Tokuji Ikenaka

Chemical Structure and Inhibitory Activities of Soybean Proteinase Inhibitors

One of the most fundamental studies to understand the inhibition mechanism of proteinase inhibitors is to elucidate the amino acid sequence of the inhibitors. Soybean contains several proteinase inhibitors, of which two inhibitors were well characterized and studied on their chemical and physicochemical properties. Kunitz soybean trypsin inhibitor, isolated and crystallized by Kunitz [2], consists of 181 amino acid residues including two disulfide bridges and the complete amino acid sequence of the inhibitor was determined by us (Fig. 1) [3–5]. Another inhibitor, Bowman-Birk soybean proteinase inhibitor, found by Bowman [6] and characterized by Birk [7] consists of 71 amino acid residues including fourteen half-cystine residues. The unusual stability of this inhibitor against heat, acid and proteolytic digestion are thought to be caused by the rigid tertiary structure constructed by a large number of disulfide bridges. This inhibitor contains two different and independent inhibitory sites for trypsin (Lys-Ser at residues 16 and 17) and chymotrypsin (Leu-Ser at residues 43 and 44) as shown in Fig. 2 [8, 9]. The most noteworthy feature of its amino acid sequence is the striking similarity between the sequences around the two reactive sites. Especially, the distribution of five half-cystine residues around the trypsin inhibitory site is absolutely identical with that around the chymotrypsin inhibitory site as shown in Fig. 3 [9]. Since disulfide bridges are known to play an important role in protein architecture, it is of much interest to determine the pairings of these half-cystine residues.

N- and C-terminal amino acid sequences of a γ-heavy chain disease protein YOK

Abstract The N- and C-terminal peptides of a γ-heavy chain disease protein YOK were isolated by cyanogen bromide cleavage of the protein followed by reduction, carboxymethylation and gelfiltration, and their sequences were determined by the conventional methods. Protein YOK consisted of Fc-fragments of IgG 1 and had heterogeneous N-termini, serine, threonine and histidine, which corresponded to positions 215, 223 and 224 respectively, from the N-terminal end of the gamma chain of immunoglobulin EU. A new amino acid substitution was found in position 431, in which YOK contained glycine in contrast to alanine in corresponding position of known human γ 1 -chains, suggesting one structural correlation with a genetic marker. These results were compared with those of other γ-heavy chain disease proteins and discussed for the genetic implications of the sequence of this protein.

β-elimination and sulfite addition reaction of chondroitin sulfate peptidoglycan and the peptide structure of the linkage region

Pronase digestion of bovine tracheal cartilage yielded acid mucopolysaccharide - peptide complexes which were fractionated by chromatography on Dowex 1(C1-). A major fraction was eluted with 1.5 M NaC1 and presumed to by chondroitin sulfate A-peptidoglycan by cellulose acetate electrophoresis. Alkaline beta-elimination and sulfite addition reaction of this fraction yielded cysteic acid-containing peptides, two of which were obtained in an homogeneous state. The sequence determination of these two made it possible to remodel their original structures as Leu-Pro-Ser-Gly-Glu-Gly-Pro-Glu and Leu-Pro-Ser-Gly-Glu, where the serine residues carried polysaccharide chains. Together with the reported data on the polysaccharide-protein linkage region, the present result suggests that the -Ser-Gly- sequence is a minimum requisite for the glycosylation of serine residues in the protein core of various proteoglycans.

On the structure of galactosylhydroxylysine and glycopeptides derived from bovine tracheal cartilage

Abstract Galactosylhydroxylysine isolated from the alkaline hydrolysate of bovine tracheal cartilage was shown to be O-β-D-galactopyranosylhydroxylysine. The structures of several hydroxylysine-containing glycopeptides suggested the existence of a unique sequence around the glycosylated site with one exception in that an amino acid other than arginine was present in the third position from the glycosylated residue. Isolation of glycopeptides with an identical hexapeptide sequence yet with a different carbohydrate side chain suggested that the attachment of glucose to a polypeptide-bound galactose is not regulated by a short amino acid sequence in the glycopeptide region.

STRUCTURE-FUNCTION RELATIONSHIPS OF SOYBEAN DOUBLE-HEADED PROTEINASE INHIBITORS

Publisher Summary This chapter discusses the structure–function relationships of soybean double-headed proteinase inhibitors. Many of the natural proteinase inhibitors inhibit more than one proteinase at the same time, and are called multi-headed inhibitors. The most extensively investigated multi-headed inhibitors are the double-headed inhibitors of legume origin, which can bind two proteinases at their dual and independent reactive sites. The soybeans used for the purification of inhibitors are the Sode-furi variety, cropped in 1973. Soybean meals were extracted with 60% ethanol at room temperature and the inhibitor fraction was precipitated by adding a double volume of cold acetone. The sticky precipitates were collected, dissolved in water, and dialyzed against water. Fraction A was found to be BBI by its chromatographic behavior, inhibitory pattern, and the amino acid composition of the purified materials. Fraction B was purified on a DE-32 column using ammonium acetate buffer system according to Frattali. Fraction C was first purified on a DEAE-cellulose column. Two major fractions, C-I and C-II, were obtained. C-II was further purified by SP-Sephadex C-25 chromatography and finally by DEAE-cellulose chromatography.

Identification of β-aminoisobutyric acid in uremic serum

Abstract An unidentified ninhydrin-positive substance found in uremic sera but not found in normal sera was isolated by gel-filtration through Sephadex G-75 followed by high voltage paper electrophoresis (pH 3.5), and identified as β-aminoisobutyric acid using paper chromatography and automated amino acid analyzer. The quantitative determination of β-aminoisobutyric acid in serum revealed that the level of β-aminoisobutyric acid in uremic sera was much higher than that of normal sera. Gas chromatographic determination of the enantiomorphs of β-aminoisobutyric acid showed that uremic sera contain R- and S-isomers of the amino acid, but with the R-isomer as the dominating form.

Identification of β-Aspartylglycine in Uremic Serum and Its Toxicity1

: 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.