Allen W. Kirleis

The origin of lysine-containing proteins in opaque-2 maize endosperm

The reduction of zein synthesis in the maize (Zea mays L.) opaque-2 mutant is associated with an increased percentage of lysine in the endosperm protein. When expressed on an endosperm basis, we found that W64A opaque-2 contains 490 μg of lysine compared with 350 μg in W64A normal. SDS-PAGE analysis of endosperm proteins indicated that several non-zein proteins are more abundant in the mutant than in normal genotype. To determine the subcellular origin of these proteins, we separated an endosperm homogenate from developing kernels by sucrose density gradient centrifugation and used marker enzyme assays and immunoblot analyses to identify cellular components. Amino acid analysis of proteins in the gradient fractions showed that the majority of the lysine occurs in soluble proteins at the top of the gradient. To identify these proteins, we prepared a complex antiserum against the entire soluble protein fraction and used it to immunoscreen an endosperm cDNA expression library. Sequence analysis of clones identified mRNAs involved in carbohydrate metabolism, amino acid biosynthesis, and protein synthesis. RNA dot blot hybridization analysis with these clones revealed significant variation in the levels of transcripts between normal and opaque-2 endosperm, but we identified several mRNAs that are elevated in opaque-2 and that may encode proteins responsible for the enhanced lysine content.

Purification and immunocytochemical localization of kafirins inSorghum bicolor (L. Moench) endosperm

SummaryKafirins are the storage proteins of sorghum and are found in protein bodies in the seed endosperm. They have been classified as α-, β-, and γ-kafirins according to differences in molecular weight, solubility, and structure. The kafirins were purified, amino acid composition was determined, and immunolocalization methods were used to determine the organization of the protein bodies and distribution of kafirins throughout the endosperm. All three groups of kafirins were low in lysine. β-Kafirins and γ-kafirins were relatively high in cysteine, and β-kafirins were relatively high in methionine. Transmission electron microscopy showed that protein bodies in the peripheral endosperm were spheroid with concentric rings and few darkly stained inclusions. In contrast, protein bodies of the central endosperm were irregularly shaped with a higher proportion of darkly stained material. The light staining regions of the protein bodies are composed primarily of α-kafirins with minor portions of β- and γ-kafirins. The dark staining regions, however, are composed primarily of β- and γ-kafirins. Immunoelectron microscopy showed that protein bodies in the peripheral endosperm contain predominantly a-kafirin with minor amounts of β- and γ-kafirin. Central endosperm protein bodies are also predominantly α-kafirin, but have a higher proportion of β-kafirin and γ-kafirin than the peripheral endosperm protein bodies.

Isolation of a high-cysteine kafirin protein and its cross-reactivity with gamma-zein antiserum

Two methods were developed for the isolation of M r 30k kafirin protein from immature sorghum, one involving direct extraction with 20% (v/v) 2-mercaptoethanol (2-ME) and the other involving extraction with 20% (v/v) 2-ME followed by reversed-phase high performance liquid chromotography (RP-HPLC). The amino acid compositions of these isolated proteins were similar to each other and to a partially purified M r 30k kafirin from mature sorghum grain. These M r , 30k kafirins contained high levels of cysteine, proline and histidine, with low levels of aspartic acid and lysine. The molecular weight, solubility, and amino acid composition of M r 30k kafirin were similar to values published for maize gamma-zein. Structural similarity between gamma-zein and M r 30k kafirin was demonstrated through an immunological study showing cross-reactivity of the M r 30k kafirin protein and a gamma-zein antiserum. This high-cysteine M r 30k kafirin protein was named gamma-kafirin based on similarities in molecular weight, solubility and structure between it and gamma-zein of maize.

Absorption of zinc and iron by rats fed meals containing sorghum food products

Zinc and iron absorption from freeze-dried, traditionally prepared sorghum food products was studied in rats. Following a period of marginal zinc or iron depletion, groups of rats (n = 6 in each group) were fed nutrient-balanced test meals containing one of four sorghum food products (fermented aceda ; acidic to, cooked with tamarind extract; neutral to, cooked in water; alkaline to, cooked with wood ash), cooked maize gruel or an egg-white control diet, each of which was extrinsically labeled with either 65Zn or 59Fe before being added to the diets. Absorption was determined by whole-body retention of the initial radioisotope dose over a period of 19 days. Iron was highly available from all products tested (75–83%) with no significant difference in absorption between groups. Zinc from fermented aceda (97%) was more available than that from the other sorghum products (69–78%) or maize gruel (76%) (P