The Biosynthetic Pathway of (Z)-11-Hexadecenal,the Sex Pheromone Component of the Rice Stem Borer,Chilo sappressalis WALKER (Lepidoptera: Pyralidae)

Lipoxygenase-3, the major component of the enzyme in rice grain, was purified 2980-fold with a yield of 7% from embryos. The purified enzyme had a specific activity of 280 μmol O₂ formed/min per mg protein. This enzyme was inactivated by SH compounds, such as cysteine and glutathione. The inactivation was prevented by the addition of catalase or replacement of the air by N₂ gas. These two treatments were also effective for the stable storage of the purified enzyme. The molecular weights measured by sodium dodecyl sulfate gel and gradient gel electrophoresis were 93,000 and 89,000, respectively, indicating that the enzyme is a single polypeptide chain. The purified enzyme contained 0.73 Fe atom per molecule. The absorption spectrum suggested that the enzyme is a non-heme iron protein. Some similarities in amino-acid composition were observed between rice, soybean, and pea lipoxygenases. The purified enzyme specifically produced 9-d-hydroperoxy-10,12(E,Z)-octadecadienoic acid when linoleic acid was used as a substrate.     

Resolution and reconstitution of succinate-cytochrome c reductase. Preparations and properties of high purity succinate dehydrogenase and ubiquinol—cytochrome c reductase

An improved method was developed to sequentially fractionate succinate-cytochrome c reductase into three reconstitutive active enzyme systems with good yield: pure succinate dehydrogenase, ubiquinone-binding protein fraction and a highly purified ubiquinol-cytochrome c reductase (cytochrome b-c₁ III complex).An extensively dialyzed succinate-cytochrome c reductase was first separated into a succinate dehydrogenase fraction and the cytochrome b-c₁ complex by alkali treatment. The resulting succinate dehydrogenase fraction was further purified to homogeneity by the treatment of butanol, calcium phosphate gel adsorption and ammonium sulfate fractionation under anaerobic condition in the presence of succinate and dithiothreitol. The cytochrome b-c₁ complex was separated into cytochrome b-c₁ III complex and ubiquinone-binding protein fractions by careful ammonium acetate fractionation in the presence of deoxycholate.The purified succinate dehydrogenase contained only two polypeptides with molecular weights of 70 000 and 27 000 as revealed by the sodium dodecyl sulfate polyacrylamide gel electrophoretic pattern. The enzyme has the reconstitutive activity and a low K_m ferricyanide reductase activity of 85 μmol succinate oxidized per min per mg protein at 38°C.Chemical composition analysis of cytochrome b-c₁ III complex showed that the preparation was completely free of contamination of succinate dehydrogenase and ubiquinone-binding protein and was 30% more pure than the available preparation.When these three components were mixed in a proper ratio, a thenoyl-trifluoroacetone- and antimycin A-sensitive succinate-cytochrome c reductase was reconstituted.     

Novel Type of Heme-Dependent Oxygenase Catalyzes Oxidative Cleavage of Rubber (Poly-cis-1,4-Isoprene)

An extracellular protein with strong absorption at 406 nm was purified from cell-free culture fluid of latex-grown Xanthomonas sp. strain 35Y. This protein was identical to the gene product of a recently characterized gene cloned from Xanthomonas sp., as revealed by determination of m/z values and sequencing of selected isolated peptides obtained after trypsin fingerprint analysis. The purified protein degraded both natural rubber latex and chemosynthetic poly(cis-1,4-isoprene) in vitro by oxidative cleavage of the double bonds of poly(cis-1,4-isoprene). 12-Oxo-4,8-dimethyltrideca-4,8-diene-1-al (m/z 236) was identified and unequivocally characterized as the major cleavage product, and there was a homologous series of minor metabolites that differed from the major degradation product only in the number of repetitive isoprene units between terminal functions, CHO-CH₂— and —CH₂-COCH₃. An in vitro enzyme assay for oxidative rubber degradation was developed based on high-performance liquid chromatography analysis and spectroscopic detection of product carbonyl functions after derivatization with dinitrophenylhydrazone. Enzymatic cleavage of rubber by the purified protein was strictly dependent on the presence of oxygen; it did not require addition of any soluble cofactors or metal ions and was optimal around pH 7.0 at 40°C. Carbon monoxide and cyanide inhibited the reaction; addition of catalase had no effect, and peroxidase activity could not be detected. The purified protein was specific for natural rubber latex and chemosynthetic poly(cis-1,4-isoprene). Analysis of the amino acid sequence deduced from the cloned gene (roxA [rubber oxygenase]) revealed the presence of two heme-binding motifs (CXXCH) for covalent attachment of heme to the protein. Spectroscopic analysis confirmed the presence of heme, and approximately 2 mol of heme per mol of RoxA was found.     

2-Hydroxyisonicotinate dehydrogenase isolated from Mycobacterium sp. INA1

2-Hydroxyisonicotinate dehydrogenase from Mycobacterium sp. INA1 was purified 26-fold to apparent homogeneity. The enzyme is involved in isonicotinate degradation by Mycobacterium sp. INA1 and catalyzes the conversion of 2-hydroxyisonicotinate to 2,6-dihydroxypyridine-4-carboxylate. The purified protein exhibited a native molecular mass of 300 kDa and subunits of 97, 31 and 17 kDa, respectively, indicating an α₂β₂γ₂ structure. The absorption spectrum of the homogeneous enzyme was characteristic for an iron/sulfur flavoprotein. 3.8 mol of iron, 3.7 mol of acid labile sulfur, 0.94 mol of FAD and 0.75 mol of molybdenum were determined per mol of protomer. The molybdenum cofactor was identified as molybdopterin cytosine dinucleotide. 2-Hydroxyisonicotinate dehydrogenase was inactivated in the presence of cyanide. According to these basic properties the protein seems to belong to the class of molybdo-iron/sulfur flavoproteins of the xanthine oxidase family.     

Homogeneous alkylcysteine lyase of Acacia farnesiana: Fresh seedlings vs. acetone powders

Alkyleysteine lyase (EC 4.4.1.6) was purified essentially to homogeneity from both fresh hypocotyls of 5- to 8-day-old etiolated seedlings of Acacia farnesiana and acetone powders of such hypocotyls. The enzyme from the fresh material had twice the specific activity of that from the acetone powder. Sodium dodecylsulphate gel electrophoresis showed that both enzymes were composed of a subunit of M_rca 42 000. The final enzyme solutions were quite different in their absorbance spectra. The fresh hypocotyl enzyme had an absorbance maximum at 425 nm in addition to the 280 nm protein absorbance. This maximum in the visible region is due to bound pyridoxal phosphate. The acetone powder enzyme had the same maxima and in addition peaks at 498 and 340 nm. The fresh enzyme contained 1.8 mol cofactor/mol enzyme and the acetone powder enzyme 1.0 mol/mol. The KK_m for the probable natural substrate L-djenkolate was the same for both enzymes, 0.8 mM, but the V_max for the fresh was twice that of the acetone powder enzyme. The common practice of using acetone powder preparations for starting material in enzyme purifications would appear to require some caution.     

Purification and Characterization of a Dipeptidase from Streptococcus cremoris Wg2

A dipeptidase was purified to homogeneity from a crude cell extract of Streptococcus cremoris Wg2 by DEAE-Sephacel column chromatography followed by preparative disc gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 49,000. The dipeptidase is capable of hydrolyzing a range of dipeptides, but not peptides with longer chains. The enzyme was shown to be a metallo-Mn²⁺ enzyme with a pH optimum of 8 and a temperature optimum of 50°C. The enzyme is strongly inhibited by thiol-reducing reagents but not by sulfhydryl reagents. Kinetic studies indicated that the enzyme has a relatively low affinity for leucyl-leucine and alanyl-alanine (K_m, 1.6 and 7.9 mM, respectively) but can hydrolyze these substrates at very high rates (V_max, 3,700 and 13,000 μmol/min per mg of protein, respectively).     

Identification of two catalases in Azotobacter vinelandii: a KatG homologue and a novel bacterial cytochrome c catalase, CCCAv

Azotobacter vinelandii produces two detectable catalases during growth on minimal medium. The heat-labile catalase expressed during exponential growth phase was identified as a KatG homologue by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a mixed protein sample. The second catalase was heat resistant and had substantial residual activity after treatment at 90°C. This enzyme was purified by anion-exchange and size exclusion chromatography and was found to exhibit strong absorption at 407 nm, which is often indicative of associated heme moieties. The purified protein was fragmented by proteinase K and identified by LC-MS/MS. Some identity was shared with the MauG/bacterial cytochrome c peroxidase (BCCP) protein family, but the enzyme exhibited a strong catalase activity never before observed in this family. Because two putative c-type heme sites (CXXCH) were predicted in the peptide sequence and were demonstrated experimentally, the enzyme was designated a cytochrome c catalase (CCC_Av). However, the local organization of the CCC_Av heme motifs differed significantly from that of the BCCPs as the sites were confined to the C-terminal half of the catalase. A possible Ca²⁺ binding motif, previously described in the BCCPs, is also present in the CCC_Av peptide sequence. Some instability in the presence of EGTA was observed. Expression of the catalase was abolished in cccA mutants, resulting in a nearly 8,700-fold reduction in peroxide resistance in stationary phase.     

Toluene dioxygenase: purification of an iron-sulfur protein by affinity chromatography

Toluene dioxygenase, from $\text{Pseudomonas}\text{putida}$, oxidizes toluene to (+)-$\text{cis}$-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene. The oxygenase-component of this multienzyme system was purified to homogeneity by a two-step procedure that utilized affinity and ion exchange chromatography. The purified enzyme would oxidize toluene only in the presence of NADH, ferrous iron and partially purified preparations of NADH cytochrome c reductase and an iron-sulfur protein (ferredoxin_TOL). Spinach NADPH cytochrome c reductase and NADPH could substitute for the $\text{Pseudomonas}$ reductase and NADH. The molecular weight of the oxygenase-component was determined to be 151,000 and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme is composed of two subunits with molecular weights of 52,500 and 20,800. The absorption spectrum showed maxima at 550 (Shoulder), 450, 326 and 278 nm and preliminary experiments have indicated the presence of 2 gram atoms of iron and 2 gram atoms of acid-labile sulfur per mole of protein. The results indicate that the oxygenase-component of the toluene dioxygenase enzyme system is an iron-sulfur protein that has been designated ISP_TOL.     

Purification and metal requirements of 3-dehydroquinate synthase from Phaseolus mungo seedlings

Dehydroquinate synthase of Phaseolus mungo seedlings was purified 4400-fold from the (NH₄)₂SO₄ fraction of a crude extract, the specific activity being 810 nkat per mg protein. When the purified enzyme was subjected to electrophoresis with or without sodium dodecyl sulfate, a single band was observed. The MW of the enzyme was estimated to be 67 000 by Sephadex G-100 gel chromatography and the minimum MW of the enzyme 43 000 by gel electrophoresis with sodium dodecyl sulfate. Atomic absorption analysis revealed that the purified enzyme contained small amounts of copper. Cobalt was not detected, although it has been implicated as a cofactor requirement.     

Alpha glucosidases in white blood cells, with reference to the detection of acid alpha 1-4 glucosidase deficiency.

The iron-containing violet acid phosphatases from beef spleen and pig allantoic fluid have been purified to homogeneity. Molecular weight determinations by zonal gel filtration, SDS-gel electrophoresis, and ultracentrifugation support values close to 40,000 for both enzymes, necessitating reappraisal of literature values. Similarly, the equivalent weight for iron is close to 20,000 for both enzymes, indicating the presence of two iron atoms per molecule of enzyme. The enzymes also have very similar ultraviolet and visible spectra, with λ_max values close to 550 nm, and ?₅₅₀ values(in terms of iron) of 2.04 × 10³ and 2.00 × 10³ for the beef spleen and pig allantoic fluid enzymes respectively.     

Characterization of the periplasmic hydrogenase from Desulfovibrio gigas.

The hydrogenase of the sulfate-reducer $\text{Desulfovibrio gigas}$ has been purified to homogeneity. The pure enzyme shows a specific activity of 90 μmoles H₂ evolved/min./mg protein. Its molecular weight is 89,500 and its is composed of two different subunits (mol. wt. : 62,000 and 26,000) which are not covalently bound. The absorption spectrum of the enzyme is characteristic of an iron-sulfur protein. The millimolar extinction coefficients of the hydrogenase are 46.5 and 170 respectively at 400 and 280 nm. It contains about 12 iron atoms and 12 acid-labile sulfur groups per molecule and the quantitative extrusion of the Fe-S centers of the hydrogenase indicates the presence of 3 Fe₄S₄ clusters. This hydrogenase has 21 half-cystine residues per molecule and a preponderance of aromatic amino-acids.     

Purification of Hydrogenase from Chlamydomonas reinhardtii

A method is described which results in a 2750-fold purification of hydrogenase from Chlamydomonas reinhardtii, yielding a preparation which is approximately 40% pure. With a saturating amount of ferredoxin as the electron mediator, the specific activity of pure enzyme was calculated to be 1800 micromoles H₂ produced per milligram protein per minute. The molecular weight was determined to be 4.5 × 10⁴ by gel filtration and 4.75 × 10⁴ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has an abundance of acidic side groups, contains iron, and has an activation energy of 55.1 kilojoules per mole for H₂ production; these properties are similar to those of bacterial hydrogenases. The enzyme is less thermally stable than most bacterial hydrogenases, however, losing 50% of its activity in 1 hour at 55°C. The K_m of purified hydrogenase for ferredoxin is 10 micromolar, and the binding of these proteins to each other is enhanced under slightly acidic conditions. Purified hydrogenase also accepts electrons from a variety of artificial electron mediators, including sodium metatungstate, sodium silicotungstate, and several viologen dyes. A lag period is frequently observed before maximal activity is expressed with these artificial electron mediators, although the addition of sodium thiosulfate at least partially overcomes this lag.     

Partition of catalase and its peroxidase activities in human red cell membrane: effect of ATP depletion.

Partititon of catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase EC 1.11.1.6) and peroxidase (donor:hydrogen-peroxide oxidoreductase EC 1.11.1.7) activities between the red cell membrane and the cytosol were studied under various experimental conditions. A small but significant amount of catalase (1.6%) was retained on human red cell membranes prepared by hemolysing washed red cells with 30 volumes of 10 mM Tris buffer, pH 7.4. Membrane -bound catalase had a relatively higher peroxidase activity than the soluble enzyme fraction. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the solubilized membranes demonstrated catalase to be a single band with a molecular weight of 60 000. Membranes prepared from adenosine triphosphate-depleted red cells depicted a two to three-fold increase in catalase activity, as well as an increase in 60 000 molecular weight band on polyacrylamide gel electrophoresis. The extra amount of retained catalase was a less efficient peroxidase than found in fresh membranes. The binding of catalase to ATP-depleted red cell membranes was dependent upon both pH and hemolysing ratio. Red cells incubated at pH 7.1 demonstrated a decrease in bound catalase, as did membranes prepared from red cells hemolysed at 1:100 dilution. beta-Mercaptoethanol decreased the catalase activity in the membranes and increased the odianisidine peroxidase activity without any significant effect on the 60 000-dalton band.     

Purification of an Iron-Containing Superoxide Dismutase from A Citrus Plant,Citrus Limonum R

A cyanide-insensitive superoxide dismutase was purified to apparent homogeneity from lemon leaves (Citrus limonum R). The enzyme was isolated from leaf extracts by ammonium sulfate salting-out. and ion-exchange, gel filtration and hydroxylapatite column chromatography. The purified Fe-SOD had a specific activity of about 1.500 U/mg and represents approximately 1.6% of the total soluble protein in lemon leaf extracts. A molecular weight of 47,500 was determined for the enzyme. Analytical gel electro-focusing of the purified preparation revealed the presence of two isozymes with pl values of 5.13 and 4.98. Metal analysis showed the presence of I g-atom of iron and 0.5g-atom of manganese per mol of enzyme. The visible and UV absorption spectra of the Citrus enzyme were similar to those reported for other iron-containing SODS from different origins. The significance of the presence of Fe-SOD in higher plants is briefly discussed.     

Purification and subunit structure of mouse liver cystathionase

Cystathionase has been purified from mouse liver by ammonium sulfate precipitation, ethanol precipitation, column chromatography on DEAE-cellulose and on hydrox-ylapatite, as well as Sephadex G-200 gel filtration. These procedures yielded a chromatographically homogeneous enzyme which was purified more than 1000-fold relative to whole liver extract. Overall recovery was approximately 4%. The purified enzyme does not contain detectable carbohydrate and migrates as a single protein component on analytical disc gel electrophoresis. A sedimentation coefficient of 8.3 S has been determined for the active enzyme by rate zonal centrifugation in glycerol gradients. This value suggests a molecular weight for the native enzyme of approximately 160,000 g/mol, a value similar to that estimated by gel filtration. Following sodium dodecyl sulfate gel electrophoresis in the presence of reducing agent and at different gel concentrations, a single protein component with a molecular weight of 40,000 g/mol was obtained. Thus, the enzyme appears to consist of four subunits of equal size. The K_m value for cystathionine at pH 8.1, 37 °C, and in the presence of 1 mm dithioerythritol is approximately 1 mm.     

Characterization of a new type of dissimilatory sulfite reductase present in Thermodesulfobacterium commune

A new type of dissimilatory bisulfite reductase, desulfofuscidin, was isolated from the nonsporeforming thermophilic sulfate-reducing microorganism Thermodesulfobacterium commune. The molecular weight of the enzyme was estimated at 167,000 by sedimentation equilibrium, and the protein was pure by both disc electrophoresis and ultracentrifugation. The bisulfite reductase was a tetramer and had two types of subunits with an α₂β₂ structure and an individual molecular weight of 47,000. The enzyme exhibited absorption maxima at 576, 389, and 279 nm, with a weak band at 693 nm. Upon the addition of dithionite, the absorption maxima at 576 and 693 nm were weakened, and a new band appeared at 605 nm. The protein reacted with CO in the presence of dithionite to give a complex with absorption peaks at 593, 548, and 395 nm. The extinction coefficients of the purified enzyme at 576, 389, and 279 nm were 89,000, 310,000, and 663,000 M⁻¹ cm⁻¹, respectively. Siroheme was detected as the prosthetic group. The protein contains 20 to 21 nonheme iron atoms and 16 to 17 acid-labile sulfur groups per molecule. The data suggest the presence of four sirohemes and probably four (4Fe-4S) centers per molecule by comparison with desulfoviridin, the dissimilatory sulfite reductase from Desulfovibrio species. The protein contains 36 cysteine residues and is high in acidic and aromatic amino acids. The N-terminal amino acids of the α and β subunits were threonine and serine, respectively. With reduced methyl viologen as electron donor, the major product of sulfite reduction was trithionate, and the pH optimum for activity was 6.0. The enzyme was stable to 70°C and denatured rapidly above this temperature. The dependence of T. commune bisulfite reductase activity on temperature was linear between 35 and 65°C, and the Q₁₀ values observed were above 3. The presence of this new type of dissimilatory bisulfite reductase in T. commune is discussed in terms of taxonomic significance.     

Cytochrome c oxidase from the liver of bullfrog, Rana catesbeina and change in its turnover rate during metamorphosis

1. Cytochrome c oxidase was purified from the liver mitochodria of bullfrog (Rana catesbeina). The heme a content of the purified enzyme was 13.5 nmol per mg protein. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the enzyme protein was composed of nine polypeptide subunits having molecular weights of 42 000, 27 000, 25 000, 20 000, 15 000, 13 000, 8 600, 5 400 and 3 600. The purified enzyme from the adult frog was immunologically identical with that from the tadpole. 2. The rates of synthesis and degradation of cytochrome c oxidase were 5.2- and 2.0-times higher at metamorphic climax than at premetamorphic stage, respectively. The amount of the enzyme in the liver was highest at metamorphic climax.     

Presence of immunoreactive material in Niemann-Pick type A placeta using anti-sphingomyelinase rabbit gammaglobulins

Human placental sphingomyelinase was highly purified through an original six-step scheme in order to raise a specific rabbit anti-sphingomyelinase antibody. Pure enzyme preparations showed specific activities ranging between 100 and 150 μmol/h per mg protein and gave two constant silver - stained bands (M_r 70 000 and 57 000) on acrylamide after electrophoresis under denaturing conditions. These two bands were the sole areas detected by the described antibody on Western blots from normal placental preparations at various stages of purification. A similar procedure was applied to the separate study of placental sphingomyelinase from two prenatally diagnosed foetuses with confirmed Niemann-Pick disease type A. During purification, the mutant enzyme could be followed owing to its minute but measurable level of catalytic activity, and behaved normally at the various chromatographic steps. In the purified preparations, specific activities of 0.18 and 0.49 μml/h per mg protein, respectively, were reached. No alteration of the K_m value (19 μmol/l) was observed, while the V_max was 0.5–1% of normal. With immunostaining of Western blots obtained as above, results similar to those described for normal tissue were found, leading to the conclusion that immunoreactive sphingomyelinase is present in Neimann-Pick disease type A.     

Transhydroxylase of Pelobacter acidigallici: a molybdoenzyme catalyzing the conversion of pyrogallol to phloroglucinol

Trihydroxybenzenes are degraded anaerobically through the phloroglucinol pathway. In Pelobacter acidigallici as well as in Pelobacter massiliensis, pyrogallol is converted to phloroglucinol in the presence of 1,2,3,5-tetrahydroxybenzene by intermolecular hydroxyl transfer. The enzyme catalyzing this reaction was purified to chromatographic and electrophoretic homogeneity. Gel filtration and electrophoresis revealed a heterodimer structure with an apparent molecular mass of 127 kDa for the native enzyme and 86 kDa and 38 kDa, respectively, for the subunits. The enzyme was not sensitive to oxygen. HgCl₂, p-chloromercuribenzoic acid, and CuCl₂ inhibited strongly the reaction indicating an essential function of SH-groups. Transhydroxylase had a pH-optimum of 7.0 and a pI of 4.1. The apparent temperature optimum was in the range of 53°C to 58°C. The activation energy for the conversion of pyrogallol and 1,2,3,5-tetrahydroxybenzene to phloroglucinol and tetrahydroxybenzene was 31.4 kJ per mol. Purified enzyme exhibited a specific activity of 3.1 mol. m⁻¹ mg⁻¹ protein and an apparent K_m for pyrogallol and 1,2,3,5-tetrahydroxybenzene of 0.70 mM and 0.71 mM, respectively. The enzyme was found to contain per mol heterodimer 1.1 mol molybdenum, 12.1 mol iron and 14.5 mol acid-labile sulfur. Requirement for molybdenum for transhydroxylating enzyme activity was proven also by cultivation experiments. No hints for the presence of flavins were obtained. The results presented here support the hypothesis that a redox reaction is involved in this intermolecular hydroxyl transfer.     

Purification and Characterization of a Dimethoate-Degrading Enzyme of Aspergillus niger ZHY256, Isolated from Sewage

A dimethoate-degrading enzyme from Aspergillus niger ZHY256 was purified to homogeneity with a specific activity of 227.6 U/mg of protein. The molecular mass of the purified enzyme was estimated to be 66 kDa by gel filtration and 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.4, and the enzyme activity was optimal at 50°C and pH 7.0. The activity was inhibited by most of the metal ions and reagents, while it was induced by Cu²⁺. The Michaelis constant (K_m) and V_max for dimethoate were 1.25 mM and 292 μmol min⁻¹ mg of protein⁻¹, respectively.