Rat mast cell tryptase

Rat mast cell tryptase is located largely if not totally in the cell's secretory granules. When the active site reagent [3H]diisopropyl fluorophosphate was used to label tryptase and chymase simultaneously, the ratio of tryptase:chymase active sites was determined to be 0.05. In comparison to chymase and tryptase in other species and chymase in the rat, rat tryptase is poorly bound to the granule matrix as evidenced by (1) its release parallel to histamine on induction of secretion and (2) its appearance in the supernatant when isolated granules were stripped of their membranes with hypotonic medium. Tryptase on release from the granule is moderately stable at a pH of 5.0 but unstable at pH 7.5, the pH that the enzyme encounters on secretion from the cell. These several properties indicate that the role of rat mast cell tryptase extracellularly is likely to differ greatly from that of chymase.     

Reaction of cyanide with glutathione peroxidase

An oxidized form of ovine erythrocyte GSH peroxidase (Form C) that contains bound glutathione in equimolar ratio to the enzyme selenium is inactivated by cyanide. When Form C was treated with 1 or 10 mM KCN at pH 7.5, there was a rapid increase in ultraviolet absorption at 250 nm, S-cyanoglutathione was released, and the enzyme was reduced, as shown by inactivation with iodoacetate (1 mM, pH 7.5) and uptake of label from [¹⁴C]iodoacetate in equimolar ratio to enzyme selenium. These observations suggest that glutathione is bound to enzyme selenium by a selenenyl-sulfide linkage (E-Se-SG) which is cleaved by cyanide to release a selenol and S-cyanoglutathione; spontaneous oxidation of the selenol to a labile oxidized form of GSH peroxidase leads to irreversible inactivation.     

Regulation of vascular endothelial growth factor binding and activity by extracellular pH

Angiogenesis, the growth of new blood vessels, is regulated by a number of factors, including hypoxia and vascular endothelial growth factor (VEGF). Although the effects of hypoxia have been studied intensely, less attention has been given to other extracellular parameters such as pH. Thus, the present study investigates the consequences of acidic pH on VEGF binding and activity in endothelial cell cultures. We found that the binding of VEGF165 and VEGF121 to endothelial cells increased as the extracellular pH was decreased from 7.5 to 5.5. Binding of VEGF165 and VEGF121 to endothelial extracellular matrix was also increased at acidic pH. These effects were, in part, a reflection of increased heparin binding, because VEGF165 and VEGF121 showed increased retention on heparin-Sepharose at pH 5.5 compared with pH 7.5. Consistent with these findings, soluble heparin competed for VEGF binding to endothelial cells under acidic conditions. However, at neutral pH (7.5) low concentrations of heparin (0.1-1.0 microg/ml) potentiated VEGF binding. Extracellular pH also regulated VEGF activation of the extracellular signal-regulated kinases 1 and 2 (Erk1/2). VEGF165 and VEGF121 activation of Erk1/2 at pH 7.5 peaked after 5 min, whereas at pH 6.5 the peak was shifted to 10 min. At pH 5.5, neither VEGF isoform was able to activate Erk1/2, suggesting that the increased VEGF bound to the cells at low pH was sequestered in a stored state. Therefore, extracellular pH might play an important role in regulating VEGF interactions with cells and the extracellular matrix, which can modulate VEGF activity.     

The role of histidine residues in glutamate dehydrogenase

1. Glutamate dehydrogenase was subject to rapid inactivation when irradiated in the presence of Rose Bengal or incubated in the presence of ethoxyformic anhydride. 2. Inactivation in the presence of Rose Bengal led to the photo-oxidation of four histidine residues. Oxidation of three histidine residues had little effect on enzyme activity, but oxidation of the fourth residue led to the almost total loss of activity. 3. Acylation of glutamate dehydrogenase with ethoxyformic anhydride at pH6.1 led to the modification of three histidine residues with a corresponding loss of half the original activity. Acylation at pH7.5 led to the modification of two histidine residues and a total loss of enzyme activity. 4. One of the histidine residues undergoing reaction at pH6.1 also undergoes reaction at pH7.5. 5. The presence of either glutamate or NAD(+) in the reaction mixtures at pH6.1 had no appreciable effect. At pH7.5 glutamate caused a marked decrease in both the degree of alkylation and degree of inactivation. NAD(+) had no effect on the degree of inactivation at pH7.5 but did modify the extent of acylation. 6. The normal response of the enzyme towards ADP was unaffected by acylation at pH6.1 or 7.5. 7. The normal response of the enzyme towards GTP was altered by treatment at both pH6.1 and 7.5.     

Activation of NAD-linked malic enzyme in intact plant mitochondria by exogenous coenzyme A

O2 uptake by potato and cauliflower bud mitochondria oxidizing malate was progressively inhibited as the pH of the external medium was increased, in response to accumulation of oxaloacetate. Adding 0.5 mM coenzyme A to the medium reversed this trend by stimulating intramitochondrial NAD-linked malic enzyme at alkaline pH. In intact potato mitochondria, coenzyme A stimulation of malic enzyme was not observed when the external pH was above 7.5; in cauliflower mitochondria, coenzyme A stimulated even at pH 8. This difference in the response of intact mitochondria was attributed to an inherent difference in the properties of malic enzyme from the two tissues. Malic enzyme solubilized from potato mitochondria was inactive at pH values above 7.8, while that from cauliflower mitochondria retained its activity at pH 8 in the presence of coenzyme A. In potato mitochondria, coenzyme A stimulation of O2 uptake at alkaline pH was only observed when NAD+ was also provided exogenously. The results show that coenzyme A can be taken up by intact mitochondria and that pH, NAD+, and coenzyme A levels in the matrix act together to regulate malate oxidation.     

Lytic enzyme from lysates of Streptomyces venezuelae infected with actinophage MSP2

A lytic enzyme was purified 600-fold with 12% recovery from lysates of Streptomyces venezuelae S13 infected with actinophage MSP2. The purified enzyme preparation was homogeneous as shown by polyacrylamide electrophoresis. The enzyme was active over a pH range 6.0 to 9.0 with a maximum at pH 7.5. The pH profile for stability was sharp, with an optimum at pH 7.5. Maximal activity occurred between 30 and 35 C. The enzyme was stable at 20 C or less. A 30-min exposure to 25, 30, 35, 40, 45, and 50 C produced an inactivation of 3, 40, 77, 82, 93, and 100%, respectively. Lytic activity was stimulated fivefold by either 5 x 10(-3)m Mg(2+) or Mn(2+) and three- and twofold by Ca(2+) and Ba(2+), respectively. Addition of Na(+), K(+), NH(4) (+), or Li(+) to the tris(hydroxymethyl)aminomethane-hydrochloride buffer did not alter the rate of lysis. Enzyme activity was inhibited 74 and 27% by 10(-4) and 10(-5)m ethylenediaminetetraacetic acid (EDTA), respectively. The inhibition by EDTA was reversed partially by addition of Mg(2+). Lytic activity was abolished by either 5 x 10(-4)m HgCl(2) or p-hydroxymercuribenzoate, whereas 5 x 10(-4)m CuSO(4) inhibited 72%. Cell wall solubilization paralleled the release of N-terminal amino groups and reached a level of 0.23 mumole per mg of cell walls. No release of reducing power was detected in treated or untreated cell wall suspensions. Tests for proteolytic activity were negative.     

Optimization of culture conditions and properties of immobilized sulfide oxidase from Arthrobacter species

Arthrobacter species strain FR-3, isolated from sediments of a swamp, produced a novel serine-type sulfide oxidase. The production of sulfide oxidase was maximal at pH 7.5 and 30 degrees C. Among various carbon and nitrogen sources tested, glucose and yeast extract were found to be the most effective substrates for the secretion of sulfide oxidase. The sulfide oxidase was purified to homogeneity and the molecular weight of the purified enzyme was 43 kDa when estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified sulfide oxidase can be effectively immobilized in DEAE (diethylaminoethyl)-cellulose matrix with a yield of 66%. The purified free and immobilized enzyme had optimum activity at pH 7.5 and 6.0, respectively. Immobilization increases the stability of the enzyme with respect to temperature. The half-life of the immobilized enzyme was 30 min at 45 degrees C, longer than that of the free enzyme (10 min). The purified free sulfide oxidase activity was completely inhibited by 1 mM Co2+ and Zn2+ and sulfhydryl group reagents (para-chloromercuribenzoic acid and iodoacetic acid). Catalytic activity was not affected by 1 mM Ca2+, Mg2+, Na+ and metal-chelating agent (EDTA).     

Sulphur metabolism in Paracoccus denitrificans. Purification, properties and regulation of serine transacetylase, O-acetylserine sulphydrylase and beta-cystathionase.

1. Serine transacetylase, O-acetylserine sulphydrylase and beta-cystathionase were purified from Paracoccus denitrificans strain 8944. 2. Serin transacetylase was purified 150-fold. The enzyme has a pH optimum between 7.5 and 8.0, is specific for L-serine and is inhibited by sulphydryl-group reagents. The apparent Km values for serine and acetyl-CoA are 4.0 - 10(-4) and 1.0 - 10(-4) M, respectively. Serine transacetylase is strongly inhibited by cysteine. 3. O-Acetylserine sulphydrylase was purified 450-fold. The enzymes has a sharp pH optimum at pH 7.5. In addition to catalysing the synthesis of cysteine, O-acetylserine sulphydrylase catalyses the synthesis of selenocysteine from O-acetylserine and selenide. The Km values for sulphide and O-acetylserine are 2.7 - 10(-3) and 1.25 - 10(-3) M, respectively. The enzyme was stimulated by pyridoxal phosphate and was inhibited by cystathionine, homocysteine and methionine. 4. beta-Cystathionase was purified approx. 50-fold. beta-Cystathionase has a pH optimum between pH 9.0 and 9.5, is sensitive to sulphydryl-group reagents, required pyridoxal phosphate for maximum activity and has an apparent Km for cystathionine of 4.2 - 10 (-3) M. beta-Cystathionase also catalyses the release of keto acid from lanthionine, djenkolic acid and cystine. Cysteine, O-acetylserine, homocysteine and glutathione strongly inhibit beta-cystathionase activity and homocysteine and methionine represses enzyme activity. 5. O-Acetylserine lyase was identified in crude extracts of Paracoccus denitrificans. The enzyme is specific for O-acetyl-L-serine, requires pyridoxal phosphate and is inhibied by KCN and hydroxylamine. The enzyme has a high Km value for O-acetylserine (50--100 mM).     

pH-dependent substrate preference of pig heart lipoamide dehydrogenase varies with oligomeric state: response to mitochondrial matrix acidification

Cycling of intracellular pH has recently been shown to play a critical role in ischemia-reperfusion injury. Ischemia-reperfusion also leads to mitochondrial matrix acidification and dysfunction. However, the mechanism by which matrix acidification contributes to mitochondrial dysfunction, oxidative stress, and the resultant cellular injury has not been elucidated. We observe pH-dependent equilibria between monomeric, dimeric, and a previously undescribed tetrameric form of pig heart lipoamide dehydrogenase (LADH), a mitochondrial matrix enzyme. Dynamic light scattering studies of native LADH in aqueous solution indicate that lowering pH favors a shift in average molecular mass from higher oligomeric states to monomer. Sedimentation velocity of LADH entrapped in reverse micelles reveals dimer and tetramer at both pH 5.8 and 7.5, but monomer was observed only at pH 5.8. Enzyme activity measurements in reverse Aerosol OT micelles in octane indicate that LADH dimer and tetramer possess lipoamide dehydrogenase and diaphorase activities at pH 7.5. Upon acidification to pH 5.8 only the LADH monomer is active and only the diaphorase activity is observed. These results indicate a correlation between pH-dependent changes in the LADH reaction specificity and its oligomeric state. The acidification of mitochondrial matrix that occurs during ischemia-reperfusion injury is sufficient to alter the structure and enzymatic specificity of LADH, thereby reducing mitochondrial defenses, increasing oxidative stress, and slowing the recovery of energy metabolism. Matrix acidification may also disrupt the quaternary structure of other mitochondrial protein complexes critical for cellular homeostasis and survival.     

吸附-交联法固定D-泛解酸内酯水解酶的研究

选择6种吸附树脂和离子交换树脂对D-泛解酸内酯水解酶进行固定化,筛选出了固定化效果较好的大孔弱碱性丙烯酸系阴离子交换树脂D-380为载体,用先吸附后交联的方法固定化。通过实验对固定化条件进行了优化,得出最佳的固定化条件为：加酶量6U／g树脂、吸附pH7．5、吸附时间4h、吸附温度30℃、交联剂戊二醛终浓度0．1％、交联时间2h。实验表明在此条件下制得的固定化酶有很好的稳定性：固定化酶在连续20次的底物水解反应后,剩余酶活达到71％。当温度达到80℃时游离酶几乎失去酶活,而固定化酶剩余酶活为60％以上。游离酶的pH稳定性范围为pH7～8,而固定化酶为pH6．5～8．5。     

Various properties of angiotensin-converting enzyme from the seal Phoca groenlandica

It was found that the molecular mass of the angiotensin-converting enzyme from seal (Phoca groenlandica) lungs determined by electrophoresis in 7.5% PAAG in the presence of sodium dodecyl sulfate is 150 kD. The enzyme has a pH optimum with respect to hippuryl-L-histidyl-L-leucine at 7.3--7.5; KM is 1.2 mM. The enzyme is inhibited by the substrate to form a nonproductive ES2 complex with the dissociation constant (Ks') of 4.8 mM. The activation of the seal angiotensin-converting enzyme in the presence of NaCl was studied. The bradykinin-potentiating factor (SQ 20881) inhibits the seal enzyme with a high efficiency (IC50 = 2.5.10(-8) M). Monoclonal antibodies to the angiotensin-converting enzyme from human lungs do not interact with its seal lung counterpart, which points to the species specificity of the angiotensin-converting enzyme.     

Purification and covalent coupling of calf brain prolidase

We have investigated methods of stabilizing prolidase by chemical modification and covalent coupling to various supports, for use in protein hydrolysis and possible use in enzyme replacement therapy. Purified acetone powder of calf brain prolidase was further purified by gel filtration on Sephadex G-200 and chromatography on DEAE-Sephadex A25. Polyacrylamide gel electrophoresis showed that the number of bands was reduced from 11 to 2. Since yields were low, the purified (NH₄)₂SO₄ fraction was used in all experiments. Thiolation of the enzyme reduced the amount of protein coupled to AH-or CH-Sepharose 4B. Activities were highest when the protein was linked through its carboxyl groups. The coupled enzyme showed much greater thermal stability than its free counterpart. Of the bound preparations, the thiolated was less stable than the untreated. Untreated and thiolated enzymes bound to either matrix showed higher activity at low pH and less at high pH than the free material. Thiolation shifted the pH maximum from 6.8 to 7.5. The free thiolated enzyme and that bound to activated SH-Sepharose 4B showed greater thermal stability and a broader pH range of optimal activity than the bound untreated enzyme. These results show that prolidase can be immobilized by coupling to an insoluble matrix through various types of covalent bonds with retention of activity and increased stability.     

Subcellular localization and kinetic properties of arginase from the liver of Genypterus maculatus

1. From the liver of the teleost fish Genypterus maculatus, a partially purified preparation of arginase was obtained and characterized. 2. The Km value for arginine was found to be 9.1 mM at pH 7.5 and 11.5 mM at the optimum pH of 9.5. At both pH values, competitive inhibition was caused by ornithine and lysine, whereas proline, leucine, valine and isoleucine caused a non-competitive inhibitory effect. Branched chain amino acids were more inhibitory than proline. 3. The enzyme was found localized in the mitochondrial matrix of the liver of Genypterus maculatus. It is suggested that this localization would be of importance in the use of arginine as an energy source.     

Purification and characterization of l-histidine decarboxylase from mouse mastocytoma P-815 cells

Histidine decarboxylase was purified from mouse mastocytoma P-815 cells to electrophoretic homogeneity by ammonium sulfate fractionation, dialyses at pH 7.5 and 6.0, chromatographies on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Hydroxylapatite, Phenyl-Superose HPLC, Mono Q HPLC, and Diol-200 gel filtration HPLC. Under the assay conditions used, the pure enzyme exhibited a specific activity of 800 nmol/min/mg, which constituted 12,500-fold purification compared to the crude extract, with a 7% yield. The two-step dialysis turned out to be essential for removing the factor(s) which interfered with the enzyme purification. The optimum pH for the enzyme reaction was 6.6 and the isoelectric point of the enzyme was pH 5.4. The molecular mass of the enzyme was found to be approximately 53 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, 110 kDa on gel filtration, and 115 kDa on polyacrylamide gradient gel electrophoresis in the absence of sodium dodecyl sulfate. The Km value for histidine was estimated to be 0.26 mM at pH 6.8.     

An alkaline thiol proteinase in the liver mitochondria of bullfrog, Rana catesbeiana

The mitoplasts were prepared from bullfrog (Rana catesbeiana) liver mitochondria by treatment with digitonin and were then separated into the matrix and inner membrane fractions. The matrix fraction thus obtained was free of lysosomal contaminations and exhibited a distinct proteinase activity. pH dependency of the matrix proteinase activity measured in the presence and absence of iodoacetamide revealed that the matrix contained at least two kinds of proteinase, a major alkaline thiol proteinase having an optimal pH at 8.5 and a minor neutral proteinase having an optimal pH at 7.5. The major matrix proteinase activity was strongly inhibited by leupeptin, chymostatin, antipain and E64-C, an inhibitor of Ca2+-dependent thiol proteinase, while it was scarcely affected by diethylpyrocarbonate. The activity was also inhibited by DTNB and p-chloromercuribenzoate. Addition of hydrocarbon compounds such as ethylene glycol, glycerol, Triton X-100 and poly (ethylene glycol) to the reaction mixture was found to decrease the matrix proteinase activity. Neither cytochrome c nor glutamate dehydrogenase was hydrolyzed when subjected to the matrix proteinase activity in vitro. On the other hand, cytochrome c oxidase was effectively hydrolyzed, and the enzyme associated with the mitochondrial innermembrane fragments was partially hydrolyzed by the major matrix proteinase activity.     

The role of nucleoside triphosphate pyrophosphohydrolase in in vitro nucleoside triphosphate-dependent matrix vesicle calcification

Nucleoside triphosphate pyrophosphohydrolase (EC 3.6.1.8) activity is associated with matrix vesicles purified from collagenase digests of fetal calf epiphyseal cartilage. This enzyme hydrolyzes nucleoside triphosphates to nucleotides and PPi, the latter inducing precipitation in the presence of Ca2+ and Pi. An assay for matrix vesicle nucleoside triphosphate pyrophosphohydrolase is developed using beta, gamma-methylene ATP as substrate. The assay is effective in the presence of matrix vesicle-associated ATPase, pyrophosphatase, and alkaline phosphatase activities. A soluble nucleoside triphosphate pyrophosphohydrolase is obtained from matrix vesicles by treatment with 5 mM sodium deoxycholate. The solubilized enzyme induced the precipitation of calcium phosphate in the presence of ATP, Ca2+, and Pi. Extraction of deoxycholate-solubilized enzymes from matrix vesicles with 1-butanol destroys nucleoside triphosphate pyrophosphohydrolase activity while enhancing the specific activities of ATPase, pyrophosphatase, and alkaline phosphatase. In solutions devoid of ATP and matrix vesicles, concentrations of PPi between 10 and 100 microM induce calcification in mixtures containing initial Ca2+ X P ion products of 3.5 to 7.9 mM2. This finding plus the discovery of nucleoside triphosphate pyrophosphohydrolase in matrix vesicles supports the view that these extracellular organelles induce calcium precipitation by the enzymatic production of PPi. Nucleoside triphosphate pyrophosphohydrolase is more active against pyrimidine nucleoside triphosphates than the corresponding purine derivatives. The pH optimum is 10.0 and the enzyme is neither activated nor inhibited by Mg2+ or Ca2+ ions or mixtures of the two. Vmax at pH 7.5 for beta, gamma-methylene ATP is 0.012 mumol of substrate hydrolyzed per min per mg of protein and Km is below 10 microM. The enzyme is irreversibly destroyed at pH 4 and is stable at pH 10.5.     

An aminopeptidase from Agave americana,chemical properties of the enzyme

Agave aminopeptidase, a new enzyme obtained from the plant Agave americana displayed activity towards a variety of substrates. A free alphaamino group on these substrates was essential, but the enzyme did not need any metal ions for optimal activity. Aliphatic, aromatic and basic amino acids situated at the amino terminal end of substrates could be hydrolysed by the enzyme. The enzyme had no endopeptidase or other proteolytic activity. Values of the apparent Michaelis constants for different amino acid substrates, all in the range from 0.1 to 0.6 × 10^?3 M, suggested a relative wide specificity. The pK-values of the two dissociating groups on the enzyme taking part in the catalytic process were pH 6.3 to 6.8 and pH 7.5 to 7.8. These and other studies suggested that histidine plays an active role in the catalytic process. The enzyme was inhibited competitively by free amino acids and this, together with other results, implied a compulsory order of product release.     

The extraction of a neutral metalloproteinase from the involuting rat uterus, and its action on cartilage proteoglycan.

1. Homogenates of rat uteri removed 1 and 2 days post partum were centrifuged at 6000 g. Both pellets and supernatants degraded Azocoll, a general proteinase substrate, at pH 7.5. More than 80% of the total activity was in the pellet fraction. 2. Part of the pellet activity was in a latent form. Trypsin and 4-aminophenylmercuric acetate (a thiol-blocking agent) both activated this latent form, indicating that it is an enzyme--inhibitor complex. An endogenous serine proteinase activated part of the latent enzyme during the assay. 3. The enzyme activity was low before parturition and after involution; it was highest during the first 2 days post partum, when the largest losses of uterine wet weight and matrix macromolecules occur. 4. Up to 70% of the enzyme in the pellets was extracted by heating at 60 degrees C for 4 min in 0.1 M-CaCl2/0.05 M-Tris/HCl, pH 7.5. Approx. 30% of the extracted enzyme was still latent. 5. The extracted enzyme was a metalloproteinase, since it was inhibited completely by 1,10-phenanthroline, but not by inhibitors of thiol or serine proteinases. 6. The enzyme was further purified 15--30-fold by gel chromatography and precipitation with (NH4)2SO4. The apparent molecular weight, estimated by gel filtration, was 24000 for the latent form and 12000 for the active form. The pH optimum was 7--7.5. 7. The enzyme also degraded cartilage proteoglycan. This activity was studied by viscometry and the products were analysed by analytical ultracentrifugation. The major product had a mol.wt. of approx. 100000. The sites of cleavage were in the protein core, since no free oligosaccharides were detected. 8. This neutral metalloproteinase is distinct from uterine collagenase and from a uterine metal-dependent endopeptidase that hydrolyses a heptapeptide related to collagen.     

Cytosolic and microsomal glutathione-S-transferases from bovine filarial worms Setaria cervi

The work presented here deals with the status of glutathione-S-transferase (GST; E.C. 2.5.1.18), the major enzyme of the phase II detoxification pathway, in bovine filarial worms Setaria cervi. GST activity was determined in various subcellular fractions of bovine filarial worms S. cervi (Bubalus bubalis Linn.) and was found to be mainly associated with cytosolic and microsomal fractions. The respective specific activities of the enzyme from cytosolic and microsomal fractions of S. cervi females were determined to be 0.122 +/- 0.024 and 0.010 +/- 0.0052 micromol/min/mg protein, respectively. Cytosolic enzyme was found to possess optimal activity between pH 6.5 and 7.5, whereas the microsomal enzyme showed a broad pH optima, centered at pH 6.0. Kinetic studies on the cytosolic and microsomal forms of the enzyme revealed significant differences between them, thereby indicating that microsomal GST from S. cervi is quite distinct to the cytosolic protein catalyzing the same reaction.     

A novel anthraquinone ring cleavage enzyme from Aspergillus terreus

An enzyme activity which catalyzes the ring cleavage of the anthraquinone questin to form benzophenone desmethylsulochrin was found in the cell-free extract of Aspergillus terreus, a (+)-geodin producer. The product was identified as desmethylsulochrin by high-resolution mass spectroscopy and chemical carrier dilution analysis. The enzyme showed an absolute requirement of NADPH and molecular oxygen. Therefore, the enzyme, named questin oxygenase, was considered to be classified as a monooxygenase. The optimum pH was around 7.5. The enzyme was very unstable and lost its activity completely after storage overnight at 4 degrees C in 0.05 M phosphate buffer, pH 7.5. The instability of the questin oxygenase was partially overcome by the addition of polyols and the non-ionic detergent Tween 80 to the buffer. By DEAE-cellulose column chromatography, two protein fractions, named DE-I and DE-II, were obtained. Neither fraction reacted with questin by itself. However, the combination of DE-I and DE-II reconstituted the questin oxygenase system to convert questin to desmethylsulochrin. This result suggested that the system is not a simple combination of oxygenase and hydrolase, but requires some additional factor(s) such as electron transfer protein.