Human liver steroid sulphotransferase sulphates bile acids.

The sulphation of bile acids is an important pathway for the detoxification and elimination of bile acids during cholestatic liver disease. A dehydroepiandrosterone (DHEA) sulphotransferase has been purified from male and female human liver cytosol using DEAE-Sepharose CL-6B and adenosine 3',5'-diphosphate-agarose affinity chromatography [Falany, Vazquez & Kalb (1989) Biochem. J. 260, 641-646]. Results in the present paper show that the DHEA sulphotransferase, purified to homogeneity, is also reactive towards bile acids, including lithocholic acid and 6-hydroxylated bile acids, as well as 3-hydroxylated short-chain bile acids. The highest activity towards bile acids was observed with lithocholic acid (54.3 +/- 3.6 nmol/min per mg of protein); of the substrates tested, the lowest activity was detected with hyodeoxycholic acid (4.2 +/- 0.01 nmol/min per mg of protein). The apparent Km values for the enzyme are 1.5 +/- 0.31 microM for lithocholic acid and 4.2 +/- 0.73 microM for taurolithocholic acid. Lithocholic acid also competitively inhibits DHEA sulphation by the purified sulphotransferase (Ki 1.4 microM). No evidence was found for the formation of bile acid sulphates by sulphotransferases different from the DHEA sulphotransferase during purification work. The above results suggest that a single steroid sulphotransferase with broad specificity encompassing neutral steroids and bile acids exists in human liver.     

Purification and structural analysis of the modulator of the glucocorticoid-receptor complex. Evidence that modulator is a novel phosphoglyceride

Modulator is the low molecular weight heat-stable inhibitor of glucocorticoid-receptor complex activation. We have purified modulator to apparent homogeneity from heated rat liver cytosol. This was accomplished using Sephadex G-15 gel filtration, Dowex 1 anion-exchange chromatography, and preparative silica high-performance liquid chromatography. The modulator preparation was judged to be homogeneous by analytical silica high-performance liquid chromatography, two-dimensional silica thin-layer chromatography, and proton nuclear magnetic resonance spectroscopy. The apparent concentration of modulator in rat liver cytosol is 6.5 microM. The purified modulator inhibits heat activation of the rat liver glucocorticoid-receptor complex and stabilizes the steroid binding ability of the unoccupied rat liver glucocorticoid receptor in a dose-dependent manner. At a concentration of 5-6.5 microM, modulator inhibits receptor activation and stabilizes the unoccupied receptor by 50%. At a concentration of 500-630 microM, sodium molybdate also inhibits receptor activation and stabilizes the unoccupied receptor by 50%. Thus, modulator appears to be the endogenous factor that exogenous sodium molybdate mimics in vitro. Chemical analysis of the purified modulator following two-dimensional silica thin-layer chromatography indicates that modulator is an aminophospholipid. Physical analysis of the purified modulator by infrared and nuclear magnetic resonance spectroscopy, as well as mass spectrometry, demonstrates that modulator is an ether aminophosphoglyceride.     

Purification of an insulin-sensitive cyclic AMP phosphodiesterase from rat liver

A low-Km cyclic nucleotide phosphodiesterase solubilised from rat liver membranes by mild proteolysis with chymotrypsin has been purified to apparent homogeneity. The purification included chromatography on cellulose phosphate, Ecteola-cellulose, hydroxyapatite, a theophylline affinity matrix and HPLC on a DEAE-substituted column. The purified enzyme has linear kinetic plots with a Km of 0.24 microM and a Vmax of 6.2 mumol mg-1 min-1 with cyclic AMP as a substrate. It also hydrolyses cyclic GMP with a Km of 0.17 microM and a Vmax which is about a third of that with cyclic AMP. Cyclic GMP is also a competitive inhibitor of cyclic AMP hydrolysis with a Ki of 0.18 microM. The proteolytically solubilised enzyme has a subunit molecular mass of 73 kDa by SDS gel electrophoresis and of 130 kDa by HPLC size-exclusion chromatography, suggesting that it exists as a dimer. A partially purified preparation of this enzyme was used to raise antiserum in a sheep. The antiserum immunoprecipitated activity from liver and adipose tissue of rat and mouse. It had little activity against phosphodiesterase from other rat tissues or other species. Insulin-activated phosphodiesterase from both adipocytes and hepatocytes was immunoprecipitated by the antiserum suggesting that the purified enzyme was an insulin-sensitive phosphodiesterase.     

2-aminophenol 1,6-dioxygenase: a novel aromatic ring cleavage enzyme purified from Pseudomonas pseudoalcaligenes JS45.

Most bacterial pathways for the degradation of aromatic compounds involve introduction of two hydroxyl groups either ortho or para to each other. Ring fission then occurs at the bond adjacent to one of the hydroxyl groups. In contrast, 2-aminophenol is cleaved to 2-aminomuconic acid semialdehyde in the nitrobenzene-degrading strain Pseudomonas pseudoalcaligenes JS45. To examine the relationship between this enzyme and other dioxygenases, 2-aminophenol 1,6-dioxygenase has been purified by ethanol precipitation, gel filtration, and ion exchange chromatography. The molecular mass determined by gel filtration was 140,000 Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two subunits of 35,000 and 39,000 Da, which suggested an alpha2beta2 subunit structure. Studies with inhibitors indicated that ferrous iron was the sole cofactor. The Km values for 2-aminophenol and oxygen were 4.2 and 710 microM, respectively. The enzyme catalyzed the oxidation of catechol, 6-amino-m-cresol, 2-amino-m-cresol, and 2-amino-4-chlorophenol. 3-Hydroxyanthranilate, protocatechuate, gentisate, and 3- and 4-methylcatechol were not substrates. The substrate range and the subunit structure are unique among those of the known ring cleavage dioxygenases.     

Purification and characterization of rat-liver cytosolic epoxide hydrolase

Rat liver cytosolic epoxide hydrolase has been purified and characterized. The enzyme was purified from tiadenol-induced rat liver 540-fold with respect to trans-stilbene oxide as a substrate. Similar purification was obtained with the substrates trans-beta-ethyl styrene oxide and styrene 7,8-oxide, the specific activities decreasing in the order trans-beta-ethyl styrene oxide greater than styrene 7,8-oxide greater than trans-stilbene oxide. The enzyme exerts highest activity at pH 7.4 Km and Vmax of the pure enzyme for trans-stilbene oxide were 1.7 microM and 205 nmol x min-1 x mg protein-1 respectively. With trans-stilbene oxide as a substrate, the inhibition by organic solvents (2.5% by vol.) increased in the order ethanol less than methanol less than acetone less than isopropanol = N,N-dimethyl formamide less than acetonitrile less than tetrahydrofuran. The native enzyme, with a molecular mass of 120 kDa, consists of two 61-kDa subunits. Digestion of rat liver cytosolic and microsomal epoxide hydrolase by three proteases resulted in markedly different peptide maps. Western-blot analysis with antiserum against rat liver cytosolic epoxide hydrolase revealed a single band with the purified enzyme, and with liver cytosol from control and clofibrate-induced rats. No cross-reactivity was observed with purified rat microsomal epoxide hydrolase or microsomes. A positive reaction at the same molecular mass was obtained with liver cytosol of mouse, guinea pig, Syrian hamster and New Zealand white rabbit but not with that of green monkey.     

Sulphoconjugation of steroids in porcine liver: Partial purification of the cytosolic sulphotransferases for pregnenolone and 5α-androst-16-en-3β-ol

Steroid sulphotransferase activities for 5α-androst-16-en-3β-ol and pregnenolone in porcine liver cytosol have been assayed using 3′-phosphoadenosine-5′-phospho[³⁵S]sulphate as sulphate donor. 5α-Androst-16-en-3β-ol sulphotransferase activity was obtained from porcine liver cytosol by gel filtration chromatography; activity was linear with time up to about 5 min., the optimum pH was near 8.0 and optimum temperature 37°C. Pregnenolone sulphotransferase activity was partially purified from porcine liver cytosol using DEAE-cellulose chromatography with an ionic gradient of KCl. This enzyme activity was linear with time up to 10 min and had optimum pH and temperature of 8.0 and 37°C, respectively.     

Isolation of similar rolipram-inhibitable cyclic-AMP-specific phosphodiesterases from rat brain and heart

A cyclic AMP phosphodiesterase form of rat brain cytosol was purified by means of affinity chromatography on an immobilized analog of the specific inhibitor rolipram, followed by an exclusion chromatography step. The resulting preparation presented two protein bands in polyacrylamide gel electrophoresis, both with phosphodiesterase activity. Kinetics of cyclic AMP hydrolysis by the purified enzyme proved of the Michaelis type, with a Km of 3 microM, while hydrolysis of cyclic GMP displayed anomalous negatively cooperative kinetics. At micromolar concentrations, this enzyme from hydrolyzed highly specifically cyclic AMP (50-fold faster than cyclic GMP). Cyclic GMP proved a poor competitor of cyclic AMP hydrolysis (Ki 1.04 mM). The neurotropic compound, rolipram, strongly inhibited the enzyme, in a competitive manner (Ki 0.9 microM). This enzyme displayed a molecular mass of around 44 kDa as determined by exclusion chromatography, but two molecular masses of 42 kDa and 89 kDa were observable by electrophoresis on a polyacrylamide gradient gel, compatible with an equilibrium between dimeric and monomeric forms. Isoelectric focusing of the preparation gave rise to two activity peaks of pI 4.8 and 6.7, with identical properties, probably representing two charge isomers of the same protein. An enzyme prepared from rat heart cytosol by the same techniques as for brain phosphodiesterase isolation shared numerous characteristics with the enzyme of cerebral origin, suggesting identity of the rolipram-sensitive form between the two tissues. Since the rolipram-sensitive form detected in crude brain preparations markedly differs from the above-described isolated enzyme, both by its molecular mass in exclusion chromatography and by its pI, it is suggested that an alteration of the native protein, due to dissociation of putative subunits, occurs during the purification procedure.     

Bacterial expression and characterization of a cDNA for human liver estrogen sulfotransferase

A distinct human estrogen sulfotransferase (hEST-1) cDNA has been isolated from a human liver γZap cDNA library using a PCR procedure. The enzymatically active protein has been expressed in two bacterial expression systems and the kinetic and immunologic properties of the enzyme have been characterized. The full-length cDNA for hEST-1 is 994 base pairs in length and encodes a 294 amino acid protein with a calculated molecular mass of 35,123 Da. Purified hEST-1 migrated with an apparent molecular mass of 35,000 Da during SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of hEST-1 expressed in E. coli with a rabbit anti-hEST-1 antibody yields a band of approximately 35,000 Da. The anti-hEST-1 antibody also detects a single band in human liver and jejunum cytosol which migrates with the same molecular mass as expressed hEST-1. There was also no cross-reactivity of hEST-1 with rabbit anti-hP-PST or rabbit anti-hDHEA-ST antibodies upon immunoblot analysis. hEST-1 was expressed in bacteria and purified to homogeneity. Expressed hEST-1 activity has a significantly greater affinity for estrogen sulfation than that found for the other human STs which conjugate estrogens. hEST-1 maximally sulfates β-estradiol and estrone at concentrations of 20 nM. hEST-1 also sulfates dehydroepiandrosterone, pregnenolone, ethinylestradiol, and 1-naphthol, at significantly higher concentrations; however, cortisol, testosterone and dopamine are not sulfated. The results presented in this paper describe the expression and characterization of a human EST distinct from other human STs which sulfate estrogens. The high affinity of hEST-1 for estrogens indicates that this ST may be important in both the metabolism of estrogens and in the regulation of their activities.     

Purification and characterization of the hydrogen uptake hydrogenase from the hyperthermophilic archaebacterium Pyrodictium brockii.

Pyrodictium brockii is a hyperthermophilic archaebacterium with an optimal growth temperature of 105 degrees C. P. brockii is also a chemolithotroph, requiring H2 and CO2 for growth. We have purified the hydrogen uptake hydrogenase from membranes of P. brockii by reactive red affinity chromatography and sucrose gradient centrifugation. The molecular mass of the holoenzyme was 118,000 +/- 19,000 Da in sucrose gradients. The holoenzyme consisted of two subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The large subunit had a molecular mass of 66,000 Da, and the small subunit had a molecular mass of 45,000 Da. Colorometric analysis of Fe and S content in reactive red-purified hydrogenase revealed 8.7 +/- 0.6 mol of Fe and 6.2 +/- 1.2 mol of S per mol of hydrogenase. Growth of cells in 63NiCl2 resulted in label incorporation into reactive red-purified hydrogenase. Growth of cells in 63NiCl2 resulted in label incorporation into reactive red-purified hydrogenase. Temperature stability studies indicated that the membrane-bound form of the enzyme was more stable than the solubilized purified form over a period of minutes with respect to temperature. However, the membranes were not able to protect the enzyme from thermal inactivation over a period of hours. The artificial electron acceptor specificity of the pure enzyme was similar to that of the membrane-bound form, but the purified enzyme was able to evolve H2 in the presence of reduced methyl viologen. The Km of membrane-bound hydrogenase for H2 was approximately 19 microM with methylene blue as the electron acceptor, whereas the purified enzyme had a higher Km value.     

Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells.

Purine nucleoside phosphorylase (PNP) was purified from rat hepatoma cells and normal liver tissue utilizing the techniques of ammonium sulfate fractionation, heat treatment, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. Homogeneity was established by disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Purified rat hepatoma and liver PNPs appeared to be identical with respect to subunit and native molecular weight, substrate specificity, heat stability, kinetics and antigenic identity. A native molecular weight of 84,000 was determined by gel filtration. A subunit molecular weight of 29,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point was observed at pH 5.8, and the pH optimum was 7.5. Inosine, guanosine, xanthosine, and 6-mercaptopurine riboside were substrates for the enzymes. The apparent K_m for both inosine and guanosine was about 1.0 × 10^?4m and for phosphate was 4.2 × 10^?4m. Hepatoma and liver PNP showed complete cross-reactivity using antiserum prepared against the liver enzyme.     

Multiple molecular forms of mouse liver arginase

Hepatic arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is an oligomer composed of three or four subunits. The present studies indicate heterogeneity in the size and charge of arginase subunits in mouse liver. Two types of arginase subunits with molecular weights of approximately 35,000 and 38,000 have been found. These two subunits are detected in liver cytosol or in purified preparations of arginase after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Two dimensional SDS-PAGE revealed multiple ionic forms of arginase for both the 35,000 and 38,000 subunits; the subunits contain basic proteins (pI range 7.8-9.1) and acidic proteins (pI range 5.8-6.4). Limited proteolysis by trypsin eliminated the molecular weight differences between the subunits without substantially affecting either their isoelectric points or activity. Comparative peptide maps and amino acid analyses of the 35,000- and 38,000-Da subunits showed that they were very similar. The data indicate that a neutral peptide (approx 3000 Da) is responsible for the differences in subunit molecular weight and that the multiple sized and charged forms are variants of the same protein.     

Lipid requirements for the aggregation of CTP:phosphocholine cytidylyltransferase in rat liver cytosol.

Two forms of CTP:phosphocholine cytidylyltransferase were identified in rat liver cytosol by gel filtration chromatography. The low molecular weight form (L form) is the major form in fresh cytosol. The enzyme associates into a high molecular weight form (H form) upon storage of the cytosol at 4 degrees C. Aggregation of the purified L form of cytidylyltransferase is caused by total rat liver lipids, neutral lipids, diacylglycerol, or phosphatidylglycerol. Diacylglycerol was the only lipid isolated from the rat liver that caused aggregation of the purified enzyme. Although the addition of diacylglycerol to the cytosol did not change the amount of aggregation of the enzyme, a 2.5-fold increase in H form was observed in cytosol pretreated with phospholipase C, or in cytosol from rats fed a high cholesterol diet. In both of these cytosolic preparations, the concentration of diacylglycerol was elevated twofold. Phosphatidylglycerol did not seem to affect the association of the enzyme in cytosol since it is present in very low concentrations in the rat liver cytosol, and its degradation in cytosol by a specific phospholipase did not affect the rate of aggregation. The results suggest that diacylglycerol in an appropriate form is required for association of cytidylyltransferase in rat liver cytosol.     

Purification and partial characterization of protein phosphatases from rat thymus

Protein phosphatases assayed with phosphorylase alpha are present in the soluble and particulate fractions of rat thymocytes. Phosphorylase phosphatase activity in the cytosol fraction was resolved by heparin-Sepharose chromatography into type-1 and type-2A enzymes. Similarities between thymocyte and muscle or liver protein phosphatase-1 included preferential dephosphorylation of the beta subunit of phosphorylase kinase, inhibition by inhibitor-2 and retention by heparin-Sepharose. Similarities between thymocyte and muscle or liver protein phosphatase-2A included specificity for the alpha subunit of phosphorylase kinase, insensitivity to the action of inhibitor-2, lack of retention by heparin-Sepharose and stimulation by polycationic macromolecules such as polybrene, protamine and histone H1. Protein phosphatase-1 from the cytosol fraction of thymocytes had an apparent molecular mass of 120 kDa as determined by gel filtration. The phosphatase-2A separated from the cytosol of thymocytes may correspond to phosphatase-2A0, since it was completely inactive (latent) in the absence of polycation and had activity only in the presence of polycations. The apparent molecular mass of phosphatase-2A0 from thymocytes was 240 kDa as determined by gel filtration. The catalytic subunit of thymocyte type-1 protein phosphatase was purified with heparin-Sepharose chromatography followed by gel filtration and fast protein liquid chromatography on Mono Q column. The purified type-1 catalytic subunit exhibited a specific activity of 8.2 U/mg and consisted of a single protein of 35 kDa as judged by SDS-gel electrophoresis. The catalytic subunit of type-2A phosphatase from thymocytes appearing in the heparin-Sepharose flow-through fraction was further purified on protamine-Sepharose, followed by gel filtration. The specific activity of the type-2A catalytic subunit was 2.1 U/mg and consisted of a major protein of 34.5 kDa, as revealed by SDS-gel electrophoresis.     

The Salmonella typhimurium nadC gene: sequence determination by use of Mud-P22 and purification of quinolinate phosphoribosyltransferase.

The Salmonella typhimurium nadC gene and its product, quinolinic acid phosphoribosyltransferase (QAPRTase), were characterized at the molecular and biochemical levels. Fusions of Mud-lac elements isolated in the nadC gene were converted to Mud-P22 insertions. Starting with six original Mud-lac fusions, the entire sequence of the nadC gene was readily obtained. The sequence shows a long open reading frame with two potential initiator methionines, one of which is preceded by the Shine-Dalgarno sequence GGAG-7-nucleotide-ATG. The protein predicted from this second open reading frame is 297 residues in length. The nadC gene was subcloned into a T7-based expression system, allowing for facile purification of the QAPRTase (EC 2.4.2.19) protein to homogeneity. Upon gel filtration, the protein gave an M(r) of 72,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a subunit M(r) of 35,000. Automated Edman degradation of several tryptic peptides confirmed the amino acid sequence predicted from the DNA sequence. Chromatography of the apparently homogeneous enzyme on reverse-phase high-performance liquid chromatography resolved two protein species. One of these species failed to give an amino-terminal sequence, while the other yielded the amino-terminal sequence predicted by the second open reading frame and lacked the initiator methionine. The mass of the mature protein, predicted from its DNA sequence, was 32,428 Da. Electrospray mass spectrometry gave masses of 32,501 and 32,581 Da for the two peptides. Steady-state kinetics on the purified QAPRTase indicated Km values of 32 microM for 5-phosphoribosyl-1-pyrophosphate and 20 microM for quinolinate. Vmax was 0.9 U/mg, similar to values reported for this enzyme by other sources.     

Immunochemical characterization of developmental changes in rat hepatic hydroxysteroid sulfotransferase.

A major isoenzyme of hepatic androsterone-sulfating sulfotransferase (AD-ST) was purified from adult female rats. The activity was purified 122-fold over that found in the cytosol and showed a single protein band with a subunit molecular mass of 30 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme exhibited four isoelectric variants of subunits on denaturing isoelectrofocusing gels (pI = 5.8, 6.1, 6.7 and 7.2). Rabbit antiserum raised against the enzyme specifically detected AD-ST polypeptide in rat liver cytosol. Immunoblot analysis of liver cytosol from female and male rats at various ages showed good correlation between the levels of AD-ST activity and AD-ST polypeptide. Significant levels of AD-ST activity and polypeptide were detected in senescent male rats, though normal adult male rats have very low levels of AD-ST activity and protein. The relative content of the isoelectric variants of AD-ST were different in liver cytosol of weanling and adult females, indicating that age- and gender-related alterations of hepatic AD-ST activity are primarily determined by the levels of AD-ST polypeptide and the relative amounts of the four isoelectric variants of the enzyme.     

Biosynthesis of all-trans-retinoic acid from retinal. Recognition of retinal bound to cellular retinol binding protein (type I) as substrate by a purified cytosolic dehydrogenase.

An NAD-dependent rat liver cytosolic dehydrogenase accepted as substrate retinal generated in situ by microsomes from retinol bound to excess CRBP (cellular retinol binding protein, type I). This activity, which was not retained by anion-exchange chromatography at pH 9.15, was designated P1. P1 activity increased 2.5-fold, with no statistically significant change in its K or Hill coefficient, in liver cytosol from rats fed a retinoid-deficient diet. Orally dosed retinoic acid partially suppressed the increase. Activities chromatographically similar to hepatic P1 were observed in cytosols from rat kidney and testes. P1, purified from rat liver cytosol, had a pI of approximately 8.3, migrated as a tetramer (214 kDa) on a Sephadex G-200 column, and had a subunit molecular mass of 55 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With free retinal it catalyzed a maximum rate of retinoic acid synthesis of 265 nmol/min/mg of protein and exhibited allosteric kinetics with a K of 0.76 +/- 0.35 microM and a Hill coefficient of 1.5 +/- 0.13 (mean +/- S.D., n = 4). Substrate inhibition was noted with retinal concentrations greater than 6 microM. The purified enzyme not only recognized retinal generated by microsomes as substrate, but also recognized retinal bound to CRBP. The rates of retinoic acid synthesis from CRBP-retinal, with a series of increasing apoCRBP concentrations, exceeded the rates that would be supported by the free retinal present. The CRBP-retinal complex exhibited allosteric kinetics (K, 0.13 microM; Hill coefficient, 1.75; averages of duplicates) in the presence of excess apoCRBP (the ratio total CRBP/total retinal at each concentration of retinal was 2). This enzyme is likely to play a significant role in retinoic acid synthesis in vivo, because it participates in the synthesis of retinoic acid from a physiologically occurring form of retinol (holoCRBP), reflects retinoid status, and is distributed in extrahepatic tissues in addition to liver. These results also suggest a novel role for CRBP in retinoid metabolism, facilitating the conversion of retinal into retinoic acid.     

A 27.368 kDa retinal reductase in New Zealand white rabbit liver cytosol encoded by the peroxisomal retinol dehydrogenase-reductase cDNA: purification and characterization of the enzyme.

We obtained a full-length cDNA based on a sequence deposited in GenBank (accession No. AB045133), annotated as rabbit peroxisomal NADP(H)-dependent retinol dehydrogenase-reductase (NDRD). The rabbit NDRD gene, like its mouse and human homologs, harbors 2 initiation sites, one of which theoretically encodes a 29.6 kDa protein with 279 amino acids, and the other encodes a 27.4 kDa protein with 260 amino acids. The purification of a rabbit cytosolic retinol oxidoreductase with a subunit molecular mass of 34 kDa and an N terminus that is not completely identical to that of NDRD, has been reported. An enzyme responsible for the all-trans retinal reductase activity in the liver cytosol of New Zealand white rabbit was purified to homogeneity using differential centrifugation and successive chromatographic analyses. The subunit molecular mass of the purified enzyme, revealed by SDS-PAGE, was approximately 27 kDa. The intact molecular mass, measured by MALDI-TOF mass spectrometry, was 27.368 kDa. The 60 kDa relative mobility observed in size-exclusion chromatography indicates that the native protein probably exists as a dimer. The purified enzyme was positively confirmed to be the product of NDRD by peptide mass fingerprinting, tandem mass spectrometry, and N-terminal sequencing. Taken together, the results suggested that the native protein is truncated at the N terminus.     

The 1-aminocyclopropane-1-carboxylate synthase of Cucurbita. Purification, properties, expression in Escherichia coli, and primary structure determination by DNA sequence analysis

The key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14). We have partially purified ACC synthase 6,000-fold from Cucurbita fruit tissue treated with indoleacetic acid + benzyladenine + aminooxyacetic acid + LiCl. The enzyme has a specific activity of 35,000 nmol/h/mg protein, a pH optimum of 9.5, an isoelectric point of 5.0, a Km of 17 microM with respect to S-adenosylmethionine, and is a dimer of two identical subunits of approximately 46,000 Da each. The subunit exists in vivo as a 55,000-Da species similar in size to the primary in vitro translation product. DNA sequence analysis of the cDNA clone pACC1 revealed that the coding region of the ACC synthase mRNA spans 493 amino acids corresponding to a 55,779-Da polypeptide; and expression of the coding sequence (pACC1) in Escherichia coli as a COOH terminus hybrid of beta-galactosidase or as a nonhybrid polypeptide catalyzed the conversion of S-adenosylmethionine to ACC (Sato, T., and Theologis, A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6621-6625). Immunoblotting experiments herein show that the molecular mass of the beta-galactosidase hybrid polypeptide is 170,000 Da, and the size of the largest nonhybrid polypeptide is 53,000 Da. The data suggest that the enzyme is post-translationally processed during protein purification.     

A specific glutathione S-transferase isozyme occurring in hereditary hyperbilirubinuria rats

A glutathione S-transferase isozyme which is absent in normal rat liver has been isolated from the hereditary hyperbilirubinuria rat liver cytosol. The enzyme was purified to apparent homogeneity by GSH-affinity chromatography and HPLC on CM-Sepharose CL-6B. It is a heterodimer of two non-identical subunits, i.e., subunit 2 and a previously uncharacterized subunit referred to here as subunit Yx. Immunoblot analysis indicated that GST 2-Yx belongs to the alpha class. GST 2-Yx is characterized by its 4-fold higher activity towards 4-hydroxy-non-2-enal, compared to that of GST 2-2.     

Betaine:homocysteine methyltransferase from rat liver: purification and inhibition by a boronic acid substrate analog.

Betaine:homocysteine methyltransferase (BHMT) from rat liver has been highly purified by an efficient procedure requiring only two chromatographic steps: Sephadex G-100 chromatography and fast protein liquid chromatography chromatofocusing. A 170-fold purification and 7.5% overall yield were achieved. Chromatofocusing yielded three active forms of BHMT with pI values near 8.0, 7.6, and 7.0. The subunit molecular weight of each active form is 45,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the native enzyme has a molecular weight of 270,000 as determined by exclusion chromatography. The stability of the purified enzyme was found to be potentiated by the presence of 1 mM dimethylglycine and 1 mM homocysteine. Boronate analogs of betaine (pinanyl N,N,N-trimethylaminomethaneboronate) (4) and dimethylglycine (pinanyl N,N-dimethylaminomethaneboronate) were synthesized from pinanyl iodomethaneboronate (3) and trimethylamine or dimethylamine, respectively. The free acid of the betaine analog (5) was reversibly generated from (4). The inhibition of BHMT by (5) appears competitive with a Ki = 45 microM. Since the Km for betaine measured with the purified enzyme is near 0.1 mM, the boronic acid analog of betaine appears to function effectively as a substrate analog inhibitor of BHMT. The analog does not appear to act as a methyl donor to homocysteine when (5) is substituted for betaine in the enzyme reaction. In addition, an enzyme assay based upon C3-cyano reverse phase HPLC detection of the o-phthalaldehyde derivative of methionine was developed as an alternative to the standard radiochemical assay. Betaine:homocysteine methyltransferase in the picomole range can be quantitated using this assay as indicated by a linear response of enzyme activity to protein concentration.