glycosaminoglycan chain synthesis, enhanced this incorporation to 18%. Dibutyryl cyclic AMP did not have any inhibitory effect on the synthesis of chains initiated on p-nitrophenyl beta-d-xylosides. Incorporation of [(3)H]serine into heparan sulphate was not affected by dibutyryl cyclic AMP, whereas the degree of substitution of serine residues with heparan sulphate chains was less in heparan sulphate synthesized in the presence of dibutyryl cyclic AMP, suggesting that cyclic AMP exerts its effect on the metabolism of sulphated glycosaminoglycans by affecting the transfer of xylose on to the protein core.