No abstract

Urea-denatured ~-zein was almost completely hydrolyzed into small peptides by digestion with 11100 (w/w) ofthermolysin at 37°C for 3 h. The angiotensin I-converting enzyme (ACE) inhibitory activity (IC so) of the thermolysin digest of total ~-zein was 24.5 pg/ ml, and most of the peptide fractions from Z 1 9 ~-zein and total ~-zein separated by reverse-phase HPLC had more or less ACE inhibitory activity. From these fractions, thirty-six peptides, including 5 dipeptides, 14 tripeptides, 9 tetrapeptides, 5 penta peptides, and 3 hexapeptides, were purified and their amino acid sequences were determined.

The toxic lectin, ricin D, contains mannose, fucose, xylose, and N-acetylglucosamine as sugar components. Sugar chains are linked to Asn-10 of the A-chain, and to Asn-95 and Asn-135 of the B-chain (Funatsu, G. et al. (1978) Agric. Biol. Chem. 42, 501-503; Araki, T. & Funatsu, G. (1985) FEBS Lett. 191, 121-124). Asparagine-linked sugar chains of each glycopeptide from ricin D were liberated by hydrazinolysis followed by N-acetylation. The reducing end residues of the sugar chains were coupled with 2-aminopyridine and the pyridylamino (PA-) derivatives obtained were purified by gel-filtration and reversed-phase HPLC. Eight main PA-sugar chains were obtained from three glycopeptides and the structures of these sugar chains were determined by component analysis, stepwise exoglycosidase digestions, partial acetolysis, and 500 MHz 1H-NMR spectroscopy. The results show that oligomannose type sugar chains (Man6-7GlcNAc2) are linked to Asn-95; Man5-7 GlcNAc2 and M4X (structure, see below) to Asn-135 of the B-chain, and M3FX and M3X to Asn-10 of the A-chain. (Formula: see text).

No abstract

The interaction of ricin D with specific saccharides was investigated by ultraviolet difference spectroscopy. Upon binding to saccharides, ricin D displayed ultraviolet difference spectra with maxima at 280 nm and 288 nm. Such difference spectra suggest that the environment of a tyrosine residue(s) located at or near the saccharide-binding site is changed by the binding of saccharide. In addition to the two positive peaks, a small trough was observed around 300 nm in the complexes with galactose-containing saccharides but not in the complex with N-acetylgalactosamine or galactosamine, suggesting the participation of tryptophan in the binding with galactose-containing saccharides. The magnitude of the difference maxima increased with increasing concentration of saccharides until the binding site was saturated. From the variation of the maximum at 288 nm as a function of saccharide concentration, the association constants were obtained for the binding of saccharides to ricin D at various temperatures and pH's. The saccharide binding of ricin D decreased with increasing temperature and with decreasing pH below pH 6.0. It was suggested that difference maximum at 288 nm observed in the ricin D-saccharide interaction reflects the binding of saccharides to the high-affinity saccharide-binding site of ricin D.

No abstract

The complete amino acid sequence of ribonuclease (RNase MC) from the seeds of bitter gourd (Motttordica ch=anria) has been determined, This has been achieved by the sequence analysis of peptides derived by enzymatic digestion with trypsin. lysylendopeptidase. and chymotrypsin, as well as by chemical cleavage with cyanogen bromide. The protein contains 191 amino acid residues and has a calculated molecular mass of 21259 Da.Comparison of this sequence with sequences of the fungal RNases. RNase T2, and RNase Rh, revealed that there are highly conserved residues at positions 32-38 (TXHGLWP) and 81-92 (FWXHEWXKHGTC).Furthermore. the sequence of RNase MC was found to be homologous to those of Nicotiatta alutn S-glycoproleins involved in self-incompatibility sharing 41% identical residues.

Purified crystal protein from Bacillus thuringiensis subsp. dendrolimus strain T84A1gave a single polypeptide band (apparent mol. wt 145,000) on SDS-PAGEand the only N-terminal amino acid sequence was Met-Asp-Asn-Asn-Pro-Asn-Ile-. No pentose or hexose was detected. Solid phase tryptic digestion of the solubilized crystal protein bound on a DEAE-cellulose column was found to be a successful method to prepare a toxic fragment without denaturation. The resulting fragment (fragment T) was slightly basic (p/ 8.9) and the molecular weight was estimated as 58,000 on SDS-PAGE and by gel filtration without denaturing agents. The apparent number of the amono acid residues was 516 and the N-and C-terminal sequences were determined as Ile-Glu-X-Gly-Tyr-Thr-and-Leu-Arg, respectively. The molar specific toxicities of the solubilized crystal protein and fragment T were nearly identical. The fragment T retained the full toxicity and selectivity of the intact crystal protein. Location of the fragment T in the crystal protein is also discussed. Bacillus thuringiensis produces a protein crystal ((S-endotoxin) in the sporulating cell, which is specifically toxic to the larvae of certain insects. Thus, selected strains that do not produce the low-specificity fly toxin (/?exotoxin) have been used as an economically important biological insecticide.1* The crystal