Activated thiol-Sepharose 4B Research Articles

Isolation and further characterization of bovine brain hexosaminidase C

Hexosaminidase C (2-acetamido-2-deoxy-β- d-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) was partially purified from bovine brain tissue. The resulting preparation, free of its lysosomal counterparts, was used for the characterization of the enzyme and for further purification (lectin affinity chromatography, hydrophobic interaction chromatography, substrate-ligand affinity chromatography, ion-exchange chromatography, chromatography on activated thiol-Sepharose 4B). Only ion-exchange chromatography on DEAE-Sephacel appeared to improve the purity. The Michaelis constant was 0.46 mM for the substrate 4-methylumbelliferyl-2-acetamido-2-deoxy-β- d-glucopyranoside. The enzyme was not inhibited by acetate or N-acetylgalactosamine. Inhibition by N-acetylglucosamine was competitive, with a K i value of 8.0 mM. Inhibition by divalent metal ions increased in the order Fe < Zn < Cu. Dithiothreitol and β-mercaptoethanol, at an optimum concentration of about 10 mM, stimulated the activity. The enzyme is apparently not a glycoprotein since it did not bind to various lectins, nor did sialidase change its isoelectric point.

Evidence that deoxyribonucleic acid photolyase from baker's yeast is a flavoprotein.

DNA photolyase purified from baker's yeast by affinity chromatography on UV-irradiated DNA noncovalently bound to cellulose and by chromatography on activated thiol-Sepharose 4B yields a single protein band having a molecular weight of 51 000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight, 53 000, determined by gel filtration was in good agreement. Upon denaturation of photolyase by heat or 8 M urea, flavin adenine dinucleotide (oxidized) was isolated from the mixture and identified by thin-layer chromatography and spectral analysis. In contrast to flavoproteins to which flavin adenine dinucleotide (oxidized) is bound which generally exhibit two absorbance maxima between 300 and 500 nm, photolyase has only one at 380 nm. These findings and the similar characteristics of the absorbance and emission spectra of native photolyase with those of flavoproteins in which the chromophore is considered to be the 4a,5-reduced flavin have led us to propose this configuration for the photolyase chromophore. The difference in properties of yeast photolyase compared to the one reported previously supports the idea that there are two photolyases in baker's yeast.

Resolution of human fibroblast interferon into two distinct classes by thiol exchange chromatography.

Chromatography of crude and purified human fibroblast interferon on activated thiol Sepharose 4B yielded two classes of material exhibiting different stability to inactivation by mechanical stress. Interferon which bound to the gel accounted for 20--30 per cent of recoverable activity, could be eluted with reducing agents and was unstable. Unbound material did not bind on subsequent rechromatography and was stable to shear forces. Leucocyte interferon did not bind, nor did fibroblast interferon treated with thiol blocking reagents. Peak eluted fractions had a specific activity of up to 10(7.9) units per milligram of protein, representing a 1700 fold purification.

Purification of procollagen type II by covalent chromatography with activated thiol-Sepharose 4B

Procollagen type II was isolated from Swarm chondrosarcoma of rat by NaCl-extraction and DEAE-cellulose chromatography and further purified from remaining type II collagen using activated thiol-Sepharose 4B for covalent chromatography. The purity of the separated collagen fractions was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results demonstrate that this method is a useful tool for separating procollagen from other collagenous material, which does not contain thiol groups.

Production of monospecific antibodies to rat liver ornithine decarboxylase and their use in turnover studies.

Two forms of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) were purified from the livers of rats which had been treated with thioacetamide for 16 h (for details, see miniprint to Obenrader, M.F., and Prouty, W. F. (1977) J. Biol. Chem. 252, 2860-2865). The enzyme was purified over 7,000-fold from liver cytosol with an overall yield of 8%. Enzyme activity was eluted finally in two distinct fractions by chromatography on activated thiol-Sepharose 4B. Both forms appear to be dimeric proteins having molecular weights of approximately 100,000 by equilibrium sedimentation and analysis on a calibrated Sephadex G-200 column. The apparent subunits are approximately 50,000 daltons as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Since electrophoresis in the presence of detergent is the only method used here to indicate subunits, the possibility that conditions of sample preparation resulted in splitting of a labile protein cannot be excluded from consideration. Ornithine decarboxylase has a very broad pH-activity curve with an optimum that shifts from pH 7.0 to pH 7.8 as the enzyme is purified. The apparent Km values for a highly purified mixture of the two forms of enzyme for L-ornithine and pyridoxal 5'-phosphate were determined to be 0.13 mM and 0.25 micronM, respectively. Both sodium and potassium chloride were shown to inhibit enzymatic activity; 50% inhibition occurred at 270 mM for each when Km amounts or ornithine were used. Rat liver ornithine decarboxylase antiserum was prepared in rabbits using Form I of the enzyme as the antigen. The antibody was shown to precipitate quantitatively the ornithine decarboxylase activity isolated from induced rat liver and rat ventral prostate. The specificity of the antiserum was demonstrated by rocket immunoelectrophoresis and by gel electrophoresis in the presence of sodium dodecyl sulfate using immunoprecipitates obtained from enzyme preparations labeled either in vivo, with [3H]leucine, or in vitro, by reductive methylation using formaldehyde and sodium [3H]borohydride. The antibody preparation has been used in a titration method to assess the half-life of antigen in livers of rats induced for ornithine decarboxylase by injection of thioacetamide. In two experiments, the t1/2 of activity at the height of induction, following injection of cycloheximide, was 19 and 24 min, while the t1/2 of disappearance of antigen was 28 and 33 min, respectively. In each experiment the t1/2 for antigen was significantly longer than the t1/2 for loss of enzyme activity. Enzyme levels appear to be modulated primarily by synthesis and degradation of antigen. Furthermore, the observation that enzyme activity is lost with a shorter t1/2 than antigen is consistent with the idea that denaturation is an initial step in the degradation of this enzyme...

Detection of multiple forms of rat liver ornithine decarboxylase.

Rat liver ornithine decarboxylase induced by injection of thioacetamide has been separated into at least two fractions by covalent chromatography on an activated thiol-Sepharose 4B column. The two major fractions could be distinguished by ion exchange chromatography and electrophoresis on acrylamide gels. In addition, the two forms displayed different Km values for ornithine. Although the two forms are separable, they display identical antigenic properties, pH optima, and they appear to be the same molecular size. The biological significance or the relationship between multiple forms of ornithine decarboxylase is not understood.

Synthesis of metallothionein in a polysomal cell-free system

A new covalent chromatography system utilizing Activated Thiol Sepharose 4B was employed to quantitate the content of thionein chains synthesized in a polysomal cell-free system. Liver polysomes from zinc injected rats directed the translation of more thionein-like polypeptide chains than polysomes from control rats. The increase was similar to the stimulation in MT synthesis in vivo following a zinc injection. This evidence supports the concept that metallothionein synthesis is regulated by changes in the pool of translatable thionein mRNA.

Purification of Histone F3 by Covalent Chromatography

The arginine-rich histone F3 has been purified by covalent affinity chromatography. By the use of activated Thiol-Sepharose 4B for the purification of cysteine containing histone F3 a highly purified protein was obtained. The simple purification procedure offers the opportunity to get larger amounts of pure histone F3 within short time.

Preparative isolation of Band 3, the predominant polypeptides of the human erythrocyte membrane

Band 3 proteins are the predominant polypeptide components of the human erythrocyte membrane and have been implicated in various transport activities. Following extraction of membrane ghosts with dimethylmaleic anhydride to remove two polypeptides (Bands 4.2 and 6) associated with Band 3, Band 3 proteins were solubilized along with the other major membrane glycoproteins (PAS 1–3) with Triton X-100 under nondenaturing conditions. Band 3 proteins were then purified (>95%) on a large scale by chromatography via thioldisulfide interchange on activated thiol-Sepharose 4B [agarose-(glutathione-2-pyridyl disulfide) conjugate]. This procedure allows the preparation of 20 to 25 mg of purified Band 3 proteins in high yield (>80%) from ghosts in a soluble form suitable for physical, chemical, and functional characterization.