The complete amino acid sequence of barley trypsin inhibitor.

The amino acid sequence of barley trypsin inhibitor has been determined. The protein is a single polypeptide consisting of 121 amino acid residues and has Mr = 13,305. No free sulfhydryl groups were detected by Ellman's reagent, which indicates the presence of five disulfide bridges in the molecule. The primary site of interaction with trypsin was tentatively assigned to the arginyl-leucyl residues at positions 33 and 34. On comparison of the sequence of this inhibitor with those of other proteinase inhibitors, we found that the barley trypsin inhibitor could not be classified into any of the established families of proteinase inhibitors (Laskowski, M., Jr., and Kato, I. (1980) Annu. Rev. Biochem. 49, 593-626) and that this inhibitor should represent a new inhibitor family. On the other hand, this trypsin inhibitor showed a considerable similarity to wheat alpha-amylase inhibitor (Kashlan, N., and Richardson, M. (1981) Phytochemistry (Oxf.) 20, 1781-1784) throughout the whole sequence, suggesting a common ancestry for both proteins. This is the first case of a possible evolutionary relationship between two inhibitors directed to totally different enzymes, a proteinase and a glycosidase.

Protein proteinase inhibitors inactivate their target proteinases by forming tight complexes. An extremely precise geometrical fit between the reactive site of the inhibitors and the active site of the proteinases has been demonstrated by crystallographic and model building studies of the complexes (1-4). It is, therefore, not surprising that there has been no convincing evidence for an inhibitor capable of inhibiting two of the four mechanistic classes of proteinases ( i e . serine, sulfhydryl, carboxyl, and metalloproteinases). The sole exception is plasma a,-macroglobulin where an entirely different mechanism of proteinase-entrapping ( 5 ) is responsible for its broad specificity.
There are very few examples of possible divergence from a common ancestor of inhibitors against nonidentical classes of proteinases (6, 7). This divergent evolution would involve very complex processes to achieve best fitting to the respective target enzymes. During the course of the sequence determination of barley trypsin inhibitor we have noted its unexpected similarity to wheat a-amylase inhibitor in the NHzterminal regions (8). Here, we describe its complete amino acid sequence which extends this similarity to the entire molecule, suggesting a possible evolutionary relationship between inhibitors directed toward totally unrelated enzymes, trypsin and a-amylase. The result also indicates that the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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barley trypsin inhibitor is not related to any proteinase inhibitor sequenced to date.

DISCUSSION
The sequence information utilized to determine the primary structure of barley trypsin inhibitor is presented in summary form in Fig. 4. There was no indication of ambiguous residues or alignment.
Serine proteinase inhibitors of known primary structure are currently grouped into about 10 families from sequence homology (28). However, we could find no inhibitors homologous to barley trypsin inhibitor in these families. Although corn trypsin inhibitor (29) showed some similarity to the barley inhibitor in the NHZ-terminal region (Fig. 6), no further similarity was found for the remaining parts of the molecules. This unique sequence of barley trypsin inhibitor places it in an entirely new proteinase inhibitor family. On the other hand, computer analysis using the program ALIGN of the National Biomedical Research Foundation (30) based on the method of Needleman and Wunsch (31) revealed a significant degree of similarity between barley trypsin inhibitor and wheat a-amylase inhibitor (32) throughout the sequences (Fig.  7). This is particularly noticeable in the middle parts of the molecules (from residues 41 to 75). The alignment score for this comparison is 7.3 standard deviation units (30), which means that the possibility that this value could have been obtained in a comparison of randomized sequences of the same amino acid composition is below lo-". This suggests the possible divergent evolution of the two inhibitors from a common ancestor.
Protein inhibitors of serine proteinases frequently consist of several homologous inhibitory domains separated by short connecting peptides. An extreme example of these "multiheaded" inhibitors is avian ovo-inhibitor, which consists seven tandem domains belonging to the pancreatic secretory trypsin inhibitor (Kazal) family (33). Another characteristic of these proteinase inhibitors is that a single species of organism usually produces sets of closely related "iso-inhibitors" with different inhibition spectra. Although the biological driving force for these phenomena is obscure, they certainly derive from two kinds of gene duplication, discrete and contiguous, and it is likely that numerous present day serine proteinase inhibitors have diverged from a rather limited number of ancestral genes (28,34). In a very few instances, this divergent evolution appears to have given rise to inhibitors against proteinases of distinct evolutionary origins.  . (7) revealed that the sequence aligns best with the second homology region of legume double-headed serine proteinase inhibitors with all 8 half-cystine residues being conserved. But these rather exceptional divergences still remain within the class of proteinase inhibitors. Consequently, the present finding appears to be of an extraordinary divergence that led to the evolution of two inhibitors of totally different enzymes, a proteinase and a glycosidase. Most evidence indicates that new proteins arise from old ones, not by the invention of entirely new functions but mainly by the modulation of previously existing ones (35). Therefore, evolution of inhibitors of structurally and functionally different enzymes from a common ancestor (whatever the function of the ancestor might have been) must have been an extremely complex process. Further structural analysis of the members of this new protein famliy may provide a better understanding of this extraordinary divergence and of the inhibition mechanism of a-amylase inhibitors, of which nothing is yet known.

RESULTS
The flrst fracclon iT5f eIuted at the breakthrough pornt contained only free a r g m r n e , but It8 uncorrected yield exceeded 100%. Suggesting that 2 mol of free a r g l n m e per mol p r o t e m were llberated by the tryptlc dlgestmn.
The 7th pool (T12'. yield = 1011, shoved a composition ldentlcal to that of the 6th pool IT121 (data for T12' not glvenl. The different ChrOmatographlc behaviors of these peptides appeared to be d result of partial deamldatmn. mol of a r g m l n e IT51 accounted for the COmpOSltlon of the proteln.

The total ammo acid Compositions Of these 11 tryptlc peptldes and 2
SeqYence Analyses Of Tryptic Peptides-These are SummarLzed ln Table   11.

Chromatographrc
CondltionS and effluent moDIrorlng were as described for Flq. I.