Jørn Hejgaard

Barley α-amylase/subtilisin inhibitor. I. Isolation and characterization

A protein inhibitor of endogenous α-amylase 2 has been isolated from germinated barley by glycogen precipitation followed by cation-exchange chromatography. Preliminary kinetic analysis showed a mixed type mechanism of inhibition with an apparent Ki of 4×10−8M. The inhibitor formed well-defined complexes with barley malt α-amylase 2 and co-purified with the α-amylase by cycloheptaamylose affinity chromatography of glycogen precipitates. The inhibitor was inactive towards α-amylases from sorghum malt, hog pancreas, Aspergillus oryzae, and Bacillus subtilis. The amino acid composition and molecular weight near 21,000 were found to be the same as those of both “band-2 protein” previously identified in preparations of barley malt α-amylase and a specific subtilisin inhibitor from barley. Inhibition experiments confirmed that the malt α-amylase inhibitor is a strong inhibitor of subtilisin Carlsberg. Measurements of α-amylase activity in the presence of equimolar amounts of inhibitor and subtilisin showed that the inhibitor is “double headed”. The inhibitory activity towards α-amylase was lost after treatment of the inhibitor at 70 °C for 15 min. Isoelectric focusing patterns confirmed that the partially heat-labile, basic α-amylase isozymes (pI=6.6) of barley malt are complexes of α-amylase 2 (pI=6.2) and the inhibitor (pI=7.2). Evidence is presented to suggest that proteins with properties similar to those of the barley inhibitor are present in other cereals including wheat and rye. Possible in vivo functions and some practical aspects of the barley inhibitor are discussed.

Two antifungal thaumatin-like proteins from barley grain

Antifungal activity has been associated with 2 immunochemically distinct proteins, protein R and S (M r ∼23 kDa; pI 9‐10), which were isolated in pure form from barley grain. The proteins are homologous with thaumatin‐ and pathogenesis‐related proteins of the PR5 family. The proteins inhibit growth of i.a. Trichoderma viride and Candida albicans in microtiter plate assays and act synergistically with barley grain chitinase C. Like maize zeamatin, protein R and S but not chitinase C retarded fungal growth in synergism with nikkomycin Z, a nucleoside‐peptide inhibitor of fungal chitin synthesis. Although no inhibition of α‐amylases or serine proteases could be associated with protein R or S the results indicate that the homologous maize grain bifunctional inhibitor of insect α‐amylase and trypsin is very similar to or identical with maize zeamatin, which was proposed to have permeabilizing activity towards fungal membranes. Thus, in addition to the intensely sweet properties of thaumatin, multiple unrelated defense functions against insect and fungal pests can now be associated with the family of thaumatin‐homologous proteins.

Serpins in plants and green algae.

Control of proteolysis is important for plant growth, development, responses to stress, and defence against insects and pathogens. Members of the serpin protein family are likely to play a critical role in this control through irreversible inhibition of endogenous and exogenous target proteinases. Serpins have been found in diverse species of the plant kingdom and represent a distinct clade among serpins in multicellular organisms. Serpins are also found in green algae, but the evolutionary relationship between these serpins and those of plants remains unknown. Plant serpins are potent inhibitors of mammalian serine proteinases of the chymotrypsin family in vitro but, intriguingly, plants and green algae lack endogenous members of this proteinase family, the most common targets for animal serpins. An Arabidopsis serpin with a conserved reactive centre is now known to be capable of inhibiting an endogenous cysteine proteinase. Here, knowledge of plant serpins in terms of sequence diversity, inhibitory specificity, gene expression and function is reviewed. This was advanced through a phylogenetic analysis of amino acid sequences of expressed plant serpins, delineation of plant serpin gene structures and prediction of inhibitory specificities based on identification of reactive centres. The review is intended to encourage elucidation of plant serpin functions.

Characterization of a bifunctional wheat inhibitor of endogenous α-amylase and subtilisin

A bifunctional α‐amylase/serine protease inhibitor which inhibits germination‐specific cereal α‐amylases of the Graminae subfamily Festucoideae as well as bacterial subtilisins has been isolated from wheat grains. This protein has M r ≈20500 and pI ≈7.2. The amino acid composition and N‐teminal sequence (45 residues) show that the inhibitor is homologous with cereal and leguminous inhibitors of the soybean trypsin inhibitor (Kunitz) family.

Antifungal activity of chitin-binding PR-4 type proteins from barley grain and stressed leaf

Antifungal activity in vitro has been associated with barley leaf and grain proteins which are homologous with pathogenesis related proteins of type 4 (PR‐4) from tobacco and tomato and with C terminal domains of potato win and Hevea hevein precursor proteins. One protein (pI ∼9.3, M r ∼13.7 kDa) from barley grain and two very similar proteins from leaves infected with Erysiphe graminis were isolated by chitin affinity chromatography, but none of the proteins showed chitinase activity in vitro. The leaf proteins were increased several fold in response to either Erysiphe infection or NiCl2 infiltration and accumulated extracellularly. The three barley proteins were found to inhibit growth of Trichoderma harzianum in microtiter plate assays using ∼‐10 μg/ml concentrations and in lower concentrations in a synergistic way when mixed either with barley chitinase C (a PR‐3 type protein) or with barley protein R (a PR‐5 type protein). Structurally similar proteins were detected in wheat, rye and oats grain extracts.

Inhibitory Serpins from Wheat Grain with Reactive Centers Resembling Glutamine-rich Repeats of Prolamin Storage Proteins CLONING AND CHARACTERIZATION OF FIVE MAJOR MOLECULAR FORMS

Genes encoding proteins of the serpin superfamily are widespread in the plant kingdom, but the properties of very few plant serpins have been studied, and physiological functions have not been elucidated. Six distinct serpins have been identified in grains of hexaploid bread wheat (Triticum aestivum L.) by partial purification and amino acid sequencing. The reactive centers of all but one of the serpins resemble the glutamine-rich repetitive sequences in prolamin storage proteins of wheat grain. Five of the serpins, classified into two protein Z subfamilies, WSZ1 and WSZ2, have been cloned, expressed in Escherichia coli, and purified. Inhibitory specificity toward 17 proteinases of mammalian, plant, and microbial origin was studied. All five serpins were suicide substrate inhibitors of chymotrypsin and cathepsin G. WSZ1a and WSZ1b inhibited at the unusual reactive center P1-P1′ Gln-Gln, and WSZ2b at P2-P1 Leu-Arg—one of two overlapping reactive centers. WSZ1c with P1-P1′ Leu-Gln was the fastest inhibitor of chymotrypsin (k a = 1.3 × 106 m −1 s−1). WSZ1a was as efficient an inhibitor of chymotrypsin as WSZ2a (k a∼105 m −1 s−1), which has P1-P1′ Leu-Ser—a reactive center common in animal serpins. WSZ2b inhibited plasmin at P1-P1′ Arg-Gln (k a∼103 m −1 s−1). None of the five serpins inhibited Bacillus subtilisin A,Fusarium trypsin, or two subtilisin-like plant serine proteinases, hordolisin from barley green malt and cucumisin D from honeydew melon. Possible functions involving interactions with endogenous or exogenous proteinases adapted to prolamin degradation are discussed.

Sequence homology between barley endosperm protein Z and protease inhibitors of the α1-antitrypsin family

Six cDNA clones encoding parts of protein Z, a major barley endosperm albumin, have been identified. Nucleotide and amino acid sequencing have established a 180 residues long C‐terminal amino acid sequence of protein Z as well as two minor amino acid sequences (14 and 7 residues). These sequences show that barley protein Z is homologous with human α1‐antitrypsin, human otj‐antichymotrypsin, human antithrombin III, mouse contrapsin and chicken ovalbumin (26–32% of the 180 residues in the C‐terminal sequence in identical positions). The sequence homology and specific cleavage of protein Z at a bond corresponding to the reactive site of the inhibitors indicate a possible inhibitory function. Inhibition of microbial or pancreatic serine proteases could, however, not be associated with protein Z.

Complete amino acid sequence of the α-amylase/subtilisin inhibitor from barley

The complete amino acid sequence of the α-amylase/subtilisin inhibitor (BASI) isolated from barley has been determined by Edman degradation using spinning cup as well as gas-phase sequencing. Necessary fragments have been obtained from cleavage with cyanogen bromide, hydroxylamine, formic acid, clostripain, and streptococcal protease V8. The molecule consists of a single peptide chain of 181 residues with two disulphide bonds and with a molecular weight of 19,865. Homology is demonstrated with other members of the soybean trypsin inhibitor (Kunitz) family of inhibitors.

Amino acid sequence homology between a serine protease inhibitor from barley and potato inhibitor I.

A lysine-rich serine protease inhibitor, isolated from barley (H. vulgare, var. Hiproly) which inhibits subtilisin strongly, chymotrypsin weaker, but not trypsin, is shown to be homologous with potato inhibitor I (Richardson andCossins, FEBS Lett. 52, 161 (1975)) (45% of the amino acids in identical positions). The barley inhibitor seems to be the first example described of a protease inhibitor from higher plants in which the structure and reactive site is not stabilized by disulfide bonds.

Amino acid sequence of serine protease inhibitor CI-1 from barley. Homology with barley inhibitor CI-2, potato inhibitor I, and leech eglin

Three molecular forms of a protein inhibitor of chymotrypsin and microbial alkaline proteases have been isolated from Hiproly high-lysine barley.Automated Edman degradation of one of these inhibitor preparations (CI-1C) resulted in the following amino acid sequence (77 residues in total): Tyr-Pro-Glu-Pro-Thr-Glu-Gly-Ser-Ile-Gly-Ala-Ser-Gly-Ala-Lys-Thr-Ser-Trp-Pro-Glu-Val-Val-Gly-Met-Ser-Ala-Glu-Lys-Ala-Lys-Glu-Ile-Ile-Leu-Arg-Asp-Lys-Pro-Asn-Ala-Gln-Ile-Glu-Val-Ile-Pro-Val-Asp-Ala-Met-Val-Pro-Leu-Asn-Phe-Asn-Pro-Asn-Arg-Val-Phe-Val-Leu-Val (His, Lys, Ala, Thr, Thr, Val, Ala, Glx, Val, Ser, Arg) Val-Gly.The inhibitor (CI-1) is homologous with another barley inhibitor (CI-2), with potato inhibitor I and with the elastase-cathepsin G inhibitor eglin from the leech Hirudo medicinalis (30–50% of the amino acid residues in identical positions). This established «family of cystine-independent inhibitors» also showed some sequence similarities with the cystine-free yeast proteinase B inhibitors 1 and 2. In the reactive site region homologies with the cystine-rich inhibitors of the «Kazal pancreas secretory inhibitor» and the «Streptomyces subtilisin inhibitor» families were observed.