Effect of insulin on the altered production of proteoglycans in rib cartilage of experimentally diabetic rats

Costal cartilage from experimentally diabetic rats, labeled in vivo or in vitro with [35S]sulfate, was shown to incorporate less label into proteoglycans than cartilage from nondiabetic rats. Analyses of guanidine HCl cartilage extracts by gel chromatography on Sepharose CL-2B showed two major peaks at Kav approximately 0.4 and 0.8 (peaks I and II, respectively). Cartilage extracts from the diabetic rats contained predominantly peak II proteoglycans, while 60 and 55%, respectively, of the total 35S-labeled proteoglycans extracted from control cartilage labeled in vivo and in vitro with [35S]sulfate were present in peak I. After insulin treatment of the diabetic rats, the relative amount of peak I 35S-labeled proteoglycans synthesized in vivo was increased to 70%. The overall in vivo incorporation of [35S]sulfate into proteoglycans was also stimulated in diabetic rats treated with insulin to levels above those found for control rats. Thus, diabetes-induced changes in the biosynthesis of rat costal cartilage proteoglycans may be alleviated by normalization of the diabetic state by insulin treatment. However, addition of insulin (10(-5)-10(-9) M) to the culture medium did not affect the amount of 35S-labeled proteoglycans synthesized in vitro or the relative amounts of peak I proteoglycans produced by control or diabetic cartilage, suggesting that insulin does not have a direct effect on proteoglycan production. Moreover, no decrease in the amount of 35S-labeled proteoglycans produced was found when glucose at high concentrations was present in the culture medium. However, the presence of rat serum resulted in an increase in the amount of 35S-labeled proteoglycans produced by both control and diabetic cartilage, demonstrating that the cartilage explants were metabolically responsive to stimulatory factors.     

The isolation, identification and characterization of sulfated glycosaminoglycans synthesized in vitro by human eosinophils

Human eosinophils were purified to greater than 92% using 16-30% metrizamide gradients, and these cells cultured for up to 72 h in vitro to label sulfated glycosaminoglycans. Over 90% of the sulfated glycosaminoglycan-containing material was extracted in 4 M guanidine HCl and had a hydrodynamic size similar to a glycosaminoglycan marker with an approximate average molecular weight of 60,000. Treatment of this salt-extracted 35S-labeled glycosaminoglycan-containing material with 0.5 M NaOH resulted in a change in mass to approx. 20,000 daltons, suggesting that the larger molecules were proteoglycans with side chains with an approximate molecular weight of 20,000. These salt extracted presumptive 35S-labeled proteoglycans were protease insensitive and behaved in a highly charged fashion on DEAE-cellulose. The composition of 35S-labeled glycosaminoglycans from human eosinophils as identified using selected polysaccharides was 70-81% chondroitin 4-sulfate, 9-12% chondroitin 6-sulfate, and 5-12% dermatan sulfate. The predominance of chondroitin 4-sulfate in human eosinophils is similar to the predominance of chondroitin 4-sulfate in human neutrophils and human platelets.     

Sulfated polysaccharides inhibit the catabolism and loss of both large and small proteoglycans in explant cultures of tendon

This study investigated the effects of two highly sulfated polysaccharides, calcium pentosan polysulfate and heparin, on the loss of newly synthesized proteoglycans from the matrix of explant cultures of bovine tendon. The tensional region of deep flexor tendon was incubated with [35S]sulfate for 6 h and then placed in culture for up to 15 days. The amount of radiolabel associated with proteoglycans lost to the medium and retained in the matrix was determined for each day in culture. It was shown that both sulfated polysaccharides at concentrations of 1000 microg x mL(-1) inhibited the loss of 35S-labeled large and small proteoglycans from the matrix and concomitant with this was a retention of chemical levels of proteoglycans in the explant cultures. In other explant cultures that were maintained in culture in the presence of both agents for more than 5 days after incubation with [35S]sulfate, inhibition of the intracellular catabolic pathway was evident, indicating that these highly sulfated polysaccharides also interfered with the intracellular uptake of small proteoglycans by tendon cells.     

The effect of retinoic acid on proteoglycan turnover in bovine articular cartilage cultures

This paper describes proteoglycan catabolism by adult bovine articular cartilage treated with retinoic acid as a means of stimulating the loss of this macromolecule from the extracellular matrix of cartilage. Addition of retinoic acid (10(-12)-10(-6) M) to adult bovine articular cartilage which had been labeled with [35S]sulfate for 6 h after 5 days in culture, resulted in a dose-dependent increase in the rate of loss of 35S-labeled proteoglycans from the matrix of the tissue. Concomitant with this loss was a decrease in the proteoglycan content of the tissue. Incubation of cultures treated with 1 microM retinoic acid, at 4 degrees C, or with 0.5 mM cycloheximide, resulted in a significant decrease in the rate of retinoic acid-induced loss of proteoglycans and demonstrated cellular involvement in this process. Analysis of the 35S-labeled proteoglycans remaining in the matrix showed that the percentage of radioactivity associated with the small proteoglycan species extracted from the matrix of articular cartilage explants labeled with [35S]sulfate after 5 days in culture was 15% and this increased to 22% in tissue maintained in medium alone. In tissue treated with 1 microM retinoic acid for 6 days, the percentage of radioactivity associated with the small proteoglycan was 58%. Approximately 93% of the 35S-labeled proteoglycans released into the medium of control and retinoic acid-treated cultures was recovered in high density fractions after CsCl gradient centrifugation and eluted on Sepharose CL-2B as a broad peak with a Kav of 0.30-0.37. Less than 17% of these proteoglycans was capable of aggregating with hyaluronate. These results indicate that in both control and retinoic acid-treated cultures the larger proteoglycan species is lost to the medium at a greater rate than the small proteoglycan species. The effect of retinoic acid on proteoglycan turnover was shown to be reversible. Cartilage cultures maintained with retinoic acid for 1 day then switched to medium with 20% (v/v) fetal calf serum for the remainder of the culture period exhibited decreased rates of loss of 35S-labeled proteoglycans from the matrix and increased tissue hexuronate contents to levels near those observed in tissue maintained in medium with 20% (v/v) fetal calf serum throughout. Furthermore, following switching to 20% (v/v) fetal calf serum, the relative proportions of the 35S-labeled proteoglycan species remaining in the matrix of these cultures were similar to those of control cultures.     

Comparison of proteoglycans synthesized by porcine normal and polycystic renal tubular epithelial cells in vitro

Newly synthesized porcine tubular epithelial cell proteoglycans were labeled in vitro with Na2[35S]SO4. At the beginning of the labeling period (24 h) [35S] sulfate incorporated into macromolecules was measured following PD-10 chromatography. There was a significant reduction in the amount of 35S-labeled macromolecules isolated from polycystic cells compared to that from normal cells. The distribution of recovered radiolabeled material among the medium, cell surface, and intracellular fractions was similar for both normal and polycystic cells. Analysis of the proteoglycans in polycystic cells demonstrated that 86 and 73% of 35S-labeled macromolecules were of the heparan sulfate type in polycystic and normal cells, respectively. The remainder was chondroitin sulfate. Proteoglycans were characterized using DEAE-Sephacel ion-exchange chromatography, chondroitinase ABC, heparitinase, and nitrous acid digestion followed by Sepharose CL-4B gel permeation chromatography. The majority of radiolabeled material in the medium, cell surface, and intracellular fractions eluted between 0.35 and 0.39 M NaCl. However, a second peak (peak II) that eluted at 0.25 M NaCl was found in the medium from polycystic cells. This peak accounted for 27% of the total macromolecules secreted into the medium. Proteoglycans in the major peak were susceptible to nitrous acid and chondroitinase ABC digestion. A similar proportion of peak II was degraded by chondroitinase ABC. However, the remainder was only slightly susceptible to treatment with nitrous acid or heparitase. In normal cells a small amount of material eluted at a similar low charge; the proteoglycans were the same as those found in the major peak and appeared as a shoulder on this peak.     

Composition of proteoglycans synthesized by rabbit aortic explants in culture and the effect of experimental atherosclerosis

The synthesis of proteoglycans by aorta explants from rabbits with diet-induced atherosclerosis and controls was studied by 35S-incorporation. Proteoglycans were isolated under dissociative conditions from incubation medium and from arterial explants. Additionally, the tissue proteoglycans that were not extracted by 4 M guanidine-HCl were solubilized by digestion of the tissue by elastase in the presence of proteinase inhibitors. The residual tissue was hydrolyzed by papain and glycosaminoglycans were isolated. The atherosclerotic aorta tissue incorporated twice the amount of 35S into proteoglycans than observed for controls; in both groups about 70% of the label incorporated into the tissue was noted in the proteoglycans extracted by guanidine-HC;, while about 30% of the total 35S-labeled proteoglycans synthesized by the explants were found in the media. Atherosclerotic tissue incorporated 35S predominantly into chondroitin sulfate proteoglycans when compared to control tissue. The chondroitinase ABC-digestable proteoglycans that were extracted by guanidine-HCl from atherosclerotic tissues were of larger molecular size than those from control tissue, but the core proteins from these preparations were similar. The heparan sulfate proteoglycan that was obtained by dissociative extraction from atherosclerotic tissue had greater amounts of N-acetyl and lesser amounts of N-sulfate ester groups than the preparation from control tissue. Digestion of the tissue by elastase yielded heparan sulfate proteoglycan as the major constituent in both groups, although atherosclerotic tissue contained relatively small amounts of this proteoglycan. The residual tissue from both groups contained chondroitin sulfate and heparan sulfate as the major glycosaminoglycans with the latter showing a decrease with atherosclerosis. Atherosclerotic tissue secreted into the medium about two-fold more 35S-labeled proteoglycans with larger molecular size than control tissue; proteoglycans of the heparan sulfate and chondroitin sulfate types were the major constituents in the culture medium of both tissues. Thus, proteoglycans undergo both quantitative and qualitative changes in atherosclerosis, reflecting the enhanced smooth muscle cell activity. These changes are potentially important in modulating lipoprotein binding and hemostatic properties, as well as fibrillogenesis of the arterial wall.     

Compression loading in vitro regulates proteoglycan synthesis by tendon fibrocartilage.

The regulation of proteoglycan synthesis in a fibrocartilaginous tissue by mechanical loading was assessed in vitro. Discs of bovine tendon fibrocartilage were loaded daily with unconfined, cyclic, uniaxial compression (5 s/min, 20 min/day) and the synthesis of large and small proteoglycans was measured by incorporation of [35S]sulfate. All discs synthesized predominantly large proteoglycan when first placed in culture. After 2 weeks in culture nonloaded discs synthesized predominantly small proteoglycans whereas loaded discs continued to produce predominantly large proteoglycan. The turnover of 35S-labeled proteoglycan was not significantly altered by the compression regime. Increased synthesis of large proteoglycans was induced by a 4-day compression regime following 21 days of culture without compression. Inclusion of cytochalasin B during compression mimicked this induction. Autoradiography demonstrated that cell proliferation was minimal and confined to the disc edges whereas 35S-labeled proteoglycan synthesis occurred throughout the discs. These experiments demonstrate that mechanical compression can regulate synthesis of distinct proteoglycan types in fibrocartilage.     

In vivo turnover of the basement membrane and other heparan sulfate proteoglycans of rat glomerulus

The metabolic turnover of rat glomerular proteoglycans in vivo was investigated. Newly synthesized proteoglycans were labeled during a 7-h period after injecting sodium [35S]sulfate intraperitoneally. At the end of the labeling period a chase dose of sodium sulfate was given. Subsequently at defined times (0-163 h) the kidneys were perfused in situ with 0.01% cetylpyridinium chloride in phosphate-buffered saline to maximize the recovery of 35S-proteoglycans. Glomeruli were isolated from the renal cortex and analyzed for 35S-proteoglycans by autoradiographic, biochemical, and immunochemical methods. Grain counting of autoradiographs revealed a complex turnover pattern of 35S-labeled macromolecules, commencing with a rapid phase followed by a slower phase. Biochemical analysis confirmed the biphasic pattern and showed that the total population of [35S]heparan sulfate proteoglycans had a metabolic half-life (t1/2) of 20 and 60 h in the early and late phases, respectively. Heparan sulfate proteoglycans accounted for 80% of total 35S-proteoglycans, the remainder being chondroitin/dermatan sulfate proteoglycans. Whole glomeruli were extracted with 4% 3-[(cholamidopropyl)dimethy-lammonio]-1-propanesulfonate-4 M guanidine hydrochloride, a procedure which solubilized greater than 95% of the 35S-labeled macromolecules. Of these 11-13% was immunoprecipitated by an antiserum against heparan sulfate proteoglycan which, in immunolocalization experiments, showed specificity for staining the basement membrane of rat glomeruli. Autoradiographic analysis showed that 18% of total radioactivity present at the end of the labeling period was associated with the glomerular basement membrane. The glomerular basement membrane [35S]heparan sulfate proteoglycans, identified by immunoprecipitation, have a very rapid turnover with an initial phase, t1/2 = 5 h, and a later phase t1/2 = 20 h.     

Synthesis by Schwann cells of basal lamina and membrane-associated heparan sulfate proteoglycans

Primary cultures that contain only Schwann cells and sensory nerve cells synthesize basal lamina. The assembly of this basal lamina appears to be essential for normal Schwann cell development. In this study, we demonstrate that Schwann cells synthesize two major heparan sulfate-containing proteoglycans. Both proteoglycans band in dissociative CsCl gradients at densities less than 1.4 g/ml, and therefore, presumably, have relatively low carbohydrate-to-protein ratios. The larger of these proteoglycans elutes from Sepharose CL-4B in 4 M guanidine hydrochloride (GuHCl) at a Kav of 0.21 and contains heparan sulfate and chondroitin sulfate chains of Mr 21,000 in a ratio of approximately 3:1. This proteoglycan is extracted from cultures by 4 M GuHCl but not Triton X-100 and accumulates only when Schwann cells are actively synthesizing basal lamina. The smaller proteoglycan elutes from Sepharose CL-4B at a Kav of 0.44 and contains heparan sulfate and chondroitin sulfate chains of Mr 18,000 in a ratio of approximately 4:1. This proteoglycan is extracted by 4 M GuHCl or by Triton X-100. The accumulation of this proteoglycan is independent of basal lamina production.     

Intestinal mucosal mast cells from rats infected with Nippostrongylus brasiliensis contain protease-resistant chondroitin sulfate di-B proteoglycans

Rats infected with the helminth Nippostrongylus brasiliensis were injected i.p. with 2 mCi of [35S] sulfate on days 13, 15, 17, and 19 after infection. The intestines were removed from animals on day 20 or 21 after infection, the intestinal cells were obtained by collagenase treatment and mechanical dispersion of the tissue, and the 35S-labeled mucosal mast cells (MMC) were enriched to 60 to 65% purity by Percoll centrifugation. The cell-associated 35S-labeled proteoglycans were extracted from the MMC-enriched cell preparation by the addition of detergent and 4 M guanidine HCl and were partially purified by density gradient centrifugation. The isolated proteoglycans were of approximately 150,000 m.w., were resistant to pronase degradation, and contained highly sulfated chondroitin sulfate side chains. Analysis by high-performance liquid chromatography of chondroitinase ABC-treated 35S-labeled proteoglycans from these rat MMC revealed that the chondroitin sulfate chains consisted predominantly of disaccharides with the disulfated di-B structure (IdUA-2SO4----GalNAc-4SO4) and disaccharides with the monosulfated A structure (G1cUA----GalNAc-4SO4). The ratio of disaccharides of the di-B to A structure ranged from 0.4 to 1.6 in three experiments. Small amounts of chondroitin sulfate E disaccharides (GlcUA----GalNAc-4,6-diSO4) were also detected in the chondroitinase ABC digests of the purified rat MMC proteoglycans, but no nitrous acid-susceptible heparin/heparan sulfate glycosaminoglycans were detected. The presence in normal mammalian cells of chondroitin sulfate proteoglycans that contain such a high percentage of the unusual disulfated di-B disaccharide has not been previously reported. The rat intestinal MMC proteoglycans are the first chondroitin sulfate proteoglycans that have been isolated from an enriched population of normal mast cells. They are homologous to the chondroitin sulfate-rich proteoglycans of the transformed rat basophilic leukemia-1 cell and the cultured interleukin 3-dependent mouse bone marrow-derived mast cell, in that these chondroitin sulfate proteoglycans as well as rat serosal mast cell heparin proteoglycans are all highly sulfated, protease-resistant proteoglycans.     

Nonspecific interaction of proteoglycans with chromatography media and surfaces: effect of this interaction on the isolation efficiencies

Nonspecific adsorption of proteoglycans to chromatography media and surfaces is demonstrated. This adsorption is highly dependent on the nature of the chromatography media and the precise buffer conditions. For a given buffer the amount of adsorption decreases as the pH of the buffer is increased. It is also highly dependent on buffer concentration and increases as the buffer concentration is increased. The effect of salts such as LiCl, NaCl, KCl, and MgCl2 was generally small and complex so that the presence of the salt both increased and decreased the amount of adsorption depending on the buffer conditions. In contrast, the effect due to the presence of guanidine hydrochloride (Gdn-HCl) was relatively large and complex. At low Gdn-HCl concentrations there generally was a large increase in the amount of adsorption, reaching a maximum at approximately 0.5 M Gdn-HCl and decreasing with further increases in Gdn-HCl concentration. Detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) and sodium dodecylsulfate generally reduced the amount of nonspecific adsorption, although in the presence of both the detergent and Gdn-HCl, the effect due to Gdn-HCl predominated. In commonly used buffers such as 0.5 M sodium acetate (NaOAc), pH 7.0 (buffer F), and 4 M Gdn-HCl in 0.05 M NaOAc, pH 5.8 (buffer D), adsorption to surfaces and chromatography media such as Sepharose CL-2B, cellulose, and controlled pore glass (CPG) is highly significant and it is particularly large for cellulose and CPG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Turnover of proteoglycans in cultures of bovine articular cartilage

Proteoglycans in cultures of adult bovine articular cartilage labeled with [35S]sulfate after 5 days in culture and maintained in medium containing 20% fetal calf X serum had longer half-lives (average 11 days) compared with those of the same tissue maintained in medium alone (average 6 days). The half-lives of proteoglycans in cultures of calf cartilage labeled after 5 days in culture and maintained in medium with serum were considerably longer (average 21 days) compared to adult cartilage. If 0.5 mM cycloheximide was added to the medium of cultures of adult cartilage, or the tissue was maintained at 4 degrees C after labeling, the half-lives of the proteoglycans were greater, 24 and greater than 300 days, respectively. Analyses of the radiolabeled proteoglycans remaining in the matrix of the tissue immediately after labeling the tissue and at various times in culture revealed two main populations of proteoglycans; a large species eluting with Kav of 0.21-0.24 on Sepharose CL-2B, of high bouyant density and able to form aggregates with hyaluronate, and a small species eluting with a Kav of 0.63-0.70 on Sepharose CL-2B, of low buoyant density, containing only chondroitin sulfate chains, and unable to form aggregates with hyaluronate. The larger proteoglycan had shorter half-lives than the smaller proteoglycan; in cartilage maintained with serum, the half-lives were 9.8 and 14.5 days, respectively. Labeling cartilage with both [3H]leucine and [35S]sulfate showed the small proteoglycan to be a separate synthetic product. The size distribution of 35S-labeled proteoglycans lost into the medium was shown to be polydisperse on Sepharose CL-2B, the majority eluting with a Kav of 0.27 to 0.35, of high buoyant density, and unable to aggregate with hyaluronate. The size distribution of glycosaminoglycans from 35S-labeled proteoglycans appearing in the medium did not differ from that associated with labeled proteoglycans remaining in the matrix.     

Proteoglycan synthesis by fibroblast cultures initiated from regions of adult bovine tendon subjected to different mechanical forces

Fibroblast cultures were initiated from two distinct regions of the adult bovine deep flexor tendon and synthesis of 35S-labeled proteoglycans by these cultures was investigated. The proximal/tensional region of the tendon was composed of linearly arranged dense collagen bundles, and its glycosaminoglycan hexosamine content was only 0.2% of the dry weight of the tissue. The proteoglycans of this region were predominantly small (Kav = 0.5 on Sepharose CL-4B). Cells placed into culture from this region attached to the substratum readily, and the radiolabeled proteoglycans from these cultures were 90% small proteoglycans. In a more distal region of the tendon that is subjected to compressive forces, the collagen was arranged as a network of fibrils separated from each other by a matrix that stained intensely with Alcian blue. The glycosaminoglycan content of this compressed region was up to 5-fold higher than in the proximal region, and as much as 50% of the proteoglycans were large molecules (eluted from Sepharose CL-4B in the Vo). Cells placed into culture from the distal/compressed region did not attach to the substratum as readily as those from the proximal region and were characterized by the presence of numerous cytoplasmic lipid inclusions. The [35S]proteoglycans synthesized by the distal tendon fibroblast cultures were divided into two approximately equal populations of large and small proteoglycans having elution characteristics similar to the proteoglycans extracted from this tissue. The distinct profiles of proteoglycan production were maintained by the cells in culture for several weeks, although eventually the amount of large proteoglycan synthesized by the distal tendon fibroblast cultures diminished. Both regions of tendon contained predominantly type I collagen, and collagen production was about 10% of the total protein synthesized by both cell cultures. These observations indicate that adult tendon fibroblasts in culture express stable synthesis of proteoglycan populations similar to those found in the region of tendon from which they were derived.     

Identification of chondroitin sulfate E in human lung mast cells

Human lung mast cells (HLMC) enriched up to 99% purity by counter current elutriation and density gradient centrifugation were labeled with 35S-sulfate to determine cell-associated proteoglycans. The 35S-labeled proteoglycans were extracted by the addition of detergent and 4 M guanidine-HCl, and separated from unincorporated precursor by Sephadex G-50 chromatography. 35S-Proteoglycans chromatographed over Sepharose 4B with a Kav of 0.48. 35S-Glycosaminoglycans separated from the parent 35S-proteoglycans by beta-elimination and chromatographed over Sepharose 4B with a Kav of 0.63. Characterization of 35S-proteoglycans by chondroitin ABC lyase treatment revealed approximately 36% of the proteoglycan to be composed of chondroitin sulfates. Analysis by HPLC of component disaccharides liberated by chondroitin ABC lyase using an amino-cyano-substituted silica column indicated that the chondroitin sulfates consisted of the monosulfated A disaccharide (GlcUA----GaINAc4SO4) (75%) and the over-sulfated E disaccharide (GlcUA----GaINAc4,6-diSO4) (25%). Nitrous acid/heparinase-susceptible heparin proteoglycans accounted for approximately 62% of the total 35S-proteoglycans present in the HLMC. Proteoglycans remaining after exposure of the original proteoglycan extract to either heparinase or chondroitin ABC lyase were of similar size, suggesting that the majority of heparin and chondroitin sulfate glycosaminoglycans were on separate protein cores. Proteoglycans extracted from HLMC were protease insensitive. Hence, in addition to heparin proteoglycans, HLMC synthesize a hitherto unrecognized quantity of chondroitin sulfate E proteoglycans.     

Identification of carboxypeptidase and tryptic esterase activities that are complexed to proteoglycans in the secretory granules of human cloned natural killer cells

Human cloned 35S-labeled NK cells were disrupted by nitrogen cavitation, and their secretory granules were obtained by filtration through 5-micron and 3-micron membrane filters followed by Percoll density-gradient centrifugation. These granule preparations, which contained 35S-labeled chondroitin sulfate A proteoglycans, were sonicated and were analyzed for carboxypeptidase activity and tryptic serine esterase activity. A carboxypeptidase activity that digested angiotensin I to des-Leu-angiotensin I, Ile-His-Pro-Phe to Ile-His-Pro and Phe, and hippuryl-L-phenylalanine to hippuric acid and Phe was detected in the granules of these NK cells. As determined by cleavage of the tetrapeptide, the pH optimum of the carboxypeptidase was 7.0. As assessed by the cleavage of N-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLTe), the granule preparations also contained a serine esterase with trypsin-like specificity that had a pH optimum of 8.5. When the isolated secretory granules were disrupted and chromatographed on columns of Sepharose CL-2B in PBS, greater than 60% of the BLTe serine esterase activity and essentially all of the carboxypeptidase activity filtered as a macromolecular complex with approximately 8% of the 35S-labeled proteoglycans. Whereas treatment with 4 M urea or nonionic detergent failed to disrupt the macromolecular complex, the serine esterase activity was dissociated from the macromolecular complex in the presence of 3 M NaCl, demonstrating an ionic interaction with the proteoglycans. No difference was observed in the disaccharide composition of the chondroitin sulfate glycosaminoglycans of the 35S-labeled proteoglycans that were complexed with the enzymes as compared to those that were not complexed. These studies indicate that the secretory granules of human NK cells contain serine esterase activity and carboxypeptidase activity, both of which have neutral pH optima, and both of which are bound to protease-resistant chondroitin sulfate proteoglycans.     

Partial characterization of proteoglycans synthesized by human glomerular epithelial cells in culture

Confluent adult and fetal human glomerular epithelial cells were incubated for 24 h in the presence of [3H]-amino acids and [35S]sulfate. Two heparan-35SO4 proteoglycans were released into the culture medium. These 35S-labeled proteoglycans eluted as a single peak from anion exchange chromatographic columns, but were separable by gel filtration on Sepharose CL-6B columns. The larger heparan-35SO4 proteoglycan eluted with the column void volume and at a Kav of 0.26 from Sepharose CL-4B columns. The most abundant medium heparan-35SO4 proteoglycan was a high buoyant density proteoglycan similar in hydrodynamic size (Sepharose CL-6B Kav 0.23) to those previously described in glomerular basement membranes and isolated glomeruli. Heparan-35SO4 chains from both proteoglycans were 36 kDa. A smaller proportion of Sepharose CL-6B excluded dermatan-35SO4 proteoglycan was also synthesized by these cells. The predominant protein cores of both medium heparan-35SO4 proteoglycans were approximately 230 and 180 kDa. A hybrid chondroitin/dermatan-heparan-35SO4 proteoglycan with an 80-kDa protein core copurified with the smaller medium heparan-35SO4 proteoglycan. This 35S-labeled proteoglycan appeared as a diffuse, chondroitinase ABC sensitive 155-kDa fluorographic band in sodium dodecyl sulfate-polyacrylamide gels after the Sepharose CL-6B Kav 0.23 35S-labeled proteoglycan fraction was digested with heparitinase. The heparitinase generated heparan sulfate proteoglycan protein cores and the 155-kDa hybrid proteoglycan fragment had molecular weights similar to those previously identified in rat glomerular basement membrane and glomeruli using antibodies against a basement membrane tumor proteoglycan precursor (Klein et al. J. Cell Biol. 106, 963-970, 1988). Thus, human glomerular epithelial cells in culture are capable of synthesizing, processing, and releasing heparan sulfate proteoglycans which are similar to those synthesized in vivo and found in the glomerular basement membrane. These proteoglycans may belong to a family of related basement membrane proteoglycans.     

Three distinct molecular species of proteoglycan synthesized by the rat limb bud at the prechondrogenic stage

To characterize proteoglycans in the prechondrogenic limb bud, proteoglycans were extracted with 4 M guanidine HCl containing a detergent and protease inhibitors from Day 13 fetal rat limb buds which had been labeled with [35S]sulfate for 3 h in vitro. About 90% of 35S-labeled proteoglycans was solubilized under the conditions used. The proteoglycan preparation was separated by DEAE-Sephacel column chromatography into three peaks; peak I eluted at 0.45 M NaCl concentration, peak II at 0.52 M, and peak III at 1.4 M. Peaks I and III were identified as proteoglycans bearing heparan sulfate side chains. The heparan sulfate proteoglycan in peak III was larger in hydrodynamic size than the proteoglycan in peak I. The heparan sulfate side chains of peak III proteoglycan were smaller in the size and more abundant in N-sulfated glucosamine than those of peak I proteoglycan. Peak II contained a chondroitin sulfate proteoglycan with a core protein of a doublet of Mr 550,000 and 500,000. The chondroitin sulfate proteoglycan was easily solubilized with a physiological salt solution and the heparan sulfate proteoglycan in peak I was partially solubilized with the physiological salt solution. The remainder of the proteoglycan in peak I and the heparan sulfate proteoglycan in peak III could be solubilized effectively only with a solution containing a detergent, such as nonanoyl-N-methylglucamide. This observation indicates the difference in the localization among these three proteoglycans in the developing rat limb bud.     

Proteoglycan extraction of sized cartilage particles

The relationship between cartilage thickness and proteoglycan extractability was examined. Bovine nasal cartilage slices (20, 100, and 500 micron thicknesses) were extracted with low-ionic-strength buffer and 4 M guanidine hydrochloride. The extractability of proteoglycans with both solutions depended on slice thickness. Thinner slices yielded greater amounts of proteoglycans. Sixty-three percent of the total cartilage uronic acid was extracted from 20-micron cartilage slices with low-ionic-strength buffer while only 7% was extracted for 500-micron slices. Each fivefold increase in cartilage surface area led to a threefold increase in uronic acid extraction with low-ionic-strength buffer. Extraction of proteoglycan aggregates was directly proportional to the cartilage surface area whereas extraction of non-aggregated proteoglycans, per surface area, increased with increasing cartilage thickness. These data are consistent with the hypothesis that proteoglycan aggregates are extracted mainly from the cartilage surface while non-aggregated proteoglycans diffuse from deep within the cartilage. Extraction with low-ionic-strength buffer occurred in two phases. There was an initial rapid loss of proteoglycans in which 1/3 to 1/2 of all proteoglycans eluting over 6 days were extracted during the first 30 min. Subsequent extraction was much slower with decreasing amounts extracted on each consecutive day. The initial rapid loss of proteoglycans was probably due to the steep osmotic-pressure gradient existing when the cartilage was placed in the low-ionic-strength buffer.     

Biosynthesis and fate of proteoglycans in cartilage and bone during development and mineralization

Subcutaneous implantation of demineralized bone matrix in rats induces migration of host cells into the site and results in the sequential development of cartilage and bone. The biosynthesis and metabolic fate of proteoglycans in the plaques at the bone matrix implantation site were investigated by [35S]sulfate labeling in vivo. 35S-Labeled proteoglycans were extracted with 4 M guanidine HCl and purified by DEAE-Sephacel chromatography. Analysis of proteoglycans on Sepharose CL-2B chromatography showed two major peaks at Kd = 0.28 and 0.68 (peaks I and II, respectively). Peak I proteoglycan has a high buoyant density and contains chondroitin sulfate chains of average Mr = 20,000. Peak II proteoglycan has a lower average buoyant density and contains dermatan sulfate chains of average Mr = 33,000. Throughout the endochondral bone development sequence, peak II proteoglycan predominates. Peak I was low on Day 3, became prominent on Day 7 (approximately 30% of the total radioactivity), and declined after Day 9. The calculated half-lives of peak I and II proteoglycans labeled on Day 7 were about 1.8 and 2.8 days, respectively. After the initiation of osteogenesis, a species of mineral-associated proteoglycan was extracted with a 4 M guanidine HCl solvent containing 0.5 M EDTA. This proteoglycan has a small hydrodynamic size (Kd = 0.38 on Sepharose CL-6B chromatography) and shows a long half-life, about 6 days.     

Stability and folding of the ferredoxin from the hyperthermophilic archaeon Acidianus ambivalens

The ferredoxin from the thermophilic archaeon Acidianus ambivalens is a small monomeric protein containing two iron-sulfur centres, one [3Fe-4S](1+/0) and one [4Fe-4S](2+/1+). It is an intrinsically hyperstable protein, being expressed at the organism's extreme optimal growth temperature: 80 degrees C. Using spectroscopic methods we have investigated the unfolding reaction of the Acidianus ambivalens ferredoxin. No unfolding of the oxidised ferredoxin was observed at pH 7.0, even in the presence of 8 M GuHCl. Upon increasing the pH to 10.0, the unfolding transition showed a midpoint at 6.3 M GuHCl and an unfolding-free energy of 70 kJ mol(-1) in buffer (pH 10) was estimated. Kinetic-unfolding experiments showed that the polypeptide unfolding correlated with rearrangement of the iron-sulfur centres to new ones which had strong absorption maxima at 520 and 610 nm. These new, possibly linear three-iron, clusters were coordinated to the unfolded protein but degraded slowly. From thermal experiments in the presence of GuHCl we estimated the melting temperature for the Acidianus ambivalens ferredoxin in buffer (at pH 7) to be 122 degrees C. Possible structural properties that contribute to the large thermal stability of the Acidianus ambivalens ferredoxin are discussed using a three-dimensional protein model.