PURIFICATION AND CHARACTERIZATION OF THE ACIDIC AND BASIC POLYPEPTIDES OF GLYCININ

MAURILIO ALVES MOREIRA, Purdue University

Abstract

The polypeptides of the 11S storage protein from soybean seeds (Glycine max (L) Merr.) were purified and characterized in an effort to define the structural features of this complex molecule. The results demonstrated that a minimum of six acidic polypeptides (A(,1a) and A(,1b) having N-terminal phenylalanine; A(,2) leucine; A(,3) and F(,2) (2) isoleucine and A(,4) arginine) and four basic polypeptides (having glycine as the common N-terminal) were associated with the 11S storage protein from soybean cultivar CX635-1-1-1. Amino acid analyses indicated that certain acidic and basic polypeptides contained 3 to 6 times more methionine than the others. This fact may have significance in efforts toward improving soybean protein quality. Characterization of the purified polypeptides by NH(,2)-terminal sequence analysis showed that within each of the two major subunit classes (acidic and basic), individual members shared extensive sequence homology. The members of each class were distinct gene products, however, as was evident by comparing their primary structures. The data suggested that the members of each major class of polypeptides shared common evolutionary origin and were synthesized at the direction of a gene family. Based on the NH(,2)-terminal sequence homology, the basic polypeptides (M(,r) 19,000) were divided into two subfamilies. Two members of one subfamily (B(,1) and B(,2)) were identical except at position 13, while the other group (B(,3) and B(,4)) had the same N-terminal sequence except at residue 9. There was about 70% sequence similarity between the two subfamilies. The cyanogen bromide fragments of five acidic polypeptides were purified, their N-terminal sequence determined and the positions of the fragments in the native molecule deduced. The structural relationships between the acidic polypeptides can be summarized as follows: Polypeptides A(,1a), A(,1b) and A(,2) (M(,r) 37,000) had a high degree of sequence identity. Polypeptides A(,3) (M(,r) 42,000) and F(,2) (2) (M(,r) 10,000) were likewise homologous with one another, but exhibited only about 60% of sequence similarity with A(,1a), A(,1b) and A(,2) proteins. The A(,4) protein (M(,r) 37,000) was the least similar to A(,1a), A(,1b) and A(,2), although eight of the first 20 residues of A(,4) were identical to those of A(,1a). In addition to the high degree of sequence homology between polypeptides, regions of internal homology were identified in each acidic polypeptide. Antibodies were prepared against most of the purified glycinin polypeptides and used to compare their immunological features. Antibodies raised against native glycinin reacted very weakly with the purified polypeptides. Conversely, antibodies prepared against purified polypeptides reacted very poorly with native glycinin. Antiglycinin failed to react with denatured glycinin, although the denatured complex was then recognized by antibodies prepared against the individual polypeptides. A comparison of the antigenic characteristics of the purified polypeptides revealed that the extent of cross-reactivity of antibodies prepared against them reflected their degree of sequence homology. Polypeptides A(,1a), A(,1b) and A(,2) could not be distinguished from one another immunologically, but were distinct from A(,3) and A(,4). Likewise, B(,1) and B(,2) were immunologically identical but were distinct from B(,3) and B(,4). Since A(,1a), A(,1b), A(,2), B(,1) and B(,2) were the high methionine containing polypeptides, this observation may be of practical significance in screening for soybean cultivars with improved methionine content.

Degree

Ph.D.

Subject Area

Botany

COinS