The heparan sulfates of Swiss mouse 3T3 cells. The effect of transformation.

Three major pools of heparan sulfate have been isolated from cultures of Swiss mouse 3T3 and SV40-transformed 3T3 cells: cell-surface, medium, and intracellular heparan sulfates. The cell-surface heparan sulfate is a high molecular weight proteoglycan which is partially degraded by pronase. Before pronase treatment, it has a peak molecular weight (as estimated by gel filtration) of approx. 7.2 . 10(5) in contrast to only 2.4 . 10(5) after pronase treatment. The medium heparan sulfate appears to be similar in structure to the cell-surface heparan sulfate, since they coelute on Bio-Gel A-15m and DEAE-cellulose, and are both proteoglycans. In contrast, the intracellular heparan sulfate has a low molecular weight (6.0 . 10(3)) and has little if any attached protein. Both the medium and intracellular heparan sulfate exhibit the transformation-associated change in structure reported earlier for cell-surface heparan sulfate (Underhill, C.B. and Keller, J.M. )1975) Biochem. Biophys. Res. Commun. 63, 448--454). This transformation-associated change, detected by DEAE-cellulose chromatography is not the result of changes in either molecular weight or protein core. Cellulose acetate electrophoresis of the cell-surface heparan sulfate at pH 1 suggests that the transformation-associated change in structure is due to a difference in sulfate content. Both types of heparan sulfate are produced in mixed cultures of 3T3 and SV3T3 cells, indicating that neither serum factors in the culture medium nor secreted cell products are responsible for the transformation-associated change in heparan sulfate structure. The presented data are discussed with respect to the postulated role of heparan sulfate in cell social behavior.     

The effect of beta-xylosides on heparan sulfate synthesis by SV40-transformed Swiss mouse 3T3 cells.

The medium and cell surface heparan sulfates isolated from SV40-transformed Swiss mouse 3T3 cells were examined in the presence and absence of 1.0 mM p-nitrophenyl-beta-D-xyloside. Incubation of the SV3T3 cells with this beta-xyloside resulted in: (a) a 4- to 5-fold reduction in the molecular weight distribution of medium heparan sulfate, (b) a 10-fold increase in the total synthesis of medium heparan sulfate, and (c) a small reduction in cell growth. There was little, if any, change in either the total level of synthesis or the molecular weight distribution of cell surface heparan sulfate. The covalent association of the beta-xyloside to the medium heparan sulfate was demonstrated by an analysis of the medium heparan sulfate produced by cells grown in the presence of [35S]sulfate and the fluorogenic beta-xyloside, 4-methylumbelliferyl-beta-D-xyloside. Treatment of the purified radiolabeled and fluorogenic heparan sulfate with either nitrous acid or heparitinase resulted in a decrease in the molecular weight of both radiolabeled and fluorogenic material. The data presented in this paper are discussed with respect to both the structure of heparan sulfate and the putative role of heparan sulfate in cell social behavior.     

Heparan sulfates of mouse cells. Analysis of parent and transformed 3T3 cell lines.

Heparan sulfate from the surface of a variety of mouse cells at different cell densities was examined by ion-exchange chromatography. The results of this analysis show that: (1) The heparan sulfate from new isolates of Swiss 3T3 cells transformed by SV40 virus (a DNA tumor virus) elutes from DEAE-cellulose at a lower ionic strength than that from the parent cell type. This finding confirms our earlier observation with an established SV40-transformed cell line (Underhill and Keller, '75) and eliminates the possibility that this change is caused by extended passage in culture. (2) For both parent and transformed 3T3 cells, the heparan sulfates from low and high density cultures were the same as judged by chromatography on DEAE-cellulose. This result demonstrates that the transformation-dependent change which we have observed is independent of cell density. (3) The heparan sulfate from Balb/c 3T3 cells transformed with Kirsten murine sarcoma virus (an RNA tumor virus) elutes from DEAE-cellulose prior to that from parent Balb/c 3T3 cells. This result extends the transformation dependent change in heparan sulfate to the Balb/c 3T3 cell line and to cells transformed with an RNA virus.     

The binding and processing of plasminogen by Balb/c 3T3 and SV3T3 cells

The binding and processing of plasminogen by Balb/c 3T3 and SV3T3 cells was studied using ¹²⁵I-labeled canine plasminogen. Throughout a 3-day period, ¹²⁵I-plasminogen in the incubation medium bound to the cells and was degraded, first to intermediate-sized macromolecules that were the same size as the large (74,600-dalton) and small (25,000-dalton) chains of active plasmin, and to smaller fragments including 3-iodo-L-tyrosine. Binding to SV3T3 cells was independent of the protease-dependent morphological change (PDMC)¹ characteristic of these and many other transformed cells. The SV3T3, and to a somewhat lesser extent, the 3T3 cells, both accumulated and released into the incubation medium 3-iodo-L-tyrosine, a terminal lysozymal digestion product. The results of a sublethal cell-surface trypsinization assay suggest that the cell-associated plasminogen was primarily bound to the surfaces of the 3T3 and SV3T3 cells while the macromolecular degradation products including active plasmin were inside the cells. The rate of ¹²⁵I plasminogen degradation exhibited by SV3T3 cells was approximately two times greater than that of 3T3 cells, which presumably reflects differences in endocytosis or lysosomal hydrolysis, or both. The rates were unaffected by addition of pancreatic or soybean trypsin inhibitor sufficient to inhibit PDMC. In the incubation medium, plasminogen was activated to plasmin by SV3T3, but not by 3T3 cells. However, 95–100% of plasmin covalently bound to a 47,000-dalton canine serum component, which could be dissociated from plasmin by hydroxylamine: 95–100% of the plasmin was inactive to reaction with DF³²P. Thus the serum component is a plasmin inhibitor. The plasmin-containing complex in the medium had an apparent molecular weight of 212,000. Under denaturing conditions, the complex dissociated into two covalently modified plasmin-containing species of 153,000 and 127,000 daltons. In addition to forming a complex with a serum component, the plasmin is cleaved into two small fragments (～10,000 and 12,000 daltons) by as-yet uncharacterized serum factors.     

The decreased synthesis of chondroitin sulfate-containing extracellular proteoglycans by SV40 transformed Balb/c 3T3 cells

Balb/c 3T3 cells synthesize 5–10 times more ³⁵SO₄^2?-labeled extracellular proteoglycan per cell than do Balb/c 3T3 cells transformed by SV40 (SV3T3). The extracellular ³⁵SO₄^2?-labeled proteoglycans of the Balb/c 3T3 and SV3T3 cells differ markedly in their acid mucopolysaccharide composition. Extracellular Balb/c 3T3 proteoglycans contain about 70–80% chondroitin sulfate, most of which is chondroitin 4-sulfate, and small amounts of heparan sulfate and/or heparin. On the other hand, extracellular SV3T3 proteoglycans contain 65–75% heparan sulfate and/or heparin and less than 15% chondroitin sulfate. Analysis of extracellular ³⁵SO₄^2?-labeled proteoglycan by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that Balb/c 3T3 alone synthesizes a class of proteoglycans capable of migrating in a 10% separating gel. This class of proteoglycans, designated as fraction C, accounts for up to 45% of the total extracellular Balb/c 3T3 ³⁵SO₄^2?-labeled proteoglycans and contains chondroitin sulfate exclusively. It is altogether absent in the extracellular SV3T3 proteoglycans. The absence of this and other classes of chondroitin sulfate-containing proteoglycans can account for the 5–10-fold decreased synthesis of ³⁵SO₄^2?-labeled proteoglycans by SV3T3 cells when compared to Balb/c 3T3 cells.     

High molecular-weight heparan sulfate from the cell surface

Heparan sulfate fragments with molecular weight of 135,000 (as determined by equilibrium sedimentation analysis) were isolated from the trypsinate of Chinese hamster cells (line CHO) grown in culture. Evidence is presented which suggests that the intracellular heparan sulfate species with molecular weight of 10,000 to 20,000 were degradation products of the larger species. We propose that the native cell-surface heparan sulfate, in its physiological location, could serve as a nonspecific “screen” to the exposure of specific, topographically adjacent, cell-surface sites.     

Heparan sulfates from Swiss mouse 3T3 and SV3T3 cells: O-sulfate difference

A difference in the extent of sulfation between the heparan sulfate isolated from Swiss 3T3 mouse cells and that from Swiss 3T3 cells transformed by the DNA virus SV40 has been reported previously. This variance is manifested by different chromatographic and electrophoretic properties. Heparan sulfates from the two cell types were treated with nitrous acid under conditions that gave selective deaminative cleavage of glucosaminyl residues with sulfated amino groups in order to define the nature of the difference in sulfation further. The O-sulfate containing fragments from the heparan sulfates were compared by gel filtration and ion-exchange chromatography. The results showed that the 3T3 heparan sulfate contains 8% more O-sulfate than does the SV3T3 heparan sulfate. Analysis of uronic acids revealed that both types of heparan sulfates contain 45% L-iduronic acid and 55% D-glucuronic acid. These and other observations indicate that the primary difference in sulfation between the 3T3 and SV3T3 heparan sulfates lies in the extent of O-sulfation.     

Production and characterization of a low‐molecular‐weight bacteriocin from Bacillus licheniformis MKU3

Aims: Enhancing production and characterization of a low‐molecular‐weight bacteriocin from Bacillus licheniformis MKU3. Methods and Results: The culture supernatant of B. licheniformis MKU3 exhibited bacteriocin‐like activity against Gram‐positive and ‐negative bacteria and different fungi and yeast. SDS–PAGE analysis of the extracellular proteins of B. licheniformis MKU3 revealed a bacteriocin‐like protein with a molecular mass of 1·5 kDa. This bacteriocin activity was found to be stable under a pH range of 3·0–10·0 and at temperatures up to 100°C for 60 min, but inactivated by proteinase K, trypsin or pronase E. An experimental fractional factorial design for optimization of production medium resulted in a maximum activity of bacteriocin (11 000 AU ml^?1) by B. licheniformis MKU3. Conclusions: A low‐molecular‐weight bacteriocin‐like protein from B. licheniformis MKU3 exhibited a wide spectrum of antimicrobial activity against several Gram‐positive bacteria, several fungi and yeast. A 3·6‐fold increase in the production of bacteriocin was achieved using the culture medium optimized through a fractional factorial design. Significance and Impact of the Study: A bacteriocin with wide spectrum of activity against Gram‐positive bacterial pathogens, filamentous fungi and yeast suggested its potential clinical use. Statistical method facilitated optimization of cultural medium for the improved production of bacteriocin.     

The decreased synthesis of chondroitin sulfate-containing extracellular proteoglycans by SV40 transformed Balb/c 3T3 cells.

Balb/c 3T3 cells synthesize 5--10 times more 35SO2/4- -labeled extracellular proteoglycan per cell than do Balb/c 3T3 cells transformed by SV40 (SV3T3). The extracellular 35SO2/4- -labeled proteoglycans of the Balb/c 3T3 and SV3T3 cells differ markedly in their acid mucopolysaccharide composition. Extracellular Balb/c 3T3 proteoglycans contain about 70--80% chondroitin sulfate, most of which is chondroitin 4-sulfate, and small amounts of heparan sulfate and/or heparin. On the other hand, extracellular SV3T3 proteoglycans contain 65-75% heparan sulfate and/or heparin and less than 15% chondroitin sulfate. Analysis of extracellular 35SO2/4- -labeled proteoglycan by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that Balb/c 3T3 alone synthesizes a class of proteoglycans capable of migrating in a 10% separating gel. This class of proteoglycans, designated as fraction C, accounts for up to 45% of the total extracellular Balb/c 3T3 35 SO2/4- -labeled proteoglycans and contains chondroitin sulfate extracellular SV3T3 proteoglycans. The absence of this and other classes of chondroitin sulfate-containing proteoglycans can account for the 5-10-fold decreased synthesis of 35SO2/4- -labeled proteoglycans by SV3T3 cells when compared to Balb/c 3T3 cells.     

Isolation and partial characterization of a rat hepatoma heparan sulfate proteoglycan

A proteoglycan was isolated from a Morris rat hepatoma by sequential precipitations with ammonium sulfate and cetyl pyridinium chloride followed by chromatography on Sepharose CL-4B and DEAE-cellulose. The proteoglycan has a molecular weight of about 1.5 × 10⁵ with 40,000 molecular weight glycosaminoglycan side chains, identified as heparan sulfate based on resistance to chondroitinase and susceptibility to nitrous acid treatment. Immunological studies showed that the protein core of this proteoglycan is immunologically distinct from a rat yolk sac tumor chondroitin sulfate proteoglycan (Å. Oldberg, E. G. Hayman, and E. Ruoslahti, 1981,J. Biol. Chem.256, 10847–10852), but resembles a heparan sulfate proteoglycan isolated from a basement membrane-producing mouse tumor (J. R. Hassell, P.M. Robey, H.-J. Barrach, J. Wilczek, S. R. Rennard, and G. R. Martin, 1980, Proc. Nat. Acad. Sci. USA77, 4494–4498).     

Quiescent SV40 virus transformed 3T3 cells in culture

Serum counteracts low nutrient concentrations in the culture medium in SV40 virus transformed 3T3 (SV3T3) cells. The transport of [³H]-leucine into TCA soluble material in SV3T3 cells is stimulated by serum and inhibited by But₂-cAMP. When SV3T3 cells are cultured in low leucine concentrations (? 8 × 10^?6 M), the cell's morphology is similar to the one of cells incubated in complete medium in the presence of But₂-cAMP and cells become quiescent. Cells become arrested throughout the cell cycle. The results suggest that the mechanism by which But₂-cAMP inhibits growth of SV3T3 cells is by inhibiting the transport of leucine in SV3T3 cells.     

Biosynthesis of glycosaminoglycans and proteoglycans by the lymph node

Previous studies of hyaluronan uptake and catabolism by lymph nodes indicated that the nodes might also add some HA of low molecular weight to the unabsorbed fraction that passes through from afferent to efferent lymph vessels.The ability of lymph nodes to synthesise HA and proteoglycans was therefore examined (i) by perfusion of [³H] acetate through an afferent lymph vessel in vivo, and recovery of labeled products from the efferent lymph vessel and from the node after perfusion; and (ii) by tissue culture of lymph nodes with [³H] acetate.Perfusion of lymph nodes with [³H] acetate in situ yielded: (a), in outflowing lymph, small amounts of chondroitin/dermatan sulfate within the first hour which continued to be produced for up to 24[emsp4 ]h; heparin in the second hour and HA in the third. In the nodes removed 17 to 19[emsp4 ]h later, equal amounts of hyaluronan and chondroitin/dermatan sulfate and heparan sulfate proteoglycans were detected. In the tissue culture of lymph nodes: (1) HA, heparin and proteoglycans of heparan sulfate and chondroitin/dermatan sulfate were released into the medium but in the cell extract only heparan sulfate proteoglycan was detected; and (ii) molecular weight of the released hyaluronan ranged widely but was mostly less than 4–5×10⁵[emsp4 ]D; heparan sulfate proteoglycan was 2.8×10⁴ to 9.4×10⁵[emsp4 ]D; heparin 7.9×10⁴[emsp4 ]D and chondroitin sulfate 1.3×10⁴[emsp4 ]D, suggesting that the chondrotin sulfate were released from their proteoglycans core by enzymic degradation.It is concluded that lymph nodes can release HA, heparin, heparan sulfate and chondroitin/dermatan sulfate proteoglycans into efferent lymph but the amount of hyaluronan is likely to be small without immune or other stimulation and its molecular weight is lower than in other tissues.     

Biochemical Characterization of Secreted Proteases During Growth in Tetrahymena pyriformis WH-14: Comparison of Extracellular with Intracellular Proteases

Tetrahymena pyriformis strain WH-14 secreted large quantities of intracellular proteases into its culture medium during growth. Extracellular enzymes were purified to homogeneity from cell-free medium by ammonium sulfate precipitation, CM-Sephadex column chromatography, gel filtration, and DEAE-cellulose column chromatography. The DEAE-cellulose eluates were separated into four peaks (P-I, P-II, P-III, and P-IV), each of which exhibited a different specific activity toward azocasein and α-N-benzoyl-DL-arginine-ρ-nitroanilide (Bz-Arg-Nan). These four forms of the protease showed similarity in amino acid composition, molecular weight (21,000–24,000), and antigenic reactivity. They had pH optima at neutral range. P-I showed the highest specificity to azocasein whereas P-IV was most effective toward the synthetic substrates. The Km values for hydrolysis of Bz-Arg-Nan were 2.4, 1.6, 1.3, and 1.4 mM for P-I, P-II. P-III, and P-IV, respectively, and the corresponding Kcat/Km values were 5.0, 9.4, 28.5, and 114.3 S^-1.M^-1. These properties of secreted proteases were compared with those of intracellular proteases purified by the same procedure except for the initial Triton X-100 extraction. There were similarities in specific activity toward two substrates, molecular weight, Km, pH optima, and antigenic reactivity between the proteases from two sources, providing evidence that the intracellular proteases may be secreted into the extracellular medium without modification.     

Heparin releases heparan sulfate from the cell surface.

Heparan sulfate of the cell surface of cultured Chinese hamster cells (line CHO) was promptly released when the cells were incubated with balanced salt solutions containing heparin. The released heparan sulfate included multichain proteoglycan of high molecular weight. The data suggest that the cell-surface localization of heparan sulfate is dependent, at least in part, upon cell-surface receptors with binding sites for the sugar chain moieties of sulfated glycosaminoglycans.     

Effect of monensin on the sulfation of heparan sulfate proteoglycan from endothelial cells.

Monensin is a monovalent metal ionophore that affects the intracellular translocation of secretory proteins at the level of trans-Golgi cisternae. Exposure of endothelial cells to monensin results in the synthesis of heparan sulfate and chondroitin sulfate with a lower degree of sulfation. The inhibition is dose dependent and affects the ratio [35S]-sulfate/[3H]-hexosamine of heparan sulfate from both cells and medium, with no changes in their molecular weight. By the use of several degradative enzymes (heparitinases, glycuronidase, and sulfatases) the fine structure of the heparan sulfate synthesized by control and monensin-treated cells was investigated. The results have shown that among the six heparan sulfate disaccharides there is a specific decrease of the ones bearing a sulfate ester at the 6-position of the glucosamine moiety. All other biosynthetic steps were not affected by monensin. The results are indicative that monensin affects the hexosamine C-6 sulfation, and that this sterification is the last step of the heparan sulfate biosynthesis and should occur at the trans-Golgi compartment.     

Cell surface proteoglycans as a negative modulator in concanavalin a-mediated agglutination of hepatoma cells

Proteoglycan (heparan sulfate-protein conjugate) was solubilized with 8 M urea from rat liver plasma membranes after enzymic (RNAase, neuraminidase) treatments and extensively purified by chromatography and gel filtration. The final products gave an average ratio of hexuronate to protein (weight) of approx. 1.5, contained hexosamine equimolar to hexuronate and were sensitive to β-elimination (the molecular weight being reduced from 20 · 10⁴ to 3 · 10⁴ (gel filtration)).The proteoglycan fraction, when added to trypsinized and untrypsinized ascites hepatoma (AH-130F(N)) cells, inhibited the concanavalin A-mediated agglutination of the cells. However, the alkali-treated proteoglycan (β-elimination) or acid mucopolysaccharide fraction prepared from liver plasma membranes by papain digestion were less effective, and a reference preparation of heparan sulfate was almost ineffective. It was confirmed that significant amounts of proteoglycan labelled with ³⁵SO₄^2? were firmly bound to or taken up by the trypsinized ascites hepatoma cells.These results together with the sensitization of lectin-mediated agglutination by mild protease treatment of cells suggest that cell surface proteoglycans may act as a negative modulator in the lectin-mediated agglutination of cells.     

Modification of transplasma membrane oxidoreduction by SV40 transformation of 3T3 cells

Transformation of 3T3 cells by SV40 virus changes the properties of the transplasma membrane electron transport activity which can be assayed by reduction of external ferric salts. After 42 h of culture and before the growth rate is maximum, the transformed cells have a much slower rate of ferric reduction. The change in activity is expressed both by change inK _m andV _max for ferricyanide reduction. The change in activity is not based on surface charge effect or on tight coupling to proton release or on intracellular NADH concentration. With transformation by SV40 virus infection the expression of transferrin receptors increases, which correlates with greater diferric transferrin stimulation of the rate of ferric ammonium citrate reduction in transformed SV40-3T3 cells than in 3T3 cells.     

The serum growth and survival requirements of SV40-transformed 3T3 cells

Summary Simian virus 40-transformed 3T3 cells are dependent on serum for survival and growth. This growth activity can be separated on a pH 2 Sephadex G100 column into two fractions: a high molecular weight activity and a low molecular weight substance that has recently been characterized as containing as its major agent, biotin. To replace the remainder of the serum requirement, hormones and other growth factors were tested. Both insulin at high, nonphysiological concentrations (200 to 500 ng/ml) and transferrin (5×10⁻⁸ M) stimulate the growth rate in low serum medium (0.3% v/v bovine calf serum DME) individually and, when added together, are nearly as growth enhancing as 10% serum. The need for the residual serum in this medium can be eliminated by the use of crystalline trypsin during trypsinization. Under these serum-free conditions, biotin and transferrin supplementation provide for moderately good growth (20 to 30 hr population doubling time, 1×10⁶ cells/3.2-cm dish final cell density). Insulin addition further stimulates the growth rate (16 to 20 hr) and the final density (1.5×10⁶ cells). Although the protein growth factors, EGF (0.5 to 1.0 ng/ml) and FGF (4 to 10 ng/ml), also appear to enhance growth individually and additively, their effects are slight and very variable. Nevertheless, the complete serum-free medium (DME supplemented with biotin, transferrin, insulin, EGF and FGF) yields growth comparable but still inferior to 10% serum supplementation (14-versus 12-hr population doubling time, 1 to 2×10⁶ versus 2 to 3×10⁶ cells final cell density). This work was supported by NIH Grant CA 20040.     

Altered metabolism of heparan sulfate in simian virus 40 transformed cloned mouse cells.

Glycoconjugates have been analyzed from a family of closely related mouse cells: a parent clone and three daughter subclones, two of which expressed the simian virus 40 (SV40) T-antigen. The experimental procedure involved the simultaneous comparison by DEAE-cellulose chromatography of papain-digested macromolecules from the parent, labeled with [3H]glucosamine, and one of the daughter subclones, labeled with [14C]-glucosamine. Three cultures compartments (the medium, the cell surface trypsinate, and the cells) from the paired cell lines were combined at the earliest time during the harvesting of the cells. Heparan sulfate on the surface of cells and secreted into the medium from T-antigen-positive subclones was eluted at lower salt concentrations from the anion exchange column than that from the parent clone. In the viable trypsinized cells a marked reduction of heparan sulfate was detected in the T-antigen-positive subclones. These changes were highly reproducible, were observed during both logarithmic and stationary phase of growth, and neither change was observed in the T-antigen-negative sister subclone. The elution point of heparan sulfate from Sepharose 6B was unaltered. Ratios of 35S to 3H for heparan sulfate obtained from cells doubly labeled with [35S]sulfate and [3H]glucosamine were lower in the T-antigen-positive subclones. Similar changes for the 35S to 3H ratio of chondroitin sulfate were associated with only small alterations in elution from anion exchange columns. Kinetic experiments suggested a reduced rate of incorporation of [35S]sulfate with no change in turnover rate. A substantial portion of the labeled heparan sulfate was associated with the cell surface; in contrast most of the hyaluronic acid and a large proportion of the chondroitin sulfate was apparently secreted. Quantitative changes in hyaluronic acid labeling did not correlate with expression of T-antigen. Glycosaminoglycans left on the dish after detaching cells with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid were nearly completely released by subsequent trypsinization. Cell detachment by trypsinization left an insignificant amount of labeled glycosaminoglycan on the dish surface. The alterations in heparan sulfate metabolism correlated with the expression of T-antigen and with the cells' ability to grow to high densities in monolayer culture, but not with growth in suspension in viscous medium. Tumorigenicity of the subclones was essentially the same as that of the parent clone.     

Regulation of proteoglycan and hyaluronan synthesis by elevated level of intracellular cyclic adenosine monophosphate in peritubular cells from immature rat testis

The effects of an increase in intracellular cAMP concentration on proteoglycan (PG) synthesis by peritubular (PT) cells from immature rat testis were investigated. In the presence of dBcAMP for 72 h, the [³H]-hexosamine incorporation in secreted PG and in cellassociated PG was reduced, whereas [³⁵S]-sulfate radioactivity was enhanced in secreted PG and not affected in cell-associated PG. Cholera toxin and IBMX, known to generate high intracellular cAMP levels, induced similar changes. Cyclic AMP did not alter PG protein moiety synthesis but enhanced PG turnover. Cholera toxin and dBcAMP profoundly modified PG characteristics: (1) Apparent molecular weight of PG was increased. (2) This was due to an increase in glycosaminoglycans (heparan sulfate (HS) and chondroitin sulfate (CS)) length. (3) The number of glycosaminoglycan chains was presumably reduced. (4) Heparan sulfate and chondroitin sulfate chains of medium and cell layer-associated PG appeared oversulfated. (5) The pattern of cell layer associated PG was modified with a decrease in HSPG and a correlative increase in CSPG. Cholera toxin and dBcAMP also dramatically stimulated hyaluronan synthesis by possible phosphorylation induced activation of hyaluronan synthase(s).