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An unidentified ninhydrin-positive substance of acidic nature was found in the serum of uremic patients. This substance was isolated from hemodialysate by the methods of ion-exchange chromatography, gel-filtration and paper electrophoresis, and identified as beta-aspartylglycine by amino acid analysis, N-terminal amino acid determination and comparison with authentic sample synthesized in this laboratory. The quantitative determination of beta-aspartylglycine in serum revealed that the serum concentrations of beta-aspartyl-glycine in uremic patients increased much higher than those in normal subjects. The toxicity of beta-aspartylglycine in mice with acute renal failure induced by uranyl acetate was investigated. The mice given more than 1,0 g/kg body weight of beta-aspartylglycine showed behavioral alterations: low response to the stimuli and low activity, and some mice died by the injection of 4.0 g/kg body weight of the peptide. These results suggested that beta-aspartyl-glycine might be a possible factor which influences the development of uremic toxaemia.

The structures of the sugar chains of hen yolk riboflavin-binding protein (RBP) were established. Asparagine-linked sugar chains of yolk-RBP were liberated by hydrazinolysis. Free amino groups of the sugar chains were acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine. Fluorescent pyridylamino (PA-) derivatives of the sugar chains were purified by gel-filtration and reversed-phase HPLC. Seven PA-sugar chains were isolated, and the structure of each was determined by composition analysis, sequential exoglycosidase digestion, methylation analysis, and 500-mHz 1H-NMR spectroscopy. These analyses showed that the main sugar chains had sialylbiantenna and sialyltriantenna structures. PA-sugar chains of plasma-RBP were also isolated, and the structures of the PA-sugar chains of yolk- and plasma-RBPs were compared as to their elution patterns on anion-exchange chromatography and reversed-phase HPLC. The plasma RBP had almost the same sugar chains as the yolk RBP did, indicating that sugar chains are not modified during incorporation into the oocyte.

The di- and trisaccharide sequences and their conjugates with serine recently identified in the first epidermal growth factor-like domain of bovine and human blood-clotting factors VII, IX, and protein Z were synthesized in order to elucidate their biological function. The disaccharide α-D-Xylp(1→3)-D-Glcp was prepared from 2,3,4-tri-O-benzyl-α-D-xylopyranosyl fluoride and allyl 2,4,6-tri-O-acetyl-α-D-grucopyranoside (9a) using tin(II) chloride and silver perchlorate as glycosylating reagents. The trisaccharide α-D-Xylp(1→3)-α-D-Xylp(1→3)-D-Glcp was prepared from O-(2,3,4-tri-O-benzyl-α-D-xylopyranosyl)-(1→3)-2,4-di-O-benzyl-α,β-D-xylopyranosyl fluoride and 9a with the same reagents. Their conjugates with serine, α-D-Xylp(1→3)-β-D-Glcp(1→3)-L-Ser and α-D-Xylp(1→3)-α-D-Xylp(1→3)-β-D-Glcp(1→3)-L-Ser, were synthesized by the glycosylation of N-benzyloxycarbonyl-L-serine benzyl ester with the corresponding glycosyl trichloroacetimidates of the above saccharides, followed by removal of the protecting groups. T...

The di- and trisaccharide sequences and their conjugates with serine recently identified in the first epidermal growth factor-like domain of bovine and human blood-clotting factors VII, IX, and protein Z were synthesized in order to elucidate their biological function. The disaccharide α-D-Xylp(1→3)-D-Glcp was prepared from 2,3,4-tri-O-benzyl-α-D-xylopyranosyl fluoride and allyl 2,4,6-tri-O-acetyl-α-D-grucopyranoside (9a) using tin(II) chloride and silver perchlorate as glycosylating reagents. The trisaccharide α-D-Xylp(1→3)-α-D-Xylp(1→3)-D-Glcp was prepared from O-(2,3,4-tri-O-benzyl-α-D-xylopyranosyl)-(1→3)-2,4-di-O-benzyl-α,β-D-xylopyranosyl fluoride and 9a with the same reagents. Their conjugates with serine, α-D-Xylp(1→3)-β-D-Glcp(1→3)-L-Ser and α-D-Xylp(1→3)-α-D-Xylp(1→3)-β-D-Glcp(1→3)-L-Ser, were synthesized by the glycosylation of N-benzyloxycarbonyl-L-serine benzyl ester with the corresponding glycosyl trichloroacetimidates of the above saccharides, followed by removal of the protecting groups. T...

An alpha-mannosidase was purified from the magnum section of Japanese quail oviduct by ammonium sulfate precipitation, DEAE-Sephacel chromatography, Sephacryl S-300 chromatography, mannan-Sepharose 4B chromatography, and hydroxyapatite chromatography. The purified alpha-mannosidase (referred to as neutral alpha-mannosidase) showed a single band on polyacrylamide gel with or without sodium dodecyl sulfate. Its molecular weight was found to be 330,000 by gel chromatography. Neutral alpha-mannosidase hydrolyzed p-nitrophenyl alpha-D-mannopyranoside and the pyridylamino derivative of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc (Km value was 3 mM). Mannosyl alpha 1-2 linkages in the pyridylamino derivative of Man alpha 1-2 Man alpha 1-6(Man alpha 1-2Man alpha 1-3)Man alpha 1-6(Man alpha 1-2Man alpha 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc were hardly hydrolyzed. Its optimum pH was found to be 7.0. The activity of the enzyme was activated by CO2+, and was potently inhibited by Cu2+, Hg2+, swainsonine, and 1-deoxymannojirimycin.

Singly modified soybean trypsin inhibitors (STIs), Tia* [Tia cleaved at Arg(63)-Ile(64)] and Tib* [Tib cleaved at Arg(63)-Ile(64)], were prepared by limited proteolysis with trypsin at pH 3.0. These singly modified inhibitors were further modified to yield doubly modified inhibitors, Tia** and Tib**, by limited proteolysis with subtilisin BPN', which cleaved the Met(84)-Leu(85) bonds of Tia* and Tib*, respectively. The doubly modified inhibitors could be separated into two parts: protein moiety A and peptide moiety a (IRFIAEGHPLSLKFDS-FAVIM) for Tia**, and protein moiety B and peptide moiety b (IRFIAEGNPLRLKFDS-FAVIM) for Tib**. These protein and peptide moieties showed no trypsin inhibitory activities alone. However, the inhibitors can be reconstituted through the mutual exchange of the protein and peptide moieties isolated from STIs. The reconstituted inhibitor which has tyrosine at position 62 and histidine at position 71 shows the highest inhibitory activity. Its Ki value for bovine trypsin is around 10(-10) M, which is almost the same as that of Tia for bovine trypsin. The inhibitor possessing either tyrosine at position 62 or histidine at position 71 exhibits a Ki value of around 10(-9) M, which is between those of Tia and Tib. The inhibitor having phenylalanine and asparagine at positions 62 and 71, respectively, shows the weakest inhibitory activity of around 10(-8) M similar to that of Tib for bovine trypsin.

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)

We have cloned and characterized the cDNA coding for a major component of cellulase, endoglucanase (FI-CMCase), produced by Aspergillus aculeatus. The cDNA was isolated from a A. aculeatus cDNA library using synthetic oligonucleotide mixtures that correspond to the internal amino acid sequence of the mature FI-CMCase protein. Nucleotide sequence analysis of the cloned cDNA insert revealed a 711 bp open reading frame that encoded a protein of 237 amino acid residues. The primary structure of FI-CMCase deduced from the nucleotide sequence of cDNA agreed with that found by amino acid sequencing of peptide fragments obtained by digestion with several proteinases and cyanogen bromide cleavage. There may be a signal peptide sequence of 16 amino acid residues at the N-terminus. The molecular mass of the mature protein calculated from the cDNA is 24002 daltons, which compares favorably with molecular mass estimates of purified FI-CMCase obtained from SDS-PAGE (25000 Da). No distinct homology was found between the amino acid sequence of FI-CMCase and known cellulase sequences of other microorganisms. This study is the first example of cDNA cloning of an endoglucanase from the genus Aspergillus.

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.

The beta-elimination and nucleophile addition reactions of the substituted serine and threonine residues were studied using several synthesized fluorescence-labeled phosphopeptides and a salmon egg polysialoglycoprotein (PSGP). The reagents used were 1 M CH3SH-0.43 M NaOH, 1 M NaBH4-0.1 M NaOH, 1 M CH3NH2-0.1 M NaOH, and 1 M Na2SO3-0.1 M NaOH. The beta-elimination reaction of a phosphoserine peptide, Gly-Ser(PO4)-Glu-AEAP, was about 20 times faster than that of the corresponding phosphothreonine peptide. The carboxyl-side amino acid of the phosphoamino acids in peptides greatly affected the beta-elimination rate. The beta-elimination reaction rates of O-glycosyl serine and threonine in the polysialoglycoprotein were similar and were about a half of that of the phosphoserine peptide. The rates of addition of the three nucleophiles and hydrogen to alpha-aminoacrylic acid (beta-elimination product of substituted serine) in the peptide decreased in the order of CH3SH, Na2SO3, CH3NH2, and H2(NaBH4), and the addition to alpha-aminocrotonic acid (beta-elimination product of substituted threonine) in the order of Na2SO3, CH3NH2, CH3SH, and H2. These results indicated that sulfite is the most recommended nucleophile because of its high addition rate. If sulfite addition is carried out in the presence of NaBH4, sugar chains can be released as alditols, converting the sugar-attaching amino acids to beta-sulfoamino acids.

Two proteins that act as alpha-amylase inhibitors, Haim I and Paim I, were crystallized and preliminary X-ray diffraction studies on them were carried out. We also sequenced Haim I prepared from Streptomyces griseosporeus YM-25 and confirmed that it is composed of 78 amino acid residues. Crystals of Haim I were grown from ammonium sulfate solution mixed with ethanol by the vapor diffusion technique. The crystals grew as hexagonal bipyramids and diffracted X-rays beyond 2.0 A resolution. They belong to the space group P6(1)22 (or P6(5)22) with unit cell dimensions of a = b = 36.7 A, c = 192.4 A, and contain one molecule per asymmetric unit. Paim I, a protein of 39 amino acid residues produced by Streptomyces corchorusii, was crystallized under similar conditions to Haim I. The crystals diffracted X-rays beyond 2.5 A. They belong to the space group P4(1)2(1)2 (or P4(3)2(1)2) with unit cell dimensions of a = b = 65.4 A, c = 96.1 A, and contain three molecules per asymmetric unit.

The concentrations of free fucose and other sugars in urine of cancer patients and healthy subjects were analyzed by high-performance liquid chromatography. After the urine samples were dried, we coupled the sugars in the residue with 2-aminopyridine to be detected as fluorescent derivatives. We then analyzed the pyridylamino derivatives of the sugars with an anion-exchange column and borate buffer. The difference between cancer patients and healthy subjects for the mean concentrations of fucose corrected for creatinine was significant (P less than 0.001). We checked the relationship between the concentrations of other sugars and the presence of cancer. This method is highly sensitive, and neither a cleanup procedure before labeling nor purification before injection into the column is needed. Not only fucose but also other sugars can be detected simultaneously, so this method should be useful for studying any changes in sugars in urine in various diseases.

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.

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)

beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene (beta-GalMNT), a specific inhibitor of beta-galactosidase, was isolated as crystals by HPLC and its chemical and physicochemical characteristics were examined. Aspergillus oryzae beta-galactosidase was inactivated by the compound. We studied the inhibition mechanism in detail. The inhibitor was hydrolyzed by the enzyme to p-nitroaniline and an active intermediate (beta-galactopyranosylmethyl carbonium or beta-galactopyranosylmethyldiazonium), which inactivated the enzyme. The efficiency of inactivation of the enzyme (the ratio of moles of inactivated enzyme to moles of beta-GalMNT hydrolyzed by the enzyme) was 3%; the efficiency of Escherichia coli beta-galactosidase was 49%. In spite of the low efficiency, the rate of inactivation of A. oryzae enzyme was not very different from that of the E. coli enzyme, because the former hydrolyzed beta-GalMNT faster than the latter did. A. oryzae beta-galactosidase was also inactivated by p-chlorophenyl, p-tolyl, and m-nitrophenyl derivatives of beta-galactopyranosylmethyltriazene. However, E. coli beta-galactosidase was not inactivated by these triazene derivatives. The results showed that the inactivation of A. oryzae and E. coli beta-galactosidases by beta-GalMNT was an enzyme-activated and active-site-directed irreversible inactivation. The possibility of inactivation by intermediates produced nonenzymatically was ruled out for E. coli, but not for the A. oryzae enzyme.

Ten oligomannose-type sugar chains (ManGlcNAc2-Man5GlcNAc2) were prepared from various glycoproteins and fluorescence labeled with 2-aminopyridine. The fluorescent pyridylamino (PA)-sugar chains were first separated into five fractions according to their molecular sizes by HPLC on a TSK gel Amide-80 column. Each fraction was then separated into the component PA-sugar chains by reversed-phase HPLC on a Capcell Pak C18 column according to their chemical structures. The method is useful for studying the substrate specificities of alpha-mannosidases with Man5GlcNAc2-PA as a substrate.

We reported the presence of a new trisaccharide composed of two xylose and reducing terminal glucose residues linked to serine residues of bovine blood clotting factors VII and IX (Hase, S., Kawabata, S., Nishimura, H., Takeya, H., Sueyoshi, T., Miyata, T., Iwanaga, S., Takao, T., Shimonishi, Y., and Ikenaka, T. (1988) J. Biochem. (Tokyo) 104, 867-868). The present paper describes the detailed structural analysis of the trisaccharide. Glycopeptides were prepared from bovine factor IX by digestion with Pronase followed by purification by column chromatography. The trisaccharide was released from the protein by the beta-elimination reaction with hydrazine, and the reducing end of the sugar chain was tagged with 2-aminopyridine. The fluorescent pyridylamino derivative of the trisaccharide was purified by gel filtration and reversed-phase high performance liquid chromatography. The glycopeptides and pyridylamino-trisaccharide thus obtained were subjected to methylation study, 500-MHz 1H nuclear magnetic resonance spectroscopy, and periodate oxidation. Glucose and xylose belong to the D series by high performance liquid chromatography on a chiral column. From the results, the structure of the trisaccharide is proposed as: D-Xyl p alpha 1-3-D-Xyl p alpha 1-3-D-Glcp beta 1-O-Ser-53.

Recently, we determined the complete amino acid sequence of bovine factor VII (Takeya, H. et al. (1988) J. Biol. Chem. 263, 14868–14877). In the course of the studies, we found an unknown serine derivative at position 52 in the first epidermal growth factor-like domain of factor VII. A pentapeptide isolated from the S-aminoethylated factor VII contained Ser-52, which could not be identified with a gas-phase sequencer. The same results were also obtained for a pentapeptide containing Ser-53 of factor IX and protein Z. 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 s-elimination reaction and its reducing end was identified as pyridylamino-glucose. These results indicate the existence of a (Xy12)Glc-Ser structure in factors VII, IX and protein Z. Similar results were obtained for human factors VII, IX and protein Z. This is the first report of a (Xy12)-Glc-Ser structure in glycoproteins to our knowledge. The presence of the unique trisaccharide structure in factors VII, IX and protein Z leads us to anticipate its biological role in the tissue factor pathway.

(1990). Improved Method for Fluorescence Labeling of Sugar Chains with Sialic Acid Residues. Agricultural and Biological Chemistry: Vol. 54, No. 8, pp. 2169-2170.

A modification of the existing methods for measuring hydroxylysine, galactosylhydroxylysine, and glucosylgalactosylhydroxylysine is described. The method is based on analysis with an automated amino acid analyzer using a conventional separation system for basic amino acids. The prior removal of acidic and neutral amino acids was necessary. This was achieved by passing an alkaline hydrolysate of collagen through a column of Amberlite CG-120, Type II (H+) and washing the column with 8% aqueous pyridine. A basic fraction containing the hydroxylysine compounds was then recovered from the column by elution with 3 M NH4OH. Model experiments showed that hydroxylysine and its glycosides could be analyzed with an hour and that recoveries exceeded 90%. This method was applied to human tissues to investigate whether the dermal scar is different in collagen composition from normal skin. With the limited number of samples analyzed, the data suggested that long-standing scar tissues reverted to a composition similar to that of normal skin. The composition of hydroxylysine-linked carbohydrate units is also discussed on the basis of the age-related change.

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.

Seven proteinase inhibitors were isolated from winged bean seeds by ion-exchange chromatographies. These inhibitors had molecular weights of around 20,000, included four half-cystine residues, and were Kunitz-type inhibitors. Two (WTI-2 and 3) inhibited bovine trypsin strongly and four (WCI-1, 2, 3, and 4) inhibited bovine alpha-chymotrypsin, but in different ways. One mole of WCI-2 or -3 could inhibit 2 mol of alpha-chymotrypsin. The remaining inhibitor (WTCI-1) could bind both bovine trypsin and alpha-chymotrypsin at the molar ratio of 1:1, but not simultaneously. All four chymotrypsin inhibitors cross-reacted with rabbit anti-WCI-3 serum, while the other inhibitors did not.

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)

A chitinase was purified from the culture filtrate of Streptomyces erythraeus (SE). The enzyme (SE chitinase) has a molecular weight of 30,000 and pI 3.7, and shows optimal activity at pH 5.0 with an optimal ionic strength of less than 0.2 M NaCl. SE chitinase could hydrolyze chitin and its derivatives, but could not hydrolyze cell walls of Micrococcus lysodeikticus. The substrate specificity of SE chitinase was compared with those of hen egg white (HEW) and SE lysozymes. The binding mode of the chitinase to substrates was investigated using chitooligosaccharides and their derivatives. The results showed that the binding mode of SE chitinase to the substrate is similar to that of HEW and SE lysozymes.

The reducing end sugar of an oligosaccharide and 2-aminopyridine were linked by means of reductive amination with sodium cyanoborohydride. The fluorescent derivative of the oligosaccharide thus obtained, which had a positive charge, was subjected to two-dimensional paper electrophoresis. In the first direction, the sugar derivative moved according to its degree of polymerization, and in the second direction, it moved according to the structure of the borate complex. In this way fluorescent derivatives of saccharides were mapped on a sheet of paper. The method was applied to some known mono- and oligosaccharides and to the saccharides obtained by nitrous deamination of the oligosaccharide portions of glycoproteins (fetuin, Take-amylase A, and ovalbumin). The fingerprints thus obtained were characteristic of the chemical structures of the original oligosaccharides.

The trypsin and chymotrypsin inhibitory activities of two families of inhibitors, the Kunitz family and the Bowman–Birk family, were screened for in seeds of 34 leguminous species by gel filtration. Results were compared with the morphological classification of leguminous plants. There seemed to be a relationship between the inhibitors found and the evolution of leguminous plants. That is, the seeds of the more primitive leguminous species contained mainly the Kunitz family inhibitors and those of the more advanced ones had the Bowman–Birk family inhibitors. Results suggested that the Kunitz family inhibitors in leguminous seeds have gradually been replaced by the Bowman–Birk family inhibitors in the process of evolution.

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.

Four proteinase inhibitors, A-II, A-III, B-I, and B-II, were isolated from seeds of Albizzia julibrissin (silk tree) of the subfamily Mimosoideae, which is often regarded as the most primitive group of the Leguminosae plants. They were all of the high-molecular weight type (21,600 for A-II and A-III, and 19,000 for B-I and B-II), and composed of two polypeptide chains, linked together by a disulfide bond. A-II (A-III) inhibited bovine trypsin and alpha-chymotrypsin probably at an identical site. B-I (BII) inactivated bovine alpha-chymotrypsin and porcine elastase. Sequence analyses of A-II and B-II revealed a considerable homology with soybean trypsin inhibitor (Kunitz) but suggested the presence of an about 20-amino acid insertion in the A-II molecule.

The conformation of the peanut inhibitor B-III was investigated by 1H NMR at 500 MHz. Resonances for about 30% of the residues were assigned and/or identified by conventional and two-dimensional 1H NMR measurements. The conformation of the modified inhibitor B-IIIRS, obtained by the cleavage of the peptide bonds of B-III at the first reactive site, Arg(10)-Arg(11), and second reactive site, Arg-(37)-Ser(38), was also investigated. Edman degradation was used to identify resonances of the residues adjoining the scissile sites. A comparison of the behavior of the resonances corresponding to Ala(12), Tyr(15), Phe(16), and Thr(37) in B-III and B-IIIRS indicated that the only conformational differences between the two proteins were those from the S-S loops containing the two reactive sites. From the similar behavior of Leu(7), Phe(24), His(26), Ala(29), five valine, and four threonine resonances in both proteins, it is suggested that the S-S loops that do not contain a scissile bond (the core region of the inhibitor) were rigid. Thus, no conformational change of the core region was observed upon cleavage of the reactive sites.

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.

A new method is described for amino acid sequencing of a polypeptide or protein involving Edman-degradation and field-desorption mass spectrometry of its constituent peptides in mixtures. The sequences can be sought from the molecular weights and N-terminal amino acid residues of the constituent peptides and the phenylthiohydantoins released in each cycle of degradation using a computer program.

The amino acid sequences of fragment I, the N-terminal cyanogen bromide fragment, and fragment II, a fragment between the first and second methionine residues, of rat serum albumin were determined by conventional methods in consideration of the sequences of human and bovine serum albumin. These sequences were compared with those of human and bovine serum albumin.

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.

The carboxyl-terminal amino acid sequence of Streptomyces subtilisin inhibitor (SSI) was reinvestigated by analysis of the amino acid sequences of the thermolysin peptides from the C-terminal decapeptide, and the sequence -Val110-Ala-Phe-Phe113, which was reported in J. Biochem. 76, 1191-1209 (1974), was revised to -Val110-Phe-Ala-Phe113. Carboxypeptidase A digestion of SSI resulted in loss of the inhibitory activity in parallel with the release of the carboxyl-terminal four amino acid residues. The resulting modified inhibitor, des(Val110-Phe113)-SSI, possessed almost full inhibitory activity against subtilisin BPN' when the inhibitor was incubated with the enzyme in amounts less than one mol of enzyme per mol of the inhibitor. However, no inhibitor activity was observed when the molar ratio of the inhibitor to the enzyme was less than one. This phenomenon suggests that the carboxyl-terminal four amino acid residues might play an important role in the maintenance of the three-dimensional structure of SSI, which resists the action of the proteinase. The addition of more than 30-fold molar excess of SSI-(104-113)-decapeptide (C-terminal decapeptide of SSI) to the modified inhibitor resulted in refolding of the polypeptide chain, rendering it immune from proteolytic digestion.

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.

p -Nitrophenyl α-maltotetraosides and α-maltopentaosides having a carboxymethyl group at the nonreducing-end glucosyl group were prepared by the action of Bacillus macerans cyclomaltodextrin glucanotransferase on a mixture of monocarboxymethyl-substituted cyclomaltoheptaose and p -nitrophenyl α- d -glucopyranoside, followed by digestion with glucoamylase, and purification by chromatography. The modes of actions of human pancreatic and salivary α-amylases on these derivatives were studied. The maltopentaoside derivatives were suitable as substrates for assays of α-amylases coupled with glucoamylase and α- d -glucosidase.

An asparagine-linked sugar chain of a protease inhibitor from barbados pride (Caesalpinia pulcherrima Sw.) was liberated by hydrazinolysis. After N-acetylation, the reducing end residue of this carbohydrate unit was coupled with 2-aminopyridine and the pyridylamino (PA-) derivative was purified by gel-filtration and reversed-phase HPLC. The structure of the resulting PA-sugar chain was determined mainly by stepwise exoglycosidase digestions and 500 MHz 1H-NMR spectroscopy and proved to be as follows: (formula; see text).

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.

N-(2-Pyridyl) derivatives of glycine (1), DL-alanine (2), DL-valine (3), and DL-norleucine (4) were synthesized as fluorescence labeled amino acids. These amino acids were used for the synthesis of N-terminal fluorescencelabeled peptides. N-(2-Pyridyl)-1,2-ethanediamine (7) was synthesized and used for the C-terminal labeling of amino acids and peptides. With these amino acid derivatives, some peptides labeled with 2-aminopyridine were synthesized and their spectroscopic characteristics were examined.

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.

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.

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.