Purification and Partial Characterization of a Second Cysteine Proteinase Inhibitor from Ungerminated Barley (Hordeum vulgare L.)

Abstract

It was previously shown that ungerminated barley contains inhibitors that suppress the activities of green malt cysteine proteinases. This paper reports the purification and partial characterization of a second barley cysteine endoproteinase inhibitor, a protein called lipid transfer protein 2 (LTP2). The chromatographically purified inhibitor had a molecular mass of 7112. The amino acid composition and sequence data of the purified inhibitor indicated that it was a protein whose gene, but not the protein itself, was isolated earlier from barley aleurone tissue. The purified protein inhibited the activities of electrophoretically separated green malt cysteine proteinases but not the activities of the serine- or metalloproteinases. The purified LTP2 inhibited the same proteases as the LTP1 that was characterized previously but was present in the mature seed in much smaller amounts. Neither LTP1 nor LTP2 has been proven to transport lipids in vivo, and it seems possible that both serve to keep cysteine endoproteinases that are synthesized during barley seed development inactive until the plant needs them. The small amount of LTP2 in the seed made it impossible to determine whether it, like LTP1, is involved in beer foam formation. Because of its proteinase-inhibiting ability and its resistance to heat inactivation, some of the LTP2 may persist in beer.

A Review: The Role of Barley Seed Pathogenesis-Related Proteins (PRs) in Beer Production

“…The protein ns-LTP2 has not been as studied as ns-LTP1. However, there is increasing literature on cereal seed ns-LTP2 structure, stability and lipid-binding ability in the last few years 20,32,85,99,110,138 to elucidate its possible technological function in cereal processing. In contrast to ns-LTP1, which is expressed in the aleurone layer late in grain development and in the early stages of germination, ns-LTP2 is expressed during the early stages of grain development.…”

“…The inhibition activity of ns-LTP1 has been under the debate for some time. The finding that ns-LTP1 could act as a cysteine protease inhibitor, possibly serving as a regulator of proteolysis during germination 84,85 , was contrasted by speculation that the denatured form of ns-LTP1 is only a preferred substrate for barley cysteine endoprotease active in malt 28 . The fact that denaturation of ns-LTP1 occurs on wort boiling was neglected.…”

A Review: Biological and Technological Functions of Barley Seed Pathogenesis-Related Proteins (PRs)

“…Inhibitory activity towards proteases or α-amylases is ascribed to the PR-5 298 , PR-12 192 and PR-14 family 160,161 . Additional roles of these families in the control of endogenous enzyme activity, or in defense against phytophagous insects, i.e., multifunctionality of plant protease inhibitors, is discussed.…”

“…Ns-LTP1 and ns-LTP2 are present in the seeds of the most economically important cereals, barley 161,325 , wheat 72,221 , rice 193,364 and maize 52,71 . Barley seed ns-LTP1 is a 91-residue basic protein (pI 9).…”

“…Two related isoforms of ns-LTP1 have also been identified in wheat seed 72 . Barley seed ns-LTP2 is a 69-residue basic protein (pI 8) 161 . The barley ns-LTP2 gene was cloned from the barley aleurone and described 147 ; its expression reaches a maximum at grain mid-maturity 167 and it is not present in mature grain; however the protein is sufficiently stable to be present in the aleurone cell layer of the endosperm.…”