DEAE-Sepharose CL-6B Ion Exchange Research Articles

Sera from patients with poststreptococcal glomerulonephritis contain antibodies to glomerular heparan sulfate proteoglycan.

Antibodies, found in human sera from patients with poststreptococcal glomerulonephritis, against proteoglycans (PG) derived from bovine and human glomeruli were investigated. PG were isolated by 4 M guanidine-HCl extraction of whole glomeruli, followed by DEAE-Sepharose CL-6B ion exchange chromatography. The anionic fractions were further purified by chromatography on Sepharose CL-4B. Biochemical analysis of the two resulting peaks revealed the presence of high molecular weight anionic material containing protein, uronic acid, glucosamine, and galactosamine. Enzymatic and chemical susceptibilities indicated the presence of heparan sulfate PG and a galactosamine-containing PG. Immunologic studies revealed the presence of anti-PG antibodies to both PG peaks of the Sepharose CL-4B column in glomerulonephritis sera. Inhibition studies using an ELISA demonstrated that heparan sulfate was a major antigenic determinant. Cross-reactivity with both mammalian and streptococcal hyaluronate was noted. Inhibition studies also indicated the presence of a second antigenic site containing N-acetylgalactosamine, possibly representing chondroitin or dermatan sulfate PG.

Biosynthesis of 2-deoxystreptamine.

Extracts of Streptomyces fradiae 75-078, a producer of an antibiotic neomycin, were found to catalyze three reactions which are included in the proposed biosynthetic pathway to 2-deoxystreptamine; amination of deoxyinosose yielding deoxyinosamine, conversion of deoxyinosamine to 2-deoxystreptamine and deamination of 2-deoxystreptamine. Glutamine was effective as an amino-donor for both transamination reactions; conversions of deoxyinosose to deoxyinosamine and of aminodeoxyinosose to 2-deoxystreptamine. Conversion of deoxyinosamine to 2-deoxystreptamine, presumably including successive dehydrogenation and transamination at position 1, was stimulated by NAD+. On DEAE-Sepharose CL-6B ion-exchange chromatography, the enzyme activity catalyzing amination of deoxyinosose and deamination of 2-deoxystreptamine was eluted as an entity (aminotransferase), while the one converting deoxyinosamine to 2-deoxystreptamine, only if the aminotransferase is supplemented, can be eluted as a separate peak (deoxyinosamine dehydrogenase). The molecular weight of the aminotransferase was estimated to be 130,000 daltons by chromatography on Sepharose CL-6B. Enzymatic synthesis of 2-deoxystreptamine from deoxyinosose was demonstrated by the cell free extracts.

The purification of myosin long subfragment 2 by DEAE-Sepharose CL-6B ion-exchange chromatography

A new method for the purification of myosin long subfragment 2 is presented. This method is based on the fractionation, by DEAE-Sepharose CL-6B ion-exchange chromatography, of either chymotryptic hydrolysates of heavy meromyosin or tryptic hydrolysates of myosin total rod. Although emphasis is given to the purification of long subfragment 2, the method could be easily adapted to the purification of short subfragment 2.

Characterization and Localization of Bovine Epidermal Transglutaminase Substrate

The substrate was purified by DEAE-Sepharose CL-6B ion exchange chromatography, Pevikon block electrophoresis, and ACA-54 gel filtration chromatography. This modified purification procedure resulted in a 19% substrate yield. Amino acid analysis of the purified substrate was performed following treatment in SDS, alkylation in 4-vinyl pyridine, and hydrolysis with methane sulfonic acid. Results included significant levels of lysine and glutamic acid, low levels of cystine and methionine, and a high amount of an amino acid eluting in identical position with authentic citrulline. Studies of the sedimentation equilibrium of the substrate showed a monodispersed protein with a molecular weight of 36,780 +/- 350. Ultrastructural studies of insoluble high molecular weight aggregates of the substrate revealed globular, amorphous masses lacking fibrillar structure. Using Ouchterlony double diffusion techniques, the initial substrate (36,000 m.w. substrate) and the soluble crosslinked high molecular weight substrate were shown to be immunologically identical. Indirect immunofluorescence was performed on freeze-dried sections of bovine snout epidermis using substrate specific rabbit antiserum and fluorescein-conjugated goat anti-rabbit IgG. Fluorescence was seen throughout the cytoplasm and inner cell membrane of granular cells, but was limited to the inner membrane of stratum corneum cells. Epidermal transglutaminase substrate is an amorphous, globular, citrulline containing protein synthesized in the cytosol of keratinizing cells with ultimate localization in the cell membrane.

An analysis of the heterogeneity of albumin.

A simple and rapid procedure for characterization of serum albumin samples and for the isolation of fractions from fresh serum or commercial sources has been developed, based on DEAE Sepharose CL-6B ion exchange chromatography. Characterization of the fractions included sulfhydryl analysis, gel electrophoresis, and immunoelectrophoresis. Both analytical and preparative procedures were used. It has been shown that the relative content of the fractions varies from individual to individual. It has also been shown that certain fractions found in commercial preparations are artifacts which should be removed before performing serious studies with serum albumin.

Purification and partial characterization of three forms of alpha-glucosidase from the fruit fly Drosophila melanogaster.

Three forms of alpha-glucosidase, I, II, and III, have been purified from the whole body extract of adult flies of Drosophila melanogaster in yields of 2.1, 5.3, and 6.7%, respectively. The purification procedures involved ammonium sulfate fractionation, Con A-Sepharose 4B affinity chromatography, DEAE-Sepharose CL-6B ion exchange chromatography, Sephacryl S-200 gel filtration, and preparative gel electrophoresis. Each purified enzyme showed a single band on polyacrylamide gel on both protein and enzyme activity staining. The molecular weights of alpha-glucosidases I, II, and III were estimated to be 200,000, 56,000, and 76,000, respectively, by gel filtration. SDS gels indicated that alpha-glucosidases II and III were each composed of a single polypeptide chain, whereas alpha-glucosidase I was composed of two identical subunits. Both alpha-glucosidases II and III hydrolyzed sucrose and p-nitrophenyl-alpha-D-glucoside (PNPG), but alpha-glucosidase I hydrolyzed PNPG to a much lesser extent than sucrose. For sucrose the pH optima of alpha-glucosidases I, II, and III were pH 6.0, 5.0, and 6.0 and the Km values were 13.1, 8.9, and 10 mM, respectively. For PNPG the pH optima of alpha-glucosidases II and III were pH 5.5 and 6.5 and the Km values were 0.77 and 0.21 mM, respectively.