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S Fukui

Publications and source records attributed to S Fukui.

At least 19 recordsLinked to original sources
PubMed1992

A mutant alpha-amylase with enhanced activity specific for short substrates.

The 210th lysine (K210) at the active site in Saccharomycopsis fibuligera alpha-amylase was altered to arginine (R) or asparagine (N) by site-directed mutagenesis. Replacement of K210 by R strengthened the 7th and weakened the 8th subsite affinities. K210 was found to contribute to both the 8th and the 7th subsites. The catalytic activity of the K210R enzyme for the hydrolysis of maltose (G2) was three-times higher than that of the native enzyme due to an increase in the affinity of the 7th subsite adjacent to the catalytic site, whereas the activity of the K210N enzyme for G2 was decreased to 1% of that of the native enzyme by a reduction in the 7th subsite affinity.

Amylose↗
PubMed1992

Cancer-associated glycoproteins defined by a monoclonal antibody, MLS 128, recognizing the Tn antigen.

A murine monoclonal antibody, MLS 128, recognizing the Tn antigen, was established and used for characterization of glycoproteins expressing the Tn antigen. The Tn antigen was expressed on three polypeptide chains with molecular weights of 250k, 210k and 150k daltons. LS 180 cells were labeled with 3H-glucosamine or 35S-sulfate metabolically, and then the immunoprecipitate derived from the cell lysate was subjected to SDS-PAGE followed by fluorography. It was revealed that these Tn antigen glycoproteins were produced through the processing of a high molecular weight precursor. The carbohydrate moieties of the Tn antigen glycoproteins labeled with 3H-glucosamine were released with alkaline-borohydride, and the released sugars were examined by gel filtration and paper chromatography. The carbohydrates predominantly consisted of GalNAc and sialyl GalNAc (greater than 90%), with a nearly equal distribution.

Antibodies, Monoclonal↗
PubMed1992

Alteration of bond-cleavage pattern in the hydrolysis catalyzed by Saccharomycopsis alpha-amylase altered by site-directed mutagenesis.

The 210th lysine (K) residue in the Saccharomycopsis alpha-amylase (Sfamy) molecule was replaced by arginine (R) and asparagine (N) residues by site-directed mutagenesis. The influences of the replacements on the bond-cleavage pattern for several substrates were analyzed. Both mutant enzymes, K210R and K210N, cleave mainly the first glycosidic bond from the reducing end of maltotetraose (G4), while the native enzyme hydrolyzes mainly the second bond from the reducing end. We changed successfully the major cleavage point in the hydrolysis reaction of G4. The 8th subsite affinities of the K210R and K210N enzymes are calculated to be +2.52 and -0.01 kcal/mol, respectively, whereas that of the native enzyme is +3.32 kcal/mol as reported in the previous paper. These affinity values suggest that the K210 residue composes the 8th subsite, one of major subsites, and that a positively charged amino residue is necessary for the 8th subsite affinity. The K210N enzyme is found to be less active for short substrates like maltotetraose (G4) than for long substrates like amylose A (approximately G18). The reduced catalytic activity specifically for the short substrates is also attributable to the remarkable decrease in the affinity of the 8th subsite.

Amino Acid Sequence↗
PubMed1992

Molecular cloning of cDNAs for human fibroblast nucleotide pyrophosphatase.

Nucleotide pyrophosphatase (NPPase) activity is markedly elevated in cultured skin fibroblasts from patients of Lowe's syndrome. cDNA clones encoding the NPPase were isolated using synthetic oligonucleotide probes designed on the basis of partial amino acid sequence of the enzyme purified from human placenta. The complete sequences of these clones yielded a merged sequence of 3508 bases. The polypeptide chain of the enzyme was deduced to comprise 873 amino acids with a calculated molecular weight of 99,703 and had the characteristics of a class II transmembrane protein. Ten potential N-glycosylation sites were detected in the protein. RNA blot analysis showed that human fibroblasts contain two minor mRNAs of 7.0 and 8.2 kb, respectively, in addition to a major 3.6-kb species that coincides with the merged cDNA in size. A computer search of a nucleotide sequence data-base revealed that plasma cell membrane glycoprotein PC-1, whose function was unknown at the time, is identical with the NPPase. Comparison of the amino acid sequence of the NPPase with the active site sequence of bovine 5'-nucleotide phosphodiesterase allowed the assignment of a putative active site domain to the central region of the COOH-terminal extracellular domain of the NPPase. The gene for human NPPase was localized to chromosome 6 at q22-q23 by in situ hybridization with a fragment of the NPPase cDNA.

Amino Acid Sequence↗
PubMed1992

Assessment of valvular regurgitation using cine magnetic resonance imaging coupled with phase compensation technique: comparison with Doppler color flow mapping.

To elucidate whether or not a newly developed technique in cinematic-displayed (cine) magnetic resonance imaging (MRI) can improve the semiquantitative evaluation of valvular regurgitant flow, 20 patients with valvular lesions were studied. Three pulse sequences of cine MRI, ie, standard, short echo time (TE), and rephasing scans, were compared with reference obtained by Doppler color flow mapping. Short TE technique and rephasing scan technique improved image quality remarkably as compared with standard technique. Each of the three cine MRI techniques showed good correlation with the Doppler method (p < 0.001). However, short TE and rephasing scan techniques gave a faithful estimation of the extent as compared with the Doppler method, whereas standard technique overestimated the regurgitation. Thus, cine magnetic resonance imaging with phase compensation technique can be utilized for the semiquantitative assessment of valvular regurgitation in a manner similar to that of Doppler color flow mapping.

Aged↗
PubMed1992

Cloning and molecular analysis of cDNA encoding a carboxymethylcellulase of the yeast Cryptococcus flavus.

A cDNA copy for carboxymethylcellulase (CMCase 1) of the yeast Cryptococcus flavus was cloned by screening an expression cDNA library with anti-CMCase 1 antibody. The sequence of the cDNA had an open reading frame of 1023 bp that encoded a preprotein of 341 amino acids with a molecular weight of 35,698. The putative precursor begins with a hydrophobic segment that possibly acts as a signal sequence for secretion, which is followed by a presumed prosequence and a sequence consistent with the N-terminal amino acid sequence of secreted CMCase 1. No potential N-glycosylation site was found in the sequence of putative pro-CMCase 1. Comparison of the deduced protein sequence shows that the C. flavus CMCase 1 is partially homologous to the Trichoderma reesei endoglucanase EGIII. Alignment of the cDNA copy and the chromosomal DNA showed seven putative introns of 45 to 134 bp. When introduced into E. coli, the cDNA directed the synthesis of CMCase 1 as seen by CMCase activity and Western blotting using anti-CMCase 1 antibody.

Amino Acid Sequence↗
PubMed1991

Expression of the Tn antigen on T-lymphoid cell line Jurkat.

Expression of the Tn antigen on a T-lymphoid cell line, Jurkat, was investigated using an anti-Tn monoclonal antibody, MLS 128. Immunoprecipitation or immunoaffinity chromatography of a lysate of Jurkat cells led to the isolation of a 120 kDa glycoprotein carrying the Tn antigen. This glycoprotein and leukosialin (CD43) were indistinguishable on SDS-PAGE and as to immunoreactivity with MLS 128. Leukosialin from an erythroid cell line, K562, exhibited no reactivity with MLS 128 despite that this leukosialin has several GalNAc alpha-Ser(Thr) structures. Pulse-chase experiments with the Jurkat leukosialin showed that newly synthesized leukosialin acquired the antigenecity after a lag of about 30 min, whereas incorporation of GalNAc into the leukosialin occurred earlier. These results indicate that the Tn antigen is expressed on leukosialin and that its epitopic structure is more complex than GalNAc alpha-Ser(Thr).

Amino Acid Sequence↗
PubMed1991

Characterization of mucin-type oligosaccharides with the sialyl-Le(a) structure from human colorectal adenocarcinoma cells.

Oligosaccharides with the sialyl-Le(a) structure have been isolated on an affinity column of a monoclonal antibody, MSW 113, from mucin-type glycoproteins derived from the surfaces of SW 1116 and LS 180 cells, and their secretions. The oligosaccharides were polydisperse with respect to molecular size, the oligosaccharides derived from glycoproteins in culture media being larger than those in cell lysates, as assessed by gel filtration. Some of the oligosaccharides were susceptible to degradation by endo-beta-galactosidase (E. freundii), as judged from the change in the gel filtration pattern. These results indicate that oligosaccharides with the sialyl-Le(a) structure derived from mucin-type glycoproteins produced by human colonic cancer cells are extremely large in size and complex in structure, and that some of them contain the poly-N-acetyllactosamine structure.

Adenocarcinoma↗
PubMed1991

Elucidation of an essential structure recognized by an anti-GalNAc alpha-Ser(Thr) monoclonal antibody (MLS 128).

To determine the epitopic structure for an anti-GalNAc alpha-Ser(Thr) (anti-Tn) monoclonal antibody, MLS 128, asialo-ovine submaxillary mucin was digested with various proteases, and the digests were fractionated by immunoaffinity column chromatography and high performance liquid chromatography. From the tryptic digest, a glycopeptide, GP-I, and five other glycopeptides, GP-1-5, were obtained as bound and unbound fractions, respectively, of the immunoaffinity column. By solid phase radioimmunoassaying, it was found that GP-I was strongly immunoreactive, whereas GP-1-5 were poorly immunoreactive. On treatment with V8 protease, GP-I was converted to two glycopeptides, one with poor reactivity and the other with intermediate reactivity. From the thermolysin digest, the smallest fragment, GP-II, was isolated, which was as strongly immunoreactive as GP-I. GP-II corresponded to a part of GP-I, its sequence being Leu-Ser*-Glu-Ser*-Thr*-Thr*-Gln-Leu-Pro-Gly, where asterisks denote amino acids to which an alpha-GalNAc residue is attached. Other anti-Tn monoclonal antibodies, NCC-LU-35 and CA 3239, showed essentially the same reactivity to these glycopeptides as MLS 128 did. The glycopeptides (GP-1-5), which exhibited poor immunoreactivity, contained various GalNAc-containing structures, such as GalNAc-Ser, GalNAc-Thr, GalNAc-Ser-(GalNAc)-Ser, and GalNAc-Thr-(GalNAc)-Thr. These results indicate that a glycopeptide including a cluster structure, Ser*-Thr*-Thr*, is an essential part of the epitope recognized by anti-Tn antibodies.

Amino Acid Sequence↗
PubMed1991

The Saccharomyces cerevisiae genes (CMP1 and CMP2) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B.

Saccharomyces cerevisiae genomic clones that encode calmodulin-binding proteins were isolated by screening a lambda gt11 expression library using 125I-labeled calmodulin as probe. Among the cloned yeast genes, we found two closely related genes (CMP1 and CMP2) that encode proteins homologous to the catalytic subunit of phosphoprotein phosphatase. The presumed CMP1 protein (62,999 Da) and CMP2 protein (68,496 Da) contain a 23 amino acid sequence very similar to those identified as calmodulin-binding sites in many calmodulin-regulated proteins. The yeast genes encode proteins especially homologous to the catalytic subunit of mammalian phosphoprotein phosphatase type 2B (calcineurin). The products of the CMP1 and CMP2 genes were identified by immunoblot analysis of cell extracts as proteins of 62,000 and 64,000 Da, respectively. Gene disruption experiments demonstrated that elimination of either or both of these genes had no effect on cell viability, indicating that these genes are not essential for normal cell growth.

Alleles↗
PubMed1991

Quantitative in vivo analysis of benzodiazepine binding sites in the human brain using positron emission tomography.

Central-type benzodiazepine binding sites were characterized in a single normal human subject, using positron emission tomography (PET) and the radiolabelled benzodiazepine antagonist, carbon-11 labelled flumazenil ([11C] Ro 15-1788). The subject was scanned using tracer alone and tracer plus 4 different concentrations of unlabelled Ro 15-1788, including one concentration of unlabelled Ro 15-1788, chosen to produce maximum displacement of [11C] Ro 15-1788 from specific binding sites. Concentrations of free, unmetabolized [11C] Ro 15-1788 in plasma were estimated using a simple extraction and ultrafiltration method. Radioactivity in the regional exchangeable pool in brain was estimated under non-saturation conditions from the ratio of radioactivity in brain to plasma, under saturation conditions and the kinetics of free ligand in plasma. The specific binding was, then, estimated by the difference between the total radioactivity in brain and exchangeable pool radioactivity. Scatchard analyses were performed to yield Bmax and Kd values under pseudo-equilibrium conditions, which was observed as an increase of specific binding/free with reduction in specific binding. In cerebral cortex and cerebellum, the Bmax values were about 62-73 nmol/l and the Kd values were 3.6-6 nM in the estimation of free ligand in plasma and 12-15 nM in the estimation of exchangeable pool in brain, as free in brain.

Adult↗
PubMed1991

Subsite structure of Saccharomycopsis alpha-amylase secreted from Saccharomyces cerevisiae.

The kinetic parameters (kcat/Km) and the cleaved-bond distributions for the hydrolysis of linear maltooligosaccharides Gn (3 less than or equal to n less than or equal to 9) by Saccharomycopsis alpha-amylase (Sfamy) secreted from Saccharomyces cerevisiae were determined at pH 5.25 and 25 degrees C. The subsite affinities of Sfamy were also evaluated from these data. The subsite structure of Sfamy is characteristic of the active site of an endo-cleavage type enzyme, consisting of internal repulsive sites with the catalytic residues and external attractive sites. Moreover, the pKa values of the catalytic residues were calculated from the pH dependence plot of the kinetic parameter (kcat/Km). The amino acid residues which contribute to the subsite affinities and the catalytic activity of Sfamy are proposed and compared with those of Taka-amylase A.

Kinetics↗
PubMed1991

Isolation and structural studies of human milk oligosaccharides that are reactive with a monoclonal antibody MSW 113.

We have determined the structures of six oligosaccharides isolated from human milk using a monoclonal antibody, MSW 113. The isolation involved affinity chromatography on a column of the immobilized monoclonal antibody and high-performance liquid chromatography. From the results of 500 and 600 mHz 1H NMR spectroscopy and fast atom bombardment-mass spectrometry their structures were deduced to be: [formula; see text] Two of these oligosaccharides, numbers 4 and 5, have not previously been described. All of them bound to MSW 113, but their reactivities are weaker than those of sialyl-Le(a) oligosaccharides. The results indicate that MSW 113 reacts with oligosaccharides with the mono- and disialyl-Le(a), and other sialyl type 1 structures.

Antibodies, Monoclonal↗
PubMed1991

Inhibition of membrane Ca2(+)-ATPase of Saccharomyces cerevisiae by mating pheromone alpha-factor in vitro.

Plasma membrane Ca2(+)-ATPase of Saccharomyces cerevisiae was solubilized and partially purified by calmodulin-affinity chromatography. The activity of Ca2(+)-ATPase isolated from MATa cells was inhibited by physiological concentrations of the mating pheromone alpha-factor in a dose-dependent manner. The enzyme prepared from a receptor-deficient sterile mutant cells (delta ste-2) was similarly inhibited by alpha-factor, but the enzyme from MAT alpha cells was resistant to the mating pheromone. We suggest that the inhibition may be involved in the alpha-factor-induced increase of Ca2+ uptake reaction of MATa cells.

Bacterial Proteins↗
PubMed1991

Blood-brain barrier transport of kynurenines: implications for brain synthesis and metabolism.

To evaluate the potential contribution of circulating kynurenines to brain kynurenine pools, the rates of cerebral uptake and mechanisms of blood-brain barrier transport were determined for several kynurenine metabolites of tryptophan, including L-kynurenine (L-KYN), 3-hydroxykynurenine (3-HKYN), 3-hydroxyanthranilic acid (3-HANA), anthranilic acid (ANA), kynurenic acid (KYNA), and quinolinic acid (QUIN), in pentobarbital-anesthetized rats using an in situ brain perfusion technique. L-KYN was found to be taken up into brain at a significant rate [permeability-surface area product (PA) = 2-3 x 10(-3) ml/s/g] by the large neutral amino acid carrier (L-system) of the blood-brain barrier. Best-fit estimates of the Vmax and Km of saturable L-KYN transfer equalled 4.5 x 10(-4) mumol/s/g and 0.16 mumol/ml, respectively. The same carrier may also mediate the brain uptake of 3-HKYN as D,L-3-HKYN competitively inhibited the brain transfer of the large neutral amino acid L-leucine. For the other metabolites, uptake appeared mediated by passive diffusion. This occurred at a significant rate for ANA (PA, 0.7-1.6 x 10(-3) ml/s/g), and at far lower rates (PA, 2-7 x 10(-5) ml/s/g) for 3-HANA, KYNA, and QUIN. Transfer for KYNA, 3-HANA, and ANA also appeared to be limited by plasma protein binding. The results demonstrate the saturable transfer of L-KYN across the blood-brain barrier and suggest that circulating L-KYN, 3-HKYN, and ANA may each contribute significantly to respective cerebral pools. In contrast, QUIN, KYNA, and 3-HANA cross the blood-brain barrier poorly, and therefore are not expected to contribute significantly to brain pools under normal conditions.

Animals↗