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Biomedical subjects

T Ikenaka

Publications and source records attributed to T Ikenaka.

At least 91 records · Page 5Linked to original sources
PubMed1985

Comparative study on amino acid sequences of Kunitz-type soybean trypsin inhibitors, Tia, Tib, and Tic.

The amino acid sequences of three variants of the Kunitz-type trypsin inhibitors, Tia, Tib, and Tic, obtained from some cultivars of soybean were determined by conventional methods. All three inhibitors consisted of 181 amino acid residues. The differences in the amino acid sequences are as follows: Tia E12 G55 Y62 H71 S74 M114 L120 P137 L176; Tib S F N R V I T V; Tic E. The amino acid sequences of Pro(60)-Ser(61) and Asp(154)-Asp(155)-Gly(156)-His(157) of Tia reported previously (Koide & Ikenaka (1973) Eur. J. Biochem. 32, 417-431) were amended to Ser(60)-Pro(61) and His(154)-Asp-Asp-Gly(157), respectively.

Amino Acid Sequence↗
PubMed1985

Structures of sugar chains of the third component of human complement.

Human C3, the third component of human complement, contained mannose and N-acetylglucosamine as sugar components. The sugar chains were liberated from the polypeptide chains by hydrazinolysis, and the free amino groups were N-acetylated. The reducing end residues of the sugar chains thus obtained were tagged with 2-aminopyridine, and the pyridylamino (PA-) derivatives of sugar chains were separated by high-performance liquid chromatography. The structures of purified PA-sugar chains were analyzed by a combination of stepwise exoglycosidase digestions, size determination by paper electrophoresis, methylation analysis, Smith degradation, and partial acetolysis. These results showed that C3 contained two high-mannose type sugar chains ranging from Man5GlcNAc2 to Man9GlcNAc2. Analyses of the sugar chains of alpha- and beta-chains of C3 indicated that the alpha-chain contained mainly Man8GlcNAc2 and Man9GlcNAc2, while the beta-chain contained mainly Man5GlcNAc2 and Man6GlcNAc2.

Acetates↗
PubMed1984

Differential assay of human pancreatic and salivary alpha-amylases in serum using a new fluorogenic substrate.

The difference in the mode action of human pancreatic and salivary alpha-amylases on 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)-O-alpha-D-glucopyranosyl-(1----4)-D-glucitol FG6R), a fluorogenic derivative of maltohexaitol, was found. The products of the enzymatic hydrolysis were analyzed by high-performance liquid chromatography (HPLC) in 8 min. FG6R was hydrolyzed by these enzymes to O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucose (FG3) and maltotriitol, or O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-D-glucose (FG4) and maltitol. Pancreatic alpha-amylase produced more FG4 than salivary alpha-amylase. Taking advantage of the differences in action of the two amylases, a differential alpha-amylase assay in serum was performed. The method is simple and rapid and can be used for routine clinical assays of alpha-amylases.

Chromatography, High Pressure Liquid↗
PubMed1984

Reexamination of the pyridylamination used for fluorescence labeling of oligosaccharides and its application to glycoproteins.

The pyridylamination reaction of sugar chains from glycoproteins was re-investigated to raise the total yield of pyridylamino sugars. Sugar chains of glycoproteins were released by hydrazinolysis, and free amino groups were N-acetylated. The sugar chains were coupled with 2-aminopyridine, and the pyridylamino derivatives thus obtained were purified by Sephadex G-15 column chromatography and analyzed by high performance liquid chromatography. In this study, conditions for the coupling reaction (temperature, time, pH, concentration of 2-aminopyridine, and amount of the reducing reagent) were re-investigated. Under the conditions established in the present study, the total recovery of pyridylamino derivatives of sugar chains of glycoproteins was about 70%, and 0.15 nmol of a glycoprotein was enough to detect the pyridylamino derivatives of sugar chains. The stability of sialyl and fucosyl linkages under the present conditions was also studied.

Acetylation↗
PubMed1984

Characterization of hen egg white- and yolk-riboflavin binding proteins and amino acid sequence of egg white-riboflavin binding protein.

White- and Yolk-riboflavin binding proteins were isolated from hen eggs, and characterized as to their chemical properties. White- and Yolk-RBPs had almost same amino acid compositions except for glutamic acid, but their carbohydrate compositions were different from each other. The complete amino acid sequence of White-RBP was determined by conventional methods. White-RBP comprised 219 amino acid residues, and the amino-terminus was pyroglutamic acid (pyrrolidonecarboxylic acid). Two amino acids, lysine and asparagine, were found at the fourteenth residue from the amino-terminus. Carbohydrate chains were linked to asparagine residues at positions 36 and 147. Both White- and Yolk-RBPs were phosphorylated. In White-RBP either six or seven of nine serine residues between Ser(185) and Ser(197) were phosphorylated. The amino acid sequences around phosphoserines showed that phosphorylation might occur at a serine residue in one of the following sequences; Ser-X-Glu or Ser-X-Ser(P).

Amino Acid Sequence↗
PubMed1984

Inhibition mechanism of a peanut trypsin-chymotrypsin inhibitor, B-III: determination of the reactive sites for trypsin and chymotrypsin.

Peanut inhibitor B-III was found to form two types of complexes with trypsin, T2I and TI, by gel filtration HPLC. Two cleaved peptide bonds, Arg(10)-Arg(11) and Arg(38)-Ser(39), in the trypsin modified inhibitor (TM-B-III*R*S) (J. Biochem. 93, 479-485 (1983] were resynthesized by the complex formation with 2 mol of trypsin. These results suggest that the two peptide bonds may be the reactive sites for trypsin. TM-B-III*R*S inhibited bovine trypsin as well as native B-III but had little chymotrypsin inhibitory activity. The two peptide bonds, Arg(10)-Arg(11) and Arg(38)-Ser(39), in B-III were cleaved partly by prolonged incubation with a catalytic amount of chymotrypsin. But gel filtration HPLC of the chymotrypsin-inhibitor complex showed the formation of only CI complex. Incubation of TM-B-III*R*S with an equimolar amount of chymotrypsin resulted in the resynthesis of only the Arg(10)-Arg(11) bond. These findings suggest that Arg(10)-Arg(11) may be a true reactive site for chymotrypsin. An inhibition mechanism of B-III against trypsin and chymotrypsin was proposed from the results obtained by the present studies.

Amino Acid Sequence↗
PubMed1983

Amino acid sequence of a trypsin-chymotrypsin inhibitor, B-III, of peanut (Arachis hypogaea).

The amino acid sequence of peanut trypsin-chymotrypsin inhibitor, B-III, was determined by conventional methods. The limited proteolysis of B-III with trypsin indicated the reactive sites of B-III for trypsin to be Arg(10)-Arg(11) and Arg(38)-Ser(39). Comparison of the established sequence of B-III with those of other Bowman-Birk type double-headed protease inhibitors indicated that B-III has four amino acid insertions and one amino acid deletion. It is especially interesting that the amino acid residue in the P1' position (No. 11) of the first reactive site for trypsin is arginine instead of serine, which seems to be conserved at the P1' position of the reactive sites of all Bowman-Birk type protease inhibitors.

Amino Acid Sequence↗
PubMed1983

Preparation of a new fluorogenic substrate of alpha-amylases and a simple alpha-amylase assay by HPLC.

A new substrate of alpha-amylases, O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, was prepared using dextrin as a starting material. Compared with other substrates so far reported, the fluorogenic substrate is unique in that it is resistant to exo-alpha-glucosidases due to the blocking group introduced into the non-reducing end glucose residue. The product of alpha-amylase digestion was rapidly separated from the substrate and was detected very sensitively by HPLC and a fluorescence detector. This method for alpha-amylase assay was also applied for determination of alpha-amylase in human serum.

Amylases↗
PubMed1983

Purification and characterization of the neutral endopeptidase from human kidney.

The presence of an endopeptidase hydrolyzing succinyl trialanine-p-nitroanilide [Suc(Ala)3-pNA] to Suc(Ala)2 and Ala-pNA in human kidney and its partial characterization have been reported (Ishida et al. (1981) Biochem. Int. 3, 239-246). This neutral metallo-endopeptidase was separated into two fractions (A and B) on Sephacryl S-300 and fraction B was further purified to an electrophoretically pure state. The fraction B enzyme had a molecular weight of 100,000 and was inhibited by metal chelators such as EDTA, o-phenanthroline and phosphoramidon, but not by serine protease inhibitors. The enzyme was found to hydrolyze peptide bonds preferentially at the amino sides of hydrophobic amino acids such as Leu and Phe, when its specificity was studied using insulin B chain and angiotensin I. Fraction A seems to be a tetramer of fraction B, judging from its molecular weight, pI, substrate specificity and immunological properties.

Amino Acids↗
PubMed1983

Amino acid sequences of trypsin-chymotrypsin inhibitors (A-I, A-II, B-I, and B-II) from peanut (Arachis hypogaea): a discussion on the molecular evolution of legume Bowman-Birk type inhibitors.

The amino acid sequences of four peanut protease inhibitors (A-I, A-II, B-I, and B-II) were determined by conventional methods and by comparison of peptide maps of their tryptic digests with that of B-III on HPLC. A-I, A-II, B-I, and B-III had the same amino acid sequence except for differences in their N-terminal regions. This suggests that the four inhibitors would be derived from an original inhibitor with a longer N-terminal amino acid sequence by proteolysis of its N-terminal region. But B-II possessed an extremely different amino acid sequence from those of the other peanut inhibitors and was thought to be biosynthesized from a gene different from that of the other inhibitors. A phylogenetic tree of legume double-headed inhibitors was constructed on the basis of the matrix of amino acid differences among their sequences. The double-headed inhibitors whose sequences have been determined were classified into four groups.

Amino Acid Sequence↗
PubMed1983

Amino acid sequences of two trypsin inhibitors from winged bean seeds (Psophocarpus tetragonolobus (L)DC.).

The trypsin inhibitor (WTI-1) purified from winged bean seeds is a Kunitz type protease inhibitor having a molecular weight of 19,200. WTI-1 inhibits bovine trypsin stoichiometrically, but not bovine alpha-chymotrypsin. The approximate Ki value for the trypsin-inhibitor complex is 2.5 X 10(-9) M. The complete amino acid sequence of WTI-1 was determined by conventional methods. Comparison of the sequence with that of soybean trypsin inhibitor (STI) indicated that the sequence of WTI-1 had 50% homology with that of STI. WTI-1 was separated into 2 homologous inhibitors, WTI-1A and WTI-1B, by isoelectric focusing. The isoelectric points of WTI-1A and WTI-1B were 8.5 and 9.4, respectively, and their sequences were presumed from their amino acid compositions.

Amino Acid Sequence↗
PubMed1983

Difference in transglycosylation between human pancreatic and salivary alpha-amylases.

Transglycosylation reactions of alpha-amylases from human pancreatic juice and saliva were examined by using O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose as a substrate and O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-1-deoxy-1-[(2-pyridyl)amino]-D-glucitol as an acceptor. The transfer reaction was estimated by quantitation of O-alpha-D-glucopyranosyl-(1 leads to 4)-1-deoxy-1-[(2-pyridyl)amino]-D-glucitol produced by the enzymes from the transfer products, because the acceptor was not hydrolyzed. The amount of O-alpha-D-glucopyranosyl-(1 leads to 4)-1-deoxy-1-[(2-pyridyl)amino]-D-glucitol in the digest with pancreatic alpha-amylase was six times that in the digest with salivary alpha-amylase at the stage when the substrate was completely consumed, and the difference increased gradually on further incubation. The phenomenon can be applied to differentiate the two alpha-amylases in human serum.

Carbohydrate Conformation↗
PubMed1982

Identification of the trimannosyl-chitobiose structure in sugar moieties of Japanese quail ovomucoid.

Ovomucoid isolated from egg white of japanese quail contained mannose, galactose, glucosamine, and sialic acid (8.6 : 1.3 : 6.6 : 0.5 mol/mol) as sugar components. Sugar chains were liberated from the polypeptide portion by hydrazinolysis, N-acetylated and fractionated on Bio-Gel P-2. The reducing ends of the thus obtained sugar moieties were pyridylaminated. Chemical and exoglycosidase digestion studies of the fluorescent pyridylamino derivative of a major sugar fraction indicated the following structure for one of the major sugar chains of japanese quail ovomucoid; Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc.

Animals↗
PubMed1982

Purification and characterization of protease inhibitors from peanuts (Arachis hypogaea).

Five protease inhibitors were isolated from peanut seeds and named A-I, A-II, B-I, B-II, and B-III. These inhibitors seemed to be Bowman-Birk type inhibitors judging from their low molecular weights and high cystine contents. All the inhibitors inhibited both bovine trypsin and chymotrypsin at ratios of 1:2 and 1:1, respectively, but not simultaneously. The complexes of the inhibitors and trypsin no longer inhibit chymotrypsin. On the other hand, their complexes with chymotrypsin inhibit trypsin with a slow release of chymotrypsin.

Amino Acids↗
PubMed1982

Studies on heterogeneity of Taka-amylase A: isolation of an amylase having one N-acetylglucosamine residue as the sugar chain.

Crystalline Taka-amylase A, prepared from Takadiastase, was fractionated into four fractions by DEAE-Sephacel and Concanavalin A-Sepharose column chromatography. The relative weight ratio of the fractions was 90 : 4 : 4 : 2. They had similar molecular weights (51,000), amino acid compositions, and hydrolytic activity against soluble starch, but different phenyl maltosidase activities and electrophoretic mobilities on polyacrylamide gel electrophoresis. Three of the fractions mainly had the high mannose type sugar chain with the sugar composition of Man5-GlcNAc2, but the other fraction had only one N-acetylglucosamine residue as the sugar chain. These results suggested that Taka-amylase A was heterogeneous both in the sugar portions and in the polypeptide portions.

Acetylglucosamine↗