Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

Purification and characterization of 7 beta-hydroxysteroid dehydrogenase from Ruminococcus sp. of human intestine

7 beta-Hydroxysteroid dehydrogenase (7 beta-HSD) was produced by Ruminococcus sp. PO1-3 obtained from among human intestinal bacteria. The enzyme was purified from a crude extract by ammonium sulfate fractionation, and Butyl-Toyopearl 650M, Sephadex G-150, Matrex Red A and Octyl-Sepharose chromatographies. The purified enzyme was obtained as a single band on polyacrylamide gel electrophoresis with enzyme activity staining and as one band corresponding to a molecular weight of 30,000 on SDS-polyacrylamide gel electrophoresis. On gel filtration, its apparent molecular weight was estimated to be 60,000. The enzyme had a sulfhydryl group(s) in its active site. Substrate specificity studies revealed that the enzyme showed absolute specificity for the beta-configuration of a hydroxyl group at the 7 position of bile acids, and required NADP+ and NADPH as cosubstrates. The Km values for ursodeoxycholic acid, 7-k etolithocholic acid, NADP+, and NADPH were 5.0, 8.5, 7.7, and 24 microM, respectively.     

Inactivation of soluble 17 beta-hydroxysteroid dehydrogenase of human placenta by fatty acids

The sensitivity of soluble, 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) of human placenta to inactivation by fatty acids was examined. Exposure to the unsaturated fatty acids oleic, arachidonic, linoleic and linolenic acid resulted in the loss of activity. Methyl and ethyl esters of oleic acid, the saturated fatty acid, stearic acid and prostaglandins E2 and F2 alpha were without effect. Inactivation by oleic acid required the fatty acid at levels above its critical micelle concentration, 50 microM, as estimated by light-scattering. Steroid substrates and inhibitors did not protect against inactivation. NAD+, NADH, NADP+ and NADPH did protect. The concentrations of NADP+, 50 microM, and NAD, 1.5 mM, necessary for complete protection were significantly greater than their respective Michaelis constants, 0.16 microM and 15.2 microM. The data suggest that soluble 17 beta-HSD can bind to fatty acid micelles and that the binding site(s) on the enzyme are at or near pyridine nucleotide binding sites.     

Isolation of novel microbial 3 alpha-, 3 beta-, and 17 beta-hydroxysteroid dehydrogenases. Purification, characterization, and analytical applications of a 17 beta-hydroxysteroid dehydrogenase from an Alcaligenes sp

By selecting for growth on testosterone or estradiol-17 beta as the only source of organic carbon, we have isolated a number of soil microorganisms which contain highly active and novel, inducible, NAD-linked 3 alpha-, 3 beta-, and 17 beta-hydroxysteroid dehydrogenases. Such enzymes are suitable for the microanalysis of steroids and of steroid-transforming enzymes, as well as for performing stereoselective oxidations and reductions of steroids. Of particular interest among these organisms is a new species of Alcaligenes containing 17 beta-hydroxysteroid dehydrogenase, easily separable from 3 beta-hydroxysteroid dehydrogenase. Unlike any of the other isolated organisms, this Alcaligenes sp. contained no 3 alpha-hydroxysteroid dehydrogenase activity. A large-scale purification (763-fold) to homogeneity of the major induced 17 beta-hydroxysteroid dehydrogenase was achieved by ion-exchange, hydrophobic, and affinity chromatographies. The enzyme has high specific activity for the oxidation of testosterone (Vmax = 303 mumol/min/mg of protein; Km = 3.6 microM) and reacts almost equally well with estradiol-17 beta (Vmax = 356 mumol/min/mg; Km = 6.4 microM). It consists of apparently identical subunits (Mr = 32,000) and exists in polymeric form under nondenaturing conditions (Mr = 68,000 by gel filtration and 86,000 by polyacrylamide gel electrophoresis). The isoelectric point is pH 5.1. The enzyme is almost completely specific for 17 beta-hydroxysteroids which may be delta 5-olefins or ring A phenols or have cis or trans A/B ring fusions. Substituents at other positions are tolerated, although the presence of a 16 alpha- or 16 beta-hydroxyl group blocks the oxidation of the 17 beta-hydroxyl function. 3 beta-Hydroxysteroids (A/B ring fusion trans, but not cis, or delta 5-olefins) are very poor substrates. The application of this highly active, specific, and stable 17 beta-hydroxysteroid dehydrogenase to the microestimation of steroids by enzymatic cycling of nicotinamide nucleotides and for the stereospecific oxidation of steroids is demonstrated.     

Purification and characterization of 7beta-hydroxysteroid dehydrogenase from rabbit liver microsomes

7beta-Hydroxysteroid dehydrogenase (7beta-HSD), a specific enzyme active in the metabolization of 7beta-hydroxycholesterol, was purified about 300-fold from male rabbit liver microsomes using ion exchange, hydroxylapatite, 2'5'ADP Sepharose 4B, and high-performance liquid chromatography on the basis of its catalytic activity. The specific activity of the purified enzyme was 276 nmol/min/mg protein. The molecular weight of the purified enzyme was 34,000. The preferred coenzyme was beta-NADP+. The optimum pH for oxidation was around 7.7 in potassium phosphate buffer, and 11.0 in glycine-NaOH buffer. The purified enzyme catalyzed the synthesis of not only 7beta-hydroxycholesterol but also corticosterone and hydrocortisone. Enzyme activities toward these three substrates accompanied all purification steps of 7beta-HSD. The amino acid sequence of the N-terminal of the purified enzyme showed that 7beta-HSD had sequence similarity to rabbit type I 11beta-hydroxysteroid dehydrogenase (11beta-HSD), indicating that 7beta-HSD may belong to the rabbit type I 11beta-HSD family and may play the same role in the metabolism of 11-hydroxysteroids and 7-hydroxysterols.     

Purification and characterization of leucine-specific aminopeptidase from the soluble fraction of human placenta

A soluble aminopeptidase distinct from two enzymes described previously was isolated from human placenta and some of its properties were investigated. The three aminopeptidases were separated by DEAE-cellulose chromatography. The newly found aminopeptidase exhibits specific hydrolysis of leucine derivatives among various synthetic substrates. However, a broad substrate specificity was observed toward some natural bioactive peptides.     

Purification and characterization of a soluble beta-fructofuranosidase from Daucus carota.

Soluble beta-fructofuranosidase with an intracellular location and an isoelectric point of 3.8 (isoenzyme I) was purified and characterized from dry seeds and seedlings of carrot (Daucus carota). The enzyme hydrolyzed sucrose with a Km of 5 mM and a broad pH optimum around 5.0. The purified protein, which was N-glycosylated with high-mannose-containing and high-xylose-containing complex glycans, eluted as a monomeric polypeptide with a molecular mass of 68,000 from a gel-filtration column. On SDS/PAGE, the protein separated in the presence of SDS and 2-mercaptoethanol into three polypeptides with molecular masses of 68, 43 and 25 kDa. The amount of the 68-kDa polypeptide was highest in dry seeds and decreased with increasing age of carrot seedlings. Amino acid sequence analysis and immunological studies showed that the 43-kDa and 25-kDa polypeptides were N-terminal and C-terminal proteolytic fragments of the 68-kDa polypeptide. A comparison of partial amino acid sequences of the soluble beta-fructofuranosidase with the complete sequence of carrot cell-wall beta-fructofuranosidase showed that their N-terminal sequences were different, whereas some of the internal tryptic peptide sequences were up to 70% identical.     

Purification and partial characterization of a soluble hemagglutinin from Yersinia pseudotuberculosis

A soluble hemagglutinin (HA) produced by Yersinia pseudotuberculosis strain Inoue, serotype 5b, was purified by ammonium sulfate precipitation, gel filtration on Sepharose CL-6B and high performance liquid chromatography on a DEAE-5PW anion-exchange column. The purified HA was a 14.5 kDa protein with an isoelectric point of 4.5. Amino acid analysis indicated that the HA consisted of 133 residues, corresponding to the molecular weight of 14,100. The amino acid sequence of N-terminal 38 amino acid residues showed no homology with that of several fimbrial proteins from Escherichia coli.     

Studies on the activity of 17 beta-hydroxysteroid dehydrogenase in human adipose tissue

The activity of 17 beta-hydroxysteroid dehydrogenase has been investigated in human subcutaneous adipose tissue. Using oestrone as substrate, oestradiol formation was linear with time and the concentration of protein in the tissue homogenate. The optimum pH was 8.0 and the Km for oestrone was 2.5 x 10(-6) M. With NADH, the production of oestradiol was about 30% of that with NADPH. Oestradiol was also a substrate for the enzyme although under the experimental conditions used reduction of oestrone appeared to be favoured in adipose tissue. In the presence of progesterone (31.8 x 10(-6) M) the Km for oestrone was increased fivefold.     

Purification and characterization of human neuropeptide Y from adrenal-medullary phaeochromocytoma tissue.

Human neuropeptide Y was isolated from acid extracts of adrenal-medullary phaeochromocytoma tissue. After (NH4)2SO4 fractionation, the neuropeptide Y-like immunoreactivity was purified from the resolubilized 80%-saturation-(NH4)2SO4 peptide-rich precipitate, by gel filtration, cation-exchange chromatography and reverse-phase high-pressure liquid chromatography. Amino acid analysis of the peptide revealed a composition almost identical with that of the pig peptide, the exception being the loss of one leucine residue and its replacement with methionine. Tryptic digestion of the peptide and subsequent amino acid analysis of the fragments further confirmed the identity of the peptide. Carboxypeptidase Y digestion of the (1-19)-peptide tryptic fragment has shown the methionine to be located at position 17 in human neuropeptide Y.     

Characterization of a glucuronyltransferase: neolactotetraosylceramide glucuronyltransferase from rat brain

The properties of a rat brain glucuronyltransferase, which is presumed to be associated with the biosynthesis of the HNK-1 epitope on sulfoglucuronyl glycolipids, are described. The enzyme required divalent cations for reaction, with maximal activity at 10mm Mn²⁺, and exhibited a dual optimum at pH 4–5 and pH 6 depending upon the buffer used, with the highest activity at pH 4.5 in MES buffer. This enzyme strictly recognized the Gal1-4GlcNAc terminal structure, and was highly specific for neolacto (type 2) glycolipids as acceptor. The enzyme was localized specifically in the brain, and was barely detected in other issues, including the thymus, spleen, liver, kidney, lung, and sciatic nerve fibres. Phosphatidylinositol and phosphatidylserine increased the enzymatic reaction 4.4- and 2.3-fold, respectively, whereas phosphatidylcholine slightly decreased the rate.Abbreviations GlcA glucuronic acid - Lc-PA₁₄ lactotetraose-phenyl-C₁₄H₂₉ - nLc-PA₁₄ neolactotetraose-phenyl-C₁₄H₂₉ - nLcOse₄-Cer neolactotetraosylceramide - NP-40 Nonidet P-40 - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SGGL sulfoglucuronyl glycolipid     

Purification and partial characterization of plasminogen activator from human uterine tissue.

A procedure was developed for the purification of a plasminogen activator from human uterine tissue. It involves six consecutive steps: (1) extraction of the plasminogen activator from delipidated uterine tissue with 0.3 M potassium acetate buffer, pH 4.2; (2) ammonium sulphate precipitation; (3) zinc chelate-agarose chromatography; (4) n-butyl-agarose chromatography; (5) concanavalin A-agarose chromatography; and (6) gel filtration on Sephadex G-150. The specific activity of the final plasminogen activator preparation was increased by a factor 4500 as compared with the crude extract. The purified plasminogen activator showed a strong tendency to adsorb to surfaces. This could be effectively prevented by Tween-80. The molecular weight of the plasminogen activator was 64 000 as estimated by gel filtration in 1.0 M NaCl and 69 000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The plasminogen activator consisted of two chains (molecular weights 31 000 and 38 000) connected by disulphide bridges. The smallest chain contained the serine residue of the active site as deduced from the incorporation of the tritium label of [3H]diisopropylphosphofluoridate.     

Purification and characterization of a soluble polyphosphatase from mitochondria of Saccharomyces cerevisiae

A polyphosphatase with the specific activity 2.2 U/mg was purified to apparent homogeneity from a soluble preparation of mitochondria of Saccharomyces cerevisiae. The polyphosphatase is a monomeric protein of approximately 41 kD. The purified enzyme hydrolyzes polyphosphates with an average chain length of 9 to 208 phosphate residues to the same extent, but its activity is approximately 2-fold higher with tripolyphosphate. ATP, PPi, and p-nitrophenyl phosphate are not substrates of this enzyme. The apparent Km values are 300, 18, and 0.25 microM obtained at hydrolysis of polyphosphates with a chain length of 3, 15, and 188 phosphate residues, respectively. Several divalent cations stimulated the enzyme activity 1.2-27-fold (Mg2+ = Co2+ = Mn2+ > Zn2+). Determination of the protein N-terminal sequence and its comparison with the EMBL data library indicates that the soluble polyphosphatase of mitochondria of S. cerevisiae is not encoded by the gene of the major yeast polyphosphatase PPX1.     

Purification and characterization of recombinant human soluble guanylate cyclase produced from baculovirus-infected insect cells

Soluble guanylate cyclase (sGC) has been purified from 100 L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni²⁺-NTA, and POROS Q columns obtained approximately 100 mg of protein. The sGC obtained by this procedure was already about 90% pure and suitable for various studies which include high throughput screening (HTS) and hit follow-up. However, in order to obtain enzyme of greater homogeneity and purity for crystallographic and high precision spectroscopic and kinetic studies of sGC with select stimulators, the sGC solution after the POROS Q step was further purified by GTP-agarose affinity chromatography. This additional step led to the generation of 26 mg of enzyme that was about 99% pure. This highly pure and active enzyme exhibited a M_r = 144,933 by static light scattering supportive of a dimeric structure. It migrated as a two-band protein, each of equal intensity, on SDS–PAGE corresponding to the α (M_r 77,000) and β (M_r 70,000) sGC subunits. It showed an A₄₃₀/A₂₈₀ = 1.01, indicating one heme per heterodimer, and a maximum of the Soret band at 430 nm indicative of a penta-coordinated ferrous heme with a histidine as the axial ligand. The Soret band shifted to 398 nm in the presence of an NO donor as expected for the formation of a penta-coordinated nitrosyl-heme complex. Non-stimulated sGC had k_cat/K_m = 1.7 × 10⁻³ s⁻¹ μM⁻¹ that increased to 5.8 × 10⁻¹ s⁻¹ μM⁻¹ upon stimulation with an NO donor which represents a 340-fold increase due to stimulation. The novel combination of using the TIPS method for co-expression of a heterodimeric heme-containing enzyme, along with the application of a reproducible ligand affinity purification method, has enabled us to obtain recombinant human sGC of both the quality and quantity needed to study structure–function relationships.     

Activation of 17beta-hydroxysteroid dehydrogenase from human red blood cells

Experiments designed to elucidate the nature of 17β-hydroxysteroid dehydrogenase from human red blood cells have shown that NADP⁺ activates and protects the enzyme, while also serving as substrate for the reaction. Enzyme activity was measured by the conversion of 17β-estradiol to estrone and by the production of NADPH with 17β-estradiol-3-sulfate as substrate. It appears that the reaction sequence is first, binding with NADP⁺ and second, binding with the steroid. The binding with NADP⁺ is essentially irreversible: the activated enzyme is completely protected against loss of activity by dilution. On dilution of the unactivated enzyme, much of the activity is lost. The bireactant rate equation of the sequential type has been restated for the case of activation by one of the reactants. Since it has been found that activation of enzyme is linear with NADP⁺ concentration, it follows that the Michaelis constant for the steroid substrate is independent of the concentration of NADP⁺ activating the enzyme. This is substantiated by the determination of the Michaelis constant for 17β-estradiol-3-sulfate from data on double-reciprocal plots of activated and unactivated enzyme with limiting amounts of steroid. The activating effect increases linearly up to a concentration of 1.2 × 10^?5m of NADP⁺ and then levels off. The activation is highly specific for NADP⁺; neither NAD⁺, ATP, NADPH, nicotinic acid, ncr nicotinamide prevent the loss of activity after storing the enzyme for 1 hr at 37 °C. The steroid substrate appears to interfere with the activation of NADP⁺.     

Purification and characterization of a ribonuclease from human liver

The major ribonuclease of human liver has been isolated in a four-step procedure. The protein appears homogeneous by several criteria. The amino acid composition and the amino-terminal sequence of the enzyme indicate that the protein is related to human pancreatic ribonuclease and to angiogenin, and that it may be identical with an eosinophil-derived neurotoxin and to a ribonuclease that has been isolated from urine. The catalytic activity of the liver ribonuclease and its sensitivity to iodoacetic acid inactivation also relate the enzyme to the pancreatic RNases, but the liver protein is clearly differentiated by immunological measurements. Antibodies to the liver ribonuclease inhibit its activity, but not that of the human pancreatic enzyme; cross-reactivity in a radioimmunological assay is small but measurable. Immunochemical measurements have been used to examine the distribution of the liver-type protein in other tissues. Inhibition of enzyme activity by anti-liver ribonuclease shows that a cross-reactive enzyme is predominant in extracts of spleen and is a significant component in kidney preparations, while the liver-type protein is almost absent in brain or pancreas homogenates. Cross-reactive ribonuclease is present in serum, but levels are not correlated with any of the disease states examined.