The proteinase (proteinase F) responsible for the initial proteolysis of the mung bean (Vigna radiata) trypsin inhibitor (MBTI) during germination has been purified 1400-fold from dry beans. The enzyme acts as an endopeptidase, cleaving the native inhibitor, MBTI-F, to produce the first modified inhibitor form, MBTI-E. The cleavage of the Asp76-Lys77 peptide bond of MBTI-F occurs at a pH optimum of 4.5, with the tetrapeptide Lys-Asp-Asp-Asp being released. Proteinase F exhibited no activity against the modified inhibitor forms MBTI-E and MBTI-C. Vicilin, the major storage protein of the mung bean, does not serve as a substrate for proteinase F between pH 4 and 7. Proteinase F is inhibited by phenylmethylsulfonyl fluoride, chymostatin, p-hydroxymercuribenzoate, and p-chlorophenylsulfonate, but not by iodoacetate and CuCl2. It is not activated by dithiothreitol, and is stable for extended periods of time (10 months, 4°C, pH 4.0) in the absence of reducing agents. An apparent molecular weight of 65,000 was found for proteinase F by gel filtration. Subcellular fractionation in glycerol suggests that greater than 85% of the proteinase F activity is found in the protein bodies of the ungerminated mung bean. The same studies indicate that at least 56% of the MBTI of the seed is also localized in the protein bodies.

Characterization of the Proteinase that Initiates the Degradation of the Trypsin Inhibitor in Germinating Mung Beans (Vigna radiata).

α-Mannosidase (EC 3.2.1.24) from rice dry seeds was purified to homogeneity. Optimum pH and Km for pNP-α-Man hydrolysis were pH 4.3–4.5 and 1.04 mM, respectively. The enzyme digested mannobioses such as Manα-1,2Man, Manα-1,6Man, Manα-1,3Man but Manα-1,4Man. Zn2+ ion was required for the activity, whereas EDTA and swainsonine inhibited the activity by 80 and 96%, respectively. The rice storage protein, glutelin was prepared and its basic subunits were shown to have high mannose-type sugar chains by two-dimensional mapping using NH2-P and C18 silica columns. They were Man9GlcNAc2, Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2 and Man5GlcNAc2. All these oligosaccharides were digested by the purified α-mannosidase, and Man-GlcNAc2 and mannose were formed. Glycopeptides, having these high mannose-type sugar chains, could also be digested by the α-mannosidase. Subunits were prepared from glutelin basic subunit and the richest subunit among them, subunit 2 (isoform 2), was digested by the α-mannosidase. Isoform 2 was digested by V8 protease only partially and slowly. However, isoform 2, pre-treated with the α-mannosidase, was rapidly and completely digested by V8 protease.

Rice α‐mannosidase digesting the high mannose glycopeptide of glutelin

The total salt-extractable protein from seeds of important cultivated legumes and sunflower kernels has a tryptophan content ranging from 0.91% (by wt.) for chick pea (Cicer arietinum) to 1.99% (by wt.) for green gram (Vigna radiata). In species with substantial albumin fractions, the tryptophan content of the albumin fraction is higher than that of the globulin fraction by factors ranging between 2.3 (pigeon pea, Cajanus cajan) and 20.3-fold (pea, Pisum sativum, cv. Telephone). Although the protein content of Acacia seeds is relatively low (5-14% of seed wt.), the tryptophan content of Acacia seed proteins is usually greater than that of the cultivated legumes (1.50-2.66% by wt.).

Colette M. McGee

Papers

Localization of Acid Glycosidases in Protein Bodies of Pea Cotyledons

Acid Glycosidase Activities in the Cotyledons of Pisum sativum L.