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This paper focuses on the active cell balancing of lithium-ion battery packs. An improved single-input, multioutput, bi-switch flyback converter was proposed to achieve effective balancing. The proposed topology simplifies the control logic by utilising a single MOSFET switch for energy transfer and two complementary pulses to control the cell-selecting switches. The proposed topology can decrease the number of switching devices as well as the size and cost of the system. The bi-switch flyback converter eliminates the need for a separate buffer circuit to minimise leakage and electromagnetic inductance. Losses and energy efficiency were analysed at each end of the proposed topology. The appropriate MATLAB simulations investigated the balancing characteristics of various state of charge (SOC) imbalances. A comparison is made between the balancing speed and energy transfer efficiency of the proposed topology and a conventional topology that employs a multi-input and multi-output flyback converter in a static mode. The results of the MATLAB simulation were validated by the OPAL-RT (OP5700) real-time simulator. The balancing data of the proposed topology were compared using MATLAB simulation and real-time simulation. This work may reduce the time required to assemble and commission the hardware for the proposed topology’s real-time implementation.

Li-ion batteries have increased dramatically in the automobile industry over the past few decades. Lithium-ion batteries are viewed more favourably due to their high energy density, specific energy, etc. Optimising the performance of lithium-ion batteries requires accurate modelling and estimation of the state of charge (SOC). This article introduces an efficient second-order resistor-capacitor (2RC) network battery model and an optimised extended Kalman-Bucy filter (EKBF) as the most effective way to model a battery and estimate its state of charge (SOC). The operating parameters of a lithium-ion battery are measured with the least-squares curve fitting method. At a temperature of 20 °C, the proposed method for estimating SOC has a mean absolute error (MAE) of 0.72 % and a root mean square error (RMSR) of 0.71 %. In addition, the operational characteristics of the proposed EKBF were validated by simulating its operation at temperatures ranging from 0 °C to 40 °C. Under all conditions, the proposed method maintained an average error rate of 0.40%. The output of the proposed topology was validated using OPAL-RT (OP5700) hardware-in-the-loop real-time simulator. Consequently, the proposed method is preferable for accurate and dependable SOC estimation in real-time battery management system (BMS) applications.

The pathogenesis of the amyloid deposition diseases is poorly understood. The CE/J mouse, which is naturally protected from amyloid A (AA) protein amyloidosis, has provided a tool to study mechanisms that may be implicated in amyloid deposition diseases by means of comparison of findings with those in an AA-susceptible mouse strain. We have compared proteoglycan/glycosaminoglycan accumulationin vivoin amyloid-protected CE/J mouse strain and in AA-susceptible CBA/J mouse strain in homeostasis and when injected with amyloid-inducing agents. Results indicate that there is an overall increase in[35S]proteoglycan/glycosaminoglycan accumulation in the spleens of both strains of mice, but with a specific increase in heparan sulfate in only CBA/J mouse spleens that are rich in amyloid. Further, we report the absence of heparan sulfate in the splenic perifollicular areas of amyloid-free CE/J mouse, whereas in the amyloid-laden CBA/J mouse there is co-localization of heparan sulfate with the AA deposits. We have also examined the glycosaminoglycan disaccharide products in both these strains of mice for their sulfation positions and found no differences in the disaccharide composition of chondroitin/dermatan sulfate and heparan sulfate isolated from the control CBA/J and control CE/J mice. There were no differences in chondroitin/dermatan sulfate in both strains after experimental induction. However, analysis of the heparan sulfate disaccharides by means of capillary high-performance liquid chromatography linked to microelectrospray ionization time-of-flight mass spectrometry indicated that the disaccharide composition of the splenic heparan sulfate obtained from the treated CBA/J mice that had developed amyloid was markedly different from that obtained from the control CBA/J mice and the treated amyloid-resistant CE/J mice. These findings suggest that unique heparan sulfates play a fundamental role in the pathogenesis of amyloid. [ABSTRACT FROM AUTHOR]

Elevations in urinary hyaluronan have been used as the principal laboratory indicator for diagnosis of Hutchinson-Gilford Progeria Syndrome (HGPS). Previous reports have provided evidence suggesting that children with HGPS have altered hyaluronan metabolism as indicated by a mean 17-fold increase in urinary hyaluronan over normal values. In addition, adults with Werner's syndrome have elevated urinary hyaluronan and even more prominent elevations in serum hyaluronan over age-matched controls. It is not known whether serum hyaluronan is elevated or whether serum hyaluronan levels correlate with urinary hyaluronan levels in children with HGPS. In a large cohort of 19 HGPS patients, we sought to confirm elevations in urinary hyaluronan concentration, to establish whether serum hyaluronan is elevated, to measure the size of urinary hyaluronan, and to determine whether serum or urine hyaluronidase levels are altered. We have analyzed urinary and serum hyaluronan levels in patients with HGPS and control patients (1) by using an enzyme-linked immunosorbent assay (ELISA)-like method in which sample hyaluronan in solution and hyaluronan in solid phase compete for a solution of biotinylated hyaluronan-binding protein, and (2) by fluorophore-assisted carbohydrate electrophoresis. The size of urinary hyaluronan was measured by using Sepharose CL-6B size exclusion chromatography. Serum and urinary hyaluronidases were evaluated quantitatively, by using ELISA, and qualitatively, by using a gel detection method. HGPS patients did not show a significant elevation in either urinary or serum hyaluronan. We detected no difference in the size of urinary hyaluronan between HGPS children and age-matched controls. Serum and urinary hyaluronidase levels were not significantly different in normal and HGPS patients. These studies indicate that neither serum nor urinary hyaluronan concentration is a reliable diagnostic or prognostic marker for HGPS and underscore a difference between adult and childhood progerias.

The objective of this study was to determine the characteristics of proteoglycans synthesized by canine annulus fibrosus cells expanded in number in monolayer culture through passage 4 and subsequently grown in a type I collagen-glycosaminoglycan matrix to be employed for tissue engineering. Newly synthesized [35S]sulfate-labeled proteoglycans were analyzed by gel chromatography, including sequential digestion with enzymes and nitrous acid. After 1 week in culture, the percentage of cell-associated, aggregated proteoglycans synthesized in type I collagen-glycosaminoglycan matrices was 52% compared with 38% by the cells in monolayer. The percentage of aggregated proteoglycan in each group increased only slightly with the addition of exogenous hyaluronic acid, but remained significantly different from each other. There were at least three different hydrodynamic sizes of proteoglycans both in the collagen-glycosaminoglycan matrix and in monolayer; the average size was larger in the collagen matrices and the glycosaminoglycan chains were longer. The proteoglycans contained chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate. The results provide a foundation for future investigations of collagen-glycosaminoglycan matrices for intervertebral disc tissue engineering.

Chondroitin sulfate and dermatan sulfate are synthesized as galactosaminoglycan polymers containing N-acetylgalactosmine alternating with glucuronic acid. The sugar residues are sulfated to varying degrees and positions depending upon the tissue sources and varying conditions of formation. Epimerization of any of the glucuronic acid residues to iduronic acid at the polymer level constitutes the formation of dermatan sulfate. Chondroitin/dermatan glycosaminoglycans are covalently attached by a common tetrasaccharide sequence to the serine residues of core proteins while they are adherent to the inner surface of endoplasmic reticulum/Golgi vesicles. Addition of the first sugar residue, xylose, to core proteins begins in the endoplasmic reticulum, followed by the addition of two galactose residues by two distinct glycosyl transferases in the early cis/medial regions of the Golgi. The linkage tetrasaccharide is completed in the medial/trans Golgi by the addition of the first glucuronic acid residue, followed by transfer of N-acetylgalactosamine to initiate the formation of a galactosaminoglycan rather than a glucosaminoglycan. This specific N-acetylgalactosaminyl transferase is different from the chondroitin synthase involved in generation of the repeating disaccharide units to form the chondroitin polymer. Sulfation of the chondroitin polymer by specific sulfotransferases occurs as the polymer is being formed. All the enzymes in the pathway for synthesis have been cloned, with the exception of the glucuronyl to iduronyl epimerase involved in the formation of dermatan residues.

Cultured peritoneal macrophages obtained from azocasein-injected mice were found to produce several fold more cell-associated and medium proteoglycans than peritoneal macrophages from untreated mice. Since serum amyloid A (an acute-phase protein) is also upregulated following injections of azocasein, we questioned whether its production was the immediate agent stimulating proteoglycan formation. Cultured peritoneal macrophages from untreated mice were then incubated with varying concentrations of SAA, resulting in a similar dose-dependent several fold increase in proteoglycan production. Of particular note was a disproportionate increase in cell-associated heparan sulfate proteoglycans in both experimental groups and of dermatan sulfate and chondroitin sulfate proteoglycans when cells were incubated in the presence of SAA in the culture medium. These results indicate a potentially important function of SAA in directing specific modifications in inflammatory conditions where increase in macrophage proteoglycans may play direct roles., (Copyright 1999 Academic Press.)

A partial-length human cDNA with a predicted amino acid sequence homologous to a previously described heparan sulfate iduronyl 2-sulfotransferase (Kobayashi, M., Habuchi, H., Yoneda, M., Habuchi, O., and Kimata, K. (1997) J. Biol. Chem. 272, 13980-13985) was obtained by searching the expressed sequence-tagged data bank. Northern blot analysis was performed using this homologous cDNA as a probe, which demonstrated ubiquitous expression of messages of 5.1 and 2.0 kilobases in a number of human tissues and in several human cancer cell lines. Since the human lymphoma Raji cell line had the highest level of expression, it was used to isolate a full-length cDNA clone. The full-length cDNA was found to contain an open reading frame that predicted a type II transmembrane protein composed of 406 amino acid residues. The cDNA in a baculovirus expression vector was expressed in Sf9 insect cells, and cell extracts were then incubated together with 3'-phosphoadenosine 5'-phospho[35S]sulfate and potential glycosaminoglycan acceptors. This demonstrated substantial sulfotransferase activity with dermatan sulfate, a small degree of activity with chondroitin sulfate, but no sulfotransferase activity with desulfated N-resulfated heparin. Analysis of [35S]sulfate-labeled disaccharide products of chondroitin ABC, chondroitin AC, and chondroitin B lyase treatment demonstrated that the enzyme only transferred sulfate to the 2-position of uronyl residues, which were preponderantly iduronyl residues in dermatan sulfate, but some lesser transfer to glucuronyl residues of chondroitin sulfate.

Microsomal membranes from chick embryo epiphyseal cartilage were fractionated by equilibrium sucrose-density-gradient centrifugation and assayed for GlcA (glucuronic acid) transferase I (the enzyme that transfers GlcA from UDP-GlcA to Gal-Gal-Xyl of proteochondroitin linkage region), for comparison with GlcA transferase II (the GlcA transferase of chondroitin polymerization). Gal(beta1-3)Galbeta1-methyl (disaccharide) and GalNAc(beta1-4)GlcA(beta1-3)GalNAc(beta1-4) GlcA(beta1-3)GalNAc(pentasaccharide) were used respectively as acceptors of [14C]GlcA from UDP-[14C]GlcA. Distributions of the two GlcA transferase activities in the sucrose-density-gradient fractions were compared with each other and with the previously reported distribution of the activities of Gal transferases (UDP-Gal to ovalbumin, and to xylose of the proteochondroitin linkage region) and GalNAc (N-acetylgalactosamine) transferase II of chondroitin polymerization. The linkage-region GlcA transferase I had a dual Golgi distribution similar to that of chondroitin-polymerizing GlcA transferase II and distinctly different from the distribution of linkage-region Gal transferases I and II, which were found exclusively in the heavier fractions. Solubilized GlcA transferase I was partly purified by sequential use of Q-Sepharose, heparin-Sepharose and wheatgerm agglutinin-agarose and was accompanied at each step by some of the GlcA transferase II activity. Both GlcA transferase I and II bound to the Q-Sepharose as though they were highly anionic. However, treatment with chondroitin ABC lyase eliminated the binding while markedly decreasing enzyme stability. The enzyme activities could not be reconstituted by adding chondroitin or chondroitin pentasaccharide to the chondroitin ABC lyase-treated enzymes. Incubation of the partly purified enzymes with both UDP-GlcA and UDP-GalNAc resulted in a 40-fold greater incorporation than with just one sugar nucleotide, indicating the presence of bound, nascent proteochondroitin serving as the acceptor for chondroitin polymerization. These results, together with the membrane co-localization, indicate that GlcA transferase I and GlcA transferase II occur closely together with nascent proteochondroitin at the site of synthesis and that this complex with the nascent proteochondroitin stabilizes both enzymes during purification.

A photoaffinity analogue, [beta-32P]5-azido-UDP-GlcA, was used to photolabel the enzymes that utilize UDP-GlcA in cartilage microsomes and rat liver microsomes. SDS-polyacrylamide gel electrophoresis analysis of photolabeled cartilage microsomes, which are specialized in chondroitin sulfate synthesis, showed a major radiolabeled band at 80 kDa and other minor radiolabeled bands near 40 and 60 kDa. Rat liver microsomes, which are enriched for enzymes of detoxification by glucuronidation, had a different pattern with multiple major labeled bands near 50-60 and 35 kDa. To determine that the photolabeled 80-kDa protein is the GlcA transferase II, we have purified the enzyme from cartilage microsomes. This membrane-bound enzyme, involved in the transfer of GlcA residues to non-reducing terminal GalNAc residues of the chondroitin polymer, has now been solubilized, stabilized, and then purified greater than 1350-fold by sequential chromatography on Q-Sepharose, heparin-Sepharose, and WGA-agarose. The purified enzyme exhibited a conspicuous silver-stained protein band on SDS-polyacrylamide gel electrophoresis that coincided with the major radiolabeled band of 80 kDa. SDS-polyacrylamide gel analysis of photoaffinity-labeled active fractions from the Q-Sepharose, heparin-Sepharose, and WGA-agarose also indicated only the single radiolabeled band at 80 kDa. Intensity of photolabeling in each of the fractions examined coincided with enzyme activity. The photolabeling of this 80-kDa protein was saturable with the photoprobe and could be inhibited by the addition of UDP-GlcA prior to the addition of the photoprobe. Thus, the photolabeling with [beta-32P]5-azido-UDP-GlcA has identified the GlcA transferase II as an 80-kDa protein. The purified enzyme was capable of transferring good amounts of GlcA residues to chondroitin-derived pentasaccharide with negligible transfer to pentasaccharides derived from hyaluronan or heparan.

Hs 198.St cells (a line derived from normal human gastric tissue), Hs 746T cells (a line derived from human gastric adenocarcinoma), and HeLa cells were used together with 3H-labelled Helicobacter pylori, strain NCTC 11637 to determine if cell surface glycosaminoglycans could act as initial receptors for adherence of the bacteria. Although as much as 40% of the 3H-labelled bacteria adhered to monolayers of the cultured cells, removal of glycosaminoglycans by prior treatment of the cells with heparitinase, heparinase, or chondroitin ABC lyase had no effect in modifying the adherence. Prior addition of heparan sulfate, heparin, or chondroitin/dermatan sulfate to bacteria had no effect on adherence, nor were bacteria released when these same glycosaminoglycans or these same enzymes were added to cultures already containing adherent bacteria. These results indicated that neither heparan sulfate nor chondroitin/dermatan sulfate are involved as receptors in the initial adherence step of H. pylori to these cultured cells.

A soluble sulfotransferase that could 6-sulfate both chondroitin sulfate and corneal keratan sulfate was purified 27,500-fold using a sequence of affinity chromatographic steps with heparin-Sepharose, wheat germ agglutinin-agarose, and 3',5'-ADP-agarose. The essentially pure enzyme had a specific activity 40 times greater than the most purified chondroitin 6-sulfotransferase previously reported and exhibited a single sharp Coomassie Blue-stained and a heavy silver-stained protein band of 75 kDa on SDS-polyacrylamide gel electrophoresis. Chromatography of the purified enzyme on Sephacryl demonstrated a size of 150 kDa, which indicated that the native enzyme exists as a dimer. In addition to 6-sulfation of nonsulfated GalNAc, the purified serum enzyme had the ability to sulfate GalNAc 4-sulfate residues to give GalNAc 4,6-disulfate residues. The purified enzyme exhibited a Km of 40 microM for adenosine 3'-phosphate 5'-phosphosulfate when either chondroitin sulfate or corneal keratan sulfate were used as the acceptors. Use of both chondroitin sulfate and keratan sulfate in the same experiment demonstrated mutual competition, establishing that the sulfation of these substrates is by the same enzyme. Photoaffinity labeling of the purified enzyme with 2-azidoadenosine 3',5'-di[5'-32P]phosphate occurred only with the 75-kDa protein, confirming that this is the chondroitin 6-sulfotransferase/keratan sulfotransferase.

Mouse mastocytoma cells were cultured in medium containing [3H]GlcN and concentrations of [35S]sulphate varying from 0.01 to 0.5 mM. Intracellular [35S]sulphate incorporation increased severalfold from the lowest concentrations, reaching a maximum at 0.1-0.2 mM, whereas incorporation of [3H]hexosamine remained constant at all sulphate concentrations. Proteo[3H]-chondroitin [35S]sulphate was isolated and incubated with chondroitin ABC lyase, yielding 35S-labelled and/or 3H-labelled delta Di-0S and delta Di-4S disaccharide products. The increasing percentage of delta Di-4S was consistent with the increasing sulphate incorporation at each higher [35S]sulphate concentration. Examination of proteochondroitin [35S]sulphate size by Sepharose CL-6B chromatography indicated a range consistent with various numbers of glycosaminoglycan chains on the protease-resistant serglycin core protein. Alkali-cleaved chondroitin [35S]sulphate products indicated similar size distributions at all sulphate concentrations with no indication of preferential sulphation being related to smaller or larger size. DEAE-cellulose chromatography of [3H]chondroitin [35S]sulphate glycosaminoglycans indicated a random undersulphation as [35S]sulphate concentration was lowered. Addition of 4-methylumbelliferyl beta-D-xyloside to the cultures resulted in a 2-2.5-fold stimulation of [3H]chondroitin [35S]sulphate synthesis with formation of beta-xyloside-[3H]chondroitin [35S]sulphate which was much smaller, as estimated by Sepharose CL-6B chromatography, than the decreased amount of [3H]chondroitin [35S]sulphate derived from proteo[3H]chondroitin [35S]sulphate. Much higher concentrations of sulphate were necessary to produce sulphation of the beta-xyloside-[3H]chondroitin comparable with that of proteo[3H]-chondroitin, as indicated by chondroitin ABC lyase products and DEAE-cellulose chromatography. The specific radioactivities of the [3H]GalN in the proteo[3H]chondroitin [35S]sulphate and beta-xyloside-[3H]chondroitin [35S]sulphate were calculated from the 3H and 35S c.p.m. of isolated dual-labelled delta Di-4S from each, and indicated that the presence of the beta-xyloside resulted in a dilution of the [3H]GlcN by endogenous GlcN that was 4 times higher than that of cultures lacking the beta-xyloside. The higher sulphate concentrations needed for sulphation of beta-xyloside-chondroitin suggests that the membrane-bound nature of the proteochondroitin acceptor in juxtaposition to a chondroitin sulphate-synthesizing enzyme complex effectively reduces the apparent Km for adenosine 3'-phosphate 5'-phosphosulphate.

The effects of the non-ionic detergent Triton X-100 on 6-sulphation of two species of endogenous nascent proteochondroitin by a chick-embryo cartilage microsomal system was examined. Sulphation of the larger (Type I) species with adenosine 3'-phosphate 5'-phosphosulphate was slightly diminished when Triton X-100 was present, whereas sulphation of the smaller (Type II) species was slightly enhanced. An ordered rather than random pattern of sulphation was obtained for the smaller proteoglycan, but with a considerably lower degree of sulphation than that of the larger proteochondroitin. These differences were consistent with other differences between these two species as described previously. Sulphation of exogenous [14C]chondroitin and exogenous proteo[3H]chondroitin by the microsomal system with Triton X-100 present produced ordered rather than random sulphation patterns. When a 100,000 g supernatant fraction was utilized for sulphation of [14C]chondroitin or proteo[3H]chondroitin, Triton X-100 was not needed, and ordered sulphation was still obtained. When hexasaccharide was used, sulphation of multiple N-acetylgalactosamine residues of the individual hexasaccharides resulted. This was relatively independent of Triton X-100 or the concentration of the hexasaccharide acceptors. With soluble enzyme, sulphation of multiple N-acetylgalactosamine residues on the individual hexasaccharide molecules was even greater, so that tri-sulphated products were found. This suggests that ordered rather than random sulphation of chondroitin with these enzyme preparations is due to enzyme-substrate interaction rather than to membrane organization.

Membranes from brefeldin A-treated and untreated chick embryo epiphyseal cartilage were fractionated separately by equilibrium sucrose density gradient centrifugation. Fractions were assayed for Gal I transferase, Gal II transferase, Gal ovalbumin transferase, chondroitin polymerization on endogenous acceptors, GalNAc transfer to exogenous chondroitin hexasaccharide, and sulfate transfer to exogenous chondroitin. Gal I transferase and Gal II transferase activities were found in heavier cis- and medial-Golgi fractions, but with distributions different from each other. Brefeldin A had no effect on either their distribution or their total activity. Gal ovalbumin transferase activity in fractions from untreated cartilage was found as a dual peak in medial- and trans-Golgi areas. The latter peak was diminished in the fractions from the brefeldin A-treated cartilage, whereas the former peak was correspondingly increased. A similar dual medial- and trans-Golgi distribution for chondroitin polymerization on endogenous acceptors was seen with fractions from untreated cartilage. This was modified in fractions from brefeldin A-treated cartilage with a complete loss of synthesis in the trans-Golgi peak and a slight increase in synthesis in the medial-Golgi peak. However, the distribution of GalNAc transferase activity using exogenous chondroitin hexasaccharide indicated that considerable chondroitin-synthesizing activity still remained in these trans-Golgi fractions. This demonstrated that brefeldin A had caused a block in movement of endogenous proteochondroitin acceptors to the trans-Golgi site of synthesis. Sulfotransferase activity was also found in a dual distribution similar to that of the chondroitin polymerization and GalNAc transferase, with a small reduction in activity in the trans-Golgi fractions of brefeldin A-treated cartilage. Thus, treatment of cartilage with brefeldin A resulted in the loss of considerable trans-Golgi chondroitin sulfate-synthesizing enzyme activity and a block in the transport of one form of proteochondroitin precursor to the trans-Golgi membranes.

Mouse mastocytoma cells were cultured with brefeldin A in medium containing [35S]sulfate and [3H]glucosamine in order to determine the effects of this fungal metabolite on the formation of chondroitin 4-sulfate by these cells. There was a marked reduction in the incorporation of [35S]sulfate into the glycosaminoglycan which was approximately equal to the reduction in the incorporation of [3H]hexosamine into the same molecule. The chondroitin 4-sulfate chain size was greatly diminished, while the number of chains appeared to remain relatively constant, indicating that the brefeldin A partially disrupted the polymerizing system, but had little effect upon movement of the nascent proteochondroitin to the site for chondroitin polymerization and sulfation.

The potential relationship of an intact membrane organization to the synthesis of chondroitin was examined before and after modification of a chick-embryo cartilage microsomal system with the non-ionic detergent Triton X-100. Incubations with labelled UDP-GlcA and UDP-GalNAc indicated that Triton X-100 had little effect on the amount of chondroitin synthesized to form one species of large proteochondroitin (Type I). However, Triton X-100 had a marked stimulatory effect on the formation of another smaller species of proteochondroitin (Type II). Presence of this detergent during chondroitin polymerization also resulted in chains that were slightly smaller. Neither of the two proteochondroitin species were collagenase-sensitive, nor did they contain dermatan-like regions. Thus in these respects they were unlike the small proteochondroitins (PG-Lb or PG-Lt) that have been found in chick-embryo cartilage. They also differed greatly in size from these small proteoglycans as well as from the large aggregatable proteochondroitin (PG-H) from the same source. Synthesis of the larger (Type I) proteochondroitin species was not affected by prior treatment of the microsomes with chondroitin ABC lyase at concentrations sufficient for elimination of synthesis of most of the smaller (Type II) proteochondroitin species. Use of chondroitin ABC lyase subsequent to synthesis of the chondroitin also resulted in preferential degradation of the smaller species. Thus there were differences in formation and limitation in access of the chondroitin ABC lyase to the two species, consistent with other differences described previously. These results indicate that there are separate loci within the microsomal membranes for synthesis of the two species.

Membranes from chick embryo epiphyseal cartilage were fractionated by equilibrium sucrose density gradient centrifugation and assayed for galactosyl xylose transferase, chondroitin polymerization and sulfation as well as the marker enzymes glucose-6-phosphatase, NADH cytochrome c reductase, galactosyl ovalbumin transferase, and sialyltransferase. The order of distribution of chondroitin sulfate synthesis from dense to light membranes correlated with the established sequence of events for its synthesis. The linkage region enzyme, viz. galactosyl xylose transferase, distributed with NADH cytochrome c reductase in an earlier and heavier cis compartment. Chondroitin polymerization and sulfation had a dual distribution similar to the galactosyl ovalbumin transferase and sialyltransferase in separate later and lighter medial and trans compartments, or in an extended medial or trans compartment. The galactosyl xylose transferase had a distribution distinctly different from that of the galactosyl ovalbumin transferase indicating that these distinct enzymes showed no cross-reactivity with their respective acceptor substrates. The dual distribution of chondroitin sulfate synthesis was consistent with our previous demonstration of the two nascent proteochondroitin populations produced by microsomal preparations from the same source. The results indicated separate subcellular locations for synthesis of the two forms.

A microsomal preparation from chondroitin 4-sulfate-synthesizing cultured mouse mastocytoma cells was incubated with UDP-[3H]GalNAc, UDP-GlcA, and 3'-phosphoadenylylphosphosulfate (PAPS) for 30 s at 10 degrees C and with UDP-[14C]GlcA, UDP-GalNAc, and PAPS for 4 h at 37 degrees C for synthesis of 3H- and 14C-labeled chondroitin/chondroitin sulfate. The latter incubation provided more than 100 times as much product as did the short incubation at 10 degrees C. Upon chromatography of the isolated labeled glycosaminoglycans on a Sepharose CL-6B column, most of the [14C]glycosaminoglycan from the 4 h, 37 degrees C incubation was excluded from the column, indicating that this nascent glycosaminoglycan had been polymerized fully. In contrast, most of the [3H]glycosaminoglycan from the 30 s, 10 degrees C incubation was mostly retarded upon cochromatography on this same column, indicating that the nascent glycosaminoglycan was still growing in size. The labeled fractions representing chondroitin/chondroitin sulfate of varying sizes were analyzed for degree of sulfation by degradation with chondroitin ABC lyase followed by paper electrophoresis of the products. Results indicated that the [14C]chondroitin/chondroitin sulfate formed in the 4-h incubation was 60-70% sulfated. Incomplete chains of [3H]chondroitin/chondroitin sulfate formed in the 30-s incubation were also sulfated as much as 20-25%. As the size of the [3H]chondroitin/chondroitin sulfate increased, there was a concomitant increase in sulfation. These results demonstrate that in this microsomal system sulfation takes place while the nascent chondroitin glycosaminoglycan chains are still actively growing in length, although the sulfation lags somewhat behind the polymerization. This not only indicates a common membrane location for both polymerization and sulfation of chondroitin but also demonstrates that the sulfation of chondroitin by these mastocytoma cells may occur during the process of glycosaminoglycan polymerization rather than subsequent to completion of the glycosaminoglycan chains.

Microsomal preparations from chondroitin 6-sulfate-producing chick embryo epiphyseal cartilage, and from chondroitin 4-sulfate-producing mouse mastocytoma cells, were incubated with UDP-[14C]glucuronic acid and UDP-N-acetylgalactosamine to form non-sulfated proteo[14C]chondroitin. Aliquots of the incubations were then incubated with 3'-phosphoadenylylphosphosulfate (PAPS) in the presence or absence of various detergents. In the absence of detergents, there was good sulfation of this endogenous proteo[14C]chondroitin by the original microsomes from both sources. Detergents, with the exception of Triton X-100, markedly inhibited sulfation in the mast cell system but not in the chick cartilage system. These results indicate that sulfation and polymerization are closely linked on cell membranes and that in some cases this organization can be disrupted by detergents. When aliquots of the original incubation were heat inactivated, and then reincubated with new microsomes from chick cartilage and/or mouse mastocytoma cells plus PAPS, there was no significant sulfation of this exogenous proteo[14C] chondroitin with either system unless Triton X-100 was added. Sulfation of exogenous chondroitin and chondroitin hexasaccharide was compared with sulfation of endogenous and exogenous proteo[14C]chondroitin. Sulfate incorporation into hexasaccharide and chondroitin decreased as their concentrations (based on uronic acid) approached that of the proteo[14C]chondroitin. At the same time, the degree of sulfation in percent of substituted hexosamine increased. However, the degree of sulfation did not reach that of the endogenous proteo[14C]chondroitin. Hexasaccharide and chondroitin sulfation were stimulated by the presence of Triton X-100. However, in contrast to the exogenous proteo[14C]chondroitin, there was some sulfation of hexasaccharide and chondroitin in the absence of this detergent. These results indicate that the intact microsomal system was not accessible to the larger substrates, and that even with detergents exogenous substrates were not sulfated as effectively as newly formed proteo[14C]chondroitin in an intact microsomal system. When the proteo[14C]chondroitin formed by the chick cartilage microsomal system was incubated together with the mast cell microsomal system and PAPS, sulfation only occurred at the 4-position. When the proteo[14C]chondroitin formed by the mouse mast cell microsomal system was incubated together with the chick cartilage microsomal system and PAPS, sulfation only occurred at the 6-position.(ABSTRACT TRUNCATED AT 400 WORDS)

Microsomal preparations from chondroitin 6-sulfate-producing chick embryo epiphyseal cartilage and from chondroitin 4-sulfate-producing mouse mastocytoma cells were incubated with varying concentrations of 3'-phosphoadenylylphospho[35S]sulfate and chondroitin hexasaccharide in the presence or absence of Triton X-100. [35S]Sulfate incorporation into hexasaccharide and into endogenous microsomal chondroitin 6-sulfate or endogenous microsomal chondroitin 4-sulfate was measured. With both microsomal systems, Triton X-100 increased the incorporation of [35S]sulfate into hexasaccharide but had much less effect on the incorporation into endogenous chondroitin sulfate. Higher concentrations of hexasaccharide inhibited the incorporation of [35S]sulfate into endogenous chondroitin sulfate. The apparent Km for 3'-phosphoadenylylphosphosulfate for both the 6-sulfotransferase and 4-sulfotransferase with hexasaccharide and with endogenous chondroitin sulfate in the presence or absence of Triton X-100 were all similar. However, the apparent Km for hexasaccharide was lower in the presence of Triton X-100 for both the microsomal sulfotransferases. This is consistent with solubilization of sulfotransferases, and indicates that hexasaccharide access to these enzymes had been limited in the particulate system. Examination of 35S-oligosaccharide products formed with each system demonstrated good 6-sulfation or 4-sulfation of penultimate GalNAc at the non-reducing end of the chondroitin hexasaccharide. However, no 6-sulfation of terminal GalNAc at the non-reducing end of a chondroitin pentasaccharide derived from hexasaccharide was observed, and there was only minimal 4-sulfation of this terminal GalNAc. Concurrent addition of GalNAc to hexasaccharide resulting in heptasaccharide did not appear to promote significant 6-sulfation or 4-sulfation of newly added non-reducing terminal GalNAc.

The potential relationship of an intact membrane organization for the synthesis of chondroitin and chondroitin 4-sulfate was examined after modification of a mouse mast cell microsomal system with the nonionic detergent, Triton X-100. The results indicated that Triton X-100 had no effect on the rate of polymerization but had a slight effect on the size of glycosaminoglycan chains. An "all or nothing" pattern of sulfation of newly formed chondroitin was obtained in both the presence and the absence of Triton X-100, and this pattern did not change whether sulfation was initiated concurrent with or subsequent to polymerization. Sulfation of exogenous [14C]chondroitin and exogenous proteo[3H]chondroitin by the microsomal system required Triton X-100 but still produced an all or nothing pattern rather than a random sulfation pattern. When a 100,000 x g supernatant fraction was utilized for sulfation of [14C]chondroitin or proteo[3H]chondroitin, Triton X-100 was not needed, and a partial sulfation pattern was obtained. However, it was similar to the all or nothing pattern in that it still produced two populations, with some chains nonsulfated and others approximately 50% sulfated. When chondroitin hexasaccharide was used with 3'-phosphoadenylylphospho[35S]sulfate, multiple GalNAc residues of the individual hexasaccharides were found to be sulfated. This was relatively independent of Triton X-100 or the concentration of the hexasaccharide acceptors. With soluble enzyme, sulfation of multiple GalNAc residues on the individual hexasaccharide molecules was even greater, so that trisulfated products were found. These results suggest that efficient sulfation of chondroitin is related to enzyme-substrate interaction more than to membrane organization.

Chondrocytes derived from normal human adult articular cartilage were established and maintained for over 5 months in a completely defined medium without the addition of serum or any other growth factors. At the end of 5 months, these cells were still metabolically active. The cells incorporated [3H] thymidine into DNA, incorporated [35S] sulfate into proteoglycans, and exhibited lysosomal enzyme activities. The 35S-labeled proteoglycans isolated from the culture medium had elution profiles on high pressure liquid chromatography (HPLC) similar to those observed for proteoglycans from other mammalian sources. This self-contained growth competence may reflect a need produced by the unusual avascular and alymphatic character of articular cartilage.

Formation of two species of [14C]proteochondroitin sulfate has previously been demonstrated with UDP-D-[14C]glucuronic acid and UDP-N-acetylgalactosamine as substrates with a microsomal preparation from chick-embryo epiphyseal cartilage. A large species of [14C]proteoglycan that appeared at an earlier stage of synthesis was excluded on Sepharose CL-2B, indicating that it was larger than proteoglycans found in cartilage matrix. Another newly synthesized, smaller [14C]proteoglycan species also formed was retarded on Sepharose CL-2B, and appeared to be at a later stage of synthesis. A 6-h pulse-chase experiment using UDP-[14C]GlcA and UDP-GalNAc followed by cold UDP-GlcA demonstrates that there was no conversion of the large [14C]proteoglycan to the small [14C]proteoglycan. Sulfation of the newly formed large and small [14C]proteoglycans with adenylyl sulfate 3'-phosphate also did not alter their chromatographic patterns, indicating that sulfation did not trigger any post-synthetic size modification. Synthesis with lower concentrations of the sugar nucleotides resulted in a disproportionate diminution in formation of the large proteoglycan. The apparent Km values for UDP-GlcA for the formation of large and small proteoglycans were 0.055 and 0.015 mM, respectively. Concentration requirements for UDP-GalNAc also showed a similar 4-fold difference. These results indicate that, even though the large proteoglycan was at an earlier stage of synthesis, it was not a precursor to the small proteoglycan, and that these proteoglycans represent two separately synthesized species.