Purification of a peptidylglycine alpha-amidating enzyme from transplantable rat medullary thyroid carcinomas

A peptidyl glycine alpha-amidating activity has been isolated from total tissue extracts of rat medullary thyroid carcinoma (MTC). Purification of the activity by ammonium sulfate fractionation, Sephacryl S-300 chromatography, and strong anion-exchange chromatography at pH 6.0 has resolved at least four peaks of activity. The activity associated with peak III has been further purified to apparent homogeneity by strong anion-exchange chromatography at pH 8.0. The purified peak III enzyme has an apparent molecular mass of 75,000 Da as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The identity of the 75,000-Da band as the alpha-amidating enzyme has been confirmed by recovery of activity from a nondenaturing polyacrylamide gel. The enzyme is catalytically active as a monomer, exhibits a pH optimum between 5.0 and 5.5, and has a turnover number of 300 min-1 for N-dansyl-Tyr-Val-Gly amidation at pH 5.5. The larger size, more acidic pH optimum, and higher specific activity distinguish the purified peak III rat MTC enzyme from the enzymes isolated from bovine and porcine pituitary or from frog skin.     

Guanosine cyclic monophosphate-dependent protein kinase from foetal calf heart. Purification, general properties and catalytic subunit.

Cyclic GMP-dependent protein kinase was purified from foetal calf hearts, and its general properties and subunit structure were studied. The enzyme was purified over 900-fold from the heart extract by pH 5.3-isoelectric precipitation, DEAE-cellulose chromatography, Sephadex G-200 filtration and hydroxyapatite treatment. The purified myocardial enzyme, free from cyclic AMP-dependent protein kinase contamination, exhibited an absolute requirement of stimulatory modulator (or crude modulator containing the stimulatory modulator component) for its cyclic GMP-stimulated activity. Inhibitory modulator (protein inhibitor) of cyclic AMP-dependent protein kinase could not stimulate nor inhibit the cyclic GMP target enzyme. The enzyme had Ka values of 0.013, 0.033 and 3.0 micronM for 8-bromo cyclic GMP, cyclic GMP and cyclic AMP respectively. The cyclic GMP-dependent enzyme required Mg2+ and Co2+ for its activity, with optimal concentrations of about 30 and 0.5 mM respectively. The pH optimum for the enzyme activity ranged from 6 to 9. Histones were generally effective substrate proteins. The enzyme exhibited a greater affinity for histones than did the cyclic AMP-dependent class of protein kinase. The holoenzyme (apparent mol.wt. 150 000) of the myocardial cyclic GMP-dependent protein kinase was dissociated into a cyclic GMP-independent catalytic subunit (apparent mol.wt. 60 000) by cyclic GMP and histone. The catalytic subunit required the stimulatory modulator for its activity, as in the case of the holoenzyme in the presence of cyclic GMP.     

Purification of adenine phosphoribosyltransferase from Brassica juncea

Adenine phosphoribosyltransferase was purified from Brassica juncea leaves approximately 4000-fold, to homogeneity. The native enzyme is a homodimer, with a Mr of 54,000. The purification involved (NH4)2SO4 fractionation, differential ultracentrifugation, and anion-exchange, hydrophobic, dye-ligand, and affinity chromatography. The purified enzyme has a pH optimum of 9.15 and a temperature optimum of 60 degrees C. Activity of the enzyme is stimulated by Mg2+ and is inhibited by sulfhydryl reagents. At the optimum pH and 37 degrees C, the apparent Km values for adenine and 5-phosphoribosyl-1-pyrophosphate were 3.8 and 15 microM, respectively. Analysis of the purified protein by isoelectric focusing revealed the presence of two isozymes with approximate isoelectric points of 5.3 and 5.4.     

人胃腺苷脱氨酶的分离纯化及性质研究

用硫酸铵分级沉淀、ＤＥＡＥ－纤维素离子交换层析、免疫亲和层析、ＳｅｐｈａｄｅｘＧ１００凝胶柱层析从人胃组织中提取出腺苷脱氨酶,酶纯化１９３２４倍,比活力为５７９７Ｕ／ｍｇ蛋白．提取酶液经ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和等电聚焦只呈现一条区带。测得该酶的分子量为４１．２ｋＤ,等电点为ｐＨ４．８．氨基酸组成分析表明该酶由３８８个氨基酸残基组成,Ｎ端氨基酸为精氨酸。酶的最适ｐＨ为６．５,ｐＨ小于５．０或大于９．０时不稳定;最适温度为３７℃,对热不太稳定,以腺苷及２－脱氧腺苷作为底物,其Ｋｍ分别为８７μｍｏｌ／Ｌ和４１μｍｏｌ／Ｌ。     

The beta-oxidation system in catalase-free microbodies of the filamentous fungus Neurospora crassa. Purification of a multifunctional protein possessing 2-enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase activities.

A trifunctional beta-oxidation protein, designated TFP, was purified to apparent homogeneity from oleate-induced mycelia of Neurospora crassa. 2-Enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase activities copurified in constant ratios with this protein when crude extracts were subjected to cation-exchange, dye-ligand, and adsorption chromatography. Trifunctionality was substantiated by coinciding enzyme activity ratios during the last two purification steps and additional chromatographic steps. The enzyme was shown to be a 365-kDa tetramer of subunits with a molecular mass of 93 kDa. Several lines of evidence suggest that these subunits are identical. Monospecific antibodies raised against the homogenous protein specifically precipitated the three enzymatic activities of TFP. Immunoblotting of fractions obtained after sucrose density gradient centrifugation of a crude extract indicated that TFP was exclusively localized in glyoxysome-like microbodies. The beta-oxidation system of N. crassa is structurally related to those of peroxisomes despite the presence of an acyl-CoA dehydrogenase rather than an acyl-CoA oxidase. A mitochondrial 2-enoyl-CoA hydratase activity was separated from TFP and purified to apparent homogeneity. The absence of all other beta-oxidation activities from mitochondria suggests that this organelle and its 2-enoyl-CoA hydratase are not involved in fatty acid degradation in N. crassa.     

Purification and characterization of dipeptidyl peptidase IV from Streptococcus salivarius HHT.

Dipeptidyl peptidase IV (DAP IV) was purified from Streptococcus salivarius HHT by anion-exchange chromatography, gel filtration and affinity chromatography after lysis of cell walls with N-acetylmuramidase. DAP IV was purified 114-fold with a yield of 16.6% from total activity of the crude extract. The purified enzyme was shown to be homogeneous by disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 109,000 by gel filtration and 47,000 by sodium dodecylsulphate SDS-polyacrylamide gel electrophoresis, suggesting that the native enzyme is a dimeric form. The optimum pH for the reaction was 8.7 in Gly-NaOH buffer, and the isoelectric point of the enzyme was pH 4.2. The enzyme hydrolysed specifically N-terminal X-Pro from X-Pro-p-nitroanilides. The enzyme activity was hardly affected by various cations, sulphydryl-blocking reagents and metal chelators. The enzyme activity was markedly inhibited by 1 mM diisopropylfluoride, and the desialysed enzyme was attacked by proteinases.     

Purification and thermodynamic characterization of glucose oxidase from a newly isolated strain of Aspergillus niger

An intracellular glucose oxidase (GOD) was isolated from the mycelium extract of a locally isolated strain of Aspergillus niger NFCCP. The enzyme was partially purified to a yield of 28.43% and specific activity of 135 U mg(-1) through ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The enzyme showed high specificity for D-glucose, with a K(m) value of 25 mmol L(-1). The enzyme exhibited optimum catalytic activity at pH 5.5. Optimum temperature for GOD-catalyzed D-glucose oxidation was 40 degrees C. The enzyme displayed a high thermostability having a half-life (t(1/2)) of 30 min, enthalpy of denaturation (H*) of 99.66 kJ mol(-1), and free energy of denaturation (G*) of 103.63 kJ mol(-1). These characteristics suggest that GOD from A. niger NFCCP can be used as an analytical reagent and in the design of biosensors for clinical, biochemical, and diagnostic assays.     

Characterization of a new cobalt-containing nitrile hydratase purified from urea-induced cells of Rhodococcus rhodochrous J1

A new cobalt-containing nitrile hydratase was purified from extracts of urea-induced cells from Rhodococcus rhodochrous J1 in seven steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. Nitrile hydratase was a 500-530-kDa protein composed of two different subunits (alpha subunit 26 kDa, beta subunit 29 kDa). The enzyme contained approximately 11-12 mol cobalt/mol enzyme. A concentrated solution of highly purified nitrile hydratase exhibited a broad absorption spectrum in the visible range, with an absorption maxima at 410 nm. The enzyme had a wide substrate specificity. Aliphatic saturated or unsaturated nitriles as well as aromatic nitriles, were substrates for the enzyme. The optimum pH of the hydratase was pH 6.5-6.8. The enzyme was more stable than ferric nitrile hydratases. The amino-terminal sequence of each subunit of R. rhodochrous J1 enzyme was determined and compared with that of ferric nitrile hydratases. Prominent similarities were observed with the beta subunit. However, the amino acid sequence of the alpha subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes.     

Isolation,Purification and Some Properties of Invertase from Leaves of Mandarin Orange

Highly active acid invertase was found in the young leaf extract of mandarin orange Citrus reticulata Blanco). The invertase was isolated and purified from the young leaf extract of mandarin orange through the procedures of ammonium sulphate precipitation, DEAE-Sepharose column chromatography and Sephacryl S-200 gel filtration. 6.4% of the invertase activity was recovered. Invertase was 179.2-folds purified. The purified invertase preparation was homogeneous as shown in polyacrylamide gel electrophoresis and Sephacryl S-200 molecular sieve chromatography. The molecular weight of the native invertase determined by gel filtration was 80 kD. The invertase consists of two identical subunits with apparent equal subunit weight of 40 kD as determined on SDS-PAGE. The invertase followed typical Michaelis-Menten Kinetics with apparent Km Of 1. 6 × 10-2 mol/L for sucrose. Vmax of the invertase was 100 mg reducing sugar · mg-1 protein · h-1 The optimum pH was 5.0 (stable from 4.5—5.5). The optimum temperature was 55℃.     

Phosphatidylinositol biosynthesis in Saccharomyces cerevisiae: purification and properties of microsome-associated phosphatidylinositol synthase

The membrane-associated phospholipid biosynthetic enzyme phosphatidylinositol synthase (cytidine 5'-diphospho-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) was purified 1,000-fold from the microsomal fraction of Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of the microsomal membranes, CDPdiacylglycerol-Sepharose (Larson et al., Biochemistry 15:974-979, 1976) affinity chromatography, and chromatofocusing. The procedure resulted in the isolation of a nearly homogeneous protein preparation with an apparent minimum subunit molecular weight of 34,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Phosphatidylinositol synthase was dependent on manganese and Triton X-100 for maximum activity. The pH optimum was 8.0. Thioreactive agents inhibited enzyme activity. The energy of activation was found to be 35 kcal/mol (146,540 J/mol). The enzyme was reasonably stable at temperatures of up to 60 degrees C.     

The production, purification and characterisation of two novel alpha-D-mannosidases from Aspergillus phoenicis

1,6-alpha-D-Mannosidase from Aspergillus phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 74 kDa by SDS-PAGE and 81 kDa by native-PAGE. The isoelectric point was 4.6. 1,6-alpha-D-Mannosidase had a temperature optimum of 60 degrees C, a pH optimum of 4.0-4.5, a K(m) of 14 mM with alpha-D-Manp-(1-->6)-D-Manp as substrate. It was strongly inhibited by Mn(2+) and did not need Ca(2+) or any other metal cofactor of those tested. The enzyme cleaves specifically (1-->6)-linked mannobiose and has no activity towards any other linkages, p-nitrophenyl-alpha-D-mannopyranoside or baker's yeast mannan. 1,3(1,6)-alpha-D-Mannosidase from A. phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 97 kDa by SDS-PAGE and 110 kDa by native-PAGE. The 1,3(1,6)-alpha-D-mannosidase enzyme existed as two charge isomers or isoforms. The isoelectric points of these were 4.3 and 4.8 by isoelectric focussing. It cleaves alpha-D-Manp-(1-->3)-D-Manp 10 times faster than alpha-D-Manp-(1-->6)-D-Manp, has very low activity towards p-nitrophenyl-alpha-D-mannopyranoside and baker's yeast mannan, and no activity towards alpha-D-Manp-(1-->2)-D-Manp. The activity towards (1-->3)-linked mannobiose is strongly activated by 1mM Ca(2+) and inhibited by 10mM EDTA, while (1-->6)-activity is unaffected, indicating that the two activities may be associated with different polypeptides. It is also possible that one polypeptide may have two active sites catalysing distinct activities.     

Purification and characterization of cytosolic glyceraldehyde-3-phosphate dehydrogenase from the dromedary camel

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12),a key enzyme ofcarbon metabolism,was purified and characterized to homogeneity from skeletal muscle of Camelusdromedarius.The protein was purified approximately 26.8 folds by conventional ammonium sulphatefractionation followed by Blue Sepharose CL-6B chromatography,and its physical and kinetic propertieswere investigated.The native protein is a homotetramer with an apparent molecular weight of approximately146 kDa.Isoelectric focusing analysis showed the presence of only one GAPDH isoform with an isoelectricpoint of 7.2.The optimum pH of the purified enzyme was 7.8.Studies on the effect of temperature onenzyme activity revealed an optimal value of approximately 28-32 ℃ with activation energy of 4.9 kcal/mol.The apparent K_m values for NAD~ and DL-glyceraldehyde-3-phophate were estimated to be 0.025±0.040mM and 0.21±0.08 mM, respectively. The V_(max) of the purified protein was estimated to be 52.7±5.9 U/mg.These kinetic parameter values were different from those described previously, reflecting protein differencesbetween species.     

Partial purification and properties of phosphatidylserine synthase from Clostridium perfringens.

The membrane-associated phospholipid biosynthetic enzyme cytidine 5'-diphospho-1,2-diacyl-sn-glycerol:L-serine O-phosphatidyltransferase (phosphatidylserine synthase; EC 2.7.8.8) was partially purified 337-fold from a cell-free extract of the gram-positive pathogenic anaerobe Clostridium perfringens (ATCC 3624). The purification procedure included extraction from the cell envelope with the nonionic detergent Triton X-100, followed by affinity chromatography on cytidine 5'-diphosphate-diacylglycerol-Sepharose. When the partially purified enzyme was subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two major bands were evident with apparent minimum molecular weights of 39,000 and 31,000. Activity of phosphatidylserine synthase was dependent on the addition of manganese ions (3 mM) and Triton X-100 (2.7 mM) for maximum activity. The rate of catalysis was maximal at 40 degrees C (with rapid thermal inactivation above this temperature), and the pH optimum was 8.5. The apparent Km values for cytidine 5'-diphosphate-diacylglycerol and L-serine were 0.24 and 0.26 mM, respectively. The synthetic (forward) reaction was favored, as indicated by an equilibrium constant of 82, and the energy of activation was found to be 18 kcal/mol (75,362 J/mol).     

Wound-inducible pinene cyclase from grand fir: purification, characterization, and renaturation after SDS-PAGE.

The major wound-inducible monoterpene synthase (cyclase) of grand fir (Abies grandis) stems transforms geranyl pyrophosphate to both (-)-alpha-pinene (40%) and (-)-beta-pinene (60%). The enzyme was purified to apparent homogeneity by anion-exchange and hydrophobic interaction chromatography, coupled to discontinuous native polyacrylamide gel electrophoresis at neutral pH and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (also at neutral pH) followed by renaturation in 1% Tween 20 (polyoxyethylenesorbitan monolaurate). The renatured enzyme produced a mixture of isomeric pinenes from geranyl pyrophosphate identical to that generated by the native form. The protein exhibited a molecular weight of 63,000 by gel permeation chromatography and of 62,000 by denaturing gel electrophoresis, indicating that the monomer is active. The enzyme required Mn2+ (Km = 30 microM) for activity, exhibited a Km value of 6 microM for the substrate geranyl pyrophosphate, showed a pH optimum at 7.8 and temperature optimum at 42 degrees C, and was inhibited by pyrophosphate (I50 = 0.17 mM), orthophosphate (I50 = 51 mM), and alpha-pinene, as well as by the histidine-directed reagent diethylpyrocarbonate (I50 = 0.64 mM) and the cysteine-directed reagent p-hydroxymercuribenzoate (I50 = 1.9 microM). Although similar in many respects to constitutive monoterpene cyclases of herbaceous species, this inducible cyclase, the first enzyme of this type to be purified to homogeneity from a conifer, is distinguished by the relatively high pH optimum, and the strict specificity and high affinity for the divalent metal ion cofactor.     

A new mechanism for regulation of tyrosine 3-monooxygenase by end product and cyclic AMP-dependent protein kinase

Tyrosine 3-monooxygenase activity of the crude extract from rat striatum had a sharp pH optimum at pH 5.4 and showed almost no activity at or above pH 7. When the crude extract was partially purified by pH precipitation and chromatography on DEAE-cellulose, the enzyme showed a high activity in the pH range of 5.8 to 7.4. Incubation of the partially purified enzyme with catecholamines such as dopamine, norepinephrine, and epinephrine resulted in a remarkable decrease in the enzyme activity, as assayed at a neutral pH. This suppression of the enzyme activity by catecholamines differed from the well-known feedback inhibition which is competitive with respect to the pterin cofactor; the former occurred at a very much lower concentration of catecholamines even in the presence of a near-saturating concentration of a pterin cofactor, and the former was a time-dependent reaction. The enzyme, the activity of which had been suppressed by the incubation with dopamine, was remarkably activated by the incubation with the catalytic subunit of cyclic AMP-dependent protein kinase in the presence of an ATP-generating system. These results suggest that the activity of tyrosine 3-monooxygenase may be suppressed by its end products in a normal state and it may be stimulated by cyclic AMP-dependent protein kinase as occasion demands.     

Purification and characterization of acid-stable protopectinase produced by Aspergillus awamori in solid-state fermentation

Aspergillus awamori IFO 4033 produced an acid-stable protopectinase in solid-state fermentation using wheat bran as the medium. The enzyme was purified to a homogeneous preparation with anion-exchange, hydrophobic, and size-exclusion chromatography. The enzyme was a monomeric protein of 52 kDa, by SDS-PAGE analysis, with an isoelectric point of pH 3.7. The optimum pH for enzyme activity was 2.0, and it was most active at 50 degrees C (at pH 2.0) and was stable up to 50 degrees C (at pH 2.0). The enzyme showed pectin-releasing activity toward protopectins from various origins, especially on lemon protopectin. An outstanding characteristic of the enzyme was its extreme stability in acidic conditions: the enzyme activity was not lost after incubating at pH 2.0 and 37 degrees C for 24 h.     

Purification and Characterization of S-adenosylmethionine Synthetase from Wheat Embryos: Subunit Structure and Molecular Properties

S-adenosylmethionine synthetase from wheat embryos was purified to electrophoretic homogeneity. The mol wt of the enzyme was 174,000 as determined by molecular sieve chromatography on Sephacryl S-200. A single subunit of purified AdoMet synthetase was observed on SOS-PAGE with a mol wt of 84,000 suggesting that the enzyme is a homodimer. The apparent Km of purified enzyme with ATP and methionine is 80 μM and 100 μM, respectively. The pH optimum of the enzyme is 7.75. The enzyme requires MgCb, KCI and reduced glutathione for optimum activity. The ³H-labelled putative S-adenosylmethionine reaction product was converted into ³H-labelled 5′-methyl-thioadenosine by heat treatment (100°C, 10 min, pH 7.0). This proved the authenticity of the reaction product of the AdoMet synthetase in wheat embryos.     

PEG-重组酵母尿酸酶结合物的基本特性研究

重组Candida utilis尿酸酶由含PET-Uricase表达质粒的重组E.coli JM109(DE3)经乳糖诱导表达,菌体破碎后依次经过硫酸铵沉淀、阴离子交换层析和凝胶过滤层析可以获得纯度95%的重组尿酸酶。还原性SDS-PAGE和HPLC测得其亚基表观分子量和天然分子量分别约为33 kDa和130 kDa。获得的纯酶与20 kDa (mPEG)2 -Lys-NHS在特定的条件下反应合成PEG-重组酵母尿酸酶结合物,考察了重组酵母尿酸酶PEG化前后的基本性质,结果显示PEG化尿酸酶的最适pH为7.5,较修饰前下降了1个pH单位,酸碱稳定范围与修饰前类似,都在pH 6-10范围内稳定;修饰前后最适温度均为40℃,重组酵母尿酸酶的热稳定性和抗蛋白酶水解能力较PEG修饰前有较大提高;PEG化尿酸酶可保留修饰前酶活力的87.5%;在最适条件下,PEG-尿酸酶结合物的Km为3.57×10-5 mol/L,而修饰前测得的Km为3.91×10-5 mol/L。研究结果为深入探讨PEG化尿酸酶的结构与功能奠定了基础。     

Isolation and characterization of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine liver

6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities were copurified to homogeneity from bovine liver. The purification scheme consisted of polyethylene glycol precipitation, anion-exchange and Blue-Sepharose chromatography, substrate elution from phosphocellulose, and gel filtration. The bifunctional enzyme had an apparent molecular weight of 102,000 and consisted of two subunits (Mr 49,000). The kinase had a Km for ATP of 12 microM and a S0.5 for fructose 6-phosphate of 150 microM while the bisphosphatase had a Km for fructose 2,6-bisphosphate of 7 microM. Both activities were subject to modulation by various effectors. Inorganic phosphate stimulated both activities, while alpha-glycerolphosphate inhibited the kinase and stimulated the bisphosphatase. The pH optimum for the 6-phosphofructo-2-kinase activity was 8.5, while the fructose-2,6-bisphosphatase reaction was maximal at pH 6.5. Incubation of the purified enzyme with [gamma-32P]ATP and the catalytic subunit of the cAMP-dependent protein kinase resulted in 32P incorporation to the extent of 0.7 mol/mol enzyme subunit with concomitant inhibition of the kinase activity and activation of the bisphosphatase activity. The mediation of the bisphosphatase reaction by a phosphoenzyme intermediate was suggested by the isolation of a stable labeled phosphoenzyme when the enzyme was incubated with fructose 2,6-[2-32P]bisphosphate. The pH dependence of hydrolysis of the phospho group suggested that it was linked to the N3 of a histidyl residue. The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine liver has properties essentially identical to those of the rat liver enzyme, suggesting that hepatic fructose 2,6-bisphosphate metabolism is under the same control in both species.     

Purification and properties of pentachlorophenol hydroxylase, a flavoprotein from Flavobacterium sp. strain ATCC 39723.

A pentachlorophenol (PCP) hydroxylase which catalyzed the conversion of PCP to 2,3,5,6-tetrachlorohydroquinone and released iodide from triiodophenol in the presence of NADPH and oxygen was identified. The enzyme was purified by protamine sulfate precipitation, ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography, gel filtration chromatography, and crystallization. The enzyme was a monomer with a molecular weight of 63,000. Under certain conditions, dimer and multimer conformations were also observed. The pI of the enzyme was pH 4.3. The optimal conditions for activity were a pH of 7.5 to 8.5 and a temperature of 40 degrees C. Each enzyme molecule contained one flavin adenine dinucleotide molecule. The Km for PCP was 30 microM and the Vmax was 16 mumol/min/mg of protein. The enzymatic reaction required 2 mol of NADPH per mol of halogenated substrate. On the basis of the data we present, it is likely that PCP hydroxylase is a flavoprotein monooxygenase. The addition of flavins to the reaction mixture did not stimulate the enzymatic reaction; however, we identified the photodegradation of triiodophenol and tribromophenol, but not PCP, by flavin mononucleotide or riboflavin and light.