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Biomedical subjects

K Omichi

Publications and source records attributed to K Omichi.

At least 37 records · Page 2Linked to original sources
PubMed1993

Identification of the characteristic amino-acid sequence for human alpha-amylase encoded by the AMY2B gene.

Human salivary and pancreatic alpha-amylases (HSA and HPA) are the respective gene products of the AMY1 and AMY2A genes. AMY2B is a newly found human alpha-amylase gene. The presence of the AMY2B gene product (HXA) in the urine of healthy humans was examined. A mixture of alpha-amylases that seemed to contain HXA, judging from the substrate specificity, was purified from urine of healthy volunteers by affinity adsorption on starch and then by ion-exchange chromatography. The mixture was reduced and S-alkylated, and the product was digested with trypsin. The digest was separated by reversed-phase HPLC. LVGLLDLALEKDYVR and LVGLLDLALEK, which were found in the digest, are peptides of HXA, but not of HSA and HPA. The detection of these characteristic peptides of HXA demonstrates the presence of HXA in the urine of healthy humans.

Amino Acid Sequence↗
PubMed1993

Structural analysis of O-linked sugar chains in human blood clotting factor IX.

Type and structural analysis of O-linked sugar chains in human blood clotting factor IX was performed by the pyridylamination method developed for O-linked sugar chains [Kuraya, N. & Hase, S. (1992) J. Biochem. 112, 122-126]. O- and N-linked sugar chains were released with hydrazine, and then N-acetylated, followed by pyridylamination. The type of sugar chain was determined by reducing-end analysis of the pyridylaminated (PA-) sugar chains. Sugar chains with PA-GalNAc at the reducing terminal and that with PA-Fuc [Nishimura, H. et al. (1992) J. Biol. Chem. 267, 17520-17525] were obtained besides known sugar chains with PA-Glc from the Xyl-Glc-Ser type and those with PA-GlcNAc from asparagine-linked sugar chains. The sugar chains with PA-GalNAc were identified as mono- and disialyl Gal beta 1-3GalNAc by two-dimensional HPLC mapping. The structure of the sugar chain with PA-Fuc was Neu5Ac alpha 2-6Gal beta 1-4GlcNAc beta 1-3Fuc, as determined by exoglycosidase digestion, methylation analysis, and Smith degradation.

Carbohydrate Conformation↗
PubMed1992

Studies on the active site of human alpha-amylases: examination of the third subsite S3' of the aglycone-binding site by control of substrate binding mode.

Modified maltooligosaccharides, IG-G-G-G-AG-G (IG: 6-deoxy-6-iodo-D-glucopyranose residue, G: D-glucopyranose residue, AG: 6-amino-6-deoxy-D-glucopyranose residue, -: alpha-1,4-glycosidic linkage), IG-G-G-G-AG-M (M: methyl), and IG-G-G-G-AG-phi (phi: phenyl) were prepared by the use of cyclodextrin glucanotransferase in order to examine the third subsite (S3') of the aglycone-binding site of human salivary and pancreatic alpha-amylases. Human alpha-amylases hydrolyzed the modified maltooligosaccharides to IG-G-G and G-AG-G, IG-G-G and G-AG-M, and IG-G-G and G-AG-phi. This implied that G, M, and phi fit into S3'. There was no difference in the rate parameters between the two enzymes. The Km values for the hydrolysis of IG-G-G-G-AG-G by both enzymes were the same as those for IG-G-G-G-AG-M, and twice those for IG-G-G-G-AG-phi. The results showed that S3' of the two enzymes has no affinity for the glucose residue and is not a subsite but a hydrophobic environment.

Bacillus↗
PubMed1992

Detection of human urinary alpha-amylase encoded by the AMY2B gene using a fluorogenic substrate, FG5P.

The existence of alpha-amylase (HXA) encoded by alpha-amylase gene AMY2B in healthy humans was examined using a fluorogenic substrate, FG5P (FG-G-G-G-G-P: FG, 6-deoxy-6-[(2-pyridyl)amino]-D-glucose residue; G, glucose residue; P, p-nitrophenyl residue; -, alpha-1,4-glycosidic bond). Chromatofocusing of urine from a healthy human was carried out. FG5P was digested with the fractions exhibiting alpha-amylase activity and each digest at an early stage was analyzed by HPLC. FG5P was hydrolyzed to FG3 (FG-G-G) and p-nitrophenyl alpha-maltoside (G-G-P), and to FG4 (FG-G-G-G) and p-nitrophenyl alpha-glucoside (G-P). The molar ratios of FG4 to FG3 (FG4/FG3) in the digests with basic fractions were larger than those in the digests of human pancreatic alpha-amylase (HPA, 1.11) and human salivary alpha-amylase (HSA, 0.51). Considering that the value for the AMY2B gene product with yeast (yHXA) is 1.88, a value of more than 1.11 implies that HXA exists. The amount of HXA was determined after removal of HSA on an anti-human salivary alpha-amylase antibody bound column. The FG4/FG3 values for six urine samples free from HSA were 1.23-1.26. Assuming that the FG4/FG3 value for HXA is the same as that for yHXA, the ratios of HXA and HPA were estimated to be 1:5.4-4.1. The results obtained showed that the AMY2B gene is usually expressed as HXA in healthy humans.

Carbohydrate Sequence↗
PubMed1991

Immunohistochemical, ultrastructural and biochemical studies of an amylase-producing breast carcinoma.

We describe a breast cancer with ectopic production of amylase, found in the patient's serum, urine and in the tumour. Clinically, serum amylase levels reflected both the progression of the disease and regression induced by various therapies. Using agarose gel electrophoresis and a wheat protein inhibitor assay, the predominant serum amylase appeared to be identical to pancreatic-type isoenzyme. However, the action mode analysis using a new fluorogenic substrate revealed that the serum contained non-salivary, non-pancreatic amylase. The tumour had microscopic features of invasive ductal carcinoma with some argyrophilic differentiation. The component cells stained positively for amylase, and ultrastructurally numerous secretory granules were seen.

Adult↗
PubMed1991

Examination of aglycone-binding site of human salivary alpha-amylase by means of transglycosylation reactions.

The active site of human salivary alpha-amylase is composed of tandem subsites (S3, S2, S1, S1',S2', etc.) geometrically complementary to several glucose residues, and the glycosidic linkage of the substrate is split between S1 and S1'. As a matter of convenience, the subsites to which the non-reducing-end part (glycone) and the reducing-end part (aglycone) of the substrate being hydrolyzed are bound are named the glycone-binding site (S3, S2, S1) and the aglycone-binding site (S1', S2'), respectively. The features of the aglycone-binding site of human salivary alpha-amylase were examined by means of transglycosylation reaction using phenyl alpha-maltoside (GG phi: G-G-phi) and its derivatives (GAG phi: G-AG-phi, GCG phi: G-CG-phi, AGG phi: AG-G-phi, and CGG phi: CG-G-phi) in which one of the glucose residues (G) has been converted to 6-amino-6-deoxy-glucose (AG) or glucuronic acid (CG) residue as the acceptor. A fluorogenic derivative of maltotetraose, p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D -glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG4P, FG-G-G-G-P), was used as the substrate. HSA catalyzed both hydrolysis of FG4P to FG3 (FG-G-G) and p-nitrophenyl alpha-glucoside (G-P) and transfer of the FG3 residue of FG4P to the acceptors. Transfer to GAG phi occurred more effectively than to GG phi. Transfers to GCG phi and CGG phi were less than to GG phi and very little transfer to AGG phi occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1991

Investigation of the active site of Bacillus macerans cyclodextrin glucanotransferase by use of modified maltooligosaccharides.

The active site of Bacillus macerans cyclodextrin glucanotransferase (CGTase) was examined by use of derivatives of phenyl alpha-maltopentaoside and phenyl alpha-glucoside as the substrates and acceptors, respectively. The active site of this enzyme is considered to be composed of tandem subsites (S4, S3, S2, S1, S1', S2', etc.) geometrically complementary to several glucose residues, and the alpha-1,4-glycosidic linkage of a substrate is split between S1 and S1'. The features of subsites S3 and S4 of the glycon binding site were estimated from the modes of the enzymatic action on phenyl alpha-maltopentaoside (G-G-G-G-G-phi; G, glucose residue; phi, phenyl residue; -, alpha-1,4-glycosidic bond) and its derivatives in which the CH2OH groups of the non-reducing-end glucose residues were converted to CH2I (IG-G-G-G-G-phi; IG, 6-deoxy-6-iodo-D-glucose residue), CH2NH2 (AG-G-G-G-G-phi; AG, 6-amino-6-deoxy-D-glucose residue), or COOH (CG-G-G-G-G-phi; CG, glucuronic acid residue). p-Nitrophenyl alpha-glucopyranoside (G-P; P, p-nitrophenyl residue) was used as an acceptor. HPLC analysis of the digests revealed that the CG residue of CG-G-G-G-G-phi was excluded from subsite S3, while it was accommodated in subsite S4. The Km and Vmax values for CG-G-G-G-G-phi were remarkably larger and smaller, respectively, than those for any other substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus↗
PubMed1990

Identification of a novel alpha-amylase by expression of a newly cloned human amy3 cDNA in yeast.

A novel amylase gene (amy3) that differs in nucleotide sequence from salivary amylase gene (amy1) and pancreatic amylase gene (amy2) has been described [Tomita et al., Gene 76 (1989) 11-18], but whether this gene can ever code for an active enzyme has not been shown. We prepared cDNA of this gene from an mRNA obtained from lung carcinoid tissue, and expressed it in Saccharomyces cerevisiae under the control of an acid phosphatase promoter. The product was secreted into culture media, and showed enzymatic activity, demonstrating that this novel alpha-amylase gene (amy3) can code for a functional isozyme. We purified this enzyme, and compared its biological properties with those of salivary and pancreatic human amylases similarly expressed in yeast. We observed that the novel amylase isozyme is more heat-sensitive than others, and that its substrate specificity is different from the other two isozymes.

Acid Phosphatase↗
PubMed1990

Differentiation of human non-salivary, non-pancreatic alpha-amylase from salivary and pancreatic alpha-amylases by use of FG5P.

Human non-salivary, non-pancreatic alpha-amylase (yHXA) is the gene product of a newly found human alpha-amylase gene expressed in yeast. Its mode of action on a fluorogenic derivative of p-nitrophenyl alpha-maltopentaoside, FG5P (FG-G-G-G-G-P), was examined at various pH values to elucidate the difference between yHXA and pancreatic or salivary alpha-amylase. The product analysis of the digests by HPLC showed that the enzyme hydrolyzed FG5P to FG3 (FG-G-G) and p-nitrophenyl alpha-maltoside (G-G-P) and to FG4(FG-G-G-G) and p-nitrophenyl alpha-glucoside (G-P), and the ratio of the two reactions changed with pH. The three enzymes differed from each other in the mode of action at pH 5.5. The molar ratio of FG4 to FG3 in the digest with yHXA was the largest. This suggested that the expression of the new gene in human can be detected by the use of FG5P as the substrate in the alpha-amylase assay.

Binding Sites↗
PubMed1989

Actions of three human alpha-amylases expressed in yeast on modified substrates and the amino acid residues causing their different actions.

The mode of action of an alpha-amylase (yHXA) which was the gene product of a newly found human alpha-amylase gene expressed in yeast on synthetic substrates was compared with those of the gene products (yHSA and yHPA) of human salivary and pancreatic alpha-amylase gene in yeast. The substrates used were phenyl alpha-maltopentaoside (G5 phi) and its derivatives in which the CH2OH groups of the non-reducing-end glucose residues were converted to CH2NH2 (AG5 phi), COOH (CG5 phi), or CH2I (IG5 phi). The digests were subjected to HPLC to determine the amounts of products. The HPLC analysis revealed that yHXA and yHSA bound G5 phi to their active sites in similar manners to give the same products, while yHPA hydrolyzed it in a different way. Modifications of the non-reducing-end glucose of G5 phi caused change of the binding mode to the active sites of the enzymes. AG5 phi and CG5 phi were hydrolyzed by the enzymes to give more phenyl alpha-glucoside (G phi) and less phenyl alpha-maltoside (G2 phi), while IG5 phi gave more G2 phi and less G phi, compared with G5 phi. The substrate binding mode of yHXA changed more extensively than that of yHSA. The results suggested that there exists an amino acid replacement between yHXA and yHSA. The amino acid residues replaced are neither acidic nor basic, are located in subsite S3, and interact with the CH2OH residue of the non-reducing-end glucose residue of G5 phi.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Alpha-amylase assay with use of a benzyl derivative of p-nitrophenyl alpha-maltopentaoside, BG5P.

p-Nitrophenyl O-(6-O-benzyl)-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)-alpha-D-glucopyranoside (BG5P) is hydrolyzed by both human salivary alpha-amylase (HSA) and human pancreatic alpha-amylase (HPA) to O-(6-O-benzyl)-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl- (1----4)-alpha-D-glucopyranose (BG3) and p-nitrophenyl alpha-maltoside (G2P). Glucoamylase and alpha-glucosidase cannot hydrolyze BG5P because of the modification of the OH group of the 6-position of the non-reducing-end glucose residue with the benzyl group. Taking advantage of these characteristics of the substrate, BG5P, we developed a method to assay the total alpha-amylase activity in human fluids using glucoamylase and alpha-glucosidase as the coupled enzymes. This method is simple and can be used as the standard method for routine clinical assays of alpha-amylase activity.

Chromatography, High Pressure Liquid↗
PubMed1988

Synthesis of p-nitrophenyl 6(5)-O-benzyl-alpha-maltopentaoside, a substrate for alpha amylases.

p-Nitrophenyl alpha-maltopentaoside, having a benzyl group on O-6 of the terminal (nonreducing) D-glucosyl group was prepared by use of a reductive ring-opening reaction. Highly regioselective reduction of p-nitrophenyl O-(2,3-di-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl)-(1----4)- tris[O-(2,3,6-tri-O-benzoyl-alpha-D-glucopyranosyl)-(1----4)]-2,3,6-tri- O- benzoyl-alpha-D-glucopyranoside by dimethylamine-borane and p-toluenesulfonic acid, followed by debenzoylation, gave p-nitrophenyl O-(6-O-benzyl-alpha-D-glucopyranosyl)-(1----4)-tris[O-alpha-D-glucopyran osyl- (1----4)]-alpha-D-glucopyranoside. An experiment was done on the mode of action of human pancreatic and salivary alpha amylases on this derivative. The compound is suitable as a substrate for the assay of alpha amylase when used with glucoamylase and alpha-D-glucosidase as coupling enzymes.

Carbohydrate Conformation↗
PubMed1988

Separation of human salivary alpha-amylase isozymes by high-performance liquid chromatography with a continuous monitor system of the activity.

Human salivary alpha-amylase isozymes were rapidly separated from each other by high-performance liquid chromatography with a postcolumn assay. The eluate from the HPLC column was mixed continuously with an intramolecularly quenched fluorescent substrate, p-nitro-phenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside delivered by a pump. The mixture was incubated in a reaction coil, and the fluorescence intensity was continuously measured by a fluorescence detector. The assay was based on the marked increase in fluorescence with the enzymatic cleavage of the glycosidic bond of the substrate that links the fluorogenic and quenching moieties.

Chromatography, High Pressure Liquid↗
PubMed1988

Simple assay for sialyltransferase activity with a new fluorogenic substrate.

A new fluorogenic acceptor for sialyltransferase, 2-[(2-pyridyl)amino]ethyl O-beta-D-galactopyranosyl-(1----4)-beta-D-glucopyranoside, was prepared from lactose as a starting material. Sialyltransferase activity was assayed by incubation of the enzyme with the acceptor and CMP-N-acetylneuraminic acid, separation of the fluorogenic sialylated product from the enzymatic reaction mixture by HPLC, and measurement of the product. Compared to assays so far reported that use radioactive substrates, this assay is simple and rapid. This method was used to assay sialyltransferase activity in human serum.

Chromatography, Gas↗
PubMed1988

Investigation of the active site of human salivary alpha-amylase from the modes of action on modified maltooligosaccharides.

The modes of action of two isozymes of human salivary alpha-amylase on phenyl alpha-maltopentaoside, phenyl alpha-maltotetraoside, and their derivatives which have an iodo or an amino or a carboxyl group at their first or penultimate glucopyranosyl residues from the non-reducing-end were examined. It is conceivable that the active site of this enzyme is composed of tandem subsites (S4,S3,S2,S1,S1',S2', and S3') geometrically complementary to several glucose residues, and that the glucosidic bonds of the substrates are split between S1 and S1'. Product analysis of each digest strongly suggested the presence of a hydrophobic amino acid residue at subsite S3 in the active site of the enzyme. No difference in the modes of action on the substrates was found between the two isozymes, indicating that the three-dimensional structures of their active site areas are, at the least, similar.

Binding Sites↗
PubMed1988

A comparison of the modes of actions of human salivary and pancreatic alpha-amylases on modified maltooligosaccharides.

The actions of three isozymes of human pancreatic alpha-amylase (HPA) on phenyl alpha-maltopentaoside, phenyl alpha-maltotetraoside, and their derivatives which have an iodo, an amino, or a carboxyl group at their first or penultimate glucopyranosyl residue from the non-reducing-end were examined. The results revealed that there was no difference in the actions of the three isozymes on the modified substrates and suggested the presence of five subsites (S3, S2, S1, S1', and S2') and a hydrophobic amino acid residue at subsite S3 in the active site of HPA. As compared with the action of human salivary alpha-amylase (HSA) on the same substrates, HPA had a tendency to release more phenyl alpha-glucoside from every substrate; however, an iodo, an amino, and a carboxyl group of the substrates had the same effects on the binding modes of the substrates to the active site of HPA as seen in the case of the salivary enzyme. This result indicates that the three-dimensional structures of the active sites of both alpha-amylases are quite similar except for some minor changes at subsites S3 and S2'.

Amylases↗