Impaired intracellular migration and altered solubility of nonglycosylated glycoproteins of vesicular stomatitis virus and Sindbis virus.

Tunicamycin, an antibiotic which prevents the glycosylation of newly synthesized proteins, inhibits the replication of both vesicular stomatitis virus and Sindbis virus. In tunicamycin-treated infected cells, all of the viral proteins are synthesized but the glycoproteins are devoid of carbohydrate. The nonglycosylated glycoproteins could not be detected on the outside of the plasma membrane by lactoperoxidase labeling, indirect immunofluorescence staining, or chymotrypsin treatment of intact cells, whereas the glycosylated glycoproteins were readily detected by all three methods. These results indicate that the bulk of the nonglycosylated glycoproteins are unable to undergo the normal migration to the cell surface. In contrast to the normal glycosylated viral glycoproteins, the nonglycosylated glycoproteins were insoluble in nonionic detergents such as Triton X-100. The nonglycosylated glycoprotein of vesicular stomatitis virus could be solubilized using a combination of 6 M guanidine hydrochloride and 0.2% Triton X-100, but precipitated when the 6 M guanidine was removed by dialysis. These results suggest that the lack of carbohydrate alters the properties of the glycoproteins, which may explain their impaired mobility through the intracellular membranous system.     

Study of detergent-mediated liberation of hepatitis B virus-like particles from S. cerevisiae homogenate: identifying a framework for the design of future-generation lipoprotein vaccine processes

Virus-like particles (VLPs) are expressed intracellularly in Saccharomyces cerevisiae and the recovery process involves the use of a detergent, which facilitates the release of VLP from host cell components. The detergent-mediated liberation of VLPs is a critical step in primary recovery and is responsible for setting the backdrop for subsequent purification in terms of product yield and characteristics of the process stream. In this paper the use of Triton X-100 detergent for the recovery of lipid envelope VLPs, using the hepatitis B surface antigen (HBsAg) as the VLP model, was investigated. To develop a framework that can be adopted in process design for future generation VLP vaccine candidates, the impact of Triton X-100 was characterized via different response factors: (i) recovery and activity of the HBsAg; (ii) level of protein and lipid contamination from the host cell; and (iii) indirect impact on the performance of an ultrafiltration step following primary recovery. Our studies identified that an increase in detergent concentration favors recovery of HBsAg only to a specific threshold, 0.5% v/v Triton X-100. Further increase in detergent results in delipidation of HBsAg leading to loss in antigenic activity. The level of contamination due to host protein and lipid co-liberation is in proportion with the amount of detergent employed. Greater membrane resistance during ultrafiltration was observed for samples generated using higher concentrations of detergent due to the increase in membrane fouling by the contaminants. Based on this study, Triton X-100 concentrations in the range of 0.2-0.5% v/v appears to be most suitable for recovery of native HBsAg. Choosing between 0.2-0.5% v/v would involve identifying a suitable tradeoff between desired product yield and the level of contamination that can be tolerated by downstream operations.     

A cytoskeleton-associated plasma membrane heparan sulfate proteoglycan in Schwann cells

Schwann cells cocultured with sensory neurons in a serum-free medium accumulate a single species of radiolabeled heparan sulfate proteoglycan (HS-PG) during incubation in medium containing 35SO4. This HS-PG was poorly extracted from cultures by solutions containing 1% Triton X-100 in low salt buffer or by solutions containing 1 M KCl, 4 M urea plus dithiothreitol, 1 mM Tris-HCl, 5 mM EDTA, or 100 micrograms/ml of heparin. The HS-PG was efficiently extracted, however, by 1% Triton X-100 in the presence of 1 M KCl or by 1% deoxycholate. These treatments solubilize both cell membranes and the Schwann cell cytoskeleton. In intact cells the HS-PG was digested by trypsin, indicating it was at least partially exposed on the cell surface. When solubilized HS-PG was applied to a column of octyl-sepharose CL-4B, more than 90% was retained by the column, but was quantitatively eluted by a solution containing 1% Triton X-100. In addition, the solubilized HS-PG could be incorporated into artificial phospholipid vesicles. These results indicate the HS-PG is an integral plasma membrane protein. The inability of low ionic strength solutions containing Triton X-100 to solubilize the HS-PG suggested it was bound to an additional structure. To determine whether the HS-PG was associated with the cytoskeleton we isolated cytoskeletons by detergent lysis of cells and centrifugation. The major protein components of isolated cytoskeletons were spectrin (Mr 225,000), vimentin (Mr 58,000), and actin (Mr 45,000). When 35SO4-labeled cells were used to prepare cytoskeletons approximately 80% of the total HS-PG was recovered in the cytoskeleton fraction. These results suggest the HS-PG is an externally exposed integral plasma membrane protein that is anchored to the Schwann cell cytoskeleton.     

Mechanism of autolysis of Neisseria gonorrhoeae.

The major autolysin(s) of Neisseria gonorrhoeae was solubilized from envelopes by extraction with 2% Triton X-100 containing 0.5 M NaCl. Neither Triton X-100 nor NaCl alone could effectively release the autolysin(s). The major autolysin is N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28). The pH optimum for this reaction was broad, ranging from 5.5 to 8.5. Optimal hydrolysis of peptidoglycan occurred in 2% Triton X-100 in 0.1 M KCl. Attempts to purify the autolysin were unsuccessful. A rapid assay for enzyme activity was developed using radioactive cell walls as a substrate ([3H]diaminopimelic acid).     

Recovery of a foreign protein from the periplasm of Escherichia coli by chemical permeabilization.

We have applied the technique of protein release by chemical permeabilization to recover a foreign protein in active form from the periplasm of a recombinant strain of Escherichia coli. The two agents used in our chemical permeabilization scheme, guanidine hydrochloride and Triton X-100, have different modes of action, allowing selectivity in protein release based on intracellular location under different treatment conditions. Specifically, treatment of E. coli C600-1 cells by guanidine alone resulted in 40-fold purification of recombinant beta-lactamase, which is periplasmically expressed in this host. Achieving such high purification in the cell disruption stage could alleviate some of the problems associated with recovery of intracellular products, such as low expression or the need to solubilize cytoplasmic inclusion bodies. Recovery of periplasmic proteins by chemical permeabilization is simpler than by osmotic shock and is less expensive than using enzymatic digestion.     

Use of resistant mutants to study the interaction of triton X-100 with Staphylococcus aureus.

Staphylococcus aureus mutants resistant to the nonionic detergent Triton X-100, isolated from the wild-type strain H and the autolysin-deficient strain RUS3, could grow and divide in broth containing 5% (vol/vol) Triton X-100, while growth of the parental strains was markedly inhibited above the critical micellar concentration (0.02%) of the detergent. Growth-inhibitory concentrations of Triton X-100 killed wild-type cells without demonstrable cellular lysis. Triton X-100 stimulated autolysin activity of S. aureus cells under nongrowing conditions, and this lytic response was markedly reduced in energy-poisoned cells. In contrast, the detergent had no effect on the activity of autolysins in cell-free systems, and growth in the presence of Triton X-100 did not alter either the cellular autolysin activity or the susceptibility of cell walls to exogenous lytic enzymes. Treatment with either Triton X-100 or penicillin G in the growth medium stimulated release of predominantly acylated intracellular lipoteichoic acid and sensitized staphylococci to Triton X-100-induced autolysis. There was no significant difference in the cell wall and membrane compositions or Triton X-100 binding between the parental strains and the resistant mutants. The resistant mutant TXR1, derived from S. aureus H, had a higher level of L-alpha-glycerophosphate dehydrogenase activity, and its oxygen uptake was more resistant to inhibition by a submicellar concentration (0.008%) of Triton X-100. Growth in the presence of subinhibitory concentrations of Triton X-100 rendered S. aureus H cells phenotypically resistant to the detergent and greatly stimulated the level of oxygen uptake. Membranes isolated from such cells exhibited enhanced activity of the respiratory enzymes succinic dehydrogenase and L-alpha-glycerophosphate dehydrogenase.     

Structure of the Proteolipid Protein Extracted from Bovine Central Nervous System Myelin with Nondenaturing Detergents

As a basis for attempts to define the structures of the proteins within myelin, methods have been developed for their extraction and isolation in solutions of non-denaturing detergents. With use of solutions of deoxycholate or Triton X-100, up to 90% of the protein has been extracted from bovine CNS myelin, along with most of the phospholipid. The proteolipid protein has been purified in deoxycholate solutions by chromatography on a blue dye-ligand column, which retained all of the basic protein and 2',3'-cyclic nucleotide-3'-phosphodiesterase, and then on Sephacryl S300, which separated proteolipid protein from phospholipid and high-molecular-weight proteins. The proteolipid protein was isolated from Triton X-100 extracts of myelin by adsorption onto phosphocellulose resin, with subsequent elution by 0.5 M sodium chloride. Gel permeation chromatography was used as the final purification step. Sedimentation equilibrium experiments gave a monomer molecular weight of 134,000 +/- 8000 in deoxycholate and 145,000 +/- 17,000 in Triton X-100 solutions. On the basis of an apparent subunit molecular weight of 23,500 it was deduced that the native protein is probably hexameric. Above 0.2 gL-1 in Triton X-100 solutions and 0.5 gL-1 in deoxycholate solutions the protein aggregated. In deoxycholate solutions the protein adopts the highly helical conformation expected for an intrinsic membrane protein.     

Detergent-resistant variants of Bacillus subtilis with reduced cell diameter.

Variants of Bacillus subtilis resistant to the detergent Triton X-100 may exhibit: (i) normal cell morphology, (ii) reduced cell diameter, or (iii) helical cell shape. One variant of type ii was studied in some detail. Triton resistance, cell diameter reduction, and poor sporulation all may have resulted from a single mutation. High concentrations of Triton caused rapid lysis of wild-type cells. B. subtilis adapted to low Triton concentrations such that, upon subsequent exposure to higher concentrations, growth continued, although it bacame inhibited at very high concentrations. The variant studied retained its sensitivity to Triton-induced lysis but, after adaptation, grew at very high Triton levels. In this strain, cell diameter and cross-sectional area were reduced to about 73 and 50%, respectively, of those of wild type, yet the cells grew at normal rates, and DNA/protein/RNA ratios were largely unaltered. Peptidoglycan content per unit of cell surface area was higher in the variant than in the wild type under at least certain growth conditions.     

Triton solubilization of proteins from pig liver mitochondrial membranes

Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X-100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well-defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined.     

Rapid and efficient extraction of soluble proteins from gram-negative microorganisms without disruption of cell walls

The ability of buffer solutions containing low concentrations of nonionic detergents (Triton X-100, Tween 20, Brij 58, and Lubrol PX) and the anionic detergent sodium deoxycholate, as well as mixtures of these detergents with chaeotropes (urea and guanidine hydrochloride), to extract intracellular proteins of Gram-negative microorganisms (Escherichia coli and Pseudomonas aeruginosa) was studied. It was established that the solutions containing Triton X-100 and sodium deoxycholate and the mixtures of these detergents with urea are the most effective. It was shown that the extraction of proteins from bacterial cells under the studied conditions is not accompanied by a release of DNA into solution but is associated with extraction of low-molecular RNAs. The level of protein extraction reaches 80%. No disruption of the bacterial cell wall occurs during the extraction, and proteins probably permeate through meshes of the murein network. The efficiencies of our buffer mixtures are close to or higher than that of the commercial reagent CelLytic B (Sigma, United States). The practical uses of the chaeotropic mixtures developed are discussed.     

Sulfated proteoglycans and sulfated proteins in guinea pig megakaryocytes and platelets in vivo. Relevance to megakaryocyte maturation and platelet activation

This study has examined changes in proteoglycan synthesis during megakaryocyte maturation in vivo. Guinea pigs were injected with Na235SO4, and megakaryocytes and platelets were isolated from 3 h to 5 days later. The proteoglycans and other sulfated molecules in both cells were characterized at each time point by gel filtration, ion-exchange chromatography, gel electrophoresis, and chemical and enzymatic digestions. Two populations of chondroitin 6-sulfate proteoglycans were found by DEAE-Sephacel chromatography. The major fraction was eluted with 4 M guanidine hydrochloride and the minor fraction with 4 M guanidine HCl, 2% Triton X-100. The Kav of the major proteoglycan peak in the platelets at 1 day after injection was 0.18-0.20 on Sepharose CL-6B and decreased gradually to 0.12 by 3 days, when proteoglycan radioactivity per cell was maximal. The peak for megakaryocyte proteoglycans at 3 h was broad, with Kav = 0.1-0.2. The appearance of different portions of the proteoglycan peak in platelets coincided with their disappearance from megakaryocytes. Proteoglycan size was a function of glycosaminoglycan chain length. The proteoglycans eluted with Triton X-100 from DEAE-Sephacel (Kav = 0.04-0.07 on Sepharose CL-6B) were not labeled in platelets until 2 days after injection. Our data suggest that megakaryocytes synthesize different-sized chondroitin sulfate proteoglycans at different stages of development. The proteoglycans of the major fraction were released from platelets in response to thrombin, and a small amount was released by ADP. The proteoglycans of the Triton X-100 eluate were not released by thrombin or ADP. About 20% of the sulfate radioactivity was incorporated into molecules that appear to be sulfated proteins and were not released by thrombin or ADP.     

Improvement of the extraction of penicillin acylase from Escherichia coli cells by a combined use of chemical methods

Several techniques for protein extraction were tested for recovering penicillin acylase from a recombinant strain of Escherichia coli. These techniques include chemical [guanidine hydrochloride, Triton X-100, ethylenediaminetetraacetic acid (EDTA), ethanol/toluene], physical (sonication, freeze-and-thawing), and enzymatic (lysozyme) treatments. Best results were obtained with the combined use of guanidine and EDTA. This extraction procedure was optimized, and it was found that 95% of the enzyme was extracted after a 10 m/M EDTA plus 10 mM guanidine treatment at room temperature for 10 h. The purification factor was 25 when compared to disruption by sonication. This extraction method could avoid purification steps for particular applications. (c) 1994 John Wiley & Sons, Inc.     

Vesicle release from rat red cell ghosts and increased association of cell membrane proteins with cytoskeletons induced by cadmium

When rat red cell ghosts were incubated with 0.1-0.5 mM CdCl2 in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, they became irregular in shape and released small vesicles. The release of vesicles was dependent on the incubation temperature and Cd2+ concentration. The maximum release occurred at 37 degrees C in the presence of 0.2 mM Cd2+. The protein composition of Cd2+-induced vesicles was similar to that of the vesicles released from ATP-depleted red cells. Upon incubation with 0.1-0.2 mM Cd2+, more than 90% of the Cd2+ added to the incubation buffer was recovered in ghosts and 15-20% of the ghost Cd2+ was located on the cytoskeletons prepared by washing ghosts with 0.5% Triton X-100 solution containing 0.1 M KCl and 10 mM Tris-HCl (pH 7.4). Moreover, the cytoskeletons prepared from Cd2+-treated ghosts markedly contained cell membrane proteins, bands 2.1, 3, 4.2 and 4.5, and glycophorins. The association of bands 3 and 4.2 with cytoskeletons increased with increasing concentrations of Cd2+ added to the incubation buffer and saturated at 0.2 mM Cd2+. The solubilization of cytoskeletal proteins, bands 1, 2 and 5, from ghosts at low ionic strength was almost completely suppressed by preincubation of ghosts with 0.1 mM Cd2+. HgCl2, PbCl2 and ZnCl2 at 0.2 mM each also produced an increased association of cell membrane proteins with cytoskeletons, whereas CaCl2 and MgCl2 did not.     

Inactivation of the calcium-transport ATPase in the sarcoplasmic reticulum by the combined effect of lasolocid and Triton X-100

The calcium-transport ATPase of the sarcoplasmic reticulum membranes is irreversibly inactivated by the combined action of Lasolocid and Triton X-100 at concentrations which separately do not interfere with the enzyme's activity. In the presence of Lasolocid the enzyme is most susceptible to inactivation when the Triton X-100 concentration just exceeds its critical micellar concentration, approximately, 0.2 mg X ml-1. Lasolocid becomes effective at a concentration of 10 microM and produces rapid inactivation at 100 microM. Phosphoprotein formation is less affected than phosphate liberation. The influence of the ATPase protein on the fluorescence intensity of Lasolocid passes a distinct maximum at the most effective Triton X-100 concentration of 0.2 mg X ml-1.     

Release kinetics of ATP in cells exposed to nonionic detergents

We have previously shown that the protein binding of intracellular ATP could be examined by monitoring the ATP release kinetics from Triton X-100 and Brij 58 nonionic detergent permeabilized cells. We have now analysed the protein binding of ATP in an isotonic medium using intact and partially ATP depleted Brij 58 treated human erythrocytes. The effects of Triton X-100 below the critical micelle concentration (CMC) was studied in normal and tumorous tissue culture cells and human red blood cells. Our results showed that the protein association of ATP was altered in the partially ATP depleted erythrocytes. Below the CMC value, but above a critical level Triton X-100 treatment was effective in mobilizing the intracellular ATP in both cell types. The ATP release curves were sigmoidal and an ‘all or none’ type of response was observed, especially in erythrocytes. The use of Triton X-100 (< CMC) delays the detergent-induced cell decomposition time thus providing a new approach to investigating the physical state of intracellular ATP.     

Rapid and efficient extraction of soluble proteins from Gram-negative microorganisms without disruption of cell walls

The ability of buffer solutions containing low concentrations of nonionic detergents (Triton X-100, Tween 20, Brij 58, and Lubrol PX) and the anionic detergent sodium deoxycholate, as well as mixtures of these detergents with chaeotropes (urea and guanidine hydrochloride), to extract intracellular proteins of Gram-negative microorganisms (Escherichia coli and Pseudomonas aeruginosa) was studied. It was established that the solutions containing Triton X-100 and sodium deoxycholate and the mixtures of these detergents with urea are the most effective. It was shown that the extraction of proteins from bacterial cells under the studied conditions is not accompanied by a release of DNA into solution but is associated with extraction of low-molecular RNAs. The level of protein extraction reaches 80%. No disruption of the bacterial cell wall occurs during the extraction, and proteins probably penetrate through meshes of the murein network. The efficiencies of our buffer mixtures are close to or higher than that of the commercial reagent CelLytic B (Sigma, United States). The practical uses of the chaeotropic mixtures developed are discussed.     

Role of phospholipid and protein-protein associations in activation and stabilization of soluble Ca2+-ATPase of sarcoplasmic reticulum

The effect of increasing concentrations of the nonionic detergent Triton X-100 on catalytic activity, stability, phospholipid content, and aggregational state of solubilized Ca2+ ion activated adenosinetriphosphatase (Ca2+-ATPase) of sarcoplasmic reticulum has been investigated. Increasing concentrations of Triton X-100 in the range 0.2-0.6% (w/v) inhibited ATP hydrolysis and p-nitrophenyl phosphate hydrolysis in parallel to the extent of 50% and 95%, respectively. Inactivation of p-nitrophenyl phosphate hydrolysis by preincubation in excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at 25 degrees C was monophasic and first order at all concentrations of Triton X-100. The rate constant for inactivation increased sharply in the range 0.1-0.6% Triton X-100. At higher concentrations, the increase was less marked. Protein-protein associations of the solubilized ATPase were assessed by glutaraldehyde cross-linking and by ultracentrifugation in sucrose gradients. Both methods indicated a decrease in these associations in the 0.1-0.5% range. Cross-linking studies established that above 0.5% Triton X-100 the enzyme is greater than 90% monomeric. The amount of phospholipid associated with the ATPase, recovered from sucrose gradients, decreased from about 50 mol of phospholipid/mol of ATPase at 0.1% Triton X-100 to about 3 mol of phospholipid/mol of ATPase at 0.5% and higher concentrations. Monomeric ATPase and aggregated ATPase isolated from equilibrium mixtures of these components had similar phospholipid/protein ratios. The results indicated that with increasing Triton X-100 concentrations, inhibition of catalysis, destabilization, loss of protein-protein associations, and loss of phospholipid occur concurrently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interrelationships between protein kinases and spectrin phosphorylation in human erythrocytes

Casein kinase and histone kinase(s) are solubilized from human erythrocyte membranes by buffered ionic solutions (0.1 mM EDTA and subsequent 0.8 M NaCl, pH 8) containing 0.2% Triton X-100. Casein kinase is separated from histone kinase(s) by submitting the crude extracts directly to chromatography on a phosphocellulose column, eluted with a continuous linear gradient of potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100. Under these conditions, the membrane-bound casein kinase activity is almost completely recovered into a quite stable preparation, free of histone kinase activity. In contrast, it undergoes a dramatic loss of activity when the extraction and the subsequent phosphocellulose chromatography are carried out with buffers which do not contain Triton X-100. Isolated spectrin, the most abundant membrane protein, is phosphorylated, in the presence of [gamma-32P]ATP, only by casein kinase while histone kinase is ineffective. Only the smaller subunit (band II) of isolated spectrin (and not the larger one (band I) is involved in such a phosphorylation process, as in the endogenous phosphorylation occurring in intact erythrocytes.     

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.     

Isolation of the major chondroitin sulfate/dermatan sulfate and heparan sulfate proteoglycans from embryonic chicken retina

A technique is presented for the preparation of three major proteoglycans from 14-day embryonic chicken retinas following their culture overnight with [35S]sulfate and either [3H]glucosamine or [3H]serine. Homogenization of the tissue in saline permitted extraction of heterogeneous soluble proteoglycans separately from most of the heparan sulfate proteoglycans. The latter were extracted from the 140,000g pellet with 0.5% Triton X-100 in 8 M urea. The medium plus the saline and urea-detergent extracts were separated from low-molecular-weight contaminants, and fractionated into two peaks of radioactivity on Sephacryl S-300 in saline with 3 M urea and 0.5% Triton X-100. The proteoglycans were isolated directly from these fractions on DEAE-Sephacel, and subjected to ultrafiltration concentration and then further purification on cesium chloride density gradient centrifugation in 4 M guanidine hydrochloride. A further step involving cetylpyridinium chloride precipitation was examined, but it resulted in essentially no further purification. The fractionations separated a large chondroitin sulfate/dermatan sulfate proteoglycan from the culture medium that was excluded from S-300 and of low buoyant density; a large heparan sulfate proteoglycan from the urea-detergent extract that was also excluded from S-300 and of low buoyant density; and two smaller and possibly related heparan sulfate proteoglycans. One was found in the medium and showed low to intermediate buoyant density; the other was isolated from the urea-detergent extract and showed a significantly higher buoyant density, associated with a lower protein content. The saline extract contained both of the two larger proteoglycans and only minor amounts of the smaller molecules.