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Investigates the inhibitory properties and primary structure of a novel serine proteinase inhibitor from the fruiting body of the basidiomycete, Lentinus edodes. Examination of the mammalian and microbial serine proteinase; Suggestion on the chemical modification studies; Composition of the complete primary structure.

A 9000-Mr protein isolated from a 60% ethanolic extract of soybean (Glycine max) seeds has been characterized and fully sequenced. The protein consists of 80 amino acid residues with four disulfide bonds. It contains a large number of hydrophobic residues and lacks methionine, phenylalanine, tryptophan, lysine and histidine residues. The protein readily crystallizes from water but is quite soluble in aqueous organic solvents like 95% 1-propanol. It aggregates to form large molecules (above 80 kDa) under ordinary denaturing conditions, such as 6 M guanidine· HCl and 8 M urea. Sequence analysis showed that the amino-terminal four-fifths is extremely hydrophobic and most of the acidic residues exist as their amide forms, and only the carboxyl-terminal short segment is rather hydrophilic. A computer search for homology detected an unexpected similarity of this protein to rat prolactin; however, its significance could not be assessed and this protein appears to represent a hitherto unknown protein family. Although no biochemical activity could be detected, the existence in relatively high abundance (approx. 200 mg from 1 kg seeds) of this novel protein may suggest its physiological significance in the plant. [ABSTRACT FROM AUTHOR]

Soybean trypsin inhibitor (Kunitz) was divided into four peptide fragments by the combination of limited hydrolysis with trypsin at acidic pH, chemical cleavage of methionyl bonds with cyanogen bromide, and reduction and carboxymethylation of disulfides in protein or peptide. Each fragment was separated and purified by gel filtration on Sephadex G-50, G-75 or G-100, and referred to as fragments A, B, C, and D from the amino- to the carboxyl-terminal region of the inhibitor. Soybean trypsin inhibitor (Kunitz) consisted of 181 ammo acid residues and its molecular weight was proved to be 20 100. Fragments A, B, C, and D consisted of 63,21, 30, and 67 amino acid residues, respectively. One of the two disulfide bridges in the inhibitor was involved in fragment D, and the other linked fragment A with fragment C. [ABSTRACT FROM AUTHOR]

For the elucidation of the amino-acid sequence of the carboxyl-terminal region of soybean trypsin inhibitor (Kunitz), fragments C and D were digested with trypsin, and the resulting peptides were separated by ion-exchange chromatography on Dowex 50X2 or by gel filtration on Bio-Gel P-4. Further fractionation and purification of the peptides were performed by ion-exchange chromatography on Dowex 1X2, by gel filtration on Bio-Gel P-2 or by high-voltage paper electrophoresis at pH 1.9 and 3.6. Three peptides were obtained in pure form from fragment C and ten peptides from fragment D, and their amino-acid sequences were determined by the direct Edman method and by carboxy- peptidase digestion technique. Overlapping peptides necessary for the alignment of the tryptic peptides from fragment D were obtained from a chymotryptic hydrolysate of fragment D. Nine main peptides and rune minor peptides were obtained. The amino acid composition and the partial amino-acid sequence of the 18 chymotryptic peptides of fragment D made it possible to establish the amino-acid sequence of the carboxyl-terminal region of the inhibitor. The complete amino-acid sequence of soybean trypsin inhibitor (Kunitz) deduced here was compared with that of Bowman-Birk inhibitor, another well-known soybean proteinase inhibitor. [ABSTRACT FROM AUTHOR]

For the elucidation of amino acid sequence around the reactive site of soybean trypsin inhibitor (Kunitz), fragments A and B were digested with trypsin, and the resulting peptides were separated by ion-exchange chromatography on Dowex 50X2 or by gel filtration on Bio-Gel P-4. Further purification of the peptides was carried out by gel filtration on Sephadex G-25 or by high-voltage paper electrophoresis at pH 3.6 and 6.5. Nine peptides were obtained in pure form from fragment A and three peptides from fragment B, and their amino acid sequences were determined by the direct Edman method and by carboxy-peptidase digestion technique. Overlapping peptides necessary for the alignment of the tryptie peptides from fragments A and B were obtained from a chymotryptic hydrolysate of fragment AB by gel filtration on Bio-Gel P-4, and their ammo acid compositions and sequence analyses of some peptides made it possible to establish the amino acid sequence of an amino-terminal region of the inhibitor consisting of AB amino acid residues. The reactive site (Arg63-Ile64) of the inhibitor to trypsin was involved in this region. [ABSTRACT FROM AUTHOR]