Search PubMed⌕ Search

Biomedical subjects

S Gasa

Publications and source records attributed to S Gasa.

At least 55 records · Page 3Linked to original sources
PubMed1992

Components and proteolytic processing sites of arylsulfatase B from human placenta.

Previous studies have shown that mature arylsulfatase B purified from human sources is composed of two non-identical chains with apparent molecular masses of 43 kDa and 8 kDa. Arylsulfatase B purified from human placenta in the present study, however, included another 7 kDa component that could be detected only by carbohydrate staining on reducing SDS-PAGE employing the Tris-Tricine system. The 43 kDa and 7 kDa components contained a carbohydrate moiety, but the 8 kDa one did not, as demonstrated by periodic acid-Schiff staining, Con-A lectin blotting, endo-glycosidase treatment and in vitro phosphorylation by UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. The purified arylsulfatase B migrated as a single polypeptide of 58 kDa on non-reducing SDS-PAGE, indicating that the three chains are linked by disulfide bonds. In order to determine the origin of the components, N-terminal sequencing of the isolated polypeptides was performed. As a result, the 43, 7 and 8 kDa components were found to commence with Ala-41, Ala-424 and Asp-466, respectively. These results suggest that after removal of the signal peptide, human arylsulfatase B undergoes proteolytic processing on at least two sites during maturation.

Amino Acid Sequence↗
PubMed1992

Binding specificity of lung surfactant protein SP-D for glucosylceramide.

The specificities of the binding of lung surfactant protein SP-D to glycolipids were examined using 125I-labeled SP-D as a probe. When the binding study was performed on TLC plates, SP-D bound exclusively to GlcCer, whereas it failed to bind to GalCer, GM1, GM2, asialo-GM1, asialo-GM2, sulfatide, Forssman antigen, ceramide dihexoside, ceramide trihexoside, globoside, paragloboside or ceramide. Excess native SP-D competed with 125I-SP-D for the binding to GlcCer. Antibody to rat SP-D inhibited 125I-SP-D binding to GlcCer. Ca2+ was absolutely required for the binding of SP-D to GlcCer; Mg2+ failed to substitute for Ca2+. SP-D bound to ceramide monohexoside in glycolipids isolated from rat lung and bronchoalveolar lavage fluids of rats.

Animals↗
PubMed1992

Proteolytic processing of human lysosomal arylsulfatase A.

Arylsulfatase A purified from human placenta contained an unreported component with an apparent molecular mass of 7 kDa in addition to the two known components with apparent molecular masses of 58 and 50 kDa. The detailed relationship between the 58 kDa component and the 50 kDa component is as yet unknown. The present study was undertaken to define the structure of the subunits of the sulfatase. The N-terminal sequence of the 50 kDa component was identical to that of the 58 kDa component. Furthermore, the peptide maps of the 50 kDa component, which was separately digested with trypsin and Achromobacter proteinase I, were quite similar to those of the 58 kDa one. Through sequence analysis of the incompatible peaks in the peptide maps, the 50 kDa component was found to lack a sequence from Val-445 to the C-terminus. On the other hand, the N-terminal sequence of the 7 kDa component began with Ala-448, though there was a minor sequence commencing with Thr-449. These observations suggest that the 50 and 7 kDa components were produced by limited proteolysis near the C-terminus of the 58 kDa component. Through analysis using unreducing SDS-PAGE, the 58 and the 7 kDa components were found to be linked by disulphide bonds. Arylsulfatase A purified from human liver was also composed of the same subunits as the placental one. This finding suggests that human arylsulfatase A undergoes similar proteolytic processing regardless of the tissue involved.

Amidohydrolases↗
PubMed1992

Biosynthesis of blood group I and i antigens in rat tissues. Identification of a novel beta 1-6-N-acetylglucosaminyltransferase.

The beta-galactoside beta 1-6- and beta 1-3-N-acetylglucosaminyltransferases (beta 1-6GnT and beta 1-3GnT) that synthesize blood group I and i antigens were identified in rat tissues, using pyridylaminated lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-PA) as an acceptor. The products of the transferase reactions were separated on high performance liquid chromatography. The products of the transferase reactions were identified by 1H NMR as (formula; see text) and GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-PA. The product for beta 1-6GnT was also identified by methylation analysis. Kinetic experiments were carried out using rat serum for beta 1-3GnT and partially purified enzyme from rat intestine for beta 1-6GnT. beta 1-3GnT has a pH optimum of 7.5 and requires Mn2+ for optimal activity. beta 1-6GnT has a pH optimum of 7.0 and does not require Mn2+. Studies on the substrate specificity of each enzyme indicated that the preferred substrate for beta 1-3GnT had the general structure Gal beta 1-4GlcNAc-OR and, for beta 1-6GnT, Gal beta 1-4GlcNAc beta 1-3Gal-OR where R = sugar. This is the first demonstration that the beta 1-6GnT acts on an internal galactose of lacto-N-neotetraose and paragloboside, and the enzyme appears to be a novel enzyme in terms of substrate specificity.

Animals↗
PubMed1992

Interaction between heat acclimation and exogenous insulin in brown adipose tissue of rats.

Seventy-one male Wistar strain rats (7 weeks old) were kept at 5, 25, or 34 degrees C, respectively, for 2 weeks with or without insulin administration. Insulin (Novo Lente MC) was given subcutaneously in a dose of 3.62 nmol/125 microliters saline per 100 g body weight. An apparent effect of insulin treatment was noted only in heat-exposed rats, resulting in a remarkable gain in interscapular brown adipose tissue (BAT) mass of heat-acclimated, insulin-treated rats in terms of weight or weight per unit body weight. The BAT from heat-acclimated, insulin-treated rats had significantly higher levels of protein, DNA, RNA, and triglyceride than BAT from heat-acclimated, saline-treated rats. Therefore, it seems likely that the growth of BAT in heat-acclimated, insulin-treated rats was mostly due to the anabolic effects of insulin. The uncoupling protein mRNA was, however, present in BAT of heat-acclimated, insulin-treated rats at rather a depressed level, explaining a corresponding decrease in cold tolerance. On the other hand, the expression of insulin receptor mRNA was attenuated in BAT of rats from all the insulin-treated groups, possibly due to the down-regulation of insulin. Thus, there appeared to be some linkage among BAT, heat acclimation, and insulin.

Acclimatization↗
PubMed1992

Binding of pulmonary surfactant protein A to galactosylceramide and asialo-GM2.

The binding of pulmonary surfactant protein A (SP-A) to glycolipids was examined in the present study. The direct binding of SP-A on a thin-layer chromatogram was visualized using 125I-SP-A as a probe. 125I-SP-A bound to galactosylceramide and asialo-GM2, but failed to exhibit significant binding to GM1, GM2, asialo-GM1, sulfatide, and Forssman antigen. The study of 125I-SP-A binding to glycolipids coated onto microtiter wells also revealed that SP-A bound to galactosylceramide and asialo-GM2. SP-A bound to galactosylceramides with non-hydroxy or hydroxy fatty acids, but showed no binding to either glucosylceramide or galactosylsphingosine. Excess native SP-A competed with 125I-SP-A for the binding to asialo-GM2 and galactosylceramide. Specific antibody to rat SP-A inhibited 125I-SP-A binding to glycolipids. In spite of chelation of Ca2+ with EDTA or EGTA, SP-A retained a significant binding to glycolipids. Inclusion of excess monosaccharides in the binding buffer reduced the glycolipid binding of SP-A, but failed to achieve complete abolishment. The oligosaccharide isolated from asialo-GM2 is also effective at reducing 125I-SP-A binding to the solid-phase asialo-GM2. From these data, we conclude that SP-A binds to galactosylceramide and asialo-GM2, and that both saccharide and ceramide moieties in the glycolipid molecule are important for the binding of SP-A to glycolipids.

Animals↗
PubMed1992

Proteolytic processing sites producing the mature form of human cathepsin D.

1. The proteolytic processing sites of human lysosomal aspartic protease cathepsin D at which the intermediate single-chain form was converted into the mature two-chain form were determined. 2. The two chains were isolated by reversed-phase HPLC in order to investigate the cleavage sites of the enzyme. 3. Protein sequencing of the heavy chain, which was presumed to be derived from the C-terminal side in the single-chain enzyme, gave an N-terminal Leu 105. In addition, it revealed that there were also minor sequences, which commenced with Gly 106 and Gly 107. 4. A small C-terminal peptide was isolated from the light chain, which had been digested with two kinds of exogenous proteases. Sequence determination of this peptide, which was characterized as a nonapeptide by mass spectrometry, suggested that the C-terminus of the light chain was Ser 98. 5. These results indicate that a Ser 98-Ala 99 bond and an Ala 104-Leu 105 bond are cleaved to release 6 amino acid residues between the two chains.

Amino Acid Sequence↗
PubMed1992

Characterization of the subunits and sugar moiety of human placental and leukemic beta-glucuronidase.

beta-Glucuronidase purified from human placenta and chronic myelogenous leukemic cells was composed of three components of 18, 64 and 80 kDa, though the relative contents of the components were different between the sources. Analysis of their N-terminal amino-acid sequences showed that the 18-kDa and 64-kDa components were derived from the 80-kDa component by cleavage between Val159 and Gly160. Furthermore, the enzyme was found to be glycosylated at Asn173 and Asn420 with high mannose-type oligosaccharides, based on the electrophoretic mobility of the components as well as the endopeptidic peptides before and after endoglycosidase treatment. The enzyme purified from leukemic cells was poorly phosphorylated by N-acetylglucosamine 1-phosphotransferase as compared to the placental enzyme.

Amino Acid Sequence↗
PubMed1992

Inconsistent expression of glycolipid sulfotransferase activity between hepatoma and serum.

Glycolipid sulfotransferase activity in human and rat hepatocellular carcinoma tissues was studied, since an elevated level of the enzyme activity in serum had been demonstrated in cancer patients. The level of the enzyme activity in carcinoma tissues, however, could not be distinguished from that of normal controls. Similar observations were made for rat hepatoma. A higher level of enzyme activity was observed in the female than in the male. Inconsistent expression between hepatoma tissue and serum suggests that humoral factor(s) derived from hepatoma tissue stimulates production of the sulfotransferase, which is released into the blood-stream in the host.

Animals↗
PubMed1992

Improved preparation method for lysogangliosides.

Lyso-GM3 and -GM1 gangliosides were prepared from the corresponding N,N'-dideacylated gangliosides using N-trifluoroacetylation at the sphingosine moiety, followed by N-acetylation and mild saponification. The blocking reaction was performed using a water-ether bilayer system at alkaline medium, in which the N-trifluoroacetylation occurred predominantly at the lipid moiety. Through the procedure, lysoGM3 and lysoGM1 were obtained with higher yields from the corresponding dideacylated gangliosides than through the previous method using 9-fluorenylmethoxycarbonyl chloride as a blocking group or of direct N-acetylation of it on liposomes containing starting ganglioside and other lipid. Chemical structures of the lysogangliosides and the synthetic intermediates were confirmed by the proton nuclear magnetic resonance spectrometry and negative fast atom bombardment-mass spectrometry.

Acetates↗
PubMed1992

Galactosylceramide containing omega-amino-fatty acids: preparation, characterization, and sulfotransferase acceptor.

For preparation of an affinity ligand, an N-fatty acyl moiety of galactosylceramide (GalCer) was chemically replaced with omega-amino-fatty acid including amino-n-hexanoic acid or amino-n-dodecanoic acid to obtain omega-aminoGalCer. For the synthesis of the compound, galactosylsphingosine (GalSph) was coupled with N-trifluoroacetyl omega-amino-fatty acid which was prepared by a reaction with S-ethyltrifluorothioacetate. After removal of the N-trifluoroacetyl group in a mild alkaline solution, in which an N-fatty acyl group was retained, aminoGalCer composed of an N-hexanoyl or an N-dodecanoyl group was obtained with an overall yield of 90%. Their chemical structures were confirmed by proton nuclear magnetic resonance and fast atom bombardment-mass spectrometries. These aminoGalCers and GalSph as well as immobilized aminoGalCer were sulfated by a glycolipid sulfotransferase from rat kidney. Furthermore, immobilized aminoGalCer on gel matrix was used for affinity chromatography of the sulfotransferase, resulting in an excellent increase in the purification (14,000-fold) with a recovery rate of 40%.

Aminocaproic Acid↗
PubMed1992

Use of immunoadsorbent column chromatography for improved purification of arylsulfatase B from human placenta.

A simple and rapid procedure involving immunoadsorbent column chromatography has been developed for the isolation of lysosomal arylsulfatase B from human placenta. Using this method, we purified the enzyme over 20,000-fold with better recovery (16%) compared to that achieved by the conventional procedure. The enzyme appeared to be homogeneous and had an apparent molecular weight of 58,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. The purified enzyme migrated as two bands with apparent molecular weights of 43,000 and 8,000 by reductive SDS-PAGE.

Chondro-4-Sulfatase↗
PubMed1991

Elevated serum level of glycolipid sulfotransferase in patients with hepatocellular carcinoma.

Activity of glycolipid sulfotransferase (cerebroside sulfotransferase) in serum was elevated in 21 (33%) of 63 patients with hepatocellular carcinoma (HCC, mean +/- S.E., 349 +/- 32 pmol/ml per h, n = 63, P less than 0.001) compared to healthy subjects (172 +/- 12, n = 85). Ho significant elevation of the sulfotransferase level was observed in liver cirrhosis (219 +/- 28, n = 10) in which many of biochemical HCC markers increase concomitantly. The elevation of sulfotransferase was independent of the production of alpha-fetoprotein and of aminotransferase levels in HCC, providing complementary value for alpha-fetoprotein-negative HCC cases. However, the sulfotransferase levels (234 +/- 21, n = 32, P less than 0.01) in sera from patients with renal cell carcinoma, in whose involved tissues the enzyme was demonstrated to increase markedly, were less than in HCC.

Carcinoma, Hepatocellular↗
PubMed1991

Distinctive glycolipid patterns in Wilms' tumor and renal cell carcinoma.

Glycolipid patterns were analysed chromatographically in Wilms' tumor and renal cell carcinoma tissues and compared with those of uninvolved tissue. Ganglioside GM3 was found to be increased in both cancer tissues, whereas sulfatides accumulated only in renal cell carcinoma, as reported earlier. Neolactotetraosylceramide was detected in both cancer tissues, but not in the uninvolved kidney tissues. In four cases of Wilms' tumors, only a low level of sulfotransferase towards galactosylceramide was found in one case, while no activity was detected in the three other cases. Present results show that the increased sulfatide(s) in the renal cell carcinoma and the deficiency of the sulfatides in Wilms' tumors appear to be biochemical characteristics of histologically different carcinomas.

Carcinoma, Renal Cell↗
PubMed1991

Human erythrocyte multicatalytic proteinase: activation and binding to sulfated galacto- and lactosylceramides.

Chymotrypsin-like activity of multicatalytic proteinase (MCP) purified from human erythrocytes was selectively activated 2.5--3.5-fold by sulfated glycolipids such as galactosylceramide sulfate (SM4) and lactosylceramide sulfate (SM3) but not by other glycolipids including galactosylceramide (GalCer), lactosylceramide (LacCer), GD1a, GM1 and GM3. Heparin also selectively activated trypsin-like activity 2.5-fold, while other mucopolysaccharides did not. This proteinase molecule bound specifically and with high affinity to both SM4 and SM3, but not to GalCer, LacCer and GM3. The binding of SM4 and SM3 to the enzyme molecule was also confirmed by thin layer chromatography.

Amino Acid Sequence↗
PubMed1991

Glycolipids in rat cochlea.

In recent years, the functions of glycolipids have been intensively studied. Before the research of the roles of glycolipids in the inner ear, it seemed to be necessary to demonstrate the composition of glycolipids at first. Therefore, rat cochlea has been examined for glycolipid composition in the present study. Glycolipids extracted from 200 cochlea samples were separated into neutral and acidic glycolipid fractions. Each fraction was analyzed by thin-layer chromatography. The neutral glycolipid was almost exclusively galactosylceramide (cerebroside) with trace amounts of globoside and unidentified glycolipids. In acidic glycolipids, sulfated galactosylceramide (cerebroside sulfate) was most abundant. Of the gangliosides (sialic acid-containing glycolipids), ganglioside GM3 (51.8%) was the predominant component, and GM2 (6.0%), GM1 (8.8%), GD3 (10.7%), GD1a (8.7%), GD1b (5.9%), and GT1b (3.6%) were also detected. In addition to these, rat cochlea also contained several, minor, unidentified gangliosides. This pattern of the cochlear acidic glycolipids is very similar to that of the renal acidic glycolipids. This finding seems to be very interesting when considering that the toxic side effects of aminoglycosides occur only in the kidney and the inner ear.

Animals↗
PubMed1991

Elevated carbohydrate phosphotransferase activity in human hepatoma and phosphorylation of cathepsin D.

To determine the cause of the increased content of carbohydrate-bound phosphate in tumour lysosomal hydrolases, the activity and kinetics in human hepatocellular carcinoma of two enzymes involved in the formation of mannose-6-phosphate in lysosomal hydrolases UDP-GlcNAc: lysosomal enzyme GlcNAc alpha l-phosphotransferase (GlcNAc-phosphotransferase) and phosphodiester glycosidase were studied. The activity level of the phosphotransferase with artificial and natural substrates was elevated (P less than 0.025 and P less than 0.001, respectively) in hepatoma compared to that in uninvolved tissue, while the phosphodiester glycosidase of hepatoma was at a level similar to that of the uninvolved tissue. To verify a previous observation that cathepsin D of human hepatoma contained increased GlcNAc-phosphomannose, the protease was examined for carbohydrate phosphorylation by the GlcNAc-phosphotransferase. The protease from normal human liver was much more phosphorylated than hepatoma protease, confirming the previous observation. The predominant phosphorylation of the protease occurred in one of two major heavy subunits, with some phosphorylation in one of two minor light subunits.

Adenocarcinoma↗