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.     

Purification, properties and regulation of urocaninase from rat liver]

Urocaninase (EC 4.2.1.4.9) from rat liver homogenate has been purified, using protein precipitation at pH 4,8, ammonium sulfate fractionation, gel-filtration through Sephadex G-200 and chromatography on DEAE-cellulose. Upon DEAE-cellulose chromatography urocaninase is separated from the proteins possessing the activity of 3',5'-AMP-dependent protein kinase. The purified enzyme becomes activated after addition of ATP and exogenous protein kinase or one of the fractions resulting from DEAE-cellulose chromatography. Using [gamma-32P]ATP, it has been shown that such activation is accompanied by incorporation of at least one phosphate residue into the enzyme molecule. The mol. weight of urocaninase as determined by gel-filtration is about 110 000. The Km value for urocanate is 15 . 10(-6) M, the isoelectric point lies at 5,6. The mechanism of regulation of the urocaninase activity in rat liver is discussed.     

Regulation of microsomal enzymes by phospholipids VI. Abnormal enzyme-lipid interactions in liver microsomes from gunn rats

The rates of synthesis of some glucuronides by liver microsomes from the Gunn strain of rat are abnormally low, but previous investigators of the activity of the p-nitrophenol metabolizing form of UDPglucuronyltransferase (UDPglucuronate glucuronyltransferase, EC 2.4.1.17) have reported normal levels of activity in these animals. Data presented in this paper indicate, however, that this enzyme is abnormal in Gunn rats. Thus, treatment of liver microsomes from normal Wistar rats with phospholipase A (EC 3.1.1.4) or Triton X-100 increases the activity of the p-nitrophenol metabolizing form of UDPglucuronyltransferase 10- and 20-fold, respectively, but these agents do not alter activity in microsomes from homozygous Gunn rats. Similarly, phospholipase A and Triton X-100 activate the o-aminophenol and o-aminobenzoate metabolizing forms of UDPglucuronyltransferase in microsomes from normal rats, but are without effect on the enzyme in microsomes from Gunn rats. In contrast, the rates of synthesis of o-aminophenyl- and o-aminobenzoylglucuronides are increased several fold by addition of diethylnitrosamine to microsomes from Gunn rats indicating that the maximum potential activities of UDPglucuronyltransferases are constrained in liver microsomes from both normal and Gunn rats.These data indicate that assays of UDPglucuronyltransferase in native microsomes are not sufficient for delineating the full extent of the defect in the Gunn rat, that there are defects in the function of at least two proteins in liver microsomes from these animals, and that there are abnormal interrelations between some forms of microsomal UDPglucuronyltransferase and their phospholipid environments.     

Reversible ATP-induced inactivation of branched-chain 2-oxo acid dehydrogenase.

1. Antiserum was raised against purified Wistar-rat liver UDP-glucuronyltransferase. 2. UDP-glucuronyltransferase activities towards 4-nitrophenol, bilirubin, 1-naphthol and morphine were co-immunoprecipitated from solubilized Wistar-rat liver preparations. 3. UDP-glucuronyltransferase activities towards 1-naphthol, 2-aminophenol and 4-nitrophenol were precipitated from solubilized Gunn-rat liver preparations by this antiserum. 4. UDP-glucuronyltransferase activities towards 1-naphthol, 4-nitrophenol and bilirubin, from Wistar-rat liver, were slightly inhibited by antiserum, whereas 1-naphthol UDP-glucuronyltransferase activity from Gunn-rat livers was greatly inhibited. 5. Measurable Wistar-rat liver glucuronyltransferase activities in washed immunoprecipitates indicate that the enzyme(s) were not merely inhibited by antiserum. 6. Immunoglobulin G purified from this antiserum immunoprecipitated transferase activities towards 4-nitrophenol, bilirubin and 1-naphthol. 7. The washed immunoprecipitates from both rat strains, containing UDP-glucuronyltransferase activity, appear to be similar when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 8. Radial-immunodiffusion studies suggest that a smaller amount of UDP-glucuronyltransferase protein is present in Gunn-rat liver than in Wistar-rat liver. 9. The significance of these results in relation to the genetic deficiency in the Gunn rat is discussed.     

Multiple forms of (1 → 4)-α-d-glucan, (1 → 4)-α-d-glucan-6- glycosyl transferase from developing zea mays L. Kernels

Two major forms of branching enzyme from developing kernels of maize have been detected after DEAE-cellulose chromatography. Branching-enzyme I, which contained 24% of the activity based on a phosphorylase-stimulation assay, but 74% of the activity based on the branching of amylose as monitored by change in spectra of the iodine-glucan complex, eluted with the column wash and was unassociated with starch-synthase activity. Branching-enzyme II was bound to DEAE-cellulose and was coeluted with both primed and unprimed starch-synthase activities. Both fractions were further purified by chromatography on aminoalkyl-Sepharose columns. Single peaks were observed for both fractions by gel filtration on BioGel A_1.5m columns and native molecular weights were estimated at 70,000–90,000 for both enzymes. Subunit molecular weights of branching-enzymes I and II were estimated by dodecyl sodium sulfate-gel electrophoresis at 89,000 and 80,000, respectively. Thus both enzymes are primarily monomeric. Branching-enzymes I and II could be distinguished by chromatography on DEAE-cellulose or 4-aminobutyl-Sepharose, and by disc-gel electrophoresis with activity staining. Branching-enyme I had a lower ratio of activity (phosphorylase stimulation-amylose branching; based on enzyme units). The ratio varied from 30–60 as compared to about 300–500 for branching-enzyme II. Likewise, branching-enzyme I had a lower K_m value for amylose than branching- enzyme II, the values being 160 and 500 μg/ml, respectively. Both enzymes could introduce further branches into amylopectin, as decreases in the overall absorption and wavelength maxima of the iodine complexes were observed. Combined action of the branching enzymes and rabbit-muscle phosphorylase a (12:1 ratio based on enzyme units) resulted in similar patterns of incorporation of d-glucose into the growing α-d-glucan and the synthesis of high molecular-weight polymers. However, the α-d-glucans differed, as shown by spectra of iodine complexes and average unit-chain length. Branching-enzyine II was separated into two fractions (IIa and IIb) by chromatography on 4-aminobutyl-Sepharose. These Fractions differed only in the branching of amylopectin, fractional IIb being more active than IIa.     

Purification and immunochemical characterization of monoamine oxidase from rat and human liver.

1. Monoamine oxidase from rat and human liver was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 2. The enzyme activity was extracted from mitochondrial preparations by Triton X-100. The enzyme was purified by (NH4)2SO4 fractionation followed by chromatography on DEAE-cellulose, Sepharose 6B, spheroidal hydroxyapatite, and finally chromatography on diazo-coupled tyramine-Sepharose. 3. Distinct differences occur in the chromatographic behaviour of the two enzymes on both DEAE-cellulose and spheroidal hydroxyapatite. 4. It is unlikely that the purification of the enzymes on tyramine-Sepharose is due to affinity chromatography and reasons for this are discussed. 5. The purified enzymes did not oxidize-5-hydroxytryptamine and the relative activities of the enzymes with benzylamine were increased approx. 1.25-fold compared with the enzyme activities of mitochondrial preparations. 6. Immunotitration of enzyme activity in extracts of mitochondrial preparations from rat liver was carried out with 5-hydroxytryptamine, tyramine and benzylamine. The enzyme activities were completely immunoprecipitated by the same volume of antiserum. Similar results were obtained with the antiserum to the enzyme from human liver.     

The identification and purification of a mammalian-like protein kinase C in the yeast Saccharomyces cerevisiae.

We have purified a yeast protein kinase that is phospholipid-dependent and activated by Diacylglycerol (DAG) in the presence of Ca2+ or by the tumour-promoting agent tetradecanoyl-phorbol acetate (TPA). The properties of this enzyme are similar to those of the mammalian protein kinase C (PKC). The enzyme was purified using chromatography on DEAE-cellulose followed by hydroxylapatite. The latter chromatography separated the activity to three distinguishable sub-species, analogous to the mammalian PKC isoenzymes. The fractions enriched in PKC activity contain proteins that specifically bind TPA, are specifically phosphorylated in the presence of DAG and recognized by anti-mammalian PKC antibodies.     

Purification and Properties of Acid Carboxypeptidase I from Aspergillus oryzae

To elucidate the constitution of peptidases from Aspergillus oryzae, systematic separation of the enzymes was carried out by batchwise treatment with Amberlite IRC-50 and precipitation with rivanol. Proteases were separated to two fractions. They were Amberlite IRC-50 adsorbed and the non-adsorbed fractions and the latter fraction was further separated to two fractions, rivanol precipitable and non-precipitable fractions.

Acid carboxypeptidase I was purified from the rivanol non-precipitable fraction by column chromatography on DEAE-cellulose, DEAE-Sephadex A-50 and SE-cellulose. The purified enzyme was not homogeneous on disc electrophoresis, although symmetric peaks were obtained for enzyme protein and activity in Sephadex gel filtration. The optimum pH is at pH 4.0 for carbobenzoxy-l-alanyl-l-glutamic acid. The enzyme activity was inhibited by SH reagents, but not inhibited by metal chelating agents. The molecular weight of the enzyme was estimated to be about 120,000 by gel filtration.     

Identification and Purification of Sulfotransferases for 20-Hydroxysteroid from the Larval Fat Body of a Fleshfly,Sarcophaga peregrina

Sulfotransferase (ST) activity for 20-hydroxyecdysone (20E) was identified in a larval fat body lysate of the fleshfly, Sarcophaga peregrina, but not in the hemolymph. The activity was highly sensitive to 2,6-dichloro-4-nitrophenol (DCNP) (IC₅₀=0.61 μM), a specific inhibitor of phenol ST (P-ST), but insensitive to triethylamine, a hydroxysteroid ST inhibitor. These results suggest that 20E-specific ST enzymes belong to the P-ST family, despite the fact that 20E is a hydroxysteroid. In addition to 20E ST activity, a relatively high level of 2-naphthol ST activity was detected in the fat body lysate. The ST activity for both substrates transiently decreased to the 50% of maximal levels, 6 hrs after induction of pupation. The ST enzymes were separated on a DEAE-cellulose column. The 20E-ST enzymes were eluted around 50 mM KCl as two separate peaks of close proximity and the P-ST was eluted at 0.1 M KCl. The 20E ST enzymes were further purified using 3′-phosphoadenosine 5′-phosphate (PAP)-agarose affinity column chromatography. Both of the eluted active fractions demonstrated 43-kDa proteins on SDS-polyacrylamide gel. Photoaffinity labeling with [³⁵S]-3′-phosphoadenosine 5′-phosphosulfate (PAPS) showed 43-kDa bands in the fat body lysate, as well as in the purified fractions. These results suggest that the 43-kDa proteins catalyze 20E sulfation within the fat body of S. peregrina.     

Purification and characterization of 1-nitropyrene nitroreductases from Bacteroides fragilis.

We isolated four nitroreductases from Bacteroides fragilis GAI0624 and examined their physicochemical and functional properties. Two major enzyme activities were found in the adsorbed and unadsorbed fractions from DEAE-cellulose column chromatography. The adsorbed fraction was subjected to Sephadex G-200 column chromatography, and two further activities were separated. One has high nitroreductase activity (nitroreductase I), and the other has low activity and relatively high molecular weight (nitroreductase III). The nitroreductase I fraction was subjected to hydroxylapatite and chromatofocusing column chromatography, and nitroreductase I was purified about 416-fold with a yield of 6.77%. The unadsorbed fraction from DEAE-cellulose column chromatography was subjected to Sepharose 2B and Sepharose 6B column chromatography. Two enzyme activities were obtained by the Sepharose 6B column chromatography. One has high activity (nitroreductase II), and the other has low activity (nitroreductase IV). Nitroreductase II was rechromatographed by Sepharose 6B gel filtration and purified about 178-fold with a yield of 9.65%. The four enzymes (nitroreductases I, II, III, and IV) were shown to be different by several criteria. Their molecular weights, determined by gel filtration, were 52,000, 320,000, 180,000, and 680,000, respectively. The substrate specificity, the effect on mutagenicity of mutagenic nitro compounds, of nitroreductases I, III, and IV was relatively high for 1-nitropyrene, dinitropyrenes, and 4-nitroquinoline 1-oxide, respectively, but nitroreductase II had broad specificity. Nitroreductase activity required a coenzyme; nitroreductases II, III, and IV were NADPH linked, but nitroreductase I was NADH linked. All enzyme activity was enhanced by addition of flavin mononucleotide and inhibited significantly by dicumarol, p-chloromercuribenzoic acid, o-iodosobenzoic acid, sodium azide, and Cu2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of 1-nitropyrene nitroreductases from Bacteroides fragilis

We isolated four nitroreductases from Bacteroides fragilis GAI0624 and examined their physicochemical and functional properties. Two major enzyme activities were found in the adsorbed and unadsorbed fractions from DEAE-cellulose column chromatography. The adsorbed fraction was subjected to Sephadex G-200 column chromatography, and two further activities were separated. One has high nitroreductase activity (nitroreductase I), and the other has low activity and relatively high molecular weight (nitroreductase III). The nitroreductase I fraction was subjected to hydroxylapatite and chromatofocusing column chromatography, and nitroreductase I was purified about 416-fold with a yield of 6.77%. The unadsorbed fraction from DEAE-cellulose column chromatography was subjected to Sepharose 2B and Sepharose 6B column chromatography. Two enzyme activities were obtained by the Sepharose 6B column chromatography. One has high activity (nitroreductase II), and the other has low activity (nitroreductase IV). Nitroreductase II was rechromatographed by Sepharose 6B gel filtration and purified about 178-fold with a yield of 9.65%. The four enzymes (nitroreductases I, II, III, and IV) were shown to be different by several criteria. Their molecular weights, determined by gel filtration, were 52,000, 320,000, 180,000, and 680,000, respectively. The substrate specificity, the effect on mutagenicity of mutagenic nitro compounds, of nitroreductases I, III, and IV was relatively high for 1-nitropyrene, dinitropyrenes, and 4-nitroquinoline 1-oxide, respectively, but nitroreductase II had broad specificity. Nitroreductase activity required a coenzyme; nitroreductases II, III, and IV were NADPH linked, but nitroreductase I was NADH linked. All enzyme activity was enhanced by addition of flavin mononucleotide and inhibited significantly by dicumarol, p-chloromercuribenzoic acid, o-iodosobenzoic acid, sodium azide, and Cu2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of cholesterol-esterifying enzymes from rat liver cytosol by high-performance liquid chromatography

Three enzymes esterifying cholesterol with long-chain fatty acids were purified approximately 31 000-fold to apparent homogeneity from the cytosol of normal rat liver. The enzymatic activity was tested by incubation of active fractions with tritiated cholesterol and separation of newly formed esters from non-reacted cholesterol by a passage through silica gel cartridges with subsequent assay for radioactivity by liquid scintillation. For the purification of enzymes, active proteins were precipitated by (NH₄)₂SO₄ to 35% saturation. The bulk of inactive proteins was removed by size-exclusion chromatography on TSK G3000 SW. The active fraction was subsequently separated on Separon HEMA BIO 1000 DEAE in gradients of 0–500 mM KCl into three enzymatic activities differing in their retention and these proteins were finally purified by affinity HPLC on columns of cholesterol immobilized on HEMA BIO 1000 E-H. Final purified enzymes showed the same single band in polyacrylamide gel electrophoresis corresponding to 16.5 kDa. Combination of individual enzymes did not increase the overall yield of cholesteryl esters but the reaction-rate was significantly accelerated. These proteins are apparently subunits of a larger complex (M_r 65 000) that can be demonstrated by electrophoresis in the absence of 2-mercaptoethanol. Results presented in this paper indicate that because of good and rapid separation of active proteins, HPLC may be a method of choice for enzyme purifications.     

Immunochemical characterization of the androgen-dependent spermine-binding protein of the rat ventral prostate.

A solid-phase radioimmunoassay was developed to measure the level of the androgen-dependent spermine-binding protein (SBP) in the cytosol fraction of the rat ventral prostate during endocrine manipulation. The concentration of SBP and immunologically cross-reacting material (CRM) in the ventral prostate was at least 5000 times higher than the level of CRM detected in rat serum or cytosol from other rat tissues. Cytosol from the ventral prostate of intact rats was separated by DEAE-cellulose chromatography into three major fractions of CRM. One of these fractions corresponded to the elution position of SBP. Cytosol prepared from rats 48 h after castration lacked SBP and one of the two other fractions of CRM. This loss coincided with an increase in CRM in the remaining fraction. No significant difference was detected in the total level of CRM when intact and 48 h-castrated rats were compared. Injection of rats with 5 alpha-dihydrotestosterone (DHT) immediately after castration prevented these changes in the profile of CRM. Several proteins cross-reacting with antibodies to purified SBP were detected in cytosol by using an immunoblot procedure. The highest-Mr band corresponded to SBP. The effect of short- and long-term castration and subsequent DHT treatment on CRM was studied by using the immunoblot technique. Short-term castration (2 days) led to the disappearance of CRM coinciding with SBP (Mr 35 000-38 000) and an increase in smaller forms of CRM (Mr 24 000 and 22 000). Injection of rats with DHT 2 days after castration led to the reappearance of CRM corresponding to SBP, which returned to normal levels within 4 to 5 days of treatment. Long-term castration (up to 14 days) led to a gradual disappearance of all CRM; subsequent DHT treatment led to the reappearance of all forms of CRM and normal levels were attained within 5 days. We have identified SBP and the various forms of CRM as a secretory product of the rat ventral prostate by immunohistochemical staining and by DEAE-cellulose fractionation of prostatic fluid. Prostatic fluid is rich in proteolytic activity and these proteinases may be responsible for processing SBP to small forms of CRM.     

Isolation of tissue kallikrein in rat spleen by monoclonal antibody-affinity chromatography

Rat spleen kallikrein was identified and purified by DEAE-cellulose and monoclonal antibody-affinity chromatography. The purified enzyme has Tos-Arg-OMe esterase activity and kinin-releasing activity from a purified low-molecular-weight kininogen substrate. In the direct radioimmunoassay for tissue kallikrein, the splenic enzyme displays parallelism with standard curves of rat urinary kallikrein. The pH profiles of the Tos-Arg-OMe esterase activities of spleen and urinary kallikrein were identical with optima at 9.0 Rat spleen kallikrein was inhibited strongly by aprotinin and affinity-purified kallikrein antibody and weakly by soybean trypsin inhibitor. The IC₅₀ values were similar to those observed against rat urinary kallikrein. Neither the urinary nor the splenic enzyme was inhibited by lima bean trypsin inhibitor or preimmune serum immunoglobulins. Spleen kallikrein was labeled with [¹⁴]diisopropylphosphorofluoridate and visualized by fluorography on a sodium dodecyl sulfate-polyacrylamide gel. The electrophoretic mobility of the splenic enzyme was indistinguishable from that of urinary kallikrein A with an estimated Mr of approx. 38 000. With Western blot analyses using a rabbit anti-kallikrein antibody followed by ¹²⁵I-labeled protein A binding, the spleen and urinary kallikreins were again visualized at identical positions by autoradiography. The data show that there is a rat splenic tissue kallikrein which is indistinguishable from a renal kallikrein with respect to physicochemical properties, immunological character and susceptibility to inhibitors.     

Partial purification and properties of frog liver tyrosine aminotransferase.

Hepatic tyrosine aminotransferase of the frog Rana temporaria was partially purified by (NH4)2SO4 fractionation and successive chromatography on DEAE-cellulose DE-52, Ultrogel AcA-34, DEAE-cellulose DE-52 again and, finally, hydroxyapatite. During the last step, the enzyme activity separated into two fractions; traces of a third fraction were also found. The major form was purified 6000-fold to a specific activity of 200 units/mg of protein; it was about 50% pure by electrophoretic criteria. It had mol.wt. about 85 000 as determined by gel filtration on a Sephadex G-100 column. It was not activated by added pyridoxal 5'-phosphate. The enzyme was, however, inactivated by the pyridoxal phosphate reactants canaline and amino-oxyacetate. The enzyme was specific for 2-oxoglutarate as the amino group acceptor. Homogentisate inhibited the enzyme and adrenaline was an activator; both effects were seen at low concentrations of the effectors. The relationship between initial rate and tyrosine or 2-oxoglutarate concentration was abnormal and complex. Form-2 enzyme had similar or identical molecular weight, cofactor requirements, oxo acid specificity and kinetics.     

Soluble starch synthases and starch branching enzymes from developing seeds of sorghum

Soluble starch synthases and branching enzymes have been partially purified from developing sorghum seeds. Two major fractions and one minor fraction of starch synthase were eluted on DEAE-cellulose chromatography. The minor enzyme eluted first and was similar to the early eluting major synthase in citrate-stimulated activity, faster reaction rates with glycogen primers than amylopectin primers, and in K_m for ADP-glucose (0.05 and 0.08 mM, respectively). The starch synthase peak eluted last had no citrate-stimulated activity, was equally active with glycogen and amylopectin primers, and had the highest K_m for ADP-glucose (0.10 mM). Four fractions of branching enzymes were recovered from DEAE-cellulose chromatography. One fraction eluted in the buffer wash; the other three co-eluted with the three starch synthases. All four fractions could branch amylose or amylopectin, and stimulated α-glucan synthesis catalysed by phosphorylase. Electrophoretic separation and activity staining for starch synthase of crude extracts and DEAE-cellulose fractions demonstrated complex banding patterns. The colour of the bands after iodine staining indicated that branching enzyme and starch synthase co-migrated during electrophoresis.     

Purification of Oidiodendron kalrai proteases.

Oidiodendron kalrai yeast-phase cells demonstrate proteolytic activity. Some of the proteolytic enzymes of the crude extract were purified by a combination of ammonium sulfate precipitation, Sephadex G-200, and diethylaminoethyl (DEAE) cellulose column chromatography. At least six proteins exhibiting a range of proteolytic activities could be identified by these procedures. Purity of the enzyme fractions obtained from the DEAE-cellulose columns was tested by running polyacrylamide gels.     

Association of triiodothyronine binding activity to soluble adenylate cyclase in testicular preparations

Summary Cytosolic adenylate cyclase activity from rat seminiferous tubules was purified by chromatography in DEAE-cellulose, hydroxylapatite and Bio-Gel A-0.5 m as well as by centrifugation in sucrose gradients. In all these purification steps, fractions with adenylate cyclase activity also contained binding activity for L-T₃. Binding studies indicate the existence of two L-T₃ receptor components associated to adenylate cyclase activity. The component exhibiting the highest hormone affinity has the lowest binding capacity.     

Molecular weight of starch synthetase from Oryza sativa leaves

Soluble ADP-glucose: α-1,4-glucan-4-glucosyltransferase with primed activity was extracted from rice leaves and purified by (NH₄)₂SO₄ fractionation, gradient elution on DEAE-cellulose and finally by Sephadex G200 gel filtration or amylopectin-cellulose chromatography. The purified enzyme was essentially homogeneous electrophoretically, but exhibited two peaks corresponding to MW of 22 000 and 67 000 on Sephadex G200 chromatography and five distinct bands on sodium dodecyl sulfate gel electrophoresis with MW of 11·5, 20, 35, 50 and 68 × 10³.     

Identification and purification of sulfotransferases for 20-hydroxysteroid from the larval fat body of a fleshfly,Sarcophaga peregrina

Sulfotransferase (ST) activity for 20-hydroxyecdysone (20E) was identified in a larval fat body lysate of the fleshfly, Sarcophaga peregrina, but not in the hemolymph. The activity was highly sensitive to 2,6-dichloro-4-nitrophenol (DCNP) (IC50=0.61 microM), a specific inhibitor of phenol ST (P-ST), but insensitive to triethylamine, a hydroxysteroid ST inhibitor. These results suggest that 20E-specific ST enzymes belong to the P-ST family, despite the fact that 20E is a hydroxysteroid. In addition to 20E ST activity, a relatively high level of 2-naphthol ST activity was detected in the fat body lysate. The ST activity for both substrates transiently decreased to the 50% of maximal levels, 6 hrs after induction of pupation. The ST enzymes were separated on a DEAE-cellulose column. The 20E-ST enzymes were eluted around 50 mM KCl as two separate peaks of close proximity and the P-ST was eluted at 0.1 M KCl. The 20E ST enzymes were further purified using 3'-phosphoadenosine 5'-phosphate (PAP)-agarose affinity column chromatography. Both of the eluted active fractions demonstrated 43-kDa proteins on SDS-polyacrylamide gel. Photoaffinity labeling with [35S]-3'-phosphoadenosine 5'-phosphosulfate (PAPS) showed 43-kDa bands in the fat body lysate, as well as in the purified fractions. These results suggest that the 43-kDa proteins catalyze 20E sulfation within the fat body of S. peregrina.