Inhibition of human placental sterylsulfatase by synthetic analogs of estrone sulfate

Synthetic analogs of estrone sulfate which carry differently substituted sulfonyl groups at position 3 of an invariable 3-desoxyestrone (dE1) moiety were tested in vitro as inhibitors of the human placental sterylsulfatase. Using both placental microsomes and a highly purified placental sterylsulfatase preparation as the enzyme source and dehydroepiandrosterone [³⁵S]sulfate or estrone [³⁵]sulfate as the substrate, the following order of inhibitory potencies was observed: dE1–3-sulfonylchloride >dE1–3-sulfonylfluoride≈dE1–3-sulfonate>dE1–3-sulfonamide≈3-methylsulfonyl-dE1. According to the results, the association of enzyme and inhibitor appears to be favored by an electronegative substituent at the sulfur atom (-Cl, -F, -O⁻). Since, however, even the most potent synthetic inhibitor was bound by the enzyme with significantly lower affinity than was the natural substrate estrone sulfate, an oxygen function between the aromatic ring and the sulfur atom may be necessary for high affinity binding towards the sterylsulfatase. In addition to its fast reversible association with the enzyme, dE1–3-sulfonylchloride further affected the sulfatase activity in a time-dependent manner. This latter inhibitory activity which may be due to a covalent modification (alkylation) of sterylsulfatase by the analog was partially prevented in the presence of substrate.     

Isolation, characterization and N-terminal amino-acid sequence of rabbit transferrin

Rabbit serum transferrin has been isolated and purified by ion-exchange column and high-performance liquid chromatography. The N-terminal amino-acid sequence of 32 residues was determined by automatic Edman degradation in a liquid phase sequenator. Of the first twelve residues sequenced previously three identifications were corrected. Comparison with the known transferrin sequences shows 15 common amino-acid residues. Comparison to human serum transferrin revealed that 37% of amino-acid residues were exchanged. Cys9 and Cys19 which are supposed to be involved in disulphide bridges, are conserved.     

N-terminal amino-acid sequence of a sphingolipid activator protein missing in a new human Gaucher disease variant

A naturally occurring non-enzymic sphingolipid activator protein (A1a activator) shown previously to be immunochemically not detectable in a new variant of human Gaucher disease (glucosylceramide-lipidosis) without glucosylceramidase deficiency was characterized by partial sequence analysis. The N-terminal amino-acid sequence of the A1a activator--a glycoprotein with high carbohydrate content--could be determined up to position 38. About 20% of the polypeptide chain are shorter by two amino-acid residues at the N-terminal end. Position 22 seems to be occupied by a carbohydrate-binding asparagine. The N-terminus of the A1a activator does not show any homology with the activator for the enzymic sulfatide degradation.     

Human placental sterylsulfatase: immunocytochemical and biochemical localization

Human placental sterylsulfatase was localised in situ by light and electron microscope immunocytochemical techniques as well as in homogenate and tissue extract fractions by enzyme assays. Light microscope observations on frozen sections of term and preterm placenta revealed sterylsulfatase immunoactivity primarily in the syncytiotrophoblast. Electron microscope observations confirmed the light microscope findings; in addition, they showed that the sulfatase is present in the endoplasmic reticulum of endothelial cells, too. In the syncytiotrophoblast, the enzyme was detectable in the cytoplasmic membrane of the nuclear evelope, in the membranes of the rough endoplasmic reticulum, in the plasma membrane with predominant localisation in coated pits, and in the membranes of endosomes and multivesicular bodies; little or no reactivity was detectable over the membranes of the Golgi complex and of lysosomes. Sterylsulfatase immunoactivity was absent in placentas with hereditary sterylsulfatase deficiency. The observations indicate that human placental sterylsulfatase is normally present in the membranes of compartments along the secretory pathway and the endocytic route of cells lining the fetal and maternal blood. Homogenates of normal term placenta as well as membrane vesicle preparations obtained by extraction of trophoblast tissue with isotonic saline were fractionated by differential centrifugation; the fractions were assayed for specific activities of sterylsulfatase and several marker enzymes of cellular topography. In agreement with our immunocytochemical findings, the results of these biochemical localisation experiments indicate the repeatedly described association of the placental sterylsulfatase with microsomal membranes but also point to the presence of the enzyme's activity in the microvillous plasma membrane of the syncytiotrophoblast. This localisation of sterylsulfatase may have functional implications in the placental uptake of circulating steroid sulfates.     

Purification and N-terminal amino-acid sequence analysis of rat polymorphonuclear leukocyte cathepsin G

Cathepsin G was purified by single-step cation-exchange chromatography from rat polymorphonuclear leukocytes, obtained from the peritoneal cavity after induction of a mild peritonitis. The 26 N-terminal amino acids were determined and showed 73% identity to those of human cathepsin G. Total amino-acid composition demonstrated a high degree of basic amino acids in accordance with its high affinity for the cationic-exchange gel medium. The protein was found to be a glycoprotein with a glucosamine content of 7.4% of the calculated Mr28,900. On SDS/polyacrylamide-gel electrophoresis the protein showed a Mr of 28,400. It migrated as two bands in a gradient SDS/polyacrylamide-gel indicating isoforms. The pH optimum for the proteinase was determined to be 8.0-8.5 using Suc-Ala-Ala-Pro-Phe-Nan as substrate (Suc = 3-carboxypropionyl; Nan = 4-nitroanilide). Km and Kcat/Km values for Suc-Ala-Ala-Pro-Phe-Nan were 0.86mM and 280M-1S-1 and for Suc-Phe-Leu-Phe-Nan 0.24mM and 3600M-1S-1, respectively.     

The primary structure of mu-chain-disease protein BOT. Peculiar amino-acid sequence of the N-terminal 42 positions

The complete primary structure of the mu heavy-chain disease (mu-HCD) protein BOT has been determined. The monomeric HCD-mu-chain consists of 391 amino-acid residues, lacking the VH and mu CH1 domains but including the entire CH2, CH3 and CH4 domains (349 residues). The sequence of the preceding 42 N-terminal residues which we designate as the "pre-C-part" presents no homology to any known variable or constant immunoglobulin sequence, but contains an internal homology of positions 10-19 to positions 20-29. The origin of the "pre-C-part" structure and the deletion of the mu CH1 domain of protein BOT are discussed.     

N-terminal amino-acid sequence of pig kidney dipeptidyl peptidase IV solubilized by autolysis.

The N-terminal amino-acid sequence of the intrinsic membrane protein dipeptidyl peptidase IV (DP IV) was determined. The protein was isolated from pig kidney and solubilized by autolysis at pH 3.8. The first 34 amino acids were sequenced and indicated approximately 78% identity to the N-terminal sequence of rat liver DP IV.     

N-terminal sequence analysis of human placental protein 14, purified in high yield from decidual cytosol

Human placental protein 14 (PP14) has been purified in high yield from first trimester decidual cytosol. High-performance liquid chromatography on anion exchange, gel filtration and reverse-phase chromatography were used. The protein obtained is approximately 97% pure with an overall recovery of about 50% from the original tissue extract. The first 24 amino acids of the N-terminal were found to be Met-Asp-Ile-Pro-Gln-Thr-Lys-Gln-Asp-Leu-Glu-Leu-Pro-Lys-Leu-Ala-Gly-Thr-Glu-His - Glu-Met-Ala-Met. PP14 has been characterized in this study to be a dimeric glycoprotein of Mr 60,000, with homologous subunits having an Mr of 28,000.     

Human placental sterylsulfatase. Interaction of the isolated enzyme with substrates, products, transition-state analogues, amino-acid modifiers and anion transport inhibitors.

The enzymatic properties of a homogeneous sterylsulfatase preparation isolated from human term placenta were studied. The enzyme exhibited both arylsulfatase and sterylsulfatase activity: it catalysed the hydrolysis of sulfuric acid esters of (in the order of decreasing specific activity) non-steroidal phenols, of a phenolic steroid, and of neutral 3 beta-, 21- and (though at a very low rate) 17 beta-hydroxysteroids. However, among all the substrates tested only the 3-sulfates of phenolic and neutral steroids exhibited high affinity towards the sulfatase. Vitamin D3 sulfate was not hydrolysed by the sterylsulfatase but strongly inhibited its activity. The products of the catalytic reaction, free steroids or phenols as well as the sulfate anion or analogues thereof, likewise interfered with the enzyme's activity. Ki values of unconjugated steroids were ten- to hundredfold higher than Km values of the respective sulfoconjugates. Inorganic sulfate only slightly inhibited the sulfatase activity; its inhibitory potency, however, increased in a time-dependent manner when it was preincubated with the enzyme prior to assay. In contrast to sulfate, the hypothetical transition-state analogues sulfite and vanadate acted as strong inhibitors of the sulfatase activity. According to the results of an analysis of the effect of pH on sterylsulfatase kinetics, enzyme constituents with pK values of approximately 5.8 and 8.0 are involved in a general acid-base catalysed reaction. Treatment of the sulfatase with amino-acid side chain modifying reagents directed against arginine, cysteine, cystine, serine or tyrosine residues did not result in significant alteration of its activity. Diethyl-pyrocarbonate known to react primarily with histidyl groups, however, rapidly inactivated the enzyme; this inactivation reaction was markedly retarded in the presence of substrate. Histidine thus appears to be essential for the catalytic activity of the sulfatase. Taken together, the present results reveal a considerable similarity between the catalytic mechanism of human placental sterylsulfatase and the ones already proposed for the lysosomal arylsulfatases A and B. Taurocholate, salicylate, ouabain, and 4,4'-substituted stilbene-2,2'-disulfonates are well known inhibitors of carrier-mediated transport of anions across cellular membranes. With the exception of ouabain, these compounds likewise turned out to inhibit the enzymatic hydrolysis of steryl sulfates; the pattern of dose dependences of their interference with the sulfatase activity resembles the one reported for inhibition of anion transport. Since the sterylsulfatase in vivo strongly is associated with cellular membranes including the plasma membrane of the syncytiotrophoblast, this finding supports the speculation that similar molecular structures may be involved in both placental transport and hydrolysis of anionic steryl sulfates.     

The amino-acid sequence of kangaroo pancreatic ribonuclease

Red kangaroo (Macropus rufus) ribonuclease was isolated from pancreatic tissue by affinity chromatography. The amino acid sequence was determined by automatic sequencing of overlapping large fragments and by analysis of shorter peptides obtained by digestion with a number of proteolytic enzymes. The polypeptide chain consists of 122 amino acid residues. Compared to other ribonucleases, the N-terminal residue and residue 114 are deleted. In other pancreatic ribonucleases position 114 is occupied by a cis proline residue in an external loop at the surface of the molecule. Other remarkable substitutions are the presence of a tyrosine residue at position 123 instead of a serine which forms a hydrogen bond with the pyrimidine ring of a nucleotide substrate, and a number of hydrophobichydrophilic interchanges in the sequence 51-55, which forms part of an alpha-helix in bovine ribonuclease and exhibits few substitutions in the placental mammals. Kangaroo ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64). The enzyme differs at about 35-40% of the positions from all other mammalian pancreatic ribonucleases sequenced to date, which is in agreement with the early divergence between the marsupials and the placental mammals. From fragmentary data a tentative sequence of red-necked wallaby (Macropus rufogriseus) pancreatic ribonuclease has been derived. Eight differences with the kangaroo sequence were found.     

The amino-acid sequence of bovine glutathione peroxidase

The amino-acid sequence of the seleno-enzyme glutathione peroxidase from bovine erythrocytes was completely determined. Fragmentation of the carboxymethylated protein comprised cleavages with trypsin, with endoproteinase Lys-C, and with cyanogen bromide in 70% formic acid. The resulting peptides were separated by reversed-phase high-performance chromatography or by gel filtration. For sequence determination automated solid or liquid phase techniques of Edman degradation were used. The proper alignment of fragments was experimentally proven in all but one instance. In this case, consistent indirect evidence was provided. The monomer of glutathione peroxidase was shown to consist of 198 amino acids representing a molecular mass ob about 21 900 Da. The active site selenocysteine was localized at position 45. In addition, four cysteine residues were found at positions 74, 91, 111, and 152. The N-terminal part of the sequence obtained revealed a pronounced homology with a partial sequence of the rat liver enzyme. Moreover, tentative sequence data predicted from X-ray crystallographic analysis of bovine glutathione peroxidase were found to agree in about 80% of the residues with the sequence presented. Differences between the predicted and the experimentally determined sequence are discussed.