Properties of purified kidney microsomal NADPH-cytochrome c reductase

NADPH-cytochrome c reductase, solubilized by lipase digestion of microsomes prepared from perfused porcine kidney cortex, was purified about 3600-fold to give a turnover number of 1230 nmoles cytochrome c reduced per min per nmole flavin. The kinetic determination of K_m and V with respect to NADPH, cytochrome c, and NADH, resulted in values similar to those obtained with purified liver reductase. The kidney microsomal enzyme also exhibited a ping-pong kinetic mechanism for NADPH-mediated cytochrome c reduction.Spectrofluorometric measurements demonstrated the presence of equimolar amounts of FAD and FMN per mole of reductase. The molecular weight was estimated by Sephadex G-200 gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 68,000 and 71,000 g per mole, respectively.Immunochemical techniques, including Ouchterlony double-diffusion studies and inhibition of catalytic activity by antibody to the liver microsomal NADPH-cytochrome c reductase, established the similarity of the purified liver and kidney reductases.     

Molecule-size distribution of soluble hiunic compounds from different natural waters

It this study the use of Sephadex G-25, Sephadex LH-20 and CPG-10-75 for the separation of soluble humic compoutids frotn fresh water is tested. It is shown by Sephadex G-25 gel filtration that differences in molecule-size distribution of soluble humic compounds in one lake at different titnes and between lakes can be predicted by E₂₅₀ and E₃₆₅ measurements.     

Peroxidase and IAA-oxidase in crude and partially purified enzyme extracts of growing and differentiating root cells

Crude enzyme extracts from the zones of division, elongation and differentiation of cells of primary maize (Zea mays) root show peroxidase activity but lack IAA-oxidase activity. After partial purification of the extracts by gel filtration on Sephadex G-25, the specific peroxidase activity increases almost twice and a high IAA-oxidase activity appears. The partial purification of the enzyme extracts does not change the electrophoretic pattern of the peroxidase isoenzymes, but significantly improves the separation and the visualization of isoenzymes with IAA-oxidase activity. The data obtained were interpreted in connection with the different modifying effect of the low molecular compounds (mainly phenolics) on the activity and the isoenzyme patterns of the peroxidase and the IAA-oxidase.     

Stimulation of potato microsomal NADH-cytochrome c reductase by acidic phospholipids

Potato microsomal membranes were solubilized by 0.5% sodium cholate solutions. Separation of lipids from proteins was realized by two successive gel filtrations on two different Sephadex columns. Lipid-free microsomal proteins maintained a high NADH-ferricyanide reductase activity but had a lowered (20%) NADH-cytochrome c reductase activity. The latter activity was strongly stimulated when lipid-free proteins were integrated, by sonication, into phosphatidylserine or phosphatidylinositol liposomes. Some stimulation was obtained also with phosphatidylcholine-lysophosphatidylcholine (7:3) mixtures. Other phospholipids were far less active or even inhibitory. Acidic phospholipids stimulate NADH-cytochrome c reductase activity by increasing noticeably the apparent affinities of enzymatic proteins for NADH or cytochrome c.     

The oxidation mechanisms of thiosulphate and sulphide in Chlorobium thiosulphatophilum: Roles of cytochrome c-551 and cytochrome c-553

A thiosulphate-cytochrome c reductase was highly purified from Chlorobium thiosulphatophilum and its properties were studied. The enzyme catalyses reduction with Na₂S₂O₃ of c cytochromes, including cytochrome c-551 of the bacterium. Cytochrome c (555, C. thiosulphatophilum) does not react directly with the enzyme at an appreciable rate but stimulates greatly the reduction by the enzyme of cytochrome c-551 with Na₂S₂O₃. The reduction of c cytochromes catalysed by the enzyme is strongly inhibited by cyanide and sulphite.Cytochrome c (553, C. thiosulphatophilum), a c-type cytochrome with covalently bound flavin, was found to catalyse reduction with sulphide of c cytochromes, including cytochrome c-555. The reaction is strongly inhibited by cyanide. Cyanide seems to combine strongly with cytochrome c-553 probably at the flavin moiety. Thus, the absorption spectrum attributable to flavin of the haemoprotein is changed on addition of cyanide, and neither the original spectrum nor the activity reappears even after the cyanide-treated cytochrome has been subjected to gel filtration with a Sephadex G-25 column or to isoelectric focusing.     

Purification of Sulphite-Cytochrome c Reductase of Thiobacillus novellus and Reconstitution of Its Sulphite Oxidase System with the Purified Constituents

Sulphite-cytochrome c reductase (sulphite: ferricytochrome coxidoreductase, EC 1.8.2.1 [EC] ) derived from Thiobacillus novelluswas purified by chromatography on a DEAE-cellulose column andby gel filtration with a Sephadex G-100 column. Although thereductase thus purified moved as a single band both in gel filtrationand in isoelectric focusing it was always split into two bandsby polyacrylamide gel electrophoresis; the one had the enzymaticactivity and showed absorption spectrum of cytochrome, whilethe other had no activity and was colourless, in contrast withthe results reported by Charles and Suzuki [(1966) Biochim.Biophys. Acta 128: 522]. The enzymatic properties of the purifiedreductase were almost the same as those of the enzyme obtainedby Charles and Suzuki. Cytochrome c-551 free of the reductase activity was obtained.Its molecular weight was determined to be 23,000 by polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate.The cytochrome seemed to exist in the organism as a complexwith the reductase or a subunit of the enzyme. In the stateof the complex with the enzyme, the cytochrome was reduced veryquickly on addition of sulphite, while the cytochrome free ofthe reductase activity was hardly reduced by the enzyme withsulphite. A sulphite oxidase system was reconstituted with the reductase,cytochrome c-550 and cytochrome oxidase highly purified fromthe bacterium. ¹ Present address: Water Research Institute, Nagoya University,Nagoya 464, Japan ² Present address: Institute for Biological Science, SumitomoChemical Co., Ltd., Takarazuka, Hyogo 665, Japan (Received January 23, 1981; Accepted March 9, 1981)     

Effect of attached carbohydrates on the activity of Trichoderma viride cellulases

Trichoderma viride 1,4-β-d-glucan cellobiohydrolase (exo-cellobiohydrolase, 1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91) purified from a commercial product to electrophoretic homogeneity by a procedure including affinity and DEAE-Sephadex chromatography, has attached carbohydrates in addition to the glycoprotein constituents. These carbohydrates are lost by consecutive gel filtration steps in Sephadex G-25 columns, whereupon there is a rapid increase in enzymatic activity. A single gel filtration step can eliminate d-glucose or cellobiose added to a solution of this enzyme, but not the carbohydrates attached during incubation with Avicel.After free carbohydrate elimination from crude cellulase complexes by Sephadex G-25 chromatography, liberation of d-glucose following incubation at 50°C and pH 4.8 was observed. This indicates that some carbohydrates remain bound after gel filtration. The elimination of carbohydrate from whole cellulase complex [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] was favoured by a yeast treatment, with a simultaneous increase in activity, but the process is not reproducible, as a secondary inactivation process exists.     

Purification and characterization of a sulfite:cytochrome c oxidoreductase from Thiobacillus acidophilus

Cell-free extracts of Thiobacillus acidophilus prepared at neutral pH showed oxidation of sulfite to sulfate with ferricyanide as electron acceptor. Horse heart cytochrome c could be used as alternative electron acceptor; however, the observed activity was only 0.1% of that found for ferricyanide. The enzyme responsible for the oxidation of sulfite was purified to homogeneity. The purified enzyme was a monomer of 42 kDa and contained one haem c per monomer. Electron paramagnetic resonance (EPR) spectroscopical analysis of the sulfite:cytochrome c oxidoreductase showed the presence of molybdenum (V), only after reduction of the enzyme with sulfite. The pH optimum for the enzymatic reaction was 7.5 and the temperature optimum 40°C. Enzymatic activity was strongly reduced in the presence of the anions: chloride, phosphate and nitrate. In contrast to other enzymes involved in sulfur metabolism and previously isolated from T. acidophilus, sulfite:cytochrome c oxidoreductase activity is not stimulated by the presence of sulfate ions.     

Isolation and properties of membrane-bound cytochrome c-552 from photosynthetic bacterium Chromatium vinosum

A membrane-bound cytochrome c-552 was isolated and purified from the photosynthetic bacterium Chromatium vinosum by treatment with sodium cholate, sodium deoxycholate and bacterial alkaline protease followed by gel filtration. The purified cytochrome c-552, which may have been modified by the protease treatment, was electrophoretically homogeneous. Its minimal molecular weight was estimated to be 19 and 20 kdaltons, respectively by SDS polyacrylamide gel electrophoresis and by gel filtration on Sephadex G-100. Cytochrome c-552 showed the absorption maxima at 419, 523 and 552 nm in the reduced form. Reduced-minus-oxidized difference millimolar absorption coefficient was 10.6 for the wavelength pair, 552 minus 540 nm. The midpoint potential at pH 8.0 was ?130 mV. The polarity in the amino acid composition of cytochrome c-552 was 40.1% and reflected its hydrophobicity. The solubilized cytochrome c-552 was shown to be a different entity from the soluble flavocytochrome c-552 in several respects.     

Purification and Characterization of a Ferredoxin-NADP Oxidoreductase-Like Enzyme from Radish Root Tissues

An enzyme able to reduce cytochrome c via ferredoxin in the presence of NADPH, was isolated, purified from radish (Raphanus sativus var acanthiformis cultivar miyashige) roots and characterized. The enzyme was purified by DEAE-cellulose, Blue-Cellulofine, Ferredoxin-Sepharose 4B, and Sephadex G-100 column chromatography. Molecular mass of the enzyme was estimated to be 33,000 and 35,000 daltons by Sephadex G-100 gel filtration and SDS-PAGE, respectively. Its absorption spectrum suggested that the enzyme contains flavin as a prosthetic group. The K_m values for NADPH and ferredoxin were calculated to be 9.2 and 1.2 micromolar, respectively. The enzyme required NADPH and did not use NADH as an electron donor. The optimal pH was 8.4. The enzyme also catalyzed the photoreduction of NADP⁺ in the spinach leaf thylakoid membranes depleted of ferredoxin and ferredoxin-NADP⁺ oxidoreductase. The effect of NaCl and MgCl₂ concentration on the activity and amino acid composition of the enzyme were demonstrated. The results suggest that the enzyme is similar to ferredoxin-NADP⁺ oxidoreductase from chloroplasts and cyanobacteria and is the key enzyme catalyzing the electron transport between NADPH, generated by the pentose phosphate pathway, and ferredoxin in plastids of plant heterotrophic tissues.     

Radioimmunoassays for prostaglandins. I. Technical validation of prostaglandin F2α measurements in human plasma using Sephadex G-25 gelfiltration

Human plasma samples of 1 ml were processed according to three different procedures prior to Radioimmunoassay (RIA) of Prostaglandin F_2α (PGF_2α). Serial dilutions of ethyl acetate extracts as such, or combined with either silicic acid of Sephadex G-25 chromatography were assessed for linearity, homogeneity and parallelism with the corresponding standard dose response line. For plasma extracts used as such, non-parallelism is observed. Subsequent chromatography on silicic acid of such extracts gave only a limited linear and parallel portion upon serial dilution. However, purification of the extracts by gelfiltration on Sephadex G-25 results in linear and parallel lines over the full range of the standard dose response line (B/B_o 0.9-0.2).Upon comparison of separation by polyethylene glycol (PEG) and Dextran coated charcoal (DCC) in these systems, PEG proved to give the best results. It was found that in the Sephadex G-25 procedure, separation by PEG is essential. The method of gelfiltration on Sephadex G-25 is simple and reliable. Intra- and inter-assay coefficients of variation are 6% and 12%, respectively. Accuracy, as measured by recovery of added known amounts of PGF_2α is 97.6%.     

Purification and characterization of angiotensin I-converting enzyme inhibitory peptide from enzymatic hydrolysates of Styela clava flesh tissue

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from the Styela clava flesh tissue. Nine proteases (Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, pepsin, trypsin, α-chymotrypsin and papain) were used, and their respective enzymatic hydrolysates and an aqueous extract were screened to evaluate their potential ACE inhibitory activity. Among all of the test samples, Protamex hydrolysate possessed the highest ACE inhibitory activity, and the Protamex hydrolysate of flesh tissue showed relatively higher ACE inhibitory activity compared with the Protamex hydrolysate of tunic tissue. We attempted to isolate ACE inhibitory peptide from the Protamex hydrolysate of S. clava flesh tissue using ultrafiltration, gel filtration on a Sephadex G-25 column and high performance liquid chromatography (HPLC) on an ODS column. The purified ACE inhibitory peptide exhibited an IC₅₀ value of 37.1 μM and was identified as non-competitive inhibitor of ACE. Amino acid sequence of the peptide was identified as Ala-His-Ile-Ile-Ile, with a molecular weight 565.3 Da. The results of this study suggested that the peptides derived from enzymes-assisted extracts of S. clava would be useful new antihypertension compounds in functional food resource.     

Kinetic studies on the reduction of cytochrome c: reaction with organic oxy-compounds

A range of organic hydroxy compounds, many of them naturally occurring, have been assayed for their ability to reduce the electron transfer protein cytochrome c. Those with conjugated hydroxyl systems e.g. catechol, acted as reducing agents while those which were phenol-like, either by separation of conjugation e.g. resorcinol or by having only one free hydroxyl group, did not. Rapid reaction kinetic investigations of the reaction of rhodizonic acid with cytochrome c revealed rapid reduction of the protein. The dianion of rhodizonic acid is the most reactive species in agreement with results obtained with catecholato compounds. The pH-dependence of this reaction is discussed in terms of the complex solution chemistry of rhodizonic acid.     

Antioxidant Properties of the Peptides Isolated From Ganoderma lucidum Fruiting Body

Ganoderma lucidum is widely used as traditional medicine for centuries particularly in China, Japan and Korea. Many bioactive metabolites isolated from G. lucidum were therapeutically active against various diseases. The peptide isolated from water extract of G. lucidum was purified by employing Sephadex G-25, Sephadex G-50 and reverse phase HPLC column chromatography. The antioxidant property of the peptide fractions was determined by various in vitro methods. All fractions obtained from Sephadex G-25 and fraction G from Sephadex G-50 are effective antioxidants and comparably fraction C has the highest antioxidant activity. The molecular weight of purified peptide determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration chromatography and Matrix-assisted laser desorption ionization time-of-flight-mass spectrometer was found to be 2.8, 3.34 and 3.35?kDa respectively. The amino acid composition of the peptide was rich in phenylalanine, aspartic acid, proline, histidine and isoleucine. Peptide isolated in the present investigation suggests that has beneficial antioxidant properties may be due to its low molecular weight and specific amino acid composition.     

Isoenzymes of ferredoxin-NADBΔ oxidoreductase from the cyanobacterium Nostoc strain MAC

Ferredoxin-NADP⁺ oxidoreductase from the cyanobacterium Nostoc strain MAC was separated into two fractions by ion-exchange chromatography. Both were purified to electrophoretic homogeneity and exhibited diaphorase and ferredoxin-dependent cytochrome c reductase activity. The activities with three different electron carriers in this latter assay were similar for the two fractions, as were the pH optima in both assays. Each fraction, however, could be resolved into several active components by isoelectric focusing, both after initial separation and following apparent purification by gel filtration on Sephadex G-150, further chromatography on DEAE-cellulose, and use of hydroxylapatite columns.Abbreviation DCIP = phenolindo-3,6-dichlorophenol>     

Radioimmunoassays for prostaglandins. II. Measurement of prostaglandin E2 and the 13,14-dihydro-15-keto metabolites of the E and F series. Description of a reliable technique with a universal applicability

Human plasma samples of 1 ml were processed according to three different procedures prior to Radioimmunoassay (RIA) of Prostaglandin F_2α (PGF_2α). Serial dilutions of ethyl acetate extracts as such, or combined with either silicic acid of Sephadex G-25 chromatography were assessed for linearity, homogeneity and parallelism with the corresponding standard dose response line. For plasma extracts used as such, non-parallelism is observed. Subsequent chromatography on silicic acid of such extracts gave only a limited linear and parallel portion upon serial dilution. However, purification of the extracts by gelfiltration on Sephadex G-25 results in linear and parallel lines over the full range of the standard dose response line (B/B_o 0.9-0.2).Upon comparison of separation by polyethylene glycol (PEG) and Dextran coated charcoal (DCC) in these systems, PEG proved to give the best results. It was found that in the Sephadex G-25 procedure, separation by PEG is essential. The method of gelfiltration on Sephadex G-25 is simple and reliable. Intra- and inter-assay coefficients of variation are 6% and 12%, respectively. Accuracy, as measured by recovery of added known amounts of PGF_2α is 97.6%.     

Effect of crosslinking cytochrome c oxidase

Bovine heart cytochrome c oxidase and rat liver mitochondria were crosslinked in the presence and absence of cytochrome c. Biimidate treatment of purified cytochrome oxidase, which results in the crosslinkage of all of the oxidase protomers except subunit I when ? 20% of the free amines are modified, inhibits ascorbate-N,N,N′,N′-tetramethyl-p-phenylene diamine oxidase activity. Intermolecular crosslinking of cytochrome oxidase molecules, which results in the formation of large enzyme aggregates displaying rotational correlation times ? 1 ms, does not affect oxidase activity. Crosslinking of mitochondria covalently binds the cytochrome bc₁ and aa₃ complexes to cytochrome c, and inhibits steady-state oxidase activity. Addition of cytochrome c to purified cytochrome oxidase or to cytochrome c-depleted mitoplasts increases this inhibition slightly. Cytochrome c oligomers act as competitive inhibitors of native cytochrome c; however, crosslinking of cytochrome c to cytochrome c-depleted mitoplasts or purified cytochrome oxidase results in a catalytically inactive complex. These experiments indicate that cytochrome c oxidase subunit interactions are required for activity, and that cytochrome c mobility may be essential for electron transport between cytochrome c reductase and oxidase.     

Isolation of an activator for prostaglandin hydroperoxidase from bovine vesicular gland cytosol and its identification as uric acid.

The enzymatic conversion of prostaglandin G₁ to H₁ was stimulated by an activator present in the cytosol of bovine vesicular gland. The activator was purified by Sephadex G-25 gel filtration and Dowex 1 column chromatography. The purified activator was identified to be uric acid by thin layer chromatography, ultraviolet and infrared absorption spectroscopy and combined gas chromatography-mass spectroscopy. Among various purine compounds tested, only uric acid and 2,8-dihydroxyadenine were active.     

凝胶过滤层析参数对家蝇蛋白粗提液分离效果的影响

考察了凝胶过滤介质、层析柱直径、柱床高度和洗脱流速对家蝇Muscadomestica蛋白粗提液分离效果的影响,结果表明：凝胶过滤介质种类、层析柱直径、柱床高度、洗脱流速均能不同程度地影响家蝇蛋白粗提液的分离效果。在实验范围内,选择1.3 cm直径、40 cm柱床高度的Sephadex G-75,以0.4 mL/min的流速洗脱时,对家蝇蛋白粗提液的分离效果比较理想。     

Isopentenyl pyrophosphate isomerase and prenyltransferase from tomato fruit plastids

Isopentenyl pyrophosphate isomerase has been isolated from an extract of tomato fruit plastids and purified 245-fold by fractionation with ammonium sulfate, gel filtration on Bio-Gel A 1.5m, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and chromatofocusing. Gel filtration on Sephadex G-100 separated the isopentenyl pyrophosphate isomerase from a prenyltransferase fraction that catalyzed the conversion of isopentenyl pyrophosphate to acid-labile compounds in the presence of dimethylallyl, geranyl, or farnesyl pyrophosphates. The molecular weights of the isopentenyl pyrophosphate isomerase and prenyltransferase were determined to be 34,000 and 64,000, respectively, by gel filtration on Sephadex G-100. The only cofactor required by either the isomerase or the prenyltransferase was a divalent cation, either Mg²⁺ or Mn²⁺. Isopentenyl pyrophosphate isomerase could also be totally inactivated by 1 × 10^?3m iodoacetamide, and this property was utilized in the assay of prenyltransferase activity in the presence of contaminating isomerase. The inactivation of isomerase by iodoacetamide is consistent with the stabilization of isopentenyl pyrophosphate isomerase by dithiothreitol. The K_m of isopentenyl pyrophosphate isomerase for isopentenyl pyrophosphate was found to be 5.7 × 10^?6.