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.     

Beta-glucosidase activity towards a bile acid glucoside in human liver.

Human liver contains hydrolytic activity toward 3 beta-glucosido-chenodeoxycholic acid. This beta-glucosidase activity, localized predominantly in the microsomal fraction, was optimally active in the presence of divalent metal ions close to pH 5.0 and was inhibited by EDTA. Kinetic parameters and other catalytic properties of hydrolytic activity towards 3 beta-glucosido-chenodeoxycholic acid from human liver microsomes are described.     

Partial purification of human liver cytochrome P 450.

Human liver cytochrome P 450 was partially purified by hydrophobic chromatography on Octyl-Sepharose, followed by ion-exchange chromatography on DEAE-cellulose. Two fractions (A and B) were obtained; cytochrome P 450 of fraction A was purified sixfold, with an overall yield of about 6 %. Its spectral properties were similar to those previously described in animal cytochromes P 450. Moreover, p-nitroanisole-O-demethylase activity could be obtained in a reconstituted system involving cytochrome P 450 of fraction A, human NADPH-cytochrome $\text{c}$ reductase and phospholipids.     

Partial purification and some properties of gamma-glutamyl transpeptidase from human bile.

1. Gamma-Glutamyl transpepetidase ((5-glutamyl)-peptide: amino acid 5-glutamyltransferase, EC 2.3.2.2) from human bile has been partially purified using protamine sulphate treatment, DEAE-cellulose chromatography and Sephadex G-200 filtration. The procedure resulted in 150-fold increase in specific acitivity with a 37% yield. 2. The partially purified enzyme showed a single zone of enzyme activity by polyacrylamide gel electrophoresis and eluted in the inner volume of Sephadex G-200. 3. The enzyme had a pH optimum of 8.1 and Km of 1.52 mM using gamma-glutamyl p-nitroanilide as substrate. 4. The effects of cations and different gamma-glutamyl acceptors on the activity of the enzyme are reported. 5. As bile gamma-glutamyl transpeptidase appears to be soluble in the absence of detergents, it is suggested that bile may prove to be a useful source for further studies of the kinetic properties and physiological role of human gamma-glutamyl transpeptidase.     

Purification and characterization of human liver dehydroepiandrosterone sulphotransferase.

A BAL17 B lymphoma cell line bearing mu and delta chains on its surface behaves in a similar manner to normal mature B cells in terms of initial biochemical transmembrane signalling [Mizuguchi, Beaven, Ohara & Paul (1986) J. Immunol. 137, 2162-2167; Mizuguchi, Yong-Yong, Nakabayashi, Huang, Beaven, Chused & Paul (1987) J. Immunol. 139, 1054-1059]. Therefore the effects of protease inhibitors on increases in inositol phospholipid metabolism and intracellular free calcium concentration ([Ca2+]i) were examined. We show that the serine protease inhibitors Tos-Phe-CH2Cl (1-chloro-4-phenyl-3-L-tosylamidobutan-2-one-, TPCK) and Tos-Lys-CH2Cl (7-amino-1-chloro-3-L-tosylamidoheptan-2-one; TLCK) inhibit anti-IgM-mediated accumulation of inositol phosphates in a dose-dependent manner. InsP3 production induced by anti-IgM is also inhibited by pretreatment with Tos-Lys-CH2Cl or Tos-Phe-CH2Cl. Tos-Lys-CH2Cl- Tos-Phe-CH2Cl-mediated inhibition is not overcome by high concentrations of anti-IgM. Moreover, anti-IgM-mediated increases in [Ca2+]i are inhibited by pretreatment of the cells with these inhibitors. However, increases in inositol phospholipid metabolism caused by NaF, an activator of guanine-nucleotide-binding proteins (G-proteins), are approx. 10-fold more resistant to Tos-Lys-CH2Cl and Tos-Phe-CH2Cl inhibition compared with anti-IgM-induced changes. Furthermore, NaF-induced increases in [Ca2+]i are not inhibited by Tos-Lys-CH2Cl or Tos-Phe-CH2Cl pretreatment, suggesting that the inhibitors act at a step proximal to phospholipase C activation. The Tos-Lys-CH2Cl or Tos-Phe-CH2Cl treatment does not change the membrane IgM density as measured by flow cytometry, indicating that the active site of the inhibitors is distal to the membrane IgM molecule. These results indicate that serine proteases may be involved in coupling the receptor cross-linkage to G-protein.     

Identification and partial purification of a unique phenolic steroid sulphotransferase in rat liver cytosol.

Phenolic steroid sulphotransferase activity for both oestradiol and oestrone was identified in male rat liver cytosol in the 30 000-40 000 Mr fractions on gel filtration when activity was assayed at pH 5.5 (pH optimum 5.5-6.0). Activity for oestradiol but not oestrone was found in the 60 000-70 000-Mr range when assayed at pH 8.0 (pH optimum biphasic, 5.5-6.0 and 7.0-8.0). Km for oestradiol (1.3 microM) was lower than published values for hydroxysteroid sulphotransferases (15-35 microM) and previously reported oestradiol sulphotransferases (71-85 microM). At above 2 microM-oestradiol phenolic sulphotransferase activity exhibited substrate inhibition. The phenolic steroid sulphotransferase activity was found to be distinct in chromatofocusing from organic-anion-binding and bile acid-binding proteins previously identified in this Mr range. Further purification on hydroxyapatite yielded a 44-fold enriched fraction that contained two monomeric bands, Mr 32 500 and 29 500.     

Subcellular organization of bile acid amidation in human liver: a key issue in regulating the biosynthesis of bile salts

To extend our knowledge of how the synthesis of free bile acids and bile salts is regulated within the hepatocyte, bile acid-CoA:amino acid N-acyltransferase and bile acid-CoA thioesterase activities were measured in subcellular fractions of human liver homogenates. Some bile acids, both conjugated and unconjugated, have been reported to be natural ligands for the farnesoid X receptor (FXR), an orphan nuclear receptor. The conversion of [(14)C]choloyl-CoA and [(14)C]chenodeoxycholoyl-CoA into the corresponding tauro- and glyco-bile acids or the free bile acids was measured after high-pressure liquid radiochromatography. There was an enrichment of the N-acyltransferase in the cytosolic and the peroxisomal fraction. Bile acid-CoA thioesterase activities were enriched in the cytosolic, peroxisomal, and mitochondrial fractions. The highest amidation activities of both choloyl-CoA and chenodeoxycholoyl-CoA were found in the peroxisomal fraction (15-58 nmol/mg protein/min). The K(m) was higher for glycine than taurine both in cytosol and the peroxisomal fraction.These results show that the peroxisomal de novo synthesis of bile acids is rate limiting for peroxisomal amidation, and the microsomal bile acid-CoA synthetase is rate limiting for the cytosolic amidation. The peroxisomal location may explain the predominance of glyco-bile acids in human bile. Both a cytosolic and a peroxisomal bile acid-CoA thioesterase may influence the intracellular levels of free and conjugated bile acids.     

Partial purification of L-asparaginyl-tRNA synthetase from rabbit liver

Asparaginyl-tRNA synthetase activity of both a crude extract of an acetone powder of rabbit liver and also of material fractionated with ammonium sulphate eluted at the void volume when chromatographed on Sephadex G-100. Chromatography on DEAE-cellulose in presence of glycerol and mercaptoethanol led to considerable losses in activity, the loss being related to the precise conditions employed. These losses of activity were due to partial separation of two components, each with a molecular weight in the region of 35,000, required for full enzymatic activity leading to the production of asparaginyl-tRNA.     

Partial purification and biological activity of liver metallothionein

The hepatic metallothionein was isolated and partially purified from rats treated previously with stable Cl2Cd and Cl2Cd109. The livers were subjected to the extraction of proteins of low relative molecular weight by heat, ammonium sulphate, Sephadex G-25 and Sephadex G-75. The partially purified extracts were subjected to the pharmacological activity test on isolated mouse vas deferens. A specific effect on the norepinephrine synapsis was obtained through an increment of the answer to the electric stimulus. The mechanism of action could be explained by the increase of the calcium permeability through the membrane or by the increment of the norepinephrine presynaptic release.     

Partial purification and characterization of a recombinase from human cells

We describe the partial purification and characterization of a human recombinase activity from RPMI 1788 B lymphoblasts. Stoichiometric amounts of recombinase carry out a strand transfer reaction between linear duplex DNA and homologous circular single-strand DNA. The product of strand transfer by the recombinase is a joint molecule composed of a single-strand circle joined to one end of the linear duplex molecule by a region of DNA heteroduplex at least 150 bp long. Formation of DNA heteroduplexes is accompanied by strand displacement. Strand invasion initiates at the ends of the linear duplex. Finally, strand displacement by human recombinase exhibits polarity and proceeds in a 3' to 5' direction. This is the first demonstration of a strand transfer activity from a high eukaryote. We discuss similarities between our recombinase and the RecA and rec1 recombination proteins from E. coli and Ustilago maydis, respectively.     

Partial purification and characterization of human sperminogen

A recently recognized non-proacrosin zymogen referred to as sperminogen has been purified from human spermatozoa, and several of its properties have been determined. The purification procedure included acid extraction of washed ejaculated sperm at pH 3.0, followed by gel filtration of the solubilized extract over a Sephadex G-75 superfine column. The sperminogen eluted from the column in a single band that was completely separated from the proacrosin band. This separation was confirmed by a gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) zymograph. This zymograph also demonstrated that the final sperminogen preparation contained four forms of zymogen, with molecular weights between 32,000 and 36,000. At neutral pH, the sperminogen was converted into spermin, its enzymatically active form, yielding a sigmoidal curve typical of zymogen autoactivation. The effects of several factors on the rate of this autoconversion indicate specific differences between sperminogen and proacrosin. Spermin hydrolyzed N-alpha-benzoyl-L-arginine ethyl ester (BzArgOEt), and was inhibited by lima bean trypsin inhibitor, pancreatic trypsin inhibitor, N-acetyl-L-leucyl-L-leucyl-L-argininal (leupeptin), and tosyl-L-lysine chloromethyl ketone, indicating that the enzyme has a trypsin-like specificity and probably belongs to the class of trypsin-like enzymes. Since acrosin is generally believed to be the only trypsin-like enzyme in mammalian sperm, the demonstration of human sperminogen and spermin necessitates further inquiry into the functions and the relationships between sperm proteinase systems.     

Partial purification of human lymphocyte activating factor

Lymphocyte Activating Factor (LAF) is a T lymphocyte stimulant released by human monocytes cultured for 18-24 hours in tissue culture medium containing 5% human serum and the non-specific immunostimulant lipopolysaccharide. The purification of LAF is essentially the separation of low MW LAF (approximately 13,000) from the human serum proteins required for production of the activity. Hollow fiber ultrafiltration has been found to effect a rapid separation of low MW LAF from serum proteins, but with a yield of only 20% of the original activity. Isoelectric focusing (IEF) efficiently separates LAF from all traces of human serum, resulting in a purified sample containing no measurable protein and revealing no bands on polyacrylamide gels. The IEF purified material is about 2% of the low MW activity present in the unfractionated culture medium and is highly active in the biological assay system.