Red Sepharose Research Articles

Contamination of Ribosome Inactivating Proteins with Ribonucleases, Separated by Affinity Chromatography on Red Sepharose

Three preparations of type 1 ribosome inactivating proteins (RIPs), namely, agrostin, saporin, and luffin, were subjected to affinity chromatography on Red Sepharose and eluted with a linear concentration gradient of NaCl in 10 mM Tris-HCl buffer (pH 7.4). The eluate was assayed for ability to inhibit translation in a cell-free rabbit reticulocyte lysate system which measures RIP activity, and for ability to hydrolyze yeast transfer RNA which measures RNase activity. It was found that, in all three RIP preparations, the peak of RIP activity, which coincided with the peak of absorbance at 280 nm, was eluted earlier than the peak of RNase activity. It appears that RNase is a possible contaminant of ribosome inactivating protein preparations and that this contamination can be minimized by using Red Sepharose.

A carbonyl reductase-catalyzing reduction of N3-phenacyluridine in rabbit liver.

Carbonyl reductase activity for a novel hypnotic, N3-phenacyluridine, was mainly localized in the cytosol fraction of rabbit liver. The enzyme (N3-phenacyluridine reductase) which catalyzes the reduction of N3-phenacyluridine to N3-alpha-hydroxy-beta-phenethyluridine was purified from the cytosolic fraction of rabbit liver by various chromatographic techniques (DEAE-Sephacel, Red Sepharose CL-6B and hydroxylapatite). N3-Phenacyluridine reductase had a minimum molecular weight of 39kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This enzyme required reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor and its optimal pH was 7.5. Flavonoids (quercetin and quercitrin) were potent inhibitors of the enzyme, but pyrazole or barbital had little effect. The apparent Km and Vmax values of the enzyme for the reduction of N3-phenacyluridine were 0.32 mM and 8.7 units/mg protein, respectively. A variety of carbonyl compounds, including N3-phenacyluridine, were effectively reduced by the enzyme. However, the enzyme purified from rabbit liver differs in several respects from known carbonyl reductases in rabbit liver.

α-Amylase Inhibitors from Cowpea Seeds

Abstract: This work describes the first isolation and partial characterization of α-amylase inhibitors from cowpea (Vigna unguiculata) seeds. α-Amylase inhibitors were isolated using an affinity chromatography on Red Sepharose CL-6B. The bound Red Sepharose fraction was active against α-amylases from Bacillus sp., Aspergilus oryzae, V unguicu/ata seeds and also against α-amylases from Callosobruchus maculatus larvae.

Preparation of highly purified momordin II without ribonuclease activity

Momordin II, a ribosome-inactivating protein from Momordica charantia seeds, was purified by a procedure involving a series of chromatographies on S-Sepharose, Sephadex G-50, CM-Sepharose, and Red Sepharose columns. Highly purified momordin II inhibited cell-free protein synthesis, released adenine from rat liver ribosomes and from DNA, and had no RNase activity.

Studies on purification of a lectin from fruiting bodies of the edible shiitake mushroom Lentinus edodes

A lectin, with a specificity for N-acetylgalactosamine and N-acetylglucosamine and a molecular weight of 43 kDa, was isolated from fruiting bodies of the edible shiitake mushroom Lentinus edodes. The purification procedure entailed extraction with aqueous buffer, ammonium sulfate precipitation, gel filtration on Sephadex G-100 and affinity chromatography on N-acetylgalactosamine-agarose. The lectin was unique in that it was tenaciously bound on anion exchangers including DEAE-cellulose, DEAE-Sepharose, DEAE-Sephadex, Q-Sepharose, Dowex and PEI-cellulose and also on hydroxyapatite and phenyl Sepharose. It was largely unadsorbed on cation exchangers including CM-cellulose, CM-Sepharose, SP-Sepharose and Amberlite, and also on protein G-Sepharose, Red Sepharose and Affi-gel Blue gel, wheat germ lectin-Sepharose and p-aminophenyl- d-glucopyranoside agarose.

Enzymological characteristics of the hyperthermostable NAD-dependent glutamate dehydrogenase from the archaeon Pyrobaculum islandicum and effects of denaturants and organic solvents.

NAD-dependent glutamate dehydrogenase (L-glutamate:NAD oxidoreductase, deaminating; EC 1.4.1.2) was purified to homogeneity from a crude extract of the continental hyperthermophilic archaeon Pyrobaculum islandicum by two successive Red Sepharose CL-4B affinity chromatographies. The enzyme is the most thermostable NAD-dependent dehydrogenase found to date; the activity was not lost after incubation at 100 degrees C for 2 h. The enzyme activity increased linearly with temperature, and the maximum was observed at ca. 90 degrees C. The enzyme has a molecular mass of about 220 kDa and consists of six subunits with identical molecular masses of 36 kDa. The enzyme required NAD as a coenzyme for L-glutamate deamination and was different from the NADP-dependent glutamate dehydrogenase from other hyperthermophiles. The Km values for NAD, L-glutamate, NADH, 2-oxoglutarate, and ammonia were 0.025, 0.17, 0.0050, 0.066, and 9.7 mM, respectively. The enzyme activity was significantly increased by the addition of denaturants such as guanidine hydrochloride and some water-miscible organic solvents such as acetonitrile and tetrahydrofuran. When fluorescence of the enzyme was measured in the presence of guanidine hydrochloride, a significant emission spectrum change and a shift in the maximum were observed but not in the presence of urea. These results indicate that this hyperthermophilic enzyme may have great potential in applications to biosensor and bioreactor processes.

Purification and characterization of chymotrypsin-like enzyme from rat plasma

A chymotrypsin-like enzyme was purified from rat plasma, involving ammonium sulfate fractionation and chromatographgy on CM-sephadex and red sepharose. The purified enzyme effectively hydrolysed the ester substrates for chymotrypsin (N-acetyl L-tyrosine ethyl ester and N-acetyl L-tryptophan ethyl ester). The Km values for the two substrates were 2.2×10−3M and 9.0×10−3M respectively. The hydrolytic activity of the enzyme was inhibited by phenylmethyl sulfonyl fluoride and tosylphenylalanine chloromethylketone, suggesting the presence of serine and histidine at the active centre. The enzyme exhibited anionic nature and possessed a high molecular weight (MW 71,000) as observed by gel exclusion chromatography on Sephadex G-200. The enzyme was stable upon exposure to pH 7.0–9.0, but was inactivated upon heat treatment at 60°C for 5 min.

Prostaglandin-E2 9-reductase from corpus luteum of pseudopregnant rabbit is a member of the aldo-keto reductase superfamily featuring 20 alpha-hydroxysteroid dehydrogenase activity.

The prostaglandin-E2 9-reductase (PGE2 9-reductase) activity in the corpus luteum of rabbits corresponds to a cytosolic, NADPH-dependent enzyme with a molecular mass of 36 kDa. This enzyme was purified from corpora lutea on day 12 of pseudopregnancy with a 266-fold enrichment. The main purification step was affinity chromatography using Red Sepharose CL-6B. The efficiency of this column was improved by elution with 1 mM NADH prior to elution of the active fractions with 1 mM NADPH. Amino acid sequence data demonstrate that the rabbit luteal PGE2 9-reductase has to be classified as a member of the aldo-keto reductase superfamily. The enzyme revealed a wide substrate specificity comprising the reduction of aldehydes, ketones, and quinones. Apparent kinetic constants were determined using methylglyoxal, DL-glyceraldehyde, and 9,10-phenanthrenquinone as substrates. The fully purified enzyme showed two catalytic activities of particular interest: PGE2 9-reductase and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activities. The competitive inhibition of 20 alpha-HSD activity by PGE2 indicates that progesterone and PGE2 are substrates for the same enzyme. From these results, we conclude that prostaglandin and steroid metabolism are tightly linked to each other. For this reason the aldo-keto reductase could be a key enzyme in the cascade of events leading to the regression of the corpus luteum in the rabbit.

Rapid purification of native SecA from Escherichia coli: development of a new affinity chromatography procedure.

The SecA protein occupies a pivotal position in the public protein export pathway in Escherichia coli. The multifunctional SecA protein recognizes cytoplasmic factors associated with export including the presecretory protein and targets the complex to the inner membrane, where it acts in the early stages of protein translocation. The ability of SecA to bind ATP was the basis for the development of a novel, rapid purification scheme involving a single chromatographic step. Affinity chromatography was carried out on Red Sepharose CL-6B. The SecA present in crude extracts of E. coli binds strongly to this dye-ligand matrix, and active protein was purified to greater than 90% homogeneity. The protein isolated by this procedure retained the previously described ATPase and RNA-binding activities of SecA. This approach should permit the rapid purification of SecA homologs from a variety microorganisms.

Biosynthesis of heparin/heparan sulfate. Purification of the D-glucuronyl C-5 epimerase from bovine liver.

The D-glucuronyl C-5 epimerase involved in the biosynthesis of heparin/heparan sulfate was purified from the high speed supernatant fraction of a homogenate of bovine liver by chromatography on immobilized O-desulfated heparin, red Sepharose, phenyl Sepharose, and concanavalin A-Sepharose. After close to 1 million-fold purification, in 10-15% yield, the product gave a single band on SDS-polyacrylamide gel electrophoresis with silver staining and had a mobility corresponding to an M(r) of approximately 52,000. Since the epimerase assay used in the course of purification was based on release of tritium, as [3H]H2O, from a [5-3H]uronyl-labeled substrate, it was important to establish that the purified enzyme did indeed catalyze the actual conversion of D-glucuronyl to L-iduronyl residues. Upon incubation of the purified enzyme with 3H-labeled heparosan N-sulfate, prepared by metabolic labeling (with D-[1-3H]glucose) of a capsular polysaccharide from Escherichia coli K5 and subsequent chemical partial N-deacetylation and N-sulfation, approximately 30% of the D-glucuronyl residues located between two N-sulfated glucosamine units were converted to L-iduronyl units.

Human Placental ( Asymmetrical) Diadenosine 5′,5‴- P 1, P4-Tetraphosphate Hydrolase: Purification to Homogeneity and Some Properties

The diadenosine 5′,5‴- P 1, P 4-tetraphosphate ( asymmetrical) hydrolase (EC 3.6.1.17) from human placenta has been purified to homogeneity by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sephecel, gel filtration on Sephadex G-100, and affinity elution from red Sepharose. The enzyme is a single polypeptide of M r 19,200. It exhibits maximum (100%) activity at pH 7.3 in the presence of 3mM MgCl 2 and 60, 50, and 40% of the activity in 1 mM CoCl 2, 0.1 mM ZnCl 2, and 0.5 mM MnCl 2, respectively. The K m value calculated for diadenosine tetraphosphate in the presence of Mg 2+ is 10 μM and in the presence of Zn 2+ 40 μM. Adenosine 5′-tetraphosphate, guanosine 5′-tetra-phosphate, and fluoride proved to he inhibitors of the diadenosine tetraphosphate hydrolase; the I 50 values were 6, 10, and 20 μM, respectively. Diguanosine tetraphosphate, bis-2,6-diaminopurine β-D-ribofuranoside tetraphosphate, and diadenosine pentaphosphate were substrates for the hydrolase; relative velocities of hydrolysis estimated for 0.5 mM diadenosine tetraphosphate and these other substrates were 1:0.51:0.44:0.20, respectively. Diadenosine tetraphosphate analogues with P 2 P 3 bridges such as CF 2, CCI 2, and CH 2 were hydrolyzed to adenosine 5′-phosphate and the corresponding adenosine 5′-triphosphate analogue. Interaction of the human placental diadenosine tetraphosphate hydrolase with diadenosine triphosphate and its phosphonate analogues was also tested. These compounds appeared to be competitive inhibitors with K i values lower than the K m for diadenosine tetraphosphate.

Quinone reduction and redox cycling catalysed by purified rat liver dihydrodiol/ 3α-hydroxysteroid dehydrogenase

A highly active preparation of rat liver dihydrodiol/3α-hydroxysteroid dehydrogenase was obtained using a newly developed, rapid purification scheme involving affinity chromatography on Red Sepharose. Depending on the coenzyme present, the purified enzyme was found to catalyse the oxidation of dihydrodiols and steroids or the reduction of substrates with carbonyl or quinone moieties. Using a wide range of synthetic quinones derived from polycyclic aromatic hydrocarbons (PAHs), we observed a pronounced regioselectivity of the quinone reductase activity. Good substrates were the o-quinones of phenanthrene, benz( a)anthracene, chrysene and benzo( a)pyrene with the quinonoid moiety in the K-region which were reduced at rates of 1–10 μmol/min · mg enzyme. 1,4-Benzoquinone, naphthalene1, 2-quinone and benz( a)anthracene-8,9-quinone were also reduced at high rates. In contrast, alkylsubstituted quinones such as duroquinone and menadione were poor substrates for the enzyme. During the enzymatic reduction of several o-quinones, but not 1,4-benzoquinone, we observed the oxidation of large amounts of NADPH and the consumption of molecular oxygen which is indicative of a redoxcycling process. Thus, the reduction of quinones of PAHs may lead to a facilitated conjugation and excretion of these highly lipophilic compounds, but may also initiate toxic processes due to the formation of reactive oxygen species.

Distribution, purification, and characterization of thermostable phenylalanine dehydrogenase from thermophilic actinomycetes

Phenylalanine dehydrogenase (L-phenylalanine:NAD oxidoreductase, deaminating; EC 1.4.1.-) was found in various thermophilic actinomycetes. We purified the enzyme to homogeneity from Thermoactinomyces intermedius IFO 14230 by heat treatment and by Red Sepharose 4B, DEAE-Toyopearl, Sepharose CL-4B, and Sephadex G-100 chromatographies with a 13% yield. The relative molecular weight of the native enzyme was estimated to be about 270,000 by gel filtration. The enzyme consists of six subunits identical in molecular weight (41,000) and is highly thermostable: it is not inactivated by incubation at pH 7.2 and 70 degrees C for at least 60 min or in the range of pH 5 to 10.8 at 50 degrees C for 10 min. The enzyme preferably acts on L-phenylalanine and its 2-oxo analog, phenylpyruvate, in the presence of NAD and NADH, respectively. Initial velocity and product inhibition studies showed that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism. The Km values for L-phenylalanine, NAD, phenylpyruvate, NADH, and ammonia were 0.22, 0.078, 0.045, 0.025, and 106 mM, respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH was exclusively transferred to the substrate.

Purification of cytosolic 3,5,3′-triiodo-L-thyronine(T 3)-binding protein(CTBP) which regulates nuclear T 3 translocation

The NADPH-dependent cytosolic 3,5,3′-triiodo-L-thyronine(T 3)-binding protein(CTBP) was purified from rat kidney using Mono Q-Sepharose, Red sepharose and T 3 affinity chromatography. CTBP which was partially purified by Red Sepharose column chromatography was adsorbed to T 3 affinity column in the presence of 50 uM NADPH. The CTBP was eluted from the gel with the buffer which did not contain NADPH. One molecule of the purified CTBP(58 kDa) bound one molecule of T 3 with 2.44×10 9M −1 of affinity constant. The purified CTBP was activated not only by NADPH but also by NADP in the presence of dithiothreitol. The NADPH-activated form did not transfer T 3 to nuclei, whereas NADP transformed the NADPH-activated CTBP to active form which was able to transfer T 3 to nuclei. These results suggested that CTBP-dependent transport of T 3 to nucleus is controlled by NADPH and NADP.

Aldose reductase from human skeletal and heart muscle. Interconvertible forms related by thiol-disulfide exchange.

Aldose reductase was purified from human skeletal and heart muscle by a rapid and efficient scheme involving Red Sepharose chromatography, chromatofocusing on Pharmacia PBE 94, and hydroxylapatite high pressure liquid chromatography. The scheme afforded homogeneous enzyme, 65% recovery, in 2 days. All muscle samples express aldose reductase but not the closely related aldehyde reductase. Aldose reductase is isolated in one of two forms that are distinguishable by their kinetic patterns with glyceraldehyde as substrate and which are interconvertible by treatment with dithiothreitol. Both forms are capable of catalyzing the reduction of glucose (Km = 68 mM), and both are highly sensitive to inhibition by aldose reductase inhibitors. The reduction of glucose was shown to be nearly stoichiometric with production of sorbitol (92 +/- 2%). Dialysis of aldose reductase in the absence of thiols or NADP converts it into a form that shows markedly different kinetic properties, including very weak catalytic activity toward glucose and insensitivity to aldose reductase inhibitors. This modified form can be converted back into the native form by dithiothreitol. Thiol titration of the two forms of aldose reductase with Ellman's reagent indicated that two thiol groups were lost when the enzyme was dialyzed in the absence of dithiothreitol or NADP.

Aldehyde and aldose reductases from human placenta. Heterogeneous expression of multiple enzyme forms.

Aldehyde reductase (ALR1) and aldose reductase (ALR2) were purified from human placenta by a rapid and efficient scheme that included rapid extraction of both reductases from 100,000 x g supernatant material with Red Sepharose followed by purification by chromatofocusing on Pharmacia PBE 94 and then chromatography on a hydroxylapatite high performance liquid chromatography column. Expression of ALR1 and ALR2 in placenta is variable with ALR1/ALR2 ratios ranging from 1:4 to 4:1. ALR1 and ALR2 are immunochemically distinct. ALR1 shows broad specificity for aldehydes but does not efficiently catalyze the reduction of glucose due to poor binding (Km = 2.5 M). ALR1 exhibits substrate inhibition with many substrates. ALR2 also shows broad specificity for aldehydes. Although glucose is a poor substrate for ALR2 compared with other substrates, the affinity of ALR2 for glucose (Km = 70 mM) suggests that glucose can be a substrate under hyperglycemic conditions. ALR2 shows normal hyperbolic kinetics with most substrates except with glyceraldehyde, which exhibits substrate activation. Treatment of ALR2 with dithiothreitol converted it into a form that exhibited hyperbolic kinetics with glyceraldehyde. Dithiothreitol treatment of ALR2 did not alter its properties toward other substrates or affect its inhibition by aldose reductase inhibitors such as sorbinil (2,4-dihydro-6-fluorospiro-[4H-1-benzopyran-4,4'-imidazolidine]-2' ,5'- dione), tolrestat (N-[[6-methoxy-5-(trifluoromethyl)-1-naphthalenyl]thioxomethyl]-N- methylglycine), or statil (3-[(4-bromo-2-fluorophenyl)methyl]-3,4-dihydro-4-oxo-1-phthalazineac etic acid).

Purification and characterization of rho-crystallin from Japanese common bullfrog lens.

In a previous paper, we reported that the partial amino acid sequence (225 residues) from the COOH terminus of rho-crystallin from European common frog lens shows 77% similarity to that of prostaglandin (PG) F synthetase, an aldo-keto reductase, from bovine lung (Watanabe, K., Fujii, Y., Nakayama, K., Ohkubo, H., Kuramitsu, S., Kagamiyama, H., Nakanishi, S., and Hayaishi, O. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 11-15). Here rho-crystallin was purified to apparent homogeneity from the eye lens of the Japanese common bullfrog (Rana catesbeiana) by four sequential chromatographies using Sephadex G-100, Red Sepharose, and dual Mono S. Two types of rho-crystallin, RHO-I and RHO-II, named according to their elution order from a Mono S column, are essentially identical in terms of immunochemical properties, amino acid composition, and partial amino acid sequence. But the NH2-terminal Thr of RHO-I is blocked with an acyl group, while that of RHO-II is free. Both crystallins as well as PGF synthetase are monomeric proteins with a molecular weight of about 35,000 and they have the ability to bind NADPH with a stoichiometry of 0.75 mol of cofactor/mol of protein. Although rho-crystallin does not cross-react with antibody against PGF synthetase, the NH2-terminal amino acid sequence (107 residues) of rho-crystallin shows 77% similarity to that of the enzyme. However, PGD2, PGE2, 9,10-phenanthrenequinone, p-nitrobenzaldehyde, DL-glyceraldehyde, D-glucuronic acid, D-glucose, D-xylose, menadione, p-nitroacetophenone, dihydroxyacetone, succinic semialdehyde, phenylglyoxal, and testosterone were not substrates for these crystallins. PGH2 9,11-endoperoxide reductase activities of RHO-I and RHO-II were 1.3 and 1.0 milliunits/mg of protein, respectively, which are only about 2% of that of bovine lung PGF synthetase. These results indicate that the rho-crystallins RHO-I and RHO-II belong to a group of aldo-keto reductases based on primary structure, molecular properties, and NADPH-binding ability, but show only low PGH2 9,11-endoperoxide reductase activity.

Effect of king cobra venom on α2-macroglobulin and proteases in human blood plasma

Normal human blood plasma showed hydrolytic activities on several synthetic substrates for proteases, the most effective being H-D-Ile-Pro-Arg-p-nitroanilide, H-D-Pro-Phe-Arg-p-nitroanilide and H-D-Val-Leu-Arg-p-nitroanilide. When plasma was preincubated for 12 h at 37°C, there was no significant alteration of the hydrolytic activities. On incubation for 12 h with king cobra venom (2 μg for 0.1 ml plasma), there was considerable decrease in the activities and complete abolition of the protease binding capacity of α2-macroglobulin. On chromatography on Sephadex G-200, α2-macroglobulin activity and bulk of the protease activity of normal plasma were eluted in the void volume region. A minor protease peak was eluted with aVe/Vo value of 2.5. With venom treated plasma, there was no decrease with this peak. The major protease peak and α2-macroglobulin activity were drastically reduced. Chromatography on red Sepharose showed that all the α 2 − acroglobulin activity and bulk of the protease activity in normal plasma were bound to the column. In venom treated plasma there was marked reduction in the bound fraction. The data suggest that cobra venom proteases directly or through proteases generated in plasmain situ causes limited cleavage of α2-macroglobulin as well as α2-macroglobulin bound proteases, inactivating them.

Purification and characterization of an insect alpha-amylase inhibitor/endochitinase from seeds of Job's Tears (Coix lachryma-jobi).

A protein inhibitor of locust gut alpha-amylase was purified from seeds of Job's Tears (Coix lachryma-jobi) using ammonium sulphate precipitation, affinity chromatography on Red Sepharose and reversed-phase HPLC. It consisted of two major isomers, each a dimer of two closely similar or identical subunits of Mr about 26,400, and associated by inter-chain disulphide bonds. These isomers also had closely similar amino acid compositions. The major isomer showed no inhibitory activity against amylases from other sources: human saliva, porcine pancreas, Bacillus subtilis, Aspergillus oryzae and barley malt. The manual DABITC/PITC method was used to determine about half of the amino acid sequence of the major isoform. This showed a high degree of homology with previously reported sequences of endochitinase enzymes from several species (tobacco, potato, barley, bean), and endochitinase activity was demonstrated by following the release of radioactivity from [3H]chitin. This novel combination of functions may be relevant to protection of the grain from insect feeding and fungal infection.

Crystallization of recombinant human interleukin 1β

The gene for the fully processed form of human interleukin 1β was cloned from SK-hep-1 hepatoma cellular RNA and expressed at high levels in E. coli. The protein produced in E. coli. was purified to homogeneity by standard chromatographic methods, including adsorption and desorption from Procion Red Sepharose, sizing on a Superose 12 FPLC column, and anion exchange chromatography on QAE Sepharose. The result is a biologically active protein, rIL-1β, that migrates on two-dimensional gels as a single spot with a pI of 6.5 ± 0.2 and a molecular mass of 17, 500 daltons. Crystals of rIL-1β have been produced from concentrated solutions of the protein by ammonium sulfate precipitation. The crystals are tetragonal, have space group P41 or its enantiomer, have lattice constants of a = 58.46(1) Å and c = 77.02(3) Å, and scatter to at least 2 Å resolution. A structure determination ba these crystals is underway.