CM-cellulose Column Chromatography Research Articles

Purification of cathepsin B from squid liver.

Cathepsin B [EC 3. 4. 22. 1] from the liver of squid, Dorytheuthis bleekri, was purified by the following steps : homogenization, ammonium sulfate fractionation, CM-cellulose column chromatography and rechromatography, Sephadex G–75 column chromatography and iso-electric focusing. The purified enzyme appears to be homogeneous by polyacrylamide gel electrophoresis at both pH 4.3 and pH 9.5 and has an isoelectric point of 6.8. The molecular weight of the enzyme was determined to be 13,600 by Sephadex G–75 column chromatography. The optimum pH for hydrolysis of α-N-benzoyl-l-argininamide (BAA), α-N-benzoyl-dl-arginine-2-naphthylamide (BANA) was 4.5, and it was 4.2~4.7 for N, N′-dimethyl hemoglobin. The value of Km for the hydrolysis of BANA was estimated to be 1.25 mm.

Studies on the red yeast cell wall. II. Purification and some properties of red yeast cell wall lytic enzyme.

The red yeast cell wall lytic enzyme of Penicillium lilacinum No. 2093 was purified about 220 fold with a yield of 22% from the culture filtrate by ammonium sulfate fractionation, DEAE-cellulose and CM-cellulose column chromatography and Bio-Gel P-60 gel filtration. The enzyme was most active at pH 4.0_??_4.5 and 50°C. The enzyme was stable at pH value from 3.5 to 7.0 on 20 hr incubation at 37°C. The enzyme released reducing sugar from the red yeast cell walls, but Saccharomyces yeasts did not show any susceptibility to the enzyme.

Pepsin treatment of avian skin collagen: Subunit composition and aggregation properties of chromatographically-derived fractions

1. 1. Collagen, solubilized from avian skin by pepsin treatment, was purified and fractionated by CM-cellulose column chromatography. 2. 2. Salt-eluted material consisted mainly of α-chains and degradation products and could not be renatured by the methods employed. 3. 3. Urea-eluted material, which was further fractionated on the basis of solubility, contained appreciable quantities of β- and γ-chains. Some material from both fractions underwent renaturation. 4. 4. The α 1-chains from all three fractions were similar to one another in terms of electrophoretic properties and to the α 1-chain of acid-soluble collagen. 5. 5. It is concluded that the β- and γ-chains in pepsin-soluble collagen arise largely from intermolecular crosslinks present in parts of the molecule resistant to pepsin.

Identification of a chloroplast ribosomal protein altered by a chloroplast mutation in Chlamydomonas.

Direct evidence is presented that a chloroplast gene mutation in Chlamydomonas reinhardi alters one of the chloroplast ribosomal proteins. Proteins of 30 S subunits of chloroplast ribosomes from mutant strains, carrying maternally inherited antibiotic resistances, were compared with those from the wild type strain by CM-cellulose column chromatography and gel electrophoresis. When 30 S ribosomal proteins from a [3H]arginine-labeled streptomycin-resistant strain and a [14C]arginine-labeled wild type strain, or vice versa, were cochromatographed on a CM-cellulose column, one peak (Peak 17) was absent from the mutant profile. Instead, a pronounced peak was observed to elute at a slightly lower ionic strength than Peak 17 in the region of Peak 16. The molecular weights in both Peak 16 and Peak 17 regions determined by discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis were indistinguishable, approximately 18,000. Thus, a chloroplast gene mutation to streptomycin resistance has altered the chromatographic behavior of a chloroplast ribosomal protein of the 30 S subunit. We interpret the additional protein in the mutant eluting at Peak 16 as most likely the mutationally altered form of the Peak 17 protein.

Amino acid sequence of cardiotoxin-analogue I from the venom of [formula omitted

Cardiotoxin(CTX)-analogue I was obtained in a yield of about 5.3% from the venom of naja naja atra by gel filtration on Sephadex G-50, followed by CM-cellulose column chromatography. When injected intraperitoneally in mice, its LD 50 was 2.8 (2.45–3.19) μg/g body weight. This toxin was cytotoxic to Yoshida sarcoma cells and produced contracture of the skeletal muscle as did CTX. The amino acid sequence of CTX-analogue I exhibits a high degree of homology with that of CTX from the same venom but differs in 13 positions.

Partial purification of the protein system controlling the breakdown of trehalose in baker's yeast

The inactive form of trehalase as well as its activating protein have been partially purified from resting cells of baker's yeast using (NH 4) 2SO 4 fractionation and subsequent DEAE- and CM-cellulose column chromatography. For its activation by cyclic 3′,5′-AMP the system appeared to be dependent on the presence of ATP and a divalent cation such as Mg 2+, Mn 2+ or Co 2+. No sensitivity towards the pH was observed in the range 6.0 – 7.5. The amount of active trehalase formed was determined by the preincubation time and the concentration of the proteins involved. The activating protein partly lost its dependence on cyclic 3′,5′-AMP during purification. The results presented suggest that this protein may be a protein kinase and that activation of trehalase is associated with phosphorylation of the enzyme protein.

Separation of myocardial cytoplasmic acidic inhibitor proteins which inhibit hepatic protein biosynthesis in vitro

Cytoplasmic inhibitor proteins of rabbit and pig myocardium have been separated and their in-vitro effects on hepatic cell-free protein biosynthesis reported. Three protein peaks could be separated from myocardial cytoplasmic pH 5 fraction by DEAE and CM cellulose column chromatography. There was a difference in the elution pattern of the acidic inhibitor proteins obtained from pig and rabbit myocardium. The myocardial acidic inhibitor proteins inhibited a liver cell-free protein biosynthesizing system, but not a myocardial cell-free system.

Comparative micelle structure. III. The isolation and chemical characterization of caprine [formula omitted] and [formula omitted

Two pure caprine (goat) β-caseins were purified by DEAE- and CM-cellulose column chromatography in buffers containing urea and 2-mercaptoethanol. The molecular weight of each β-casein was found to be about 24 500 as determined by gel filtration on Sepharose 6B in guanidine·HCl, and each had the same amino acid composition; Asp 9, Thr 12, Ser 15, Glu 43, Pro 33, Gly 6, Ala 5, Val 21, Met 6, Ile 9, Leu 20, Tyr 4, Phe 9, His 5, Lys 12, Arg 3, Trp. Phosphate determinations showed, however, that β 1- casein (the more mobile component on electrophoresis at pH 8.6) contained six residues per molecule, whilst β 2- casein contained only five. This difference may have been caused by a gene duplication and mutation. Both caseins are similar to bovine β-casein in molecular weight and net charge.

The isolation of an easily reversible post-synaptic toxin from the venom of a sea snake, Laticauda semifasciata.

A weakly neurotoxic component (Ls-III) was isolated by CM-cellulose column chromatography from the venom of a sea snake Laticauda semifasciata. The content of component LsIII was about 10-20% of the venom as determined by u.v. absorption at 280nm. Component LsIII was homogeneous on rechromatography and disc electrophoresis, and its molecular weight was shown to be 7100 by ultracentrifugation and 7300 by sodium dodecyl sulphate-polyacrylamide-gel disc electrophoresis. The isoelectric point of component LsIII was pH7.2. Component LsIII consisted of 66 amino acid residues including 10 half-cystine residues. The LD(50) of component LsIII by intramuscular injection was 1.24mug/g body wt. for mice and 0.45mug/g for baby chicks, which is about eight to ten times less toxic than erabutoxins a, b and c, all of which are contained in the same venom. Experiments with three isolated muscle preparations from different species indicated that component LsIII was a post-synaptically acting toxin, the action of which was easily reversed by washing.

Studies on Human β-d-N-Acetylhexosaminidases: I. PURIFICATION AND PROPERTIES

Abstract β-d-N-Acetylhexosaminidase A and B were purified from human placenta. In the initial steps of purification, namely, extraction, ammonium sulfate precipitation, lyophilization, Sephadex G-200 filtration, and DEAE-cellulose column chromatography hexosaminidase A and hexosaminidase B were purified together. Hexosaminidase A was further purified by a second DEAE-cellulose column chromatography followed by ECTEOLA-cellulose column chromatography, Sephadex G-200 filtration, electrofocusing, and a third Sephadex G-200 filtration. Hexosaminidase B was further purified by CM-cellulose column chromatography, electrofocusing, and Sephadex-G-200 filtration. After the first Sephadex G-200 filtration hexosaminidase A and B were also purified by a second method. This method involved calcium phosphate gel, DEAE-Sephadex, ECTEOLA-cellulose, CM-Sephadex and CM-cellulose column chromatography, and preparative polyacrylamide disc electrophoresis. Purified hexosaminidase A and hexosaminidase B moved on polyacrylamide disc electrophoresis as single protein bands with virtually all of the enzyme activity. Hexosaminidase A and B in 10 mm phosphate buffer containing 0.1 m (NH4)2SO4 were stable at 4° for at least 4 months. The isoelectric points of hexosaminidase A and hexosaminidase B were found to be pH 5.4 and 7.9, respectively. Placental hexosaminidases were found to differ electrophoretically and antigenically from the liver and fibroblast enzymes. In contrast to the reports of others neuraminidase failed to convert heat-labile hexosaminidase A to a heatstable form of enzyme resembling hexosaminidase B.

The activation of Cls with purified Clr

The Cls activator obtained in neutral euglobulin precipitates (pH 7·5, RSC 0·04) was solubilized with 0·02 M EDTA, 0·005 M L-lysine buffer (pH 7·5, RSC 0·04) and subjected to DEAE and CM cellulose column chromatography. The final preparations showed a single major protein band on analytical acrylamide gel electrophoresis with a few minor bands near the cathode. They gave a single precipitation line on immunodiffusion against a potent horse anti-whole human serum. This line completely fused into a precipitation line made with anti-Clr and these fractions. Cls activator activity corresponded to the single major band on acrylamide gel and was associated with the hemolytic activity of Clr. The spontaneous activation in highly purified Cls (Sakai and Stroud, 1973) was minimized by treating with DFP. The stabilized Cls was still susceptible to activation by Clr. In other experiments using DFP- 32P binding to the smaller subunit of C ls was demonstrated, suggesting that this peptide chain contains the active site.

Multiple components of beta-N-acetylhexosaminidase from equine kidney. Their action on glycolipids and allied oligosaccharides.

β-N-Acetylhexosaminidase [EC 3.2.1.52] extracted from equine kidney had three components, acidic A, basic B, and a third one A'. These three components were separated by a combination of DEAE- and CM-cellulose column chromatography. Isoelectric points were: A, 6.07; A', 6.25; B, 8.20. Component A was heat labile. Component B was relatively stable against heat treatment but lost activity rapidly above 55°C. Component A' was very heat stable, and retained 75% of its activity after 30 min at 60°C. The molecular weights of A, A', and B were 125,000, 250,000, and 125,000 daltons, respectively. The Michaelis constants against phenyl β-N-acetylgalactosaminide were A: 0.147mM, A': 0.102mM, and B: 0.043mM, and those against p-nitrophenyl β-N-acetylgalactosaminide was A: 0.571 mM, A': 0.246 mM, and B: 0.086 mM. Oligosaccharide obtained from ganglioside GM2 was hydrolyzed only by A and A', not by B, and those from asialo GM2 and Globoside I were hydrolyzed by all three components. Component A preparation free from sialidase [EC 3.2.1.18] was not converted to B by sialidase from Cl. perfringens.

Isolation and Purification of Turkey Heme Proteins

Turkey heme proteins, myoglobin, hemoglobin and cytochrome c were isolated and their interactions were studied. This report describes procedures for the chromatography of myoglobins on CM cellulose, DEAE cellulose and Sephadex columns. DEAE cellulose was used to separate hemoglobin and cytochrome c.Turkey metmyoglobin was shown to display a heterogeneity as demonstrated in CM cellulose column chromatography, polyacrylamide gel electrophoresis, and isoelectric fractionation. Three electrophoretically distinct myoglobins were observed in gel electrophoresis, and seven myoglobins were separated by isoelectric fractionation. The major myoglobin fraction was found to have an isoelectric point of 7.99.Turkey myoglobin was similar to chicken myoglobin, but different from mammalian myoglobin in amino acid composition and molecular weight. Molecular weight of turkey myoglobin was determined to be 18,199.The autoxidation rate of turkey myoglobin exhibited a 6-fold slower rate in the presence of cytochrome c. The cytochrome c reduction of metmyoglobin to reduced myoglobin was the probable mechanism.The heat denaturation of turkey myoglobin is quite similar to myoglobins from other species (1/2 D = 78.0° C.). The denaturation temperature increased by approximately 1° C. as the other heme proteins were added to myoglobin, indicating a protein-protein interaction, thus allowing myoglobin to resist heat denaturation. The heat denaturation study revealed that cytochrome c resists denaturation at 105° C.

An Improved Method of the Purification of Ricin D

An improved method of the purification of ricin D was investigated. Ricin was purified by gel-filtration through Sephadex G-75 at pH 8.0, followed by either CM-cellulose column chromatography at pH 6.5 or DEAE-cellulose column chromatography at pH 8.5. The homogeneity of the purified ricin was criticized by polyacrylamide gel disc electrophoresis. The purified ricin behaved homogeneous also in ampholine electrophoresis, indicating the isoelectric point of 7.34. Ricin thus purified was identical with ricin D in electrophoretical migration and toxicity. By the measurement of optical rotatory dispersion of the purified ricin, ORD constant, λC, Moffitt-Yang parameters, a0 and b0, were evaluated to be 235 nm, — 138 and —66, respectively.

An Improved Method of the Purification of Ricin D

An improved method of the purification of ricin D was investigated. Ricin was purified by gel-filtration through Sephadex G-75 at pH 8.0, followed by either CM-cellulose column chromatography at pH 6.5 or DEAE-cellulose column chromatography at pH 8.5. The homogeneity of the purified ricin was criticized by polyacrylamide gel disc electrophoresis. The purified ricin behaved homogeneous also in ampholine electrophoresis, indicating the isoelectric point of 7.34. Ricin thus purified was identical with ricin D in electrophoretical migration and toxicity. By the measurement of optical rotatory dispersion of the purified ricin, ORD constant, λC, Moffitt-Yang parameters, a0 and b0, were evaluated to be 235 nm, — 138 and —66, respectively.

Microheterogeneity of rat liver ferritin: Comparison of electrofocusing and chromatographic fractions

Isoelectric fractionation of rat liver ferritin showed three heterogeneous peaks. The isoelectric points of three peaks were pI 5.16, 5.24, and 5.29. Using CM-cellulose column chromatography, heat supernatant of rat liver homogenate was separated to six fractions by a stepwise elution. Among them, three fractions which are eluted by acetate buffer with pH 5.1, 5.2, and 5.3 were shown to be ferritin electrophoretically and immunologically. Three ferritin fractions eluted by CM-cellulose column chromatography were applied to electrofocusing columns. The ferritin fraction which was eluted by a acetate buffer, pH 5.1, gave a single peak with isoelectric point of 5.16. The ferritin fraction which was eluted by a acetate buffer pH 5.2 gave also a single peak with isoelectric point of 5.24. The last ferritin fraction could not be applied to electrofocusing column because of a small amount of protein concentration. Microheterogeneity of rat liver ferritin on isoelectric fractionation corresponds to it on CM-cellulose column chromatography.

A thermophilic extracellular α-amylase from Bacillus licheniformis

A strain of Bacillus licheniformis isolated from soil produced an extracellular α-amylase(s) with unusual characteristics. The enzyme was purified 126-fold by starch adsorption, DEAE-cellulose treatment, and CM-cellulose column chromatography. Four active protein bands were detected by disc electrophoresis in poly-acrylamide gel although the enzyme behaved as a single peak during both ultracen-trifugation and chromatography using CM-cellulose and Sephadex G-100. The enzyme showed a very broad pH-activity curve and had substantial activity in the alkaline range. The optimal temperature was 76 °C at pH 9.O. The enzyme was stable between pH 6 and 11 at 25 °C, and below 60 °C at pH 8.0. Using Sephadex G-100 gel filtration, a molecular weight of 22,500 was estimated for the enzyme. The action pattern on amylose and amylopectin is unique in that the predominant product during all stages of hydrolysis is maltopentaose.

Amino acid replacements in proteins S5 and S12 of two Escherichia coli revertants from streptomycin dependence to independence.

Ribosomes were isolated from two E. coli revertants from streptomycin dependence to independence, N660 and d1023. After separation of subunits, proteins were extracted from ribosomal 30S subunits and separated by CM-cellulose column chromatography and gel filtration. Pure S5 and S12 proteins of the two mutants were digested with trypsin and all resulting peptides were isolated by column and paper chromatography. The amino acid compositions of the peptides from the four mutant proteins were compared with the corresponding peptides of the wild type strain A19. The amino acid sequences of non-identical peptides were determined. The following amino acid replacements were found: Glycine by arginine in peptide T2 of protein S5 from mutant N660 and glycine by aspartic acid in peptide T15 of protein S12 from the same mutant. In the other mutant, d1023, arginine in peptide T2 of protein S5 was replaced by leucine and furthermore arginine by serine in peptide T10 of protein S12. Besides the single amino acid replacements mentioned above which are compatible with alterations of single nucleotides, a rather drastic difference between peptides T15 of proteins S12 isolated from strain A19 and mutant d1023 has been detected. The results presented in this paper are compared with amino acid replacements in proteins S5 and S12 from other ribosomal mutants of E. coli.

Changes in mulitiple forms of polyphenol oxidase during maturation of tea leaves

Changes in polyphenol oxidase activity during davelopment of tea leaves were investigate by CM-cellulose column chromatography and isoelectric focusing components I-III separate by CM-cellulose chromatography, II and II decreased markedly during maturation. polyphenol oxidase activity was separated int

Purification and Properties of a Keratan Sulfate Hydrolyzing Enzyme, An Endo-β-Galactosidase

A keratan sulfate hydrolyzing enzyme was isolated from Coccobacillus, Chase, ATCC 13939, a non-gram-negative soil organism. The crude enzyme u as extracted by disruption of the cells, purified by ammonium sulfate fractionation, followed by DEAE- and CM-cellulose column chromatography and Bio-Gel P-100 gel filtration. The final preparation showed 113 times the specific activity of the original extract. The enzyme IS a basic protein, has an optimum pH at 5.5 and a temperature optimum at 32°C. It is an endo-β-D-galactosidase and splits KS§ into a series of sulfated oligosaccharides and sulfated peptido-oligosaccharides. The enzyme activity is inhibited by heavy metal ions, but not by EDTA anti dithiothreitol.The enzyme was assayed by measuring the trubidity produced by the reaction of KS with Hyamine. The enzyme unit was expressed as half turbidity, after 30 minutes or incubation with 200 μg of KS at pH 5.5 and 31°C. One unit of enzyme activity was contained in 9.7 μg of the purified enzyme fraction. The ass...