Purification and properties of protein Z – a major albumin of barley endosperm

Abstract

A major albumin of barley grain, called protein Z, has been purified from endosperm flour. Extraction with 0.05 Mβ‐mercaptoethanol and successive use of (NH 4)2SO4‐precipitation, anion exchange at pH 7.5, cation exchange at pH 4.5, and anion exchange at pH 8.0 resulted in a highly pure protein as judged from various electrophoretic and immunoelectrophoretic tests. As protein Z is a major protein component of beer, antibodies towards a protein‐rich beer fraction could be used to detect the protein during purification. Protein Z consists of at least four antigenically identical molecular forms with isoelectric points in the range 5.55–5.8, but same molecular mass near 40000. Dimer and, probably, tetramer forms were detected by gel filtration in the absence of reducing agents. Monospecific antibodies towards protein Z were prepared. Immunoelectrophoretic properties of the protein were not affected by treatment at pH 1–13 (30 min at 30°C) or up to 100°C (30 min at pH 7). Commonly grown barley varieties contained about 1.5–2.5 mg protein Z/g grain, but a much lower content (∼ 0.2 mg/g grain) was found in a few varieties. Like barley β‐amylase, protein Z was present in both salt‐extractable “free” (20–30%) and thiol‐extractable “latent” (70–80%) forms in the grain. Protein Z contains 2 cysteine and 20 lysine residues per monomer molecule and is relatively rich in leucine and other hydrophobic residues. Protein Z may contribute up to 5% of the total grain lysine in normal varieties and more than 7% in some high‐lysine barleys.

Genetics of CM-proteins (A-hordeins) in barley

“…It has been reported that B and C-hordeins are markedly decreased in Riso 1508 and to a lesser extent in Hiproly, while the salt soluble proteins are increased in both (see Miflin and Shewry 1979). Rhodes and Gilí (1980) reported a salt-soluble component that seemed to be absent in Riso 1508, and Hejgaard (1982) found that a salt-soluble protein, designated protein Z, was at a lower level in Riso 1508 but was greatly increased in Hiproly. To our knowledge, CMe-1 is the first salt-soluble protein to be described whose accumulation is totally or partially blocked both in Riso 1508 and Hiproly.…”

Inhibition of coagulation factors by recombinant barley serpin BSZx

“…All other enzymes, recombinant soluble domain of human tissue factor (sTF) and heparin were gifts from Novo Nordisk. Mono-and polyclonal antibodies towards BSZ4 [10] and BSZ7 [11] from barley grain and a polyclonal antibody against a pool of wheat serpins [12] were produced and purified according to standard procedures.…”

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

“…They are present abundantly in cereal seeds. Wheat 246 , rye 124 , oat 126 and barley 123,282 serpins are potent inhibitors of serine proteases in vitro. Serpins are composed of nine α-helices, three β-sheets and a reactive centre loop, containing a specific bait sequence for the serpin's target proteinase 101 .…”

“…The first member of the plant serpins, originally named protein Z, was isolated from barley seed 121,123 . Although it appears as a typical storage protein, its primary structure reveals a similarity to animal serpins.…”