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T Ikenaka

Publications and source records attributed to T Ikenaka.

At least 55 records · Page 3Linked to original sources
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

Amino acid sequence of winged bean (Psophocarpus tetragonolobus (L.) DC.) chymotrypsin inhibitor, WCI-3.

The complete amino acid sequence of winged bean chymotrypsin inhibitor 3 (WCI-3) was determined by the conventional methods. WCI-3 consisted of 183 amino acid residues, but was heterogeneous in the carboxyl terminal region owing to the loss of one to four carboxyl terminal amino acid residues. The sequence of WCI-3 was highly homologous with those of soybean trypsin inhibitor Tia, winged bean trypsin inhibitor WTI-1, and Erythrina latissima trypsin inhibitor DE-3. One of the reactive site peptide bonds of WCI-3 was identified as Leu(65)-Ser(66), which was located at the same position as those of the other Kunitz-family leguminous proteinase inhibitors.

Amino Acid Sequence↗
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↗
PubMed1988

Amino acid sequence of a lysozyme (B-enzyme) from Bacillus subtilis YT-25.

The amino acid sequence of a lysozyme, (B-enzyme), from Bacillus subtilis YT-25 was determined by conventional methods. B-Enzyme comprised 117 amino acid residues and had a heterogeneous sequence in the amino-terminal region. The amino acid sequence of B-enzyme was different from those of all other lysozymes the sequences of which are known. However, the partial amino acid sequence of Ser(74) to Ser(97) of B-enzyme was homologous with that of the active-site region of hen egg-white lysozyme (Ser(36) to Ser(60], which includes one of the catalytic amino acids, Asp(52). It is interesting that B-enzyme has an amino acid sequence homologous with that of the gag protein p25 of the AIDS virus ARV-2.

Amino Acid Sequence↗
PubMed1988

A new trisaccharide sugar chain linked to a serine residue in bovine blood coagulation factors VII and IX.

A new trisaccharide sugar chain was identified in bovine blood coagulation factors VII and IX. A pentapeptide isolated from factor VII contained Ser-52, which could not be identified with a gas-phase sequencer, suggesting an unknown substituent on the serine residue (Takeya, H. et al. (1988) J. Biol. Chem., in press). The same results were obtained for a pentapeptide containing Ser-53 of factor IX. Component sugar analysis revealed that the peptide contained 1 mol of glucose and 2 mol of xylose. This sugar component was also confirmed by high-resolution fast atom bombardment mass spectrometric analysis of the pentapeptide. The trisaccharide was released from the peptides by means of beta-elimination reaction and its reducing end was coupled with 2-aminopyridine. The fluorescent pyridylamino (PA-) derivative of the trisaccharide was purified by gel-filtration and reversed-phase HPLC. The sugar composition of the PA-trisaccharide was found to be 2 mol of xylose and 1 mol of PA-glucose. These results indicate the existence of a (Xyl2)Glc-Ser structure in factors VII and IX.

Amino Acids↗
PubMed1988

Inspection of human salivary alpha-amylase action by its transglycosylation action.

The course of the action of human salivary alpha-amylase (HSA) on a substrate was examined taking advantage of its transglycosylation action. IG5 phi (IG-G-G-G-G-phi), IG4 phi (IG-G-G-G-phi), and GIG4 phi (G-IG-G-G-G-phi) were used as the substrates and p-nitrophenyl alpha-glucoside (GP, G-P) as the acceptor. HSA hydrolyzes IG5 phi, IG4 phi, and GIG4 phi to IG3 (IG-G-G) and G2 phi (G-G-phi), to IG3 and G phi (G-phi), and to GIG3 (G-IG-G-G) and G phi, respectively. In the presence of GP, a part of the glycon residues, IG3 and GIG3, were transferred to the acceptor to give IG4P (IG-G-G-G-P) and GIG4P (G-IG-G-G-G-P), respectively. Whenever the enzyme attacks the substrate, G phi or G2 phi is liberated in both transglycosylation and hydrolysis. The extent of transglycosylation can be, therefore, estimated from the molar ratio of the transfer product to the liberated aglycon, G phi or G2 phi. HPLC analysis of the reaction mixtures revealed that the value of IG4P/G phi in the digest of IG4 phi was nearly equal to that of GIG4P/G phi in the digest of GIG4 phi and these values were ten times larger than that of IG4P/G2 phi in the digest of IG5 phi. These data suggested that G phi residue would fall away from aglycon binding site more rapidly than G2 phi residue after the cleavage of the alpha-1,4-glycosidic linkage to offer GP more chance to attack to the activated glycon and also indicated that the space of the glycon binding site corresponds to three glucose residues.

Binding Sites↗
PubMed1987

Fluorescence method for the structural analysis of oligomannose-type sugar chains by partial acetolysis.

Fluorescence labeling was used in the analysis of partial acetolysis products of oligomannose-type sugar chains with five to nine mannose residues. The principle of the method was the pyridylamination of fragments obtained by the partial acetolysis of pyridylamino sugar chains and the identification of the fragments with an HPLC apparatus equipped with a fluorescence spectrophotometer. The method was tested by analysis of eight oligomannose-type sugar chains with known chemical structures and was found to be effective for analysis of branching structures with samples of 0.5 nmol.

Carbohydrate Sequence↗
PubMed1987

Identification method for twelve oligomannose-type sugar chains thought to be processing intermediates of glycoproteins.

A mixture of the pyridylamino (PA) derivatives of 12 oligomannose-type sugar chains was fractionated into five fractions (mannose5N-acetylglucosamine2-PA approximately mannose9N-acetylglucosamine2-PA) by size-fractionation HPLC with a MicroPak AX-5 column. Each fraction thus obtained was then analyzed by reversed-phase HPLC with a Cosmosil 5C18-P column. In this way, the 12 PA-oligomannose-type sugar chains were completely separated from each other. The method was used to identify the structures of oligomannose-type sugar chains of human C3, the third component of human complement.

Carbohydrate Sequence↗
PubMed1987

Participation of S-S loops in inhibitory activity of peanut protease inhibitor B-III.

A peanut Bowman-Birk (BBI) type protease inhibitors B-III has two regions, 1 and 2, homologous with each other. Each region contains three S-S loops and a reactive site in its outermost loop. The inhibitor was used to investigate the contribution of the S-S loops of BBI-type inhibitors to their inhibitory activity. Two steps of Edman degradation of the native inhibitor cleaved loop III (the innermost S-S loop) of region 1 of B-III, and the antichymotryptic activity of the first reactive site decreased to about 1/4 of that of native B-III. A third step of Edman degradation split loop II and the inhibitory activity at that site became extremely low (about 1/200 of the original value). These results suggest that protease inhibitor B-III maintains its active conformation by means of the three S-S loops and that the conformation is markedly changed by the splitting of loop II.

Amino Acid Sequence↗
PubMed1987

Chemical replacement of P1' arginine residue at the first reactive site of peanut protease inhibitor B-III.

The contribution of the P1' residue at the first reactive site of peanut protease inhibitor B-III to the inhibition was analyzed by replacement of the P1' Arg(11) with other amino acids (Arg, Ser, Ala, Leu, Phe, Asp) after selective modification of the second reactive site. The Arg derivative had the same trypsin inhibitory activity as the native inhibitor (Ki = 2 X 10(-9) M). The Ser derivative inhibited more weakly (Ki = 2 X 10(-8) M). The Ala and Leu derivatives inhibited trypsin very weakly (Ki = 2 X 10(-7) M and 4 X 10(-7) M, respectively), and the Phe and Asp derivatives not at all. These results suggest that the P1' arginine residue is best for inhibitory activity at the first reactive site of B-III, although it has been suggested that a P1' serine residue at the reactive site is best for inhibitory activity of Bowman-Birk type inhibitors.

Amino Acid Sequence↗
PubMed1987

Positions of disulfide bonds in riboflavin-binding protein of hen egg white.

Riboflavin-binding protein of hen egg white (egg-white RBP) comprised 219 amino acid residues and nine disulfide bonds. To identify the locations of these bonds, the native protein was oxidized with cyanogen bromide and digested with trypsin, thermolysin, and Staphylococcus aureus V8 protease. The cystine-containing peptides were isolated by HPLC. Amino acid analyses and amino acid sequence analyses of the reduced pyridylethylated derivatives of the cystine peptides showed that seven of the disulfide bonds were as follows: Cys(24)-Cys(73), Cys(57)-Cys(138), Cys(64)-Cys(110), Cys(99)-Cys(169), Cys(116)-Cys(134), Cys(103)-Cys(152), Cys(167)-Cys(202). The other two disulfide bonds were either Cys(5)-Cys(32) and Cys(33)-Cys(77) or Cys(5)-Cys(33) and Cys(32)-Cys(77).

Amino Acid Sequence↗
PubMed1987

Possible pathway for the processing of sugar chains containing xylose in plant glycoproteins deduced on structural analyses of sugar chains from Ricinus communis lectins.

The structures of sugar chains from two lectins in seeds of the castor bean (Ricinus communis) were identified. The sugar chains were liberated from the lectins by hydrazinolysis. After N-acetylation, the reducing-end residues of the sugar chains were coupled with 2-aminopyridine. The pyridylamino derivatives thus obtained were purified by gel filtration and HPLC. The structures of the purified derivatives were identified by component sugar analysis, stepwise exoglycosidase digestion, partial acetolysis, and 500 MHz 1H-NMR spectroscopy. A new processing pathway for sugar chains in plant glycoproteins was proposed on the basis of the structures of the sugar chains.

Carbohydrate Sequence↗
PubMed1987

Conversion of peanut trypsin-chymotrypsin inhibitor B-III to a chymotrypsin inhibitor by deimination of the P1 arginine residues in two reactive sites.

The deimination of the arginine residues in peanut trypsin-chymotrypsin inhibitor B-III caused the disappearance of its trypsin-inhibitory activity. Peanut protease inhibitor B-III was incubated with peptidylarginine deiminase, resulting in the conversion of 2.5 mol of arginine to citrulline and in the loss of its trypsin-inhibitory activity. However, the ability of the deiminated inhibitor to inhibit chymotrypsin was as strong as before. Structural analysis of the deiminated B-III indicated that the P1 arginine residues at both reactive sites, Arg(10) and Arg(38), were completely modified to citrulline by the action of peptidylarginine deiminase, and that the Arg(60) in the C-terminal region of B-III was partially deiminated. These residues seem to be exposed on the surface of the molecule. The P1' arginine residue at the first reactive site, Arg(11), was not deiminated at all.

Amino Acid Sequence↗
PubMed1987

Chemical replacement of P1' serine residue at the second reactive site of soybean protease inhibitor C-II.

The P1' Ser(50) at the second reactive site of soybean protease inhibitor C-II was replaced with arginine to confirm the contribution of this residue to the inhibition. The Arg derivative had less trypsin inhibitory activity (Ki = 1 X 10(-7) M) than the Ser derivative (Ki = 2 X 10(-8) M), in contrast to the results obtained from studies on peanut protease inhibitor B-III reported in the previous paper (J. Biochem. 101, 723-728 (1987)). These results suggest that each Bowman-Birk type inhibitor has an amino acid at the P1' position inherently best suited to maintaining its inhibitory activity, and that serine is not unique for the P1' amino acid in Bowman-Birk type inhibitors.

Amino Acids↗
PubMed1987

Studies on the active site of Taka-amylase A: its action on phenyl maltooligosides with a charge at their non-reducing-ends.

Five modified moltooligosaccharides, phenyl O-6-amino-6-deoxy-alpha-D- glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)- alpha-D-glucopyransoide (AG4P), phenyl O-(alpha-D-glucopyranosyluronic acid)-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-d-glucopyran osy l- (1----4)-alpha-D-glucopyranoside (CG4P), phenyl O-6-amino-6-deoxy-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyra nos yl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)- alpha-D-glucopyranoside (AG5P), phenyl O-(alpha-D-glucopyranosyluronic acid)-(1----4)-O-alpha-D-glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)- alpha-D-glucopyranoside (CG5P), and 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)-a lph a-D- glucopyranoside (FG4P), were prepared to examine the active site of Taka-amylase A (TAA) [EC 3.2.1.1, Aspergillus oryzae]. Phenyl alpha-maltotetraoside (G4P) was predominantly hydrolyzed by TAA to maltose and phenyl alpha-maltoside (G2P). While G2P, phenyl alpha-glucoside (GP), and phenol were liberated from AG4P in the ratio of 7:63:30. G4P, phenyl alpha-maltotrioside (G3P), G2P, and GP were liberated from G5P in the ratio of 1:20:73:6, but AG5P was almost completely hydrolyzed to modified maltotriose and G2P. On the hydrolysis of CG4P and CG5P, no remarkable change was observed except for a decrease in the relative reaction rates compared with G4P and G5P, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗