Preparation and analysis of mass amounts of germin: demonstration that the protein which signals the onset of growth in germinating wheat is a glycoprotein

Abstract

(1) A rapid method (2–3 weeks) is described for preparing milligram amounts of germin, in high yield, from kilogram quantities of germinated wheat embryos. An ammonium-sulphate fraction of proteins in the low-speed supernatant from an embryo homogenate is suspended in buffer for pepsin digestion, and the resulting digest is filtered through Ultrogel AcA34 to obtain germin, virtually free of contamination (<1%) by other proteins.(2) An acid hydrolysate of germin contains a full complement of the 20 different amino acids. The proportions of half-cystine, tyrosine, and tryptophan are conspicuously low (<1.5%), the proportions of aspartic acid and glycine (each ca. 11%) as well as proline (ca. 8%) are relatively high, and the cumulative proportion of hydrophobic amino acids (valine, leucine, isoleucine, phenylalanine) is also high (ca. 25%). The N-terminal sequence of germin, as determined by Edman degradation, is enriched with respect to aspartic acid, which occupies 5 of the first 15 positions.(3) There are two forms of germin, one (G) being the dominant species in the soluble fraction of homogenates of germinated embryos and the other (G′) being the dominant species in a fraction obtained by rinsing growing embryos. Consistent differences between the amino-acid compositions of G and G′ were not observed. Similarly, wherever identification was possible, the N-terminal amino-acid sequences (residues 1–22) of G and G′ were not different.(4) The polymeric and monomeric forms of germin (G or G′) give a positive Schiff reaction, characteristic of glycoproteins, when electrophoretically separated in sodium dodecyl sulphate (SDS) – poly aerylamide. Treatment with trifluoromethanesulfonic acid affirmed the glycoprotein character of germin, as did labeling, in vivo, from exogeneously supplied tritiated sugars.(5) The G and G′ forms of germin were among the most heavily labeled proteins seen in SDS–polyacrylamide separations if wheat-embryo proteins were labeled, in vivo, from exogenous supplies of tritiated mannose, glucosamine, or fucose. Differences between the patterns of glucosamine labeling and fucose labeling of G and G′ suggest the possibility that the two forms differ with respect to their carbohydrate components.