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Amino acid residues in the metal-binding and putative substrate-binding sites of Escherichia coli methionine aminopeptidase (MAP) were mutated, and their effects on the function of the enzyme were investigated. Substitution of any amino acid residue at the metal-binding site resulted in complete loss of the two cobalt ions bound to the protein and diminished the enzyme activity. However, only Cys70 and Trp221 at the putative substrate-binding site are involved in the catalytic activity of MAP. Changing either of them caused partial loss of enzyme activity, while mutations at both positions abolished MAP function. Both residues are found to be conserved in type I but not type II MAPs.

Specific antsera were prepared against the Bowman-Birk trypsin inhibitor and four other trypsin inhibitors of low molecular weight isolated from soybeans (Glycine max L. cv. Tracy). These antisera were used to detect the presence and amount of the inhibitors in: (a) seeds and protei extracts of soybean meal; (b) (8). Based upon the fact that lectins make up from 2 to 10% of the total protein in most leguminous seed>, Liener suggested that they must play some important physiological role in the plant (16). Several recent studies (8, 9, 16) have contributed data pertinent to the influence of lectins in the rhizosphere. The present paper is an extension of the recent finding in soybean (14,15) of four new protease inhibitors in the mol wt range of the Bowman-Birk inhibitor (10, 18). The low mol wt protease inhibitors are proteins with very unusual amino acid compositions. They contain about 20% of S-containing amino acids, especially methionine, the most limited essential amino acid in soybean seeds. It is important to find out the distributions and mode of actions of these protease inhibitors in the life cycle of soybeans in order to elucidate the physiological roles of these proteins in the legume seeds. In this report, we describe the quantitative estimation of the low mol wt inhibitors by means of competitive inhibition of immunoprecipitation in the following protein sources: (a) SBM4; (b) proteins extractable from SBM by buffer (76 mm tris-HCl [pH 8] containing 5 mm 2-mercaptoethanol); (c) proteins released from seeds during the first 24 hr of imbibition; (d) proteins released from roots of seedlings into the surrounding water over a 20-day period; and (e) various plant parts, i.e. epicotyls, hypocotyls, cotyledons, and roots of 12-day-old seedlings. We also extend the recent study of the release of lectins from imbibing seeds (9) to include growing plants by determining hemagglutinating activity not only during imbibition by seeds but also during certain stages of seedling growth. MATERIALS AND METHODSFour-to 5-month-old male New Zealand rabbits were purchased from The Jackson Laboratory, Bar Harbor, Maine; goat anti-rabbit y-globulin was obtained from Cappel Laboratories, Downington, Pa.; lima bean trypsin inhibitor and Kunitz soybean trypsin inhibitor came from Worthington Biochemical Corporation, Freehold, N.J.; and [1251]sodium iodide was the product of Amersham/Searle Corp., Arlington Heights, Ill. Purified soybean protease inhibitors of low mol wt (PI I-V), 1251_ labeled antigens, and antisera were obtained as described previ-

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