Sepharose 4B Column Chromatography Research Articles

A radioactive antigen-binding assay for the measurement oof antibody to Haemophilus influenzae type b capsular polysaccharide

A new polyethylene glycol (PEG) radioimmunoprecipitation assay was developed for the detection of antibody to Haemophilus influenzae b capsular polysaccharide, polyribosylribitol phoshate (PRP). The radioactive antigen, [ 3H]PRP, with a high specific activity, was produced by growing the organism in the presence of [ 3H]ribose and was purified by hydroxylapatite and Sepharose 4B column chromatography. In the assay, PEG (12.5%) was used to separate antibody-bound [ 3H]PRP from free [ 3H]PRP. The assay covered the range of 0.5 and 20 ng antibody/assay at a maximum sensitivity of 0.5 ≈ 1.0 ng antibody/assay. With various dilutions (1–20 ng antibody/assay) of S. Klein reference antiserum, the within-run coefficient of variation (CV) of 10 replicates ranged from 3.5 to 8.5%. Average CVs of 8.9% and 11.0% were obtained in the between-run and day-to-day reproducibility studies. The binding of [ 3H]PRP to S. Klein reference antiserum was severely inhibited by a minute amount of non-radioactive PRP; however, no significant interference was found in the presence of high concentrations of polysaccharides from Escherichia coli K100 and Streptococcus pneumoniae indicating that the RIA was highly specific for antibody to H. influenzae b PRP. The present RIA is a simple, specific, sensitive and reproducible procedure for the evaluation of antibody responses of young animals and infants to H. influenzae b vaccines and infections.

Steroid hormones may regulate autophosphorylation of adenosine-3',5'-monophosphate-dependent protein kinase in target tissues.

A protein in rat liver cytosol whose phosphorylation was regulated by hydrocortisone administration in vivo was tentatively identified as the regulatory subunit of a cAMP-dependent protein kinase. Evidence that this protein, whose phosphorylation was regulated by steroid and cyclic AMP, is the regulatory subunit of type-II cAMP-dependent protein kinase included: (a) co-purification of the steroid/cAMP-regulated protein and the regulatory subunit during DEAE-cellulose, Sepharose 4B, and hydroxylapatite column chromatography, (b) co-migration of the two proteins on dodecyl sulfate/polyacrylamide slab gels during the various steps of purification, (c) specific adsorption of the two proteins onto 8(6-aminohexylamino)-cAMP--Sepharose 4B, and (d) a similar pattern of distribution of the two proteins in various subcellular fractions prepared from rat liver homogenate. By each of these criteria, it was found that the steroid/cAMP-regulated protein present in rat liver cytosol behaved identically with the regulatory subunit of type-II cAMP-dependent protein kinase in that tissue. Results qualitatively similar to those obtained in the study of the effect of hydrocortisone on rat liver were also obtained in studies of the effects of other steroid hormones on other target tissues in the rat, including uterus (17 beta-estradiol), ventral prostate and seminal vesicle (testosterone), and epididymal fat pad (hydrocortisone). The tentative identification of the steroid/cAMP-regulated protein as the regulatory subunit of the type-II cAMP-dependent protein kinase in the cytosol of several tissues indicates that autophosphorylation of the regulatory subunit of type-II protein kinase may be regulated by the steroid hormones. The fact that three different classes of steroid hormones appear to affect the phosphorylation of the regulatory subunit of type-II cAMP-dependent protein kinase in their target tissues raises the possibility that this common biochemical action may play an important role in the mechanism of steroid hormone action. It is also possible that this effect of the steroid hormones may provide a molecular basis for some of the known physiological interactions of the steroid hormones with those hormones that act through using cAMP as a second messenger.

Purification and characterization of kallikrein from plasma of patients with acute pancreatitis.

1. A kallikrein-like enzyme in plasma of patients with acute pancreatitis was further purified by successive hydroxyapatite/cellulose and Sepharose-4B column chromatography. 2. By these procedures 0.26 mg of purified enzyme with a specific activity of 215 S-2266 chromozyme units/mg of protein was obtained from 10 ml of original plasma. 3. The purified material was homogeneous as ascertained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had an apparent molecular weight of 31 000 as measured by gel filtration on Sephadex G-200. 4. It was confirmed immunologically that this enzyme was pancreatic kallikrein, which is distinct from plasma kallikrein, and that it could combine with alpha 2-macroglobulin only in the presence of trypsin.

Eigenschaften und Regulation der Enzyme des Shikimat-Pathway von Hansenula henricii: Properties and Regulation of Enzymes in the Shikimate Pathway of Hansenula henricii

Summary The following enzymes has been isolated of the yeast Hansenula henricii by means of ammonium sulfate fractionation, DEAE-Sephadex A-50 and Sepharose 6B column chromatography: 3-dehydroquinate synthase, 3-dehydroquinase-shikimate: NADP oxido-reductase complex, shikimate kinase, 5-enolpyruvyl shikimate-3-phosphate synthase, and quinate: NAD oxido-reductase. The approximate molecular weights of these enzymes have been estimated using gel filtration. Some properties of purified enzymes have been studied. Kinetic parameters and regulation of activity of enzymes were similar to those found in higher plants and bacteria. Aromatic amino acids do not regulate activity and level of the enzymes studied.

Subunit structure of glucose isomerase from Streptomyces griseofuscus, S-41.

The subunit structure of glucose isomerase isolated from Streptomyces griseofuscus, S-41 was studied using denaturants. The enzyme was quite stable to SDS under mild conditions, and dissociation into smaller subunits was dependent on pH and temperature. At acidic pH and high temperature, the enzyme showed rapid dissociation and inactivation. The dissociation into constituent subunits paralleled the loss of enzymatic activity. The enzyme was also dissociated into subunits by 6 M guanidine hydrochloride. A single symmetrical elution peak was observed in Sepharose 6B column chromatography in the presence of 6 M guanidine hydrochloride, and the eluate showed no activity. Molecular weight estimations of the subunit, using both SDS-polyacrylamide gel electrophoresis and gel nitration, were the same (43,000). The N-terminal amino acid was identified as serine, and the yield of serine was estimated to be 43 percent from analyses of PTH and DNP derivatives. The sequence from N-terminal to the third amino acid was ...

Purification and some properties of debranching enzyme of germinating rice endosperm.

A debranching enzyme was extracted from the endosperm of germinating rice seeds and purified through three steps, namely cyclohexaamylose-coupled Sepharose 6B, Ultrogel AcA-44 and Bio-Gel P-150 column chromatography. This disc-electrophoretically homogeneous enzyme showed a specific activity of 43 units/mg of protein (30°C) with a pH optimum of 5.5. The isoelectric point was 4.9, unlike that (pI 3.5) of debranching enzyme of ungerminated rice seeds. Our enzyme hydrolyzed pullulan rapidly, and glutinous rice starch and waxy corn starch moderately. The enzyme was also able to act on phytoglycogen and glycogen unlike debranching enzymes originating in some plants.

Isolation of four isotoxic proteins and one agglutinin from jequiriti bean ( Abrus precatorius)

Four isotoxic proteins and one agglutinin were purified from the seeds of Abrus precatorius by Sepharose 4B and DEAE-cellulose column chromatography. Abrin-b and abrin-c which bind weakly on the Sepharose 4B are eluted from the column in the absence of galactose while abrin-a, abrin-d and abrus agglutinin which bind strongly are eluted with 0·1M galactose. The mol. wts of abrin-a and abrin-c are 63,000 and abrin-b, abrin-d and abrus agglutinin 67,000. The lethality of the four abrins was similar; 10, 25, 16 and 31 μg/kg body wt for abrin-a, b, c and d, respectively. The amino acid compositions of four isotoxic proteins and the A and B subunit of three isotoxic proteins are similar but not identical, and they are different from those of abrus agglutinin.

Succinyl trialanine p-nitroanilide-hydrolytic enzymes in human serum. Partial purification and characterization.

The levels of two kinds of elastase-like enzymes, which are able to hydrolyze an artificial elastase substrate, suc-(Ala)3-pNA, but unable to hydrolyse a naturally occurring substrate, elastin, were found to be elevated in the sera of patients suffering from hepatobiliary disorders and other diseases accompanied by tissue damage. One of the enzymes was characterized as being sensitive to a chelating reagent, EDTA, and partially inactivated enzyme activity was recovered by the addition of calcium ion. The apparent molecular weight estimated by Sepharose 4B column chromatography showed a wide distribution from 200,000 to approximately 10,000,000, but all components were converted to a molecular weight of about 200,000 by treatment with 2% Triton X-100. The activity of this enzyme was partially reduced by the addition of anti-beta-lipoprotein antibody, showing that a part of the enzyme was affiliated with low and very low density lipoproteins in the serum. The level of the other enzyme was rarely increased in the sera of patients suffering from severe hepatic disorders. This enzyme was resistant to EDTA, and the apparent molecular weight was 150,000-200,000. It appeared not to be associated with lipid component. Both enzymes were assumed to be tissue-derived enzymes, because their activities were very low in the sera of healthy persons.

Isolation and characterization of proteodermatansulfate from rat skin.

A proteodermatansulfate was extracted from rat skin with 4 M guanidine hydrochloride containing protease inhibitors at 4 degrees C. It was separated from collagen on DEAE-cellulose in 7 M urea and was purified by cesium chloride density gradient centrifugation under associative conditions, DEAE-Sephadex column chromatography and then Sephadex G-200 gel filtration. On SDS-disc electrophoresis, the proteoglycan gave a single band staining for protein and a band of the same mobility staining with periodate-Schiff reagent. The proteoglycan contained 46% protein, and this was bound to dermatansulfate via an O-glycosidic linkage. The proteoglycan also contained 20% uronic acid and 16% hexosamine, but no detectable hydroxyproline. The ratio of galactosamine to glucosamine was 68. The dermatansulfate, the only glycosaminoglycan, had a hybrid structure containing iduronic acid and glucuronic acid as uronic acids. The content of glucuronic acid in uronic acid was much higher than that of pig skin dermatansulfate. The dermatansulfate had a slightly higher mobility on cellulose acetate electrophoresis than those from other sources. Gel chromatography on Sepharose CL-6B showed that the molecular weight of the dermatansulfate was 23,000, while that of the proteoglycan was 36,000. The elution profile of a mixture of the proteoglycan and hyaluronic acid on Sepharose 6B column chromatography was similar to that of the proteoglycan alone.

Preliminary studies of an acid-labile factor (ALF) in human sera that inactivates platelet-activating factor (PAF)

Rabbit PAF is known to be a potent inducer of in vitro platelet aggregation and secretion of platelet constituents, including serotonin, histamine, ADP, and prostaglandins. Normal rabbit serum contains an acid-labile factor (R-ALF) that rapidly inactivates PAF. Since PAF is acid-stable, it can now be recovered by rapidly acidifying blood samples obtained from IgE-sensitized rabbits experiencing antigen-induced anaphylaxis. This report describes the presence of a similar acid-labile factor in normal human serum (H-ALF). It is itself, or is associated with, lipoproteins and can be partially purified by Sepharose 6B column chromatography or by a two-step ultracentrifugation procedure. The presence of H-ALF may be of great importance during inflammation because it so rapidly degrades PAF in a manner analogous to the control of other potent mediators of inflammation such as histamine, slow-reacting substance of anaphylaxis (SRS-A), and eosinophil chemotactic factor of anaphylaxis (ECF-A).

Purification and characterization of Band 3, the major intrinsic membrane protein of the bovine erythrocyte membrane.

Band 3 from bovine erythrocyte membranes was isolated in a state of high purity by the following steps in the presence of a nonionic detergent, nonaethyleneglycol n-dodecyl ether (C12E9): (1) selective removal of Band 2.6 from ghosts by solubilization with 2% C12E9 (2) extraction of Band 3-rich fraction with 4% C12E9 from 2% C12E9-treated membrane residues, and (3) purification of Band 3 by aminoethyl-conjugated Sepharose 4B column chromatography. Human Band 3 was also purified in good yield by aminoethyl-conjugated Sepharose 4B column chromatography of erythrocyte membrane proteins solubilized with 1% C12E9 and treated with 2,3-dimethymaleic anhydride. There were no significant differences in CD spectra in C12E9, amino acid compositions, and migration mobilities in sodium dodecyl sulfate-gel electrophoresis between bovine and human Band 3. Calculations of average hydrophobicity and discriminant function demonstrated that bovine Band 3 could be categorized as a typical integral membrane protein. Bovine Band 3 showed a tendency to form a dimer and higher aggregates in 0.1% C12E9; these were resistant to dissociation into monomers in sodium dodecyl sulfate solution and, further, the protein retained residual secondary structure in highly concentrated guanidine hydrochloride solution, indicating the possible presence of an extended sequence of hydrophobic amino acid residues.

Presence in Dry Pea Cotyledons of Soluble Succinate Dehydrogenase That Is Assembled into the Mitochondrial Inner Membrane during Seed Imbibition

SUCCINATE DEHYDROGENASE (SUCCINATE: phenazine methosulfate oxidoreductase, EC 1.3.99.1) activity in crude mitochondrial fraction from pea (var. Alaska) cotyledons increased during seed imbibition to reach a maximum after about 12 hours. The increase was not inhibited by either cycloheximide or d(-)threo-chloramphenicol. The postmicrosomal fraction from dry cotyledons, but not that from fully imbibed ones, contained a soluble form of succinate dehydrogenase. The soluble enzyme was partially purified by ammonium sulfate fractionation and diethylaminoethyl-cellulose and Sepharose 6B column chromatography. The enzyme showed no succinate-coenzyme Q oxidoreductase activity and had a molecular mass of about 100,000 daltons. The soluble enzyme seemed to differ only slightly from succinate dehydrogenase solubilized from the mitochondrial inner membrane from fully imbibed cotyledons by a detergent. It is proposed that the soluble succinate dehydrogenase is associated with an inert mitochondrial inner membrane in dry cotyledons to form an active one during seed imbibition.

3,4,5,3′,4′-Pentachlorobiphenyl as a useful inducer for purification of rat liver microsomal cytochrome P448

An easy purification of rat liver microsomal cytochrome P448 was performed by using 3,4,5,3′,4′-pentachlorobiphenyl as an inducer. The cytochrome P448, a high spin form, was purified to 18.1 nmoles/mg protein with a good yield by ω-aminooctyl Sepharose 4B column chromatography followed by a hydroxyapatite column chromatography. This hemoprotein cross-reacted with antibody to cytochrome P448 from β-naphthoflavone-treated rats, but not with antibody to cytochrome P450 from phenobarbital-treated rats at all. The results of amino acid analyses suggested that this cytochrome P448 is similar to cytochrome P448 of 3-methylcholanthrene-treated rats.

Partial purification and properties of a rat brain phospholipase.

At least 90% of a membrane-bound phospholipase D was solubilized by extraction of freeze-dried rat brain with 0.8% Miranol H2M and 0.5% cholate. The bulk of base exchange reaction enzymes remained firmly bound to the particulate fraction under these conditions. The phospholipase D specific activity was enriched 240-fold by a purification protocol employing ammonium sulfate precipitation, and both Sepharose 4B and DEAE-cellulose column chromatography. The approximate molecular weight of the partially purified enzyme was calculated to be 200,000 based upon the elution profile from Sepharose 4B and Sephadex G-200 columns. The optimum pH was 6.0, and Km values for phosphatidylcholine and phosphatidylethanolamine were 0.75 mM and 0.91 mM, respectively. The enzyme activity was not dependent on the presence of divalent cation although Ca2+ and Fe2+ showed stimulatory effects.

Synthesis and characterization of a new fluorescent phospholipid.

A novel fluorescent phospholipid, whose structure was tentatively assigned as 1-(2'-thio-1'-hydroxyethyl)-2-(ethylphosphatidyl)isoindole), was synthesized by reacting O-phthalaldehyde and beta-mercaptoethanol with phosphatidylethanolamine. The fluorescent lipid product was purified by silicic acid chromatography. The purity was demonstrated by thin-layer chromatography. This fluorescent phospholipid could not form stable lipid vesicles. However, a mixture of phosphatidylcholine and this fluorescent phospholipid did form stable vesicles after sonication, as demonstrated by Sepharose 4B column chromatography and electron microscopy. The absorption and fluorescence properties of this lipid, both as aqueous micelles or incorporated into vesicles, have been determined. The potential usage of this new fluorescent phospholipid in membrane studies is discussed.

Antigenic bacterial polysaccharide in rheumatoid synovial effusions

Phenol-water extracted rheumatoid synovial fluids and synovial fluid leukocytes contain an antigen immunologically identical to the Proprionibacterium group bacteria. The antigen was identified by counter-immunoelectrophoresis in 70% of rheumatoid synovial fluid leukocyte pellets and in 60% of rheumatoid synovial fluids. It was also present in 6% of nonrheumatoid fluids and in 22% of nonrheumatoid inflammatory fluid leukocytes. Antigen was not detectable in synovial samples before extraction. Synovial and bacterial antigens were further purified by proteolytic digestion and Sepharose 4B column chromatography. Biochemical and enzymatic studies of bacterial and synovial antigens were similar and consistent with a high molecular weight polysaccharide. Serum antibody to bacterial and synovial antigens was significantly less frequent in rheumatoid sera than in normal controls. The significance of demonstrating a bacterial polysaccharide primarily in rheumatoid synovial effusions is discussed.

Studies of human carboxypeptidase B purification and properties from human small intestine

The carboxypeptidase B of human small intestine has been purified and partially characterized. The enzyme was purified by ammonium sulfate precipitation, DEAE-cellulose, CM-cellulose, and Sepharose 6B column chromatography. Purity was confirmed by disc gel electrophoresis at pH 8.5. The enzyme was purified 412-fold over the crude extract. Molecular weight of the enzyme was about 24000, as determined by Sephadex column chromatography and disc gel electrophoresis with sodium dodecyl sulfate. The enzyme was immunologically different from human pancreatic carboxypeptidase B 1 and B 2b since it does not cross-react with antiserum elicited against pure human pancreatic carboxypeptidase B 1. Extraction of the insoluble precipitate of intestinal wall yielded a carboxypeptidase which is possibly the dimer of carboxypeptidase B. The carboxypeptidase hydrolyzed HLA, HLL, and HLAa substrates. The enzyme was activated by Cd 2+ and Hg 2+. It was inhibited by heated SDS and urea. The enzymes were not inhibited or activated by Zn 2+, EDTA, and 2-mercaptoethanol.

Identification and partial purification of an ATP-stimulated alkaline protease in rat liver.

Extracts from rat liver contain a sulfhydryl-dependent endoprotease which degrades [methyl-14C]globin or 125I-hemoglobin to acid-soluble peptides. This enzyme was isolated from the 100,000 x g supernatant of the homogenate. It showed a pH optimum between 7.5 and 9.5 and very little activity below pH 7.0. The enzyme has an apparent molecular weight of 550,000 as determined on Sepharose 6B column chromatography and sucrose density gradient centrifugation. ATP, at physiological concentrations, as well as pyrophosphate, stimulated the protease activity in these partially purified preparations up to 3-fold. Nonionic detergents such as Triton X-100 increased proteolytic activity and the stimulation by ATP. Other nucleotide triphosphates and ADP also increased proteolysis but less effectively than ATP. Sodium phosphate, creatine phosphate, and EDTA had no stimulatory effect.

Fractionation of Escherichia coli isoaccepting tRNA species by sepharose 4B column chromatography

We have determined the elution profile on Sepharose 4B chromatographic column ofthe tRNA isoaccepting species of all 20 amino acids from Escherichia coli MRE 600. Further chromatography on a reversed phase column (RPC-5) is sufficient, in some cases, for a complete purification.

Studies of thyroid hormone binding in dog brain chromatin and cytosol

Intravenous infusion of labeled T3 to adult dogs followed by analysis of gradient-dialyzed 0.4 m KCl extracts of chromatin from nuclei of cerebral cortex gave in vivo evidence of soluble chromatin-associated thyroid hormone-binding proteins. Subsequent in vitro Sepharose 4B column chromatography of soluble brain chromatin extract and cytosol proteins provided evidence of specific binding of both labeled T3 and T4. Kinetic analysis of in vitro T3 and T4 binding to chromatin extract and to a Sepharose-purified cytosol fraction showed specific binding in each which appeared of low-affinity, high-capacity type. It is suggested that the soluble-binding activity demonstrated may be characterized as a relatively large, slowly equilibrating pool of intracellular thyroid hormone-binding protein.