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M Suiko

Publications and source records attributed to M Suiko.

35 records · Page 2Linked to original sources
PubMed1995

Sulphation of L-tyrosine in mammalian cells: a comparative study.

Chang liver cells, Caco-2 human intestinal epithelial cells and Madin-Darby canine kidney (MDCK) cells, labelled with [35S]sulphate in the presence of different concentrations of cycloheximide, produced 87.7-95.3%, 35.8-41.1% and 23.2-25.9%, respectively, of the amounts of free tyrosine O-[35S]-sulphate (Tyr[35S]) formed by corresponding cells labelled in the absence of cycloheximide. Homogenates prepared from the three kinds of cells showed the presence of enzymic activities catalysing the sulphation of L-tyrosine, with specific activities in the order: Caco-2 cells > MDCK cells > Chang liver cells. In all three cases, most of the tyrosine sulphotransferase' activity was found in the cytosolic fraction, indicating the enzyme to be a cysolic protein. A tyrosine-dependence experiment revealed that, for all three kinds of cells labelled with [35S]sulphate, the production of free Tyr[35S] was proportional to the concentration of L-tyrosine present in the culture medium. These results imply an involvement of sulphation in removing excess intracellular L-tyrosine.

Animals↗
PubMed1994

De novo sulfation of L-tyrosine in HepG2 human hepatoma cells and its possible functional implication.

HepG2 human hepatoma cells, labeled with [35S]sulfate in the presence of 10-30 micrograms/ml of cycloheximide, released up to 64% of the amount of free tyrosine-O-[35S]sulfate produced and released by cells labeled in the absence of cycloheximide. A time-course study revealed that, in cells incubated in medium containing [3H]tyrosine, free [3H]tyrosine-O-sulfate was produced within 5 min of incubation, whereas no [3H]tyrosine-sulfated proteins were detected until 20 min after the incubation had begun. Using 3'-phosphoadenosine, 5'-phospho[35S]sulfate as the sulfate donor, HepG2 cell homogenate was shown to contain enzymic activity catalyzing the sulfation of L-tyrosine with the formation of tyrosine-O-[35S]sulfate. Upon subcellular fractionation, the majority of the enzyme activity was found in the cytosolic fraction. The enzyme, designated tyrosine sulfotransferase, displayed the optimum activity at pH 8.0 in the presence of 10 mM Mn2+. Under optimum conditions, the apparent Km of the enzyme for L-tyrosine, at 4.5-microM concentration of 3'-phosphoadenosine, 5'-phosphosulfate, was determined to be 1.95 mM, while that for 3'-phosphoadenosine, 5'-phosphosulfate, at 1 mM L-tyrosine concentration, was 8.3 microM. The Vmax determined under these conditions was 1.05 pmol.min-1.mg protein-1. A tyrosine-dependence study showed that, for cells labeled with [35S]sulfate, the production and release of free tyrosine-O-[35S]sulfate appeared to proceed actively and increase proportionally to the L-tyrosine concentration when it was raised above a threshold level in the culture medium. These results may imply a possible involvement of sulfation in removing excess intracellular L-tyrosine.

Carcinoma, Hepatocellular↗
PubMed1994

Identification and characterization of a major bovine serum tyrosine-O-sulfate-binding protein as a complement factor H.

A major tyrosine-O-sulfate (TyrS)-binding protein present in bovine serum was purified to electrophoretic homogeneity using a combination of TyrS-Affi-Gel 10 affinity chromatography, DEAE-Bio-Gel A ion-exchange chromatography, and hydroxylapatite chromatography. The purified TyrS-binding protein migrated as doublet protein bands with apparent molecular weights of ca. 160,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. N-termini of the two forms of purified TyrS-binding protein contain most likely identical sequence for the first fifteen amino acids residues, which displays a high degree of homology to those of human and mouse complement factor H. Furthermore, the purified TyrS-binding protein exhibited immunologic cross-reactivity with anti-human complement factor H. These results indicate the identity of the purified TyrS-binding protein being bovine complement factor H. The two forms of the purified bovine factor H were investigated with respect to the sensitivity to limited trypsin digestion. The high-molecular weight form was cleaved into two fragments with apparent molecular masses of, respectively, 45 kD and 125 kD. The low-molecular weight form was cleaved in a different manner to generate three major fragments with molecular masses of 25 kD, 45 kD and 100 kD, respectively. Limited V8 protease mapping of the two forms yielded similar, yet unidentical, peptide band patterns. Purified bovine factor H appeared to bind agarose-bonded heparin through its anion-binding domain and the binding was inhibited by the presence of free heparin or dextran sulfate.

Amino Acid Sequence↗
PubMed1993

Identification of a putative tyrosine-O-sulphate (TyrS) receptor possibly functioning in the biosynthetic transport of tyrosine-sulphated proteins in Madin-Darby canine kidney cells.

By employing an affinity-gel fractionation technique coupled to Western-blot analysis, we have identified a 175 kDa tyrosine-O-sulphate (TyrS)-binding protein present in Madin-Darby canine kidney (MDCK) cells. The binding of this TyrS-binding protein to TyrS covalently bonded to Sepharose gel was found to be pH-dependent, being strong from pH 8.0 down to pH 6.5 and increasingly weak at pH 6.0 and below. Results obtained from Triton X-114 temperature-induced phase separation and sodium carbonate buffer (pH 11) extraction experiments indicated that the TyrS-binding protein is an integral membrane protein. This 175 kDa TyrS-binding protein was found to be present in association with a major tyrosine-sulphated protein, the apically secreted 80 kDa glycoprotein (gp 80), in cell lysate prepared from MDCK cells maintained under normal growth conditions. When the cell lysate used was prepared from MDCK cells pretreated with 20 mM sodium chlorate, a metabolic sulphation inhibitor, the complex formed between the two proteins could no longer be detected, indicating that the binding of the TyrS-binding protein is through the TyrS residue(s) of gp 80. Both cell-surface biotinylation and cell-surface trypsinization studies demonstrated the predominantly, if not exclusively, intracellular location of the TyrS-binding protein. Furthermore, radioactive pulse-chase experiments revealed that the newly synthesized radiolabelled fibronectin and gp 80 were present in complexes with the TyrS-binding protein in MDCK cells pulse-labelled with [35S]methionine or [35S]sulphate. Exogenous [35S]methionine-labelled gp 80 added to the medium, on the other hand, was not found to be present in association with the TyrS-binding protein in MDCK cells over a 2-h time course. These results strongly suggested the identity of the 175 kDa TyrS-binding protein as a putative 'TyrS receptor', possibly functioning in the biosynthetic transport of tyrosine-sulphated proteins in MDCK cells.

Animals↗
PubMed1993

Isolation and characterization of a novel microsomal membrane-bound phenol sulfotransferase from bovine liver.

A novel phenol sulfotransferase (PST) was detected in bovine liver microsomal membrane fraction. The enzyme was found to be capable of catalyzing the sulfation of simple phenolic compounds, with 3'-phosphoadenosine-5'-phosphosulfate as the sulfate donor. Detergent extracted PST showed a pH optimum of 5.7 and, among the simple phenols tested, the PST exhibited highest activity toward alpha-naphthol. No activities were detected when tyrosine and its derivatives were used as substrates. Both 2,6-dichloro-4-nitrophenol and chlorpromazine were capable of inhibiting the activity of the PST toward p-nitrophenol with inhibition Coefficient50 values of 100 nM and 4 mM, respectively.

Animals↗
PubMed1992

Post-translational modification of protein by tyrosine sulfation: active sulfate PAPS is the essential substrate for this modification.

In vitro tyrosine sulfation of recombinant proteins would be a valuable tool in converting those proteins expressed in prokaryotic vectors to their natural form. For this purpose tyrosylprotein sulfotransferase (TPST), the enzyme responsible for tyrosine sulfation of proteins, was characterized from a bovine liver Golgi preparation. TPST was active in a acidic environment with a pH optimum of 6.25, and displayed a stimulation by the Mn2+, with the optimum activity in the presence of 5mM MnCl2. TPST was able to sulfate recombinant hirudin variant 1 (rHV-1) expressed in Escherichia coli and the C-terminal hirudin fragment 54-65 but not the N-terminal hirudin fragment 1-15 by using 3'-phosphoadenosine 5'-phosphosulfate (PAPS), indicating its specificity for the naturally sulfated tyrosine 63. Comparison of the reaction kinetics on synthetic peptides showed that the bovine liver TPST has a higher affinity and reaction rates for those peptides with a aspartyl residue on the N-terminal side of the tyrosine when compared with a glutamyl residue.

Amino Acid Sequence↗
PubMed1992

Enzymatic synthesis of PAPS with an ATP-regeneration system.

Sulfate activating enzymes, ATP sulfurylase and APS kinase, were newly isolated from a thermophile, Bacillus stearothermophilus. Adenosine 3'-phosphate 5'-phosphosulfate (PAPS) was synthesized by these enzymes. The reaction proceeded more efficiently when an ATP-regeneration system, using acetate kinase, was coupled to the reaction system.

Acetate Kinase↗
PubMed1991

Post-translational modifications and binding properties of the apically secreted 80-kDa glycoprotein from Madin-Darby canine kidney cells: similarities to the C-terminal portion of the basolaterally secreted fibronectin.

Apically secreted 80-kDa glycoprotein (gp 80) from Madin-Darby canine kidney cells was found to be immunoprecipitated by the polyclonal antiserum against fibronectin or a monoclonal antibody specific for the fibronectin C-terminal fibrin binding domain. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), gp 80 migrated as a doublet band under nonreducing conditions. Under reducing conditions, gp 80 was resolved into three distinct bands, respectively of 45-, 40-, and 35-kDa molecular mass. Analysis by two-dimensional SDS-PAGE revealed that gp 80 exists in two molecular forms: one consisting of a 45-kDa subunit and a 40-kDa subunit, and one consisting of a 45-kDa subunit and a 35-kDa subunit. V-8 protease mapping indicated the 40 and 35-kDa subunits as being of the same homologous group and also as bearing partial homology to the 45-kDa subunit. Radioactive labeling revealed that labeled gp 80 was subjected to covalent modifications by sulfation and phosphorylation. Sulfate analysis showed that [35S]sulfate-labeled gp 80 contained ca. 2.45 +/- 0.07% tyrosine-bound [35S]sulfate with the rest being presumably carbohydrate-bound. [32P]-Phosphate-labeled gp 80, on the other hand, was found to contain serine-O-phosphate as the predominant phosphorylated amino acid residue. Employing the affinity gel fractionation technique, it was shown that gp 80 exhibited binding affinities toward heparin and fibrin. Binding of gp 80 to heparin-agarose or fibrin-Sepharose, however, was inhibited in the presence of added fibronectin or the monoclonal antibody. Tryptic peptide mapping revealed common peptide spots between fibronectin and the three subunits of gp 80. Furthermore, Western blot analysis showed that fibronectin could be recognized and bound by anti-gp 80 antibodies. These results indicate that gp 80 bears both structural and functional similarities to the C-terminal portion of the fibronectin molecule.

Animals↗
PubMed1991

Purification and characterization of a membrane-bound tyrosine-O-sulfate-binding protein from bovine liver.

A membrane-bound 175-kDa tyrosine-O-sulfate (TyrS)-binding protein from bovine liver was purified to electrophoretic homogeneity. The purified protein exhibited an apparent molecular weight of 175,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Upon SDS-PAGE under nonreducing conditions, the TyrS-binding protein migrated considerably faster than the dimeric fibronectin, indicating its presence in the monomeric form and therefore the absence of a disulfide-bonded subunit structure. The purified TyrS-binding protein was found to bind concanavalin A-Sepharose and yielded a positive reaction toward periodic acid-Schiff (PAS) staining, indicating its glycoprotein nature. The purified TyrS-binding protein displayed strong binding to TyrS, but not the unmodified tyrosine, covalently bonded to Sepharose. Using a tyrosine-sulfated cholecystokinin octapeptide (CCK-8) as the ligand in a radioimmunoassay, it was found that the binding of the TyrS-binding protein was pH-dependent, being strong from pH 8.0 down through 6.5, and becoming dramatically weaker at pH below 6.0. Divalent cations, added in the assay mixture, exerted significant promoting effects on the binding in the order Mn+2 greater than Ca2+ greater than Mg2+. Western blot analysis clearly showed that the purified TyrS-binding protein was capable of forming complexes with two tyrosine-sulfated proteins, fibronectin and fibrinogen, but not two non-tyrosine-sulfated proteins, transferrin and albumin. These results provided support to a role of the TyrS-binding protein being a putative receptor for tyrosine-sulfated proteins.

Amino Acids↗
PubMed1991

Identification of complexes between the tyrosine-O-sulphate-binding protein and tyrosine-sulphated proteins in bovine liver membrane lysates.

Rabbit antiserum against electrophoretically purified bovine liver tyrosine-O-sulphate (TyrS)-binding protein was prepared. Affinity-purified antibodies from the antiserum were found to be capable of immunoprecipitating the TyrS-binding protein from the sodium choleate extract of a bovine liver microsomal membrane fraction. Using purified specific antibodies as the probe, Western blot analysis for the presence of TyrS-binding protein/tyrosine-sulphated protein complexes in bovine liver membrane lysates was performed. It was found that the TyrS-binding protein co-precipitated with three tyrosine-sulphated proteins (fibronectin, fibrinogen and complement C4) immunoprecipitated by their respective antibodies. In contrast, for the two non-tyrosine-sulphated proteins (haptoglobin and transferrin) tested, co-precipitation of the TyrS-binding protein was not observed. On employing an affinity gel fractionation technique, it was shown that partially purified TyrS-binding protein exhibited binding affinity towards Sepharose gels covalently bonded to fibronectin or fibrinogen, but not towards Sepharose gels bonded to albumin or transferrin. These results indicate that the TyrS-binding protein formed complexes with tyrosine-sulphated proteins both in vivo and in vitro, and thus provide support for the putative role of the former being the receptor of the latter.

Animals↗
PubMed1990

Tyramine-O-sulfate, in addition to tyrosine-O-sulfate, is produced and secreted by HepG2 human hepatoma cells, but not by 3Y1 rat embryo fibroblasts.

The spent media of HepG2 human hepatoma cells and 3Y1 rat embryo fibroblasts labeled with [35S]sulfate, upon ultrafiltration, were analyzed by a two-dimensional thin-layer separation procedure. Autoradiographs of the cellulose thin-layer plate revealed the presence of tyramine-O-[35S]sulfate in addition to tyrosine-O-[35S]sulfate in spent medium from human hepatoma cells. In contrast, only tyrosine-O-[35S]sulfate was observed in spent medium of 3Y1 rat fibroblasts. Using adenosine, 3'-phosphate, 5'-phospho[35S]sulfate as the sulfate donor, sulfotransferase(s) present in HepG2 cell homogenate catalyzed the sulfation of tyramine to tyramine-O-[35S]sulfate, but not the sulfation of tyrosine to tyrosine-O-[35S]sulfate. Endogenous aromatic amino acid decarboxylase present in HepG2 homogenate was shown to catalyze the decarboxylation of [3H]tyrosine to form [3H]tyramine while attempts to use it for the decarboxylation of tyrosine-O-sulfate to form tyramine-O-sulfate were unsuccessful. These results suggest that tyramine-O-sulfate may be derived from the de novo sulfation of tyramine, instead of the decarboxylation of tyrosine-O-sulfate.

Animals↗
PubMed1988

Isolation and characterization of a bovine liver tyrosine-O-sulfate-binding protein--a putative receptor molecular for tyrosine-sulfated proteins?

Golgi-enriched microsomal membrane fraction was prepared from bovine liver. Sodium choleate extract of this membrane preparation was subjected to fractionation using Sepharose gel covalently bonded with tyrosine-O-sulfate. SDS gel electrophoresis of the fractionated sample revealed the presence of a major protein with an apparent molecular weight of 175,000. The protein appears to be specific for tyrosine-O-sulfate as it binds neither the unmodified tyrosine nor the structurally similar tyrosine-O-phosphate. pH-dependence study showed the binding of the protein to tyrosine-O-sulfate-Sepharose gel to be strong from pH 8.0 down through 6.0. At pH 5.5, the binding affinity became dramatically reduced. A similar tyrosine-O-sulfate-binding protein was also detected in the choleate extracts of the Golgi-enriched microsomal membrane fractions prepared from bovine pancreas and from both liver and pancreas of dog.

Animals↗
PubMed1988

Change in binding affinities of 3Y1 secreted fibronectin upon desulfation of tyrosine-O-sulfate.

Desulfated form of the 3Y1 secreted fibronectin was prepared by treatment with arylsulfatases. Under optimal conditions, the degrees of tyrosine-desulfation of [35S]sulfate-labeled fibronectin by arylsulfatases from Helix pomatia (Type H-1), Patalle vulgata (Type V) and Abalone entrails (Type VIII) were determined to be 55.7%, 54.9% and 76.4%. Upon desulfation of [3H]leucine-labeled fibronectin by Type H-1 or Type V arylsulfatase, gelatin-binding affinity remained unchanged; while heparin-binding affinity increased nearly 50%. Treatment with Type VIII arylsulfatase caused a considerable decrease in gelatin-binding and a slight decrease in heparin-binding affinities. Nevertheless, desulfation by all three enzymes consistently resulted in a dramatic decrease of fibrin-binding affinity, ranging from 42.1% to 64.4%.

Animals↗
PubMed1987

Studies of the decrease of tyrosine-O-sulphated proteins in Rous sarcoma-virus-transformed rat embryo fibroblasts, line 3Y1. Examination of the sulphate activation and tyrosyl-protein sulphotransferase systems.

The sulphate activation and tyrosyl-protein sulphotransferase systems in normal 3Y1 rat embryo fibroblasts and the same cells transformed by Schmidt Ruppin subgroup-A-Rous sarcoma virus (SRA-3Y1) were examined. Employing metabolic [35S]sulphate-labelling followed by PEI (polyethyleneimine)-cellulose thin-layer chromatography of the labelled cell lysates, it was found that the steady-state level of 'active' sulphate, adenosine 3'-phosphate 5'-phosphosulphate, was drastically lower in SRA-3Y1 cells compared with their normal counterparts. When the sulphate activating enzymes were tested, it appeared that the activities in 3Y1 homogenates were 2-2.5 times greater than those in SRA-3Y1 homogenates. An endogenous sulphation assay for tyrosyl-protein sulphotransferase revealed that activities in 3Y1 and SRA-3Y1 homogenates were comparable. Nearly identical patterns were observed with both sets of cells when [35S]sulphated proteins generated in the endogenous assay were separated by two-dimensional gel electrophoresis. It therefore seems that the tyrosyl-protein sulphotransferase(s) are unimpaired in SRA-3Y1 cells. While the lower (approx. 8 times) sulphate uptake remains the major cause for the decrease of tyrosine-O-sulphated proteins in SRA-3Y1 cells [Liu & Lipmann, (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3695-3698], the 2-2.5-fold lower sulphate activating enzyme activities also contribute to some extent to the difference between the SRA-3Y1 and 3Y1 cells.

Animals↗
PubMed1987

Tyrosine sulfation site is located in the C-terminal fibrin-binding domain in secreted fibronectin from rat embryo fibroblasts, line 3Y1.

Tryptic fragments of [35S]sulfate-labeled 3Y1 secreted fibronectin were fractionated by hydroxylapatite column chromatography and examined using sodium dodecyl sulfate gel electrophoresis, followed by autoradiography. Radioactive bands containing tyrosine-O-[35S]sulfate were detected at 17- and 40-kDa positions under reducing conditions. Under nonreducing conditions, the 17-kDa band was no longer present and new bands at 57- and 80-kDa positions appeared, indicating a disulfide linkage between the two smaller fragments in the native state. These fragments exhibited binding affinity toward fibrin and could be immunoprecipitated by the monoclonal antifibronectin Fib-2 domain antibody. These results suggested that the tyrosine sulfation site in 3Y1 secreted fibronectin is located in the C-terminal fibrin-binding (Fib-2) domain, being within 17 kDa of the C-terminus.

Animals↗
PubMed1987

Rapid catabolism of tyrosine-O-sulphated proteins and the formation of free tyrosine O-sulphate as an end product in rat embryo fibroblasts.

Rat embryo fibroblasts, line 3Y1, were prelabelled for 24 h with [35S]sulphate and incubated in fresh medium without [35S]sulphate. A rapid efflux of the overall 35S-labelled compounds from the cells into the medium was observed. After 9 h of incubation, about 50% of the total 35S radioactivity appeared in the medium and up to 84.3% did so at the end of a 48 h incubation. Determination of [35S]sulphated macromolecules present in both the cell-associated and the incubation-medium fractions at different time points during incubation indicated that the majority of the 35S-labelled compounds released from the cells were low-Mr products derived from digestion of the [35S]sulphated macromolecules. Further analysis for tyrosine-O-[35S]sulphated proteins, which constituted only a small fraction of the overall [35S]sulphated macromolecules, showed that, after 9 h of incubation, there was a 65% decrease in the cell-associated fraction, and only 16.4% remained after 48 h. During that time, an amount equivalent to 20.7% of the cell-associated tyrosine-O-[35S]sulphated proteins originally present was released into the medium. Free tyrosine O-[35S]sulphate was generated in the cells and excreted into the incubation medium. Its rate of increase with time, however, was slow, and could account for only 12.4% of the tyrosine-O-[35S]sulphated proteins catabolized at the end of the 48 h incubation.

Animals↗