Subtilisin inhibitor from seeds of broad bean (vicia FABA); purification, amino acid sequence and specificity of inhibition

Abstract

A potent inhibitor of microbial serine proteases, including subtilisin, has been purified 1100-fold from seeds of broad bean (Vicia faba, cv. Kleine Thtiringer). Chymotrypsin and trypsin were not inhibited, but a weak "temporary" inhibition of pancreas elastase was observed. The preparation of pure inhibitor contained one major molecular form with a blocked N-terminal (MW -10,000, pl -4.8) and a minor form ( < 10%) with slightly lower MW and pl -5.0. About 80% of the amino acid sequence of the major form was determined by automatic Edman degradation of a cyanogen bromide fragment and tryptic peptides. The inhibitor is homologous with barley, potato and leech inhibitors of the "potato inhibitor I family" (36-56% of the amino acid residues in identical positions). Cleavage studies suggest one enzyme inhibitory site at an Ala-Asp bond.

Two Kunitz-Type Proteinase Inhibitors from Potato Tubers

“…PKI-1 is homologous to a cathepsin D inhibitor isolated from potato tubers (10), being identical in residues 21 to 42, but it differs in 12 of the first 20 residues (Fig. 3A).…”

“…Other well characterized subtilisin inhibitors from dicots are members ofthe proteinase inhibitor I and II families, e.g. the inhibitors from adzuki beans (12) and broad beans (21), and an inducible microbial proteinase inhibitor from tobacco (4). In contrast to these inhibitors, PKI-2 appears to have a somewhat broad specificity, with limited but significant activity against both trypsin and chymotrypsin.…”

The amino acid sequence of a trypsin inhibitor from the seeds of Momordica charantia Linn. Cucurbitaceae

“…The N-terminal residue of the protein was not detected by usual Edman degradation, its Nterminal amino group was blocked, similar to Vicia subtilisin inhibitor (SVI) from the broad bean [5] and chymotrypsin inhibitor-2 from barley [12]. Also MCI-3 was not digested by carboxypeptidase A, B and Y under the above described conditions.…”

“…In this paper, we report the amino acid sequence determination of MCI-3. The amino acid sequence showed that MCI-3 was markedly different from the known trypsin inhibitors, but has significant homology with the 'potato inhibitor I family' inhibitors [5]. This family includes a number of homologous inhibitors from the higher plants [6,7] and one from a lower animal [8].…”

Sequence specific assignment of the proton nuclear magnetic resonance spectrum of barley serine proteinase inhibitor 2

“…The amino acid sequence has been determined using chemical amino acid sequencing (40) and recently from the DNA-sequence of the gcnc of the inhibitor (53). The amino acid sequence is homologous to the potato serine proteinasc inhibitor (41) and has been assigned to the family of inhibitors named after this, which include a number of other inhibitors of plant sources as well as the leech eglin c (42,44). In this family of inhibitors as well as in the entire group of protein inhibitors the BSPI-2 and eglin c arc unique sofar in being the only inhibitors which do not contain a disulfide bridge.…”