phenyl-Sepharose Column Chromatography Research Articles

Isolation and reconstitution of the iron-sulfur protein in ubiquinol-cytochrome c oxidoreductase complex. Phospholipids are essential for the integration of the iron-sulfur protein in the complex.

An iron-sulfur protein has been purified from beef heart ubiquinol-cytochrome c oxidoreductase (Complex III) of the mitochondrial respiratory chain by phenyl-Sepharose column chromatography and Sephacryl S-200 gel chromatography. Depletion of most of the endogenous phospholipids in the complex was a prerequisite to the dissociation of the protein from the complex in the former chromatography. The iron-sulfur protein was nearly homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained 76 ng atoms of nonheme iron and 66 nmol of acid-labile sulfide/mg of protein. When this preparation was incubated with an iron-sulfur protein-depleted complex in the presence of soybean phospholipids, the enzymic activity was restored up to 90% of that of the parent Complex III, whereas the recovery of the activity was marginal in the absence of the phospholipids. Thus it is clear that the iron-sulfur protein is integrated into the complex with the aid of phospholipids.

Characterization of lysosomal acid lipase purified from rabbit liver.

Lysosomal acid lipase from rabbit liver was solubilized with digitonin and purified 25,000-fold by Bio-Gel A-1.5 m, DEAE Bio-Gel A and phenyl Sepharose column chromatographies, preparative slab gel electrophoresis and finally Affi-Gel Blue affinity column chromatography. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The molecular weight of the acid lipase was estimated to be 42,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 40,000 by gel filtration on Bio-Gel A-0.5 m. The enzyme was a hydrophobic glycoprotein with an isoelectric point of 5.15-5.90. The purified enzyme hydrolyzed tri-, di-, and monoolein and cholesterol oleate, with apparent Vmax values of 5.41, 56.1, 21.7, and 3.25 mumol/min/mg protein, and Km values of 50, 70, 200, and 40 microM, respectively. It hydrolyzed 4-methylumbelliferyl esters with fatty acids of different lengths in the order, medium length chains greater than long chains much greater than short chains. It did not hydrolyze dipalmitoylphosphatidylcholine. Its activity was inhibited by micromolar concentrations of p-chloromercuriphenyl sulfonic acid and p-bromophenacyl bromide and millimolar concentrations of Cu2+ and diethylpyrocarbonate. The activities of the enzyme towards the five substrates listed above showed almost identical thermal stabilities, mobilities on polyacrylamide gel electrophoresis and inhibition by several inhibitors. These findings support the idea that one enzyme is involved in the hydrolysis of both acylglycerols and cholesterol esters in lysosomes.

Further Studies on myo-Inositol-1-phosphatase from the Pollen of Lilium longiflorum Thunb

myo-Inositol-1-phosphatase has been purified to homogeneity from Lilium longiflorum pollen using an alternative procedure which includes pH change and phenyl Sepharose column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis shows that the enzyme is a dimer (subunit molecular weight, 29,000 daltons). The enzyme is stable at low pH values and is inactivated only below pH 3.0. In addition to 1l-and 1d-myo-inositol-1-phosphate, it shows high specificity for 1l-chiro-inositol-3-phosphate. As observed earlier with other primary phosphate esters, d-glucitol-6-phosphate and d-mannitol-6-phosphate are hydrolyzed very slowly. No activity is observed with inorganic pyrophosphate or myo-inositol pentaphosphate as substrate. The enzyme is inhibited by fluoride, sulfate, molybdate, and thiol-directed reagents. Partial protection against N-ethylmaleimide inhibition by substrate and Mg(2+) together suggests sulfhydryl involvement at the active site.

Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L929 cells.

A procedure is described for purification of a factor (D-factor)-inducing differentiation of mouse myeloid leukemic cells (M1) into macrophages from serum-free mouse L929 cell-conditioned medium. The procedure included ammonium sulfate precipitation, DEAE-cellulose, Sephadex G-200 and phenyl-Sepharose column chromatographies, reverse-phase high-performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel-filtration column. The purified factor gave a single band of protein with a molecular weight of 62,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis which coincided with biological activity. Its half-maximal concentration for inducing differentiation of M1 cells into macrophages was 1.7 X 10(-11) M. Even at 2.6 X 10(-9) M, it did not induce colony formation of normal bone marrow cells, suggesting that it was distinct from the growth factor for normal precursors of macrophages and/or granulocytes.

Lysosomal cholesterol esterase の精製とその分子特性

Lysosomal cholesterol esterase from rabbit liver was solubilized with digitonin and purified 21, 000-fold to homogeneity by Bio-Gel A-1.5m, DEAE-Bio-Gel A, Phenyl-Sepharose column chromatography, preparative slab gel electrophoresis and finally Sephacryl S-200 column chromatography. The molecular weight of the purified enzyme, calculated by SDS-polyacrylamide gel electropholesis, was about 42, 000. The enzyme hydrolyzed both cholesterol oleate and triolein. It was activated by phospholipids (phosphatidylcholine, phosphatidylethanolamine) and lysosomal membrane. The enzyme was adsorbed on Phenyl-Sepharose and Concanavalin A-Sepharose anddissociated from them by ethylene glycol and a-methylmannoside, respectively, suggesting that it was a hydrophobic glycoprotein. We also discuss the implication of the enzyme for the accumulation of cholesterol ester in atheromatous aorta.

24] d-Glucose dehydrogenase from Pseudomonas fluorescens, membrane-bound

Publisher Summary This chapter describes an assay method for the synthesis of D-glucose dehydrogenase from Pseudomonas fluorescens . D-Glucose dehydrogenase occurs on the outer surface of the cytoplasmic membrane of oxidative bacteria, such as Pseudomonas and Gluconobacter species, and initiates a direct oxidation of D-glucose through an electron transport chain. The enzyme is solubilized from the membrane and further purified to a homogeneous state. A spectrophotometric assay at 25° measures the decrease of absorbance at 600 nm of 2,6-dichlorophenolindophenol (DCIP) mediated with phenazine methosulfate (PMS). The activity can also be measured with PMS, DCIP, Wurster's blue (WB), ferricyanide, or coenzyme Q (CoQ) as an electron acceptor. Many strains of Pseudomonas aeruginosa and P. fluorescens are suitable sources of D-glucose dehydrogenase. The steps involved in the purification procedure are (1) the preparation of membrane fraction, (2) the solubilization of enzyme, (3) polyethylene glycol fractionation, (4) ethanol fractionation, (5) hydroxyapatite column chromatography, and (6) phenyl-sepharose column chromatography. All procedures are carried out at 0–5°C. D-Glucose dehydrogenase has a dual pH optimum depending on the electron acceptor used.

Solubilization and partial purification of human placental cytochromes P-450

Human placental mitochondrial and microsomal cytochrome P-450 (P-450) was solubilized with sodium cholate in the presence of Emulgen 911 and the solubilized preparations purified by phenyl-Sepharose and DEAE-cellulose column chromatography. The final preparations exhibited specific activities between 3.0 and 7.0 nmol P-450/mg protein implying a 30–115-fold purification over the starting material. Androstenedione exhibited type I spectral interaction with the microsomal P-450 and pregnenolone a reverse type I spectrum with mitochondrial P-450. Hydrophobic column chromatography proved to be a rapid and efficient initial purification step for placental P-450s.

Two esterases released from Mycobacterium smegmatis from the hydrolysis of long chain acyl-CoAs and Tween.

An esterase activity hydrolyzing palmitoyl-CoA was released into the culture medium from Mycobacterium smegmatis. Although another esterase activity hydrolyzing Tween 20 (polyoxyethylene sorbitan monolaurate) was also found in the culture medium, the bulk of the esterase activity was retained in the cells. However, treatment of early-log phase cells with lysozyme to prepare ghosts released 80% of the Tween 20 hydrolyzing activity, indicating the localization of the esterase in the periplasmic space or the cell envelope fraction. The presence of two different esterases hydrolyzing palmitoyl-CoA and Tween 20, suggested by the above results, was confirmed by the separation of these esterases on phenyl-Sepharose column chromatography. Palmitoyl-CoA hydrolase (thioesterase) was purified 630-fold from lysozyme-treated supernatant fluid to homogeneity, by means of Sephadex G-100 gel filtration, and DEAE-cellulose, phenyl-Sepharose and Blue-Agarose column chromatographies. Its molecular weight was approximately 42,000. Tween hydrolase was partially purified 150-fold by the same purification procedure up to the step of phenyl-Sepharose chromatography and its molecular weight was found to be about 51,000. These activities were stable against heating at 60 degrees C and treatment with non-ionic detergents. Thioesterase hydrolyzed long chain acyl-CoAs (C12-C20), but not Tween 20-80 or beta-naphthyl acetate. On the other hand, Tween hydrolase hydrolyzed Tween 20-80 and beta-naphthyl acetate. On the other hand, Tween hydrolase hydrolyzed Tween 20-80 and beta-naphthyl acetate, but not acyl-CoAs. Both esterases hydrolyzed monoolein, but not diolein, triolein, or phosphatidylcholine.

Presence of an inactive protein immunologically analogous to cytochrome c oxidase in the inner membrane of sweet potato root mitochondria.

A protein, which was immunoreactive to antibody against cytochrome c oxidase, was found in the mitochondrial membrane fraction of sweet potato root tissue. The protein was associated relatively weakly with the mitochondrial inner membrane as compared with cytochrome c oxidase. It exerted no cytochrome c oxidase activity and contained no heme a. The protein was purified by phenyl-Sepharose column chromatography and polyacrylamide gel electrophoresis. The molecular weight of its polypeptide chain was 57,000. In addition, the protein decreased during aging of tissue slices. It is therefore not improbable that the protein is a precursor of cytochrome c oxidase composed of only the subunits of cytoplasmic origin, since aging of tissue slices has been shown to result in an increase in the enzyme activity which is inhibited by chloramphenicol but not by cycloheximide.

Partial purification and characterization of aldehyde dehydrogenase from sweet potato roots.

Two kinds of aldehyde dehydrogenases were found in the supernatant fraction of extracts of sweet potato roots. These enzymes had different >Km values for acetaldehyde. The major enzyme, which had a low >Km value, was partially purified from fresh sweet potato roots by several steps, including phenyl-Sepharose column chromatography. Purification increased the specific activity 240-fold compared to that at the ammonium sulfate fractionation step. The molecular weight was calculated to be close to 150,000. In potassium phosphate buffer the optimal pH was approximately 8.0, and, in Tris-HCl buffer, it was approximately 8.8. The >Km value for acetaldehyde was 7.0 µM. According to the data obtained from phenyl-Sepharose column chromatography, the enzyme was rather amphipathic in spite of its being a soluble enzyme.

Purification and characterization of nucleoside diphosphatase from rat-liver microsomes. Evidence for metalloenzyme and glycoprotein.

Nucleoside diphosphatase was purified from rat liver microsomes more than 3000-fold with a 16% yield using a procedure including concanavalin-A--Sepharose and phenyl-Sepharose column chromatography. The purified enzyme had a specific activity of 2500 units/mg protein and appeared homogeneous by gel electrophoresis. The enzyme had a sedimentation coefficient of 6.5 S by sucrose-density gradient centrifugation. The enzyme had a sedimentation coefficient of 6.5 S by sucrose-density gradient centrifugation, and a Stokes' radius of 4.8 nm was estimated by the gel filtration technique. Its molecular weight is 130,000, but only one single band of Mr 65,000 was detected after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme seems thus to be composed of two identical subunits. The purified enzyme was confirmed to be a glycoprotein containing approximately 9% carbohydrates. The enzyme had a pH optimum of 7.5, an isoelectric point of 4.85 and a Km of 2.5 mM for UDP. On the basis of direct measurement of metal content in the native enzyme, the rat liver nucleoside diphosphatase was found to be a metalloenzyme containing 0.9 mol zinc and 0.1 mol manganese/mol 65,000-Mr subunit. Metal-free nucleoside diphosphatase has been prepared. The activity of the metal-free enzyme was restored by the addition of several divalent cations, zinc being the most effective.

Purification and in vitro labeling of interferon from a human fibroblastoid cell line.

Interferon was produced from a clonal human fibroblastoid line. This cell line was derived from an established fibroblastoid culture treated with ethylmethane sulfonate and is capable of producing higher amounts human interferon than primary human fibroblast cultures. The interferon produced from this cell line was purified by concanavalin A and phenyl-Sepharose column chromatography followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified interferon was then labeled with [125I]-iodine, with [3H]5-dimethylaminonaphthalene-1-sulfonyl-chloride and with sodium [3H]borohydride after periodate oxidation. An appreciable amount of biological activity was retained after both tritium labelings but not after iodination. When subjected to electrophoresis in the presence of sodium dodecyl sulfate all three radioactive interferon preparations comigrated with the purified interferon preparation as a single protein component with a molecular weight of 19,000. Both purified and radioactive preparations were also shown to migrate with the antiviral activity in polyacrylamide gels in the absence of sodium dodecyl sulfate. The specific activity of purified interferon preparations ranged from 2 X 10(8) to 10 X 10(8) units/mg of protein.