phenyl-Sepharose Column Research Articles

Organ distribution, purification and characterization of kynureninase in Suncus murinus (Insectivora) and anthranilic acid level in the serum

1. 1. The highest kynureninase activity was measured in Suncus liver compared with other organs tested. The holo-enzyme activity detected in liver was 25%. 2. 2. The enzyme mainly localized in liver cytosol was purified to a single protein band by heat treatment, ammonium sulfate fractionation, DEAE-sepharose, hydroxyapatide and phenyl-sepharose column chromatography. 3. 3. The purified enzyme was effective for both kynurenine and 3-hydroxykynurenine, and showed the optimum pH at 8.5 against both substrates. 4. 4. K m values were 250 μM, 18 μM and 70 μM for kynurenine, 3-hydroxykynurenine and pyridoxal 5′-phosphate, respectively. Various compounds such as histidine, aspartate and nicotinamide enhanced the enzyme activities. 5. 5. Anthranilate, the product of kynureninase, was present at a concentration in Suncus serum of 1.96 μM. This value was higher than those of rat and human.

Purification of labelled antibodies by hydrophobic interaction chromatography

In order to improve the sensitivity of immunometric assays, a chromatographic technique was developed that virtually eliminates components causing non-specific background. Labelled antibodies were applied to a phenyl-Sepharose column in physiological buffer. When labelled anitbodies were purified by this technique, the non-specific background of various time-resolved immunofluorometric assays was reduced 3- to 10-fold and was very close to the instrument background. The assay sensitivity was simultaneously increased by a factor of 2 to 16. This purification method might be used to improve the results of immunometric assays in general.

27] Snowdrop lectin

Publisher Summary The snowdrop ( Galanthus nivalis ) lectin (GNA) is one of a series of mannose-binding lectins present in the tubers (bulbs) of members of the family Amaryllidaceae. Their carbohydrate-binding specificity is unique in that it is confined to nonreducing terminal α-D-mannosyl groups; internal mannosyl residues and D-glucose and N-acetyl-o-glucosamine do not interact with these lectins. This property distinguishes these lectins from lectins of the family Leguminoseae (concanavalin A and lectins from pea, lentil, and Vicia faba ). Phenyl-Sepharose column chromatography removes slight contamination with phenolic substances. The original preparation of the snowdrop lectin included an anion-exchange chromatography step, which is unnecessary. Studies on the tissue localization and biosynthesis of the snowdrop lectin were reported recently. The unique carbohydrate-binding specificity of the snowdrop makes this lectin a valuable addition to the armamentarium of the biomedical researcher.

Analysis of Toxoplasma gondii proteins after Triton X-114 solubilization and hydrophobic chromatography.

The distribution of the surface proteins of Toxoplasma gondii radiodinated were studied using the phase separation technique and ability of binding in the phenyl-Sepharose column. Eight polypeptides with Mr 22 to 180 distributed exclusively in the detergent rich-phase, while six polypeptides with mol. wt. 15,000 to 76,000 distributed exclusively in the detergent poor-phase. Two polypeptides with 15,000 and 70,000 distributed in both phase. All the polypeptides present in the detergent rich-phase binding in the phenyl-Sepharose column, and can be isolated in two peak according with their relative hydrophobicities. Two polypeptides hydrophobic with Mr 60 and 66 recognized by human serum were isolated by the association of the two technique. Our result showed that the surface proteins of T. gondii present different degrees of hydrophobicity and that the use of hydrophobic interaction chromatography after Triton X-114 extraction may be an important isolation method of membrane proteins.

S-adenosylmethionine synthetase in the thermophilic archaebacterium Sulfolobus solfataricus. Purification and characterization of two isoforms.

Two isoforms of methionine adenosyltransferase (S-adenosylmethionine synthetase), A and B, have been partially purified from Sulfolobus solfataricus, a thermophilic archaebacterium optimally growing at 87 degrees C. The chromatographic procedure, involving hydrophobic chromatography on a phenyl-Sepharose column as a major step, results in 330-fold and 150-fold purification of adenosylmethionine synthetase A and B respectively. The apparent molecular masses, estimated by gel filtration, are 180 kDa for A and 75 kDa for B. The A and B isoforms follow Michaelis-Menten kinetics with apparent Km values of 10 microM and 20 microM for L-methionine and of 50 microM and 150 microM for ATP respectively. Adenosylmethionine, a product of the reaction, acts as a powerful non-competitive inhibitor (Ki = 50 microM) of the A isoform while it inhibits only slightly the B isoform. Both isozymes exhibit tripolyphosphatase activity but only that associated with the form A is stimulated by 5 microM adenosylmethionine concentration. The two enzymes absolutely require a divalent cation for the activity, but are not affected by monovalent ions and reducing agents. The optimum temperature is 90 degrees C and no significant loss of activity is observable after incubation of the two isoforms at 100 degrees C in the presence of ATP. The Arrhenius plots observed for both isozymes are biphasic, indicating different activation energies below and above 75 degrees C. The cytoplasmic levels of ATP, methionine and adenosylmethionine are evaluated.

Characterization of multiple forms of cytochrome P-450 from an insecticide resistant strain of house fly ( Musca domestica)

Components of the microsomal monooxygenase system derived from abdominal preparations of an insecticide resistant house fly ( Musca domestica, Rutgers strain) were characterized. Cytochrome P-450 isozymes with molecular weights of 49,000, 53,000, 54,000, and 58,000 were partially purified using a procedure involving chromatography on columns of phenyl Sepharose, DEAE-cellulose (DE-52), carboxymethyl-Sepharose, hydroxylapatite, and an affinity column prepared from rat liver cytochrome b 5. Activity toward a variety of pesticides, as well as other exogenous and endogenous monooxygenase substrates, was examined using intact microsomes or purified isozymes in reconstituted systems. Oxidation of benzo[ a]pyrene in microsomal preparations was high although neither O-dealkylation of 7-ethoxyresorufin nor N-dealkylation of p-chloromethylaniline could be detected. Other substrates metabolized by abdominal microsomes from Rutgers flies included testosterone, lauric acid (primarily at in-chain positions), phorate, p-nitroanisole, and aldrin. Activity of abdominal microsomes from the susceptible CSMA strain toward benzo[ a]pyrene and lauric acid was, surprisingly, higher than that from the resistant Rutgers strain. Synergism between NADPH and NADH was noted with microsomes from either strain. Reconstitution experiments were relatively unsuccessful but activity toward lauric acid was obtained using a system that included purified cytochrome P-450 fractions, purified house fly or rat liver NADPH-cytochrome P-450 reductase, an NADPH generating system, and a phospholipid. Unlike similar systems utilizing mammalian cytochrome P-450, phosphatidylethanolamine was more active than phosphatidylcholine.

Purification and properties of the cytosolic substrate for botulinum ADP-ribosyltransferase. Identification as an Mr 22,000 guanine nucleotide-binding protein.

The substrate for ADP-ribosyltransferase from Clostridium botulinum was purified from the cytosol of bovine adrenal gland. Purification procedures consisted of ammonium sulfate fractionation, chromatographies on columns of DEAE-Sepharose and phenyl-Sepharose, gel filtration on a TSK-gel G3000SW column, and Mono Q fast protein liquid chromatography. On DEAE-Sepharose chromatography, the substrate activity was eluted in two separate peaks, and electrophoretic analyses revealed that the substrates in the two peaks are of similar molecular weight but different isoelectric points. The major peak of the substrate was further purified. It was purified about 1,800-fold with a recovery of 2.2% by the above procedures. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the final preparation showed a single protein band at Mr 22,000. The purified protein served as a substrate for botulinum ADP-ribosyltransferase and was maximally ADP-ribosylated to the extent of about 0.7 mol of ADP-ribose/mol of protein. A guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was co-purified with the ADP-ribosylation substrate, and the purified protein maximally bound about 0.5 mol of GTP gamma S/mol. GTP gamma S binding was effectively competed by GTP and GDP but not by GMP, ATP, and ADP. Thus, the ADP-ribosylation substrate is a GTP-binding protein. This protein, designated Gb (b for botulinum), is widely distributed in various tissues. It was rich in brain, pituitary, and adrenal glands, and poor in heart, smooth, and skeletal muscles.

A pathogenesis-related protein in cucumber is a chitinase

A pathogenesis-related (PR) protein was found in both the infected and the uninfected leaves of cucumber plants inoculated on the first true leaf with a fungal, a bacterial or a viral pathogen. This host-coded protein was detected up to five leaves above the infected leaf. The protein was purified from the intercellular fluid by ion-exchange chromatography and by high performance liquid chromatography on ion-exchange and phenyl-sepharose columns. The purified PR-protein was shown to be a chitinase with a molecular mass of 28 000 as determined by SDS-polyacrylamide gel electrophoresis and by gel filtration.

A new preparation of S-100 protein from rat and bovine brains.

The S-100 nervous system protein was purified from bovine and rat brains by a modification of the original procedure. The main modification consisted in substituting a step of calcium-dependent binding of S-100 to a phenyl-Sepharose column for the original step of chromatography on G-200 Sephadex. The proteins were pure as determined by SDS gel electrophoresis. HPLC on a reversed phase and on a size-separation column, and by immunological criteria. The bovine S-100 behaved as previously described, during calcium binding, by displaying a conformational change as evidenced by increase in native fluorescence.

Improved purification of human liver alkaline phosphatase by phenyl-Sepharose column chromatography

Improved purification of human liver alkaline phosphatase by phenyl-Sepharose column chromatography

Ca 2+-dependent protein kinase from alfalfa ( Medicago varia): Partial purification and autophosphorylation

A calcium-dependent protein kinase (CDPK) was purified to 1400-fold from the soluble fraction of alfalfa ( Medicago varia) cells by ammonium sulfate fractionation, Sephacryl-300, DEAE-Sephacel, Phenyl-Sepharose and Hydroxylapatite column chromatography. The enzyme is mainly monomeric. During the course of the purification steps a 50 kDa phosphoprotein doublet and a 56 kDa phosphoprotein copurified with the CDPK activity. Mobility shift of these proteins have been shown by SDS PAGE in Ca 2+ free conditions. Tests on enzyme activity after separation by native gel electrophoresis revealed two protein kinase activities in our enzyme preparation and the phosphorylation of the 50 kDa and 56 kDa proteins. We suggest that these proteins are the autophosphorylated forms of calcium dependent protein kinases. Preincubation of the CDPK in ATP resulted in a marked increase in enzyme activity, but did not alter the Ca 2+ sensitivity of the protein kinase.

Human Lipid Transfer Proteinの精製と抗LTP IgGの作製

A core lipid transfer protein (LTP) from human plasma was purified 16, 000 fold by acid-dextran sulfate precipitation in the presence of 17% ethanol, Phenyl-Sepharose, DEAF-Sephadex, Succinylated LDL-Sepharose, and Hydroxyl Apatite column chromatographies. The purified LTP showed a single band with an apparent molecular weight of 65, 000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Anti human LTP IgG was prepared from rabbit anti-serum. There was a single precipitation line between purified LTP and anti human LTP IgG by the Ouchterlony double-diffusion method. No visible precipitation line was seen between anti LTP IgG and apo D, apo A-I, apo A-II, human serum albumin, or lecithin-cholesterol acyltransferase. Addition of anti LTP IgG to the assay mixture containing purified LTP or plasma as a LTP source resulted in 100% inhibition of lipid exchange activity.These results suggest that core lipid transfer protein in human plasma may be a single protein.

Properties of bovine erythrocyte acetylcholinesterase solubilized by phosphatidylinositol-specific phospholipase C1.

The properties of acetylcholinesterase solubilized from bovine erythrocyte membrane by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis or with a detergent, Lubrol-PX, were studied. The activity of Lubrol-PX-solubilized acetylcholinesterase was broadly distributed in the fractions having Ve/Vo = 1.0-2.0 in gel filtration on a Sepharose 6B column. The intermediary fractions (Ve/Vo = 1.3-1.7) were collected as "the middle active Sepharose 6B eluate" and characterized on the basis of enzymology and protein chemistry. When this eluate was treated with PI-specific phospholipase C, the major activity peak was obtained in the later fractions with Ve/Vo = 1.75-2.0 on the same column chromatography. Lubrol-solubilized and phospholipase C-treated acetylcholinesterase preparations were different in the thermostability, the elution profiles of chromatography on Mono Q, butyl-Toyopearl and phenyl-Sepharose columns, and the affinity to phospholipid micelles. On treatment with PI-specific phospholipase C, Lubrol-solubilized acetylcholinesterase became more thermostable. The phospholipase C-treated enzyme was eluted at lower NaCl concentration from the Mono Q column than the Lubrol-solubilized enzyme. The most important difference was observed in the hydrophobicity of these two enzyme preparations. The Lubrol-solubilized enzyme shows high affinity to phospholipid micelles and hydrophobic adsorbents such as butyl-Toyopearl and phenyl-Sepharose. However, this hydrophobicity was lost when acetylcholinesterase was solubilized from bovine erythrocyte membrane by PI-specific phospholipase C. The presence of myo-inositol was confirmed in the purified preparation of acetylcholinesterase by gas chromatography (GC)-mass spectrometry (MS).(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-sensitive, lipid-binding cytoskeletal proteins of the human placental microvillar region.

In this study we describe a group of Ca2+-sensitive proteins located in the microvillar region of the human placental syncytiotrophoblast. By following the distribution of proteins between the particulate and supernatant phases of detergent-solubilized microvilli in the presence of defined concentrations of free Ca2+, we demonstrate a class of proteins of subunit molecular weights 72,000, 69,000, 38,000, 36,000, and 32,000 that associate with both the cytoskeleton and lipid at high concentrations of free Ca2+. These proteins can be released from microvilli using EGTA-containing buffers. Although they do not bind to phenyl-Sepharose, they will bind to phospholipids immobilized on phenyl-Sepharose columns in a Ca2+-dependent manner and show a marked preference for phospholipids with negatively charged headgroups. The results provide evidence for a sequence of events which may occur within the microvillus as the localized concentration of intracellular free Ca2+ rises.

Separation and characterization of 5'- and 3'-tRNA processing nucleases from rat liver mitochondria.

The 5'- and 3'-tRNA processing nucleases have been isolated from rat liver mitochondria. The two activities co-purified through heparin-agarose and phenyl-Sepharose columns and then efficiently separated on a DEAE-cellulose column. The 5' processing nuclease was found in the flow-through fraction, and the 3' processing activity eluted with 0.5 M KCl. Both enzymes were greater than 500-fold purified over the high speed supernatant of a mitoplast extract. The 159-base pre-tRNATyr used as a substrate in this study was synthesized in vitro and contained the Escherichia coli suppressor III tRNATyr plus a 49-base leader sequence and a 25-base trailing sequence. The 5' processing nuclease converted the pre-tRNATyr into two discrete RNA species, identified as the 5'-processed intermediate and the 5' flanking fragment, by endonucleolytic cleavage at the 5' end of the mature tRNATyr sequence. The 3' processing nuclease was inactive with the intact pre-tRNATyr as substrate but efficiently converted the 5'-processed intermediate to the mature tRNATyr, indicating an obligatory order of processing in which 5' maturation was necessary before cleavage by the 3' processing nuclease could occur. The mitochondrial enzymes exhibited optimal activity in the presence of about 2 mM Mg2+, but both enzymes were nearly fully active without addition of exogenous Mg2+ to the reaction mixtures. In contrast, a partially purified 5' processing endonuclease present in the postmitochondrial cytosolic fraction required higher [Mg2+] for activity, thus providing a means for differentiating between these similar enzyme activities obtained from the cytosolic and mitochondrial fractions.

Isolation of a Ca-dependent erythrolytic protein (perforin) from cytotoxic T-lymphocytes.

A Ca-dependent erythrolytic protein (perforin) was isolated from a cytotoxic T-cell line (CTLL2). Cellular extracts were fractionated on DEAE-cellulose and hydrophobic Phenyl-Sepharose columns. Lytic activity was tightly bound to the hydrophobic column and was eluted with 50% ethyleneglycol. The erythrolytic activity was dependent on the concentration of Ca2+ ions, and heparin accelerated the lysis of erythrocytes by perforin 10-fold, with a half maximal concentration of 12 ng/ml. The activity was strongly inhibited by micromolar concentrations of heavy metal ions, such as Zn2+ and Fe2+, and glycylarginine-methylcoumarinamide (Gly-Arg-MCA) in the presence of 100 ng/ml heparin.

Purification and characterization of murine protoporphyrinogen oxidase.

The penultimate enzyme of the heme biosynthetic pathway, protoporphyrinogen oxidase (EC 1.3.3.4), has been purified to apparent homogeneity from mouse liver mitochondria. The purification involves solubilization from mitochondrial membranes with sodium cholate followed by ammonium sulfate fractionation and gel filtration on a Sepharose CL-6B column. The eluate is adjusted to 0.67 M (NH4)2SO4 and loaded onto a phenyl-Sepharose column. After salt washes, the enzyme is eluted with 0.5% sodium cholate and 0.5% Brij 35. The final step is high-pressure ion-exchange chromatography on a DEAE-5PW column. The purified protein has a molecular weight of approximately 65,000 by gel filtration chromatography on Sepharose CL-6B in the presence of 0.5% sodium cholate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a single band corresponding to a molecular weight of 65,000. The absorption spectrum of the purified enzyme shows no evidence of a chromophoric cofactor. Purified protoporphyrinogen oxidase has a Km for protoporphyrinogen IX of 5.6 microM with a Vmax of 2300 nmol mg-1 h-1. It utilizes meso- and hematoporphyrinogen at about 10% the level of protoporphyrinogen. The pH optimum is broad with a maximum at 7.1. There is no stimulation or inhibition by any tested divalent cations, and sulfhydryl reagents have no inhibitory effect on the purified enzyme.

Purification and characterization of a carboxylesterase from rabbit liver lysosomes.

Carboxylesterase [EC 3.1.1.1] was purified from rabbit liver lysosomes by means of detergent solubilization, and by hydroxyapatite, phenyl-Sepharose and chromatofocusing column chromatographies. The purified enzyme appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 58,000. This enzyme was eluted at an isoelectric point of approximately 5.8 by chromatofocusing, and exhibited a broad pH optimum of between 6.0 and 9.0. The enzyme hydrolyzed 4-methylumbelliferyl esters of saturated fatty acids (C2-C12), and it also hydrolyzed p-nitrophenylacetate, methyl butyrate, and tributyrin, but not acetanilide. Its activity was completely inhibited by diisopropyl-fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF) at 10(-4) M, but was not affected by eserine, or by alpha- or beta-naphthyl acetate at 10(-3) M. Various metal ions (Mg2+, Mn2+, Ca2+, Co2+, Cu2+, Zn2+, Ni2+) at 10(-3) M also had no effect on the enzyme activity.

Heterogeneity of bovine corneal proteokeratan sulfate.

Proteokeratan sulfate was extracted and purified from bovine corneal stroma and then characterized by chemical and biochemical analyses. It was fractionated into several fractions by affinity chromatography on a concanavalin A-Sepharose column or by hydrophobic chromatography on a phenyl-Sepharose column. These fractions differed widely from one another in carbohydrate content, though no significant differences of their amino acid compositions were observed. One fraction (ca. 25%, on a dry weight basis) tightly bound to a concanavalin A-Sepharose column, compared with another fraction (ca. 65%) weakly bound to the same column, was poor in galactose and N-acetylglucosamine, but contained mannose in a high proportion. Fractions (ca. 30%) tightly bound to a phenyl-Sepharose column, in contrast to the one (ca. 66%) weakly bound, had low carbohydrate contents, like the fraction tightly bound to a concanavalin A-Sepharose column. Additionally, the fractions tightly bound to these affinity columns exhibited strong inhibitory actions on erythrocyte-concanavalin A agglutination. To obtain further details of the carbohydrate moiety of the proteokeratan sulfate, an attempt was made to separate and characterize peptidokeratan sulfate and Asn-linked oligosaccharide derived from some proteokeratan sulfate fractions. The present work revealed that the proteokeratan sulfate contains keratan sulfate and high mannose-type oligosaccharide in an approximate chain number ratio of 3.5:1.0, the keratan sulfate content varies widely and the oligosaccharide content increases with decrease of the keratan sulfate content, and the protein core is homogeneous at least with respect to the amino acid composition.

N-acetyl-galactosamine-specific lectin, a novel lectin in the haemolymph of the ascidian Halocynthia roretzi: isolation, characterization and comparison with galactose-specific lectin

1. 1. An N-acetyl-galactosamine-specific lectin has been isolated from haemolymph of the ascidian Halocynthia roretzi in an electrophoretically homogenous form, by four chromatographic operations including affinity chromatography on p- aminophenyl-N- acetyl-β- d-galactosaminide-Sepharose . 2. 2. The lectin has the mol. wt of 500,000 and the apparent sedimentation coefficient of 16–19 S. It is composed of subunits with a mol. wt of 28,000. 3. 3. The haemagglutinating activity of the lectin is Ca 2+-dependent and inhibited with N- acetyl- d-galactosamine and its analogs. 4. 4. The lectin is adsorbed to a column of phenyl-Sepharose and eluted with 66% ethylene glycol. 5. 5. The concentration of the lectin in the haemolymph, as well as that of the galactose-specific lectin found previously in the same source, remains unchanged in response to injection of horse erythrocytes. 6. 6. Properties of the lectin is compared with those of the galactose-specific lectin.