Effect of the composition of sodium dodecyl sulfate preparations on the renaturation of enzymes after polyacrylamide gel electrophoresis

The extent of renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate depended on the source of the detergent. Analysis of commercial preparations of sodium dodecyl sulfate revealed appreciable amounts of tetradecyl and hexadecyl sulfates in some preparations. Inhibition of renaturation was correlated with the amount of hexadecyl sulfate and, to a much lesser extent, of tetradecyl sulfate present. The higher alkyl sulfates appeared to bind more tenaciously to proteins in the gel. More extensive washing was required to remove them than to remove dodecyl sulfate, and they were inhibitory to enzyme activity at lower detergent concentrations. A system is described for gas chromatographic analysis of alkyl sulfates containing chains of 10 to 16 carbon atoms in length.     

Electrophoretic separation and characterization of urinary glycosaminoglycans and their roles in urolithiasis

Urinary polyanions recovered from the urine samples of kidney stone-formers and normal controls were subjected to preparative agarose gel electrophoresis, which yielded fractions 1-5 in a decreasing order of mobility. In both groups, chondroitin sulfates were identified in the fast-moving fractions and heparan sulfates in the slow-moving fractions. Furthermore, two types of heparan sulfates were identified based on their electrophoretic mobility: slow-moving and fast-moving. The fractionated urinary polyanions were then tested in an in vitro calcium oxalate crystallization assay and compared at the same uronic acid concentration, whereby, the chondroitin sulfates of stone-formers and heparan sulfates of normals enhanced crystal nucleation. Fraction 5 of the normals, containing glycoproteins (14-97 kDa) and associated glycosaminoglycans, were found to effectively inhibit crystallization. Papainization of this fraction in stone-formers revealed crystal-suppressive effects of glycoproteins, which was not seen in similar fractions of normals. It was concluded that glycoproteins could modulate the crystal-enhancing glycosaminoglycans such as chondroitin sulfates of stone-formers but not in normals. The differing crystallization activities of electrophoretic fraction 1 of normals and stone-formers revealed the presence of another class of glycosaminoglycan-hyaluronan. Hence, in the natural milieu, different macromolecules combine to have an overall outcome in the crystallization of calcium oxalate.     

Acrylamide gel electrophoresis of acid glycosaminoglycans for the study of molecular size distribution

Chondroitin sulfates from bovine nasal septum and lamprey cartilage and keratan sulfate from human costal cartilage show a wide molecular size polydispersity by acrylamide gel electrophoresis. The number of fractions were limited only by the number of slices obtained from gel slabs after electrophoresis. The polysaccharides are probably distributed in a continuum of varying chain lengths.For the study of the size distribution of sulfated glucosaminoglycan by acrylamide gel disc electrophoresis, several buffer systems were formulated according to a general guide.     

A new electrophoretic method for the separation of disaccharides obtained by digestion of chondroitin sulfates and dermatan sulfate with chondroitinases

A new rapid and simple method has been developed for the separation of disaccharides obtained by chondroitinase digestion of chondroitin sulfates and dermatan sulfate using electrophoresis on cellulose acetate plates (Titan III cellulose acetate plates). Three disaccharides are completely separated by electrophoresis in barium acetate or calcium acetate in a short time, and less than 50 μg of glycosaminoglycan samples can be analyzed within 2 h.     

Activity of chondroitin ABC lyase on dermatan sulfate partially degraded by cupric-ion-mediated free-radical treatment

Dermatan sulfate was extracted and purified from bovine intestinal mucosa, pig intestinal mucosa and pigskin. Small differences in M_r, charge density and constituent disaccharides were detected for the three purified natural dermatan sulfates. Bovine intestinal mucosa dermatan sulfate was depolymerized by a controlled free-radical process mediated by cupric ions in the presence of hydrogen peroxide. Different low-molecular-mass dermatan sulfate fractions were produced and analysed by high-performance size-exclusion chromatography and polyacrylamide gel electrophoresis. The results obtained by this last technique strongly support the hypothesis that the free-radical process proceeds essentially via the destruction of dissacharide units. The partial degradation of dermatan sulfates by cupric-ion-mediated free-radical treatment reduces or even eliminates the capacity of chrondroitin ABC lyase to depolymerize these derivatives. This was confirmed by polyacrylamide gel electrophoresis and the time curves of enzymatic treatments evaluated by spectrophotometry.     

Highly sulfated proteochondroitin sulfates synthesized in vitro by rat glomeruli

A previous report from this laboratory (Kobayashi, S., Oguri, K., Kobayashi, K. and Okayama, M. (1983) J. Biol. Chem. 258, 12051-12057) indicated that isolated rat glomeruli synthesized three species of sulfated glycoconjugates in vitro, namely, sulfated glycoproteins, proteoheparan sulfates and proteochondroitin sulfates. In the present study, the proteochondroitin sulfates, which showed the greatest incorporation of [35S]sulfate among the three sulfated glycoconjugates, were isolated and characterized. Radiolabeled tissue proteochondroitin sulfates were clearly separated on Sepharose CL-6B into three components with partition coefficients (Kd) of 0.16, 0.22 and 0.58, and medium proteochondroitin sulfates were separated into two components with Kd values of 0.33 and 0.62. When the chondroitin sulfate chains released by alkaline borohydride treatment were analyzed by digestion with chondroitinase AC-II, chondroitinase ABC, chondro-6-sulfatase and chondro-4-sulfatase, the results showed that all the samples contained glucuronosyl-N-acetylgalactosamine (chondroitinase AC-II-susceptible sequences, 72-86%) and iduronosyl-N-acetylgalactosamine (chondroitinase ABC-susceptible sequences, 14-28%), containing 4-sulfated N-acetylgalactosamine (50-70%) and 4,6-disulfated N-acetylgalactosamine (30-50%). On two-dimensional electrophoresis on cellulose acetate, all samples gave a single spot which closely coincided with chondroitin sulfate E of squid cartilage in electrophoretic mobility. These results indicated that the chains were highly sulfated chondroitin sulfates containing glucuronic acid and iduronic acid residues.     

Structural differences of dermatan sulfates from different origins

The dermatan sulfates from hog, rat, rabbit, and beef liver, hog, rat, beef, and dog spleen, and hog skin were isolated and submitted to structural analysis. All of them migrated as single bands, close to the standard position for dermatan sulfate in agarose-gel electrophoresis. In polyacrylamide gel, however, each dermatan sulfate showed a characteristic electrophoretic migration-pattern: one, two, or three polydisperse bands, corresponding to different molecular weights, were obtained for the dermatan sulfates according to their origins. Chemical analysis showed that all of the dermatan sulfates here described are hybrid polymers composed of D-glucuronic and L-iduronic acid-containing disaccharide units. The relative position of these units in the polymer chains and the presence of 6-sulfated disaccharides were determined with the aid of chondroitinases B and AC from Flavobacterium heparinum. These studies show that each dermatan sulfate has a unique structure as regards the molecular weight, the presence of 6-sulfated disaccharide units, and also the relative amount and position of glucuronic and iduronic acid residues in the chains. These findings suggests a tissue- and species-specificity for the dermatan sulfates.     

Protein Profile of <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> Strains Exhibiting Different Levels of Tolerance to Metal Sulfates

Strains of Acidithiobacillus ferrooxidans exhibited differences in the inhibition of Fe²⁺ oxidation in the presence of 250 mM of cadmium, zinc, and manganese sulfates in respirometric assays. Strains LR and I35 were practically not inhibited, whereas strains SSP and V3 showed significant inhibition (30–70%). Analysis by SDS-PAGE of total proteins from cells grown in the absence of metal sulfates showed different profiles between the more tolerant strains (LR and I35) and the more susceptible ones (SSP and V3). Total proteins of strains LR and V3 were also resolved by two-dimensional polyacrylamide gel electrophoresis (2-DE). A set of major proteins (40, 32, 22, and 20 kDa) could be identified only in the more tolerant strain LR. Our results show that protein profiles analysis could differentiate A. ferrooxidans strains that considerably differ in the tolerance to metal sulfates and present low genomic similarity as revealed by Random Amplified Polymorphic DNA (RAPD) data obtained previously in our laboratory.     

Marked effect of cations on the separation of spinach thylakoid chlorophyll-proteins on polyacrylamide gel electrophoresis at 0 degree C in the presence of alkanolammonium dodecyl sulfates

The separation of spinach thylakoid chlorophyll-proteins on polyacrylamide gel electrophoresis at 0 degrees C in the presence of dodecyl sulfate is markedly influenced by the kind of surfactant cation in the media used for solubilization and electrophoresis. The mode of separation can thus be modulated through the cation selection. Three kinds of alkanolammonium dodecyl sulfates were tested and their abilities as to the dissociation of the molecular assemblies of chlorophyll-proteins were found to decrease in the following order: tris(hydroxymethyl)methylammonium, triethanolammonium and triisopropanolammonium. Comparison of the electrophoretic patterns obtained with different kinds of cation may help clarify the hierarchy of the molecular assemblies of chlorophyll-proteins.     

Major chondroitin sulfate proteoglycans identified in L6J1 myoblast culture

The major proteoglycans from L6J1 rat myoblast culture were identified. The proteoglycans were isolated from different constituents of cell culture: culture medium, extracellular matrix (ECM), and myoblasts. To identify their core proteins, the proteoglycans were treated with enzymes specifically digesting chondroitin/dermatan sulfates or chondroitin sulfates. Subsequent electrophoresis and mass spectrometry revealed versican, collagen XII, and inter-α-trypsin inhibitor classified as chondroitin sulfate proteoglycans and biglycan known to be chondroitin/dermatan sulfate proteoglycan. Versican was identified in ECM and the other proteoglycans in the culture medium. Such difference in localization is likely to be a consequence of different biological functions. Versican, collagen XII, and biglycan are synthesized by myoblasts and inter-α-trypsin inhibitor originates from fetal bovine serum (a culture medium component).     

Effect of various detergents on protein migration in the second dimension of two-dimensional gels.

Two-dimensional gel electrophoresis (2D)1 is a powerful technique used to separate complex protein mixtures. The technique involves the separation of proteins by charge in the first dimension and by molecular weight in the second dimension. The effect of substituting various detergents for sodium dodecyl sulfate (SDS) in the second dimension (PAGE) was investigated. Individual C-10 through C-14 alkyl sulfates, C-11 through C-14 alkyl sulfonates, sodium N-lauroyl-N-methyl-taurine, N-lauroylsarcosine, sodium laurate, or benzyldimethyl-n-hexadecylammonium chloride were substituted for SDS in equilibration buffer, gel buffer, and upper running buffer. The cationic benzyldimethyl-n-hexadecylammonium chloride system was run with reversed polarity. Dramatic effects on protein migration from human mesothelial cell extracts were observed when different detergents were utilized. The C-12 (SDS) through C-14 alkyl sulfates and sulfonates resulted in anomalous migration of the simple epithelial keratins. Unlike SDS, the C-10 and C-11 alkyl sulfates and C-11 sulfonate resulted in gels in which the keratins were separated accurately with respect to their gene sequence-determined molecular weights. However, with these shorter chain alkyl sulfates and sulfonate, resolution was compromised, especially with respect to the high-molecular-weight polypeptides. The C-12 alkyl sulfate (SDS) and alkyl sulfonate provided the best resolution of polypeptides. Mixtures of C-11 sulfate and SDS resulted in gels with better sequence molecular weight estimates and high resolution. In addition, trace amounts of sodium tetradecyl sulfate/sodium heptadecyl sulfate in commercial SDS preparations had an effect on polypeptide resolution.     

Characterization of rat and human steroid sulfatases

Rat and human steroid sulfatases were purified from liver and placenta, respectively, by the same procedure. The rat and human enzymes were solubilized with Triton X-100, and purified by immunoaffinity chromatography with a monoclonal antibody showing high binding activities to both the enzymes. They were further purified by high-pressure anion-exchange chromatography to compare their structural and catalytic properties. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that both enzymes had a molecular weight of 62,000. The enzymes had similar amino acid compositions and amino-terminal amino acid sequences. Significant differences of the optimum pH, Michaelis constant and maximum velocity were observed between these enzymes. The optimum pH of each enzyme varied from 6.0 to 8.0, depending on substrates and with or without Triton X-100. In detergent-free media, steroid sulfates competitively inhibited the ability of these enzymes to hydrolyze 4-nitrophenyl sulfate. In media containing Triton X-100, on the other hand, the inhibition types of the steroid sulfates on the hydrolyzing activities of the rat and human enzymes were noncompetitive- and mixed-types, respectively.     

Continuous fractionation of proteins and mucopolysaccharides by simultaneous two-dimensional gel filtration and electrophoresis

A method for continuous-loading Sephadex gel electrophoresis is described. Albumin, α₁, α₂, β and γ globulins, and two kininases and an amino-peptidase are completely separated and obtained at the preparative scale by this procedure. Heparan sulfates and abnormal mucopolysaccharides from the urine of patients with Hurler's syndrome were also fractionated by this method.     

Fractionation and properties of four heparitin sulfates from beef lung tissue: Isolation and partial characterization of a homogeneous species of heparitin sulfate

Several commerical batches of heparitin sulfate extracted from beef lung tissue were fractionated into at least four distinct mucopolysaccharides by a combination of polyacrylamide and agarose gel electrophoresis. The four heparitin sulfates (A, B, C and D) were distinguished from each other and from heparin by several physical and chemical properties such as electrophoretic migration, molecular weight, presence of N-acetyl, N- and )-sulfate residues, optical rotation and enzymatic degradation. Of particular significance was the isolation of a heparitin sulfate (heparitin sulfate C) with a homogeneous molecular weight.     

Comparative evaluation of the glycosaminoglycan composition in plasma membranes of normal and transformed mouse liver cells

Using electrophoresis in agarose gel, a comparative study of composition of membrane-bound glycosaminoglycans (GAG) from normal mouse liver cells and from o-aminoazatoluene-induced mouse hepatoma cells was carried out. Differences in the composition and localization of GAG were revealed. The plasma membrane fraction of hepatoma cells contained no GAG; the bulk of GAG (approximately 98%) was localized in the plasma membrane free fraction. Within this fraction GAG contained no heparan sulfates with a high electrophoretic mobility that were detected in plasma membranes of normal liver cells. The possible involvement of proteoglycans bound to cell surface in transmembrane signal transfer is discussed.     

Glycosaminoglycans of the plasma membranes of renal tubule and liver cells

Glycosaminoglycans were isolated from plasma membranes of hepatic and renal tubule cells of guinea pig. Plasmalemma of renal tubule cells contained more total glycosaminoglycans, hyaluronic acid, chondroitin-4 sulfates and chondroitin-6 sulfates, and less dermatan sulfates and heparin sulfates than liver plasma membranes. These glycocalyx components, owing to their polyanionic properties, may have a role in the transport of water, ions, and macromolecules across the cell membrane.     

Isolation and structural studies of heparan sulfates and chondroitin sulfates from three species of molluscs

The isolation and some structural features of heparan sulfates and chondroitin sulfates from three species of molluscs (Pomacea sp., Tagelus gibbus, and Anomalocardia brasiliana) are reported. It is shown that heparan sulfates with structural similarities to the ones found in mammals are present in the three molluscs analyzed. All the heparan sulfates were degraded by heparitinases I and II to four distinct unsaturated disaccharides with the same properties as the ones present in heparan sulfates of mammalian origin. This suggests that these four disaccharide units are maintained through the evolution. Furthermore, the proportion of these units varied in the heparan sulfates according to the species of origin. The chondroitin sulfates, on the other hand, exhibit different structural features according to the species of origin. For instance, by the action of chondroitinase AC, 4- and nonsulfated disaccharides are produced from Pomacea chondroitin, whereas 4- and 6-sulfated disaccharides are formed from Tagelus and Anomalocardia. The possible role of these compounds in cell recognition and/or adhesiveness is discussed in view of the present findings.     

Residual keratan sulfate in chondroitin sulfate formulations for oral administration

Chondroitin sulfate is a biomedical glycosaminoglycan (GAG) mostly used as a dietary supplement. We undertook analysis on some formulations of chondroitin sulfates available for oral administration. The analysis was based on agarose-gel electrophoresis, strong anion-exchange chromatography, digestibility with specific GAG lyases, uronic acid content, NMR spectroscopy, and size-exclusion chromatography. Keratan sulfate was detected in batches from shark cartilage, averaging ～16% of the total GAG. Keratan sulfate is an inert material, and hazardous effects due to its presence in these formulations are unlikely to occur. However, its unexpected high percentage compromises the desired amounts of the real ingredient specified on the label claims, and forewarns the pharmacopeias to update their monographs. The techniques they recommended, especially cellulose acetate electrophoresis, are inefficient in detecting keratan sulfate in chondroitin sulfate formulations. In addition, this finding also alerts the manufacturers for improved isolation procedures as well as the supervisory agencies for better audits. Analysis based on strong anion-exchange chromatography is shown to be more reliable than the methods presently suggested by standard pharmacopeias.     

A quantitative solid-phase assay for identifying radiolabeled glycosaminoglycans in crude cell extracts

Extraction of radiosulfate-labeled cell layers in denaturing urea and nonionic detergent allows the quantitative binding of GAG-containing materials from up to 96 discrete samples to a single cationic nylon blot. Free sulfate and/or sulfated lipids fail to bind. Washing the blot with differential salt concentrations discriminates between native proteoglycans and free glycosaminoglycan chains or fragments. In addition, chondroitin sulfates and heparan sulfate are identified either by prior digestion with chondroitin ABC or AC lyase, as generated disaccharides fail to bind to the blot, or by treatment of the entire blot with nitrous acid following binding. Similarly, heparan sulfate can be identified on chromatograms or Western transfers from polyacrylamide gel electrophoresis by autoradiography before and after treatment of the blot with nitrous acid.