Hydrophobic chromatographic purification of ethylene-enhanced chlorophyllase from Citrus unshiu fruits

Ethylene-enhanced chlorophyllase from Citrus unshiu fruits was purified to a homogeneous state after solubilization with sodium cholate, using acetone precipitation and hydrophobic chromatography. The enzyme adhered to phenyl Sepharose CL-4B in 3M KCl and was eluted with a linear gradient of Triton X-100 (0–0.5%). Its MW (SDS-PAGE) was 27 000. The enzyme behaved as a protein of MW 110 000 on Sephacryl S-200 gel filtration. The enzyme showed a specific activity of 0.069 μamol chlorophyllide a produced/min/ mg protein. This purification procedure is a rapid method for obtaining pure chlorophyllase.     

Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli.

Fumarate reductase has been purified 100-fold to 95% homogeneity from the cytoplasmic membrane of Escherichia coli, grown anaerobically on a defined medium containing glycerol plus fumarate. Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic-lipophilic balance (HLB) number near 13 and we routinely solubilized the enzyme with Triton X-100 (HLB number = 13.5). Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme. The enzyme whether membrane bound, in Triton extracts, or purified, had an apparent Km near 0.42 mM. Two peptides with molecular weights of 70 000 and 24 000, predent in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity. A minimal native molecular weight of 100 000 was calculated for fumarate reductase by Stephacryl S-200 gel filtration in the presence of sodium cholate. This would indicate that the enzyme is a dimer. The purified enzyme has low, but measurable, succinate dehydrogenase activity.     

Solubilization of alkyldihydroxyacetone-P synthase from Ehrlich ascites cell microsomal membranes.

The enzyme, alkyldihydroxyacetone-P synthase, has been solubilized and partially purified from microsomal preparations of Ehrlich ascites cells after treatment with Triton X-100 and phospholipase C, followed by chromatography on Sepharose 4B. When the Triton X-100 was removed after solubilization the enzyme was still active but eluted in the void volume of the Sepharose 4B column, whereas in the presence of detergent it eluted much later as a single peak of activity, indicating that the solubilized enzyme tends to aggregate unless detergent is present. The lower molecular weight form of alkyldihydroxyacetone-P synthase (in detergent) had an estimated molecular mass of 250,000–300,000 daltons.     

Kinetic properties and solubilization of microsomal cholesterol ester hydrolase from rat liver

Some kinetic properties of the microsomal cholesterol ester hydrolase (CEH) have been examined in rat liver. The reaction was linear with time up to 60 min and with enzyme concentration up to 0.3 mg/mL, and a pH optimum of 6.7 for enzyme activity was observed. Cholesterol esterase exhibited the following apparent kinetic constants: Km, 68.88 microM and Vmax, 33 Units/mg protein. Cholesteryl palmitate was hydrolyzed to a much greater extent than cholesteryl oleate by the enzyme. Product inhibition with cholesterol and palmitic acid was not apparent; however, oleic acid added to the system reduced markedly microsomal CEH activity. The present paper also reports the solubilization of cholesteryl palmitate hydrolase from the microsomal fraction by pretreating it with Triton X-100, sodium deoxycholate, and sodium dodecylsulfate. All ionic and non-ionic detergents tested are capable of making the enzyme soluble, and maximal effects were found at higher concentrations of detergents although the esterase activity was strongly inhibited. Triton X-100 was found to be more effective than sodium deoxycholate and sodium dodecylsulfate in enzyme and protein solubilization. When the direct effects of detergents on CEH activity were studied, progressive concentration-dependent inhibitions were observed.     

Purification and characterization of murine protoporphyrinogen oxidase

The penultimate enzyme of the heme biosynthetic pathway, protoporphyrinogen oxidase (EC 1.3.3.4), has been purified to apparent homogeneity from mouse liver mitochondria. The purification involves solubilization from mitochondrial membranes with sodium cholate followed by ammonium sulfate fractionation and gel filtration on a Sepharose CL-6B column. The eluate is adjusted to 0.67 M (NH4)2SO4 and loaded onto a phenyl-Sepharose column. After salt washes, the enzyme is eluted with 0.5% sodium cholate and 0.5% Brij 35. The final step is high-pressure ion-exchange chromatography on a DEAE-5PW column. The purified protein has a molecular weight of approximately 65,000 by gel filtration chromatography on Sepharose CL-6B in the presence of 0.5% sodium cholate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a single band corresponding to a molecular weight of 65,000. The absorption spectrum of the purified enzyme shows no evidence of a chromophoric cofactor. Purified protoporphyrinogen oxidase has a Km for protoporphyrinogen IX of 5.6 microM with a Vmax of 2300 nmol mg-1 h-1. It utilizes meso- and hematoporphyrinogen at about 10% the level of protoporphyrinogen. The pH optimum is broad with a maximum at 7.1. There is no stimulation or inhibition by any tested divalent cations, and sulfhydryl reagents have no inhibitory effect on the purified enzyme.     

Isolation and characterization of human erythrocyte insulin receptors

The noncovalently associated 125I-insulin-receptor complex was isolated from human erythrocyte membranes after allowing 125I-insulin to interact with the membranes followed by extraction of the 125I-insulin-receptor complex with Triton X-102 or, alternatively, by complete solubilization of the membranes with sodium dodecyl sulfate (SDS), removal of SDS, and then treatment of the solubilized sample with 125I-insulin. Sepharose CL-6B column chromatography of the 125I-insulin-receptor complex obtained by both of the above procedures yielded a highly radioactive 140,000-Da complex which was dissociated into small peptides when subjected to SDS-polyacrylamide gel electrophoresis. In contrast, when the 125I-insulin-treated membrane sample was extracted with Triton X-102, purified by DEAE-Sephacel ion exchange chromatography, covalently cross-linked with disuccinimidyl suberate, and then subjected to SDS-polyacrylamide gel electrophoresis, a highly radioactive component with Mr = 53,000 was obtained. On the other hand, when the Triton X-102-solubilized membrane receptor sample was fractionated by DEAE-Sephacel ion exchange chromatography, complexed with 125I-insulin, covalently crosslinked, and then applied to a Sepharose CL-6B column, a 95,000-Da complex with high specific radioactivity was obtained. Upon SDS-polyacrylamide gel electrophoresis, the 95,000-Da complex was dissociated into a 53,000-Da component which appeared identical with that obtained from the receptor complex described above which was obtained by direct interaction of the membranes with 125I-insulin.     

Solubilization of hyaluronic acid synthetic activity from streptococci and its activation with phospholipids

To date all hyaluronic acid synthetic systems have been of a particulate nature, and attempts at solubilization have been unsuccessful. This has hampered attempts to elucidate the mechanism by which hyaluronic acid is produced. In this paper we demonstrate that the hyaluronic acid synthetic activity from group C streptococcal membranes was solubilized using 2% digitonin and that the activity was optimized by reconstitution with cardiolipin at an optimum phospholipid/protein ratio (microgram/microgram) of 5:1. Furthermore, chromatography of the solubilized synthetase demonstrated that it eluted after the void volume of a Sepharose CL-6B column. CHAPSO, octyl glucopyranoside, sodium cholate, Triton X-100, and zwittergent 314 either inhibited or failed to solubilize the synthetic activity. Phospholipids other than cardiolipin also reconstituted the activity from the digitonin extract, particularly phosphatidylethanolamine and phosphatidylserine. In our system, the specific activity of hyaluronic acid synthetase was increased up to 63 times that of the system of the intact membrane. Furthermore, the total activity of the reconstituted system was 4.9 times greater than that of intact membranes. The soluble enzyme system showed similarities to the membrane-bound synthetase in the kinetics of production of trichloroacetic acid-soluble and -insoluble hyaluronic acid, and the hyaluronic acid produced was of comparable molecular weight.     

Aminopeptidase M from human liver. I. Solubilization, purification, and some properties of the enzyme

Aminopeptidase M [EC 3.4.11.2] was purified 772-fold to homogeneity from the microsomal fraction of human liver, with a yield of 18.9%, by a combination of solubilization with 0.5% Triton X-100 and then 1 M urea and chromatography on columns of DEAE-cellulose, hydroxylapatite, Butyl-Toyopearl, and Sephacryl S-300. The purified enzyme had a molecular weight of 140,000 by SDS-polyacrylamide gel electrophoresis and of 280,000 by gel filtration on a column of TSK gel 2000 SW. It was reconstituted into proteoliposomes with asolectin, showing its amphiphilic nature. The aminopeptidase M from liver was found to be efficiently inhibited by bile acids. The enzyme was almost completely inhibited by chenodeoxycholic acid and 70-90% inhibited by cholic acid at a concentration of 6 mM. The extent of inhibition by conjugated and unconjugated bile acids was in the order: unconjugated greater than glycoconjugated greater than tauroconjugated bile acid, independent of the nature of the substrates used. The inhibition by the various bile acids was totally reversible. Further, it was immunochemically revealed that a considerable amount of liver aminopeptidase M was released into the bile duct. The role of the aminopeptidase M on the bile canalicular membrane and of the enzyme released in the bile duct is discussed in relation to the effects of bile acids.     

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.     

A novel neutral protease(s) from monkey liver.

A novel neutral protease(s), which is presumably membrane-bound, was found in monkey liver using heat-denatured casein as a substrate and was separated from other major catheptic proteases by successive procedures of gel filtration on Ultrogel AcA 22, solubilization by deoxycholate and gel filtration on Sepharose 6B. The enzyme(s) showed maximal activity at pH 8.0, and was strongly inhibited by DFP and PMSF. Many other reagents tested, including TPCK, TLCK, pCMB, iodoacetic acid, and EDTA, were without marked effect on the activity. Activation of the enzyme(s) by NaCl was not observed.     

A comparative study of the effect of various detergents on the structure and function of sarcoplasmic reticulum vesicles

Summary The effects produced by the detergents Triton X-100, sodium dodecylsulphate and sodium cholate on sarcoplasmic reticulum vesicles have been comparatively studied. In all cases, maximal effects are found 5 min after detergent addition. Triton X-100 and SDS are approximately ten times more effective than cholate in protein and phospholipid solubilization. Both Triton X-100 and SDS maintain Ca⁺⁺ accumulation in SR vesicles at detergent concentrations below 10^–3 M; higher concentrations cause a strong inhibition. On the other hand, cholate produces a gradual inhibition of Ca⁺⁺ accumulation in the concentration range between 10^–4 M and 2.5 × 10^–2 M. Triton X-100 and SDS produce a gradual solubilization of the specific Ca⁺⁺-ATPase activity up to a 10^–3 M detergent concentration, above which a strong inactivation occurs, while the enzyme solubilization increases with the presence of cholate in the whole concentration range under study. The different behaviour of sodium cholate, when compared to SDS or Triton X-100, is discussed in relation to the surfactant molecular structures. The possibility of membrane lysis and reassembly in the presence of some detergents is also considered.Abbreviations SR sarcoplasmic reticulum - SDS sodium dodecylsulphate - DTT dithiothreitol - EGTA ethyleneglycoltetraacetate - PEP phosphoenolpyruvate     

Purification and characterization of an arginine-specific carboxypeptidase from Mycoplasma salivarium.

The carboxypeptidase which had been shown to be present exclusively in nonfermentative mycoplasmas was found to be associated with cell membranes of Mycoplasma salivarium. The enzyme was released from the membranes with Triton X-100 and purified by ion-exchange chromatography on DEAE-Sephacel, affinity chromatography on arginine-Sepharose 4B, and chromatofocusing. The purified enzyme had a molecular mass of 218 kilodaltons, as estimated by gel filtration through Sepharose CL-6B, and yielded one band of activity in analytical disc-polyacrylamide gel electrophoresis performed in the presence of 0.5% (wt/vol) Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme treated in the presence or absence of 2-mercaptoethanol revealed one band with a molecular mass of 87 kilodaltons. The enzyme catalyzed selectively the cleavage of the C-terminal arginine residue of peptides such as N-benzoylglycyl-L-arginine, tuftsin, and bradykinin and was inhibited considerably by o-phenanthroline and EDTA but only slightly by NiCl2. The inhibition of the enzyme by EDTA was fully reversed by the addition of ZnCl2, whereas the addition of CoCl2 activated the enzyme.     

Improved Method for Prufication of 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase from Bovine Cerebral White Matter

Abstract: An improved procedure of the solubilization and purification of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase) from bovine cerebral white matter is reported. To remove easily extractable protein, the tissue was homogenised in 10 vol. of 0.5 M-ammonium acetate containing 10 mM-Tris. HCI, pH 6.9, at 4°C and centrifuged at 105,000 g for 60 min. The precipitate was extracted with 10 vol. of 0.5% Triton X-100 containing 10 mM-Tris. HCI, pH 6.9, and centrifuged, By this extraction, over 70% soluble protein could be removed in the supernatant and most CNPase activity was kept in the precipitate. The precipitate was extracted with 10 vol. of 1% Triton X-100 and 1 M-ammonium acetate mixture containing 10 mM-Tris.HCI, pH 8.2, and centrifuged at 105,000 g for 60 min. The extract contained 54% of CNPase and the specific activity was fivefold that of the original homogenate. Subsequently, the extractions were carried out with 2% Triton X-100-2 M-ammonium acetate and 4% Triton X-100-4 M-ammonium acetate at pH 8.2. The recovery of CNPase was found to be nearly 90% from the original homogenate, without loss of enzyme activity during extraction, while much CNPase activity was lost when guanidinium chloride was used as the extraction medium. Using the Triton X-100-ammonium acetate extract, several column chromatography techniques were applied to purify the enzyme. In the first step, Phenyl-Sepharose CL-4B column chromatography was performed by eluting with a double-linear gradient of ammonium acetate and Triton X-100. In the second step, the fraction containing CNPase after Phenyl-Sepharose CL-4B column chromatography was applied to a Sepharose 6B column and the enzyme was eluted with 1% Triton X-100- I M-ammonium acetate, pH 8.2. The peak containing CNPase was applied to CM-Sepharose CL-6B column chromatography in the final step. The enzyme was eluted with a linear gradient of KCI. In this step, CNPase eluted as a sharp peak and the specific activity was approximately 2300 pmol 2′-AMP formed/min/mg protein. The recovery of CNPase from the original homogenate was about 50%. By the isoelectrofocusing technique, the pI of CNPase was found to be 8.6. When Reisfeld polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis were carried out on the purified CNPase, only one protein band, corresponding to CNPase activity, was detected. Its molecular weight was estimated to be approximately 51,000 as the active enzyme form. K, value of the purified enzyme for 2′,3′-CAMP calculated from a Lineweaver-Burk plot was 3.13 mM.     

Purification and properties of steroid sulfatase from human placenta.

Steroid sulfatase was purified approximately 170-fold from normal human placental microsomes and properties of the enzyme were investigated. The major steps in the purification procedure included solubilization with Triton X-100, column chromatofocusing, and hydrophobic interaction chromatography on phenylsepharose CL-4B. The purified sulfatase showed a molecular weight of 500-600 kDa on HPLC gel filtration, whereas the enzyme migrated as a molecular mass of 73 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.7 by isoelectric focusing in polyacrylamide gel in the presence of 2% Triton X-100. The addition of phosphatidylcholine did not enhance the enzyme activity in the placental microsomes obtained from two patients with placental sulfatase deficiency (PSD) after solubilization and chromatofocusing. This result indicates that PSD is the result of a defect in the enzyme rather than a defect in the membrane-enzyme structure. Amino acid analysis revealed that the purified human placental sulfatase did not contain cysteine residue. The Km and Vmax values of the steroid sulfatase for dehydroepiandrosterone sulfate (DHA-S) were 7.8 microM and 0.56 nmol/min, while those for estrone sulfate (E1-S) were 50.6 microM and 0.33 nmol/min, respectively. The results of the kinetic study suggest the substrate specificity of the purified enzyme, but further studies should be done with different substrates and inhibitors.     

Purification of the insulin receptor from human placental membranes

Insulin receptors were purified from human placental microsomal membranes by solubilisation with Triton X-100 followed by Sepharose 6B chromatography, phosphate gradient elution from hydroxyapatite and affinity chromatography on concanavalin A-Sepharose. 2000-fold purification was achieved with 63% overall recovery. The purified receptor gave a single band on 3.75% polyacrylamide (0.1% Triton X-100) gel electrophoresis. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis there was a major band at 75,000 and a minor band at 80,000 daltons. The purified receptor rechromatographed on Sepharose 6B with an apparent molecular weight of 300,000.     

Purification and characterization of an aminopeptidase from Mycoplasma salivarium.

The aminopeptidase which had been shown to be present in Mycoplasma salivarium was found to be associated with the cell membranes of the organism. The enzyme was solubilized in water by papain digestion of the membranes pretreated with Triton X-100 and purified approximately 130-fold by ion-exchange chromatography on DEAE-Sephadex A-50, affinity chromatography on L-leucylglycine-AH-Sepharose 4B, and gel filtration on Sepharose CL-6B. The purified enzyme had a molecular mass of 397 kilodaltons, estimated by gel filtration through Sepharose CL-6B, and gave two bands of activity in analytical disc polyacrylamide gel electrophoresis: a dense, diffuse band and a less dense, narrow one, accounting for 90 and 5% of stained proteins in the gel, respectively. The purified protein revealed two bands with molecular masses of 50 and 46 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzed selectively the cleavage of the N-terminal arginine and leucine residues of peptides; had a pH optimum at 8.5; and was inhibited remarkably by bestatin, o-phenanthroline, EDTA, and L-cysteine, but was activated nine- and twofold by MnCl2 and MgCl2, respectively. The enzyme pretreated with MnCl2 had much higher maximum velocity (Vmax) for L-leucine-p-nitroanilide than the one not treated. That is, the Michaelis constant (Km) and Vmax values of the pretreated enzyme were 10.5 mM and 12.1 microM/min, respectively, whereas those of the untreated enzyme were 5.8 mM and 1.6 microM/min, respectively.     

Enkephalinase from rat kidney. Purification, characterization, and study of substrate specificity

"Enkephalinase," a membrane-bound peptidase hydrolyzing the Gly3-Phe4 amide bond of enkephalins, initially characterized in brain, was purified from a rat kidney microsomal fraction. After differential solubilization with Triton X-100, the use of DEAE-Sephadex, concanavalin A, and hydroxylapatite chromatography led to a 2000-fold purification, close to homogeneity. Renal enkephalinase appears to be a glycoprotein Mr = 92,000-95,000 with catalytic properties and sensitivity to chelating agents and inhibitors (Thiorphan, phosphoramidon) very similar to those of the cerebral enzyme. The enzyme co-purified until the final step with "renal brush-border neutral proteinase" (EC 3.4.24.11) activity assayed with 125I-insulin B chain as substrate and displaying similar sensitivity to inhibitors. The specificity of the purified enkephalinase has been studied using either peptides derived from the enkephalins or model peptides of general formula (Ala)m-Tyr-(Ala)n as substrates. In all cases the bond cleaved was that involving the amino group of an aromatic residue, specificity being also defined by the nature of the neighboring residue on the COOH-terminal side. A free carboxyl in the latter residue was essential in the two series of substrates, indicating that enkephalinase more efficiently functions as a dipeptidyl carboxypeptidase than as an endopeptidase.     

Purification and characterization of a novel broad-specificity (alpha 1----2, alpha 1----3 and alpha 1----6) mannosidase from rat liver.

We have identified a mannosidase in rat liver that releases alpha 1----2, alpha 1----3 and alpha 1----6 linked manose residues from oligosaccharide substrates, MannGlcNAc where n = 4-9. The end product of the reaction is Man alpha 1----3[Man alpha 1----6]Man beta 1----4GlcNAc. The mannosidase has been purified to homogeneity from a rat liver microsomal fraction, after solubilization into the aqueous phase of Triton X-114, by anion-exchange, hydrophobic and hydroxyapatite chromatography followed by chromatofocusing. The purified enzyme is a dimer of a 110-kDa subunit, has a pH optimum between 6.1 and 6.5 and a Km of 65 microM and 110 microM for the Man5GlcNAc-oligosaccharide or Man9GlcNAc-oligosaccharide substrates, respectively. Enzyme activity is inhibited by EDTA, by Zn2+ and Cu2+, and to lesser extent by Fe2+ and is stabilized by Co2+. The pattern of release of mannose residues from a Man6GlcNAc substrate shows an ordered hydrolysis of the alpha 1----2 linked residue followed by hydrolysis of alpha 1----3 and alpha 1----6 linked residues. The purified enzyme shows no activity against p-nitrophenyl-alpha-mannoside nor the hybrid GlcNAc Man5GlcNAc oligosaccharide. The enzyme activity is inhibited by swainsonine and 1-deoxymannojirimycin at concentrations 50-500-fold higher than required for complete inhibition of Golgi-mannosidase II and mannosidase I, respectively. The data indicate strongly that the enzyme has novel activity and is distinct from previously described mannosidases.     

Partial purification and characterization of a serine endopeptidase from rat liver plasma membranes

A serine endopeptidase was partially purified from rat liver plasma membranes by using a four-step procedure: solubilization with N-lauroylsarcosine; Ultrogel AcA-34 chromatography; CM Affi-Gel blue chromatography; agarose-soybean trypsin inhibitor chromatography. This enzyme was found to hydrolyze casein and various chromogenic peptide substrates; highest activity occurred with H-D-Val-Leu-Arg-p-nitroanilide, reported to be a specific substrate for human glandular kallikreins. The enzyme was heat-sensitive, showed a pH optimum between 8.0 and 9.0 and was inhibited by D-Phe-L-Phe-L-Arg-CH2Cl, aprotinin, diisopropyl fluorophosphate (DFP), soybean trypsin inhibitor, phenylmethylsulphonyl fluoride, leupeptin, antipain and dithiothreitol. This liver plasma membrane proteinase has an apparent molecular weight of about 30 000 as determined by Ultrogel AcA-34 chromatography and by autoradiography of [3H]DFP-labelled protein electrophoresis.     

Purification of rat-liver microsomal UDP-glucuronyltransferase. Separation of two enzyme forms inducible by 3-methylcholanthrene or phenobarbital.

Glucuronidation reactions catalysed by rat liver microsomal UDP-glucuronyltransferase are differentially inducible by 3-methylcholanthrene and phenobarbital. To elucidate the molecular basis of this functional heterogeneity the enzyme was purified from livers of rats pretreated with the inducing agents. Using cholate solubilization, chromatography on Bio-Gel A-1.5m and on DEAE-cellulose in the presence of the nonionic detergent Brij 58, two enzyme forms could be separated. Both forms were subsequently purified to apparent homogeneity by affinity chromatography on UDP-hexanolamine Sepharose 4B, 3-Methylcholanthrene-inducible enzyme activity towards 1-naphthol, 4-nitrophenol, 3-hydroxybenzo(a)pyrene and N-hydroxy-2-naphthylamine copurified with one enzyme form (enzyme 1). In contrast phenobarbital-inducible enzyme activity towards morphine, chloramphenicol and 4-hydroxybiphenyl was associated with the other enzyme fraction (enzyme 2). Sodium dodecylsulfate/polyacrylamide gels showed similar molecular weights of 54000 for enzyme 1 and 56000 for enzyme 2. The results suggest the presence of at least two forms of UDP-glucuronyltransferase in rat liver. Factors affecting enzyme activity in purified and membrane-bound states are discussed.