Comparative efficiencies of isopropyl and tert-butyl alcohols for extracting zeins from maize endosperm

Protein of endosperm of maize grains originating from three wild-type inbreds and their opaque-2 versions were solubilized in diverse extracts (E) by the sequential use of 0.5 M NaCl, water (E(1,2)), alcohol plus a reducing agent (E(3)), and salt plus a reducing agent (E(4)). Zeins were isolated in extracts E(3) and E(4) obtained by using 55% (w/w) isopropyl alcohol (i-PrOH) + 0.2% dithiothreitol (DTT) followed by 0.5 M NaCl + 0.2% DTT buffered at pH 10 or 60% tert-butyl alcohol (t-BuOH) + 0.2% DTT followed by 0.5% sodium acetate + 0.2% DTT in 30% t-BuOH. For a given genotype the percentage of extracted zeins was independent of the nature of the alcohol. The latter had a slight effect on the respective magnitude of E(3) and E(4): E(3) increased at the expense of E(4) when t-BuOH was substituted to i-PrOH for their isolation. The percentage of the total endosperm nitrogen present in E(3) + E(4) was identical to that of fractions F(II) + F(III) + F(IV) isolated according to the classical Landry-Moureaux extraction procedure. SDS-PAGE analysis revealed the presence of all types of zeins (alpha, beta, gamma, and delta) in E(3) and F(III), residual zeins in E(4) isolated with t-BuOH, and streaking only in E(4) and F(IV) isolated with NaCl at pH 10. The data together with those of the literature were discussed with regard to the influence of procedure on the yield of zeins using alcoholic extraction.     

Purification and reconstitution of chloride channels from kidney and trachea

Chloride channels mediate absorption and secretion of fluid in epithelia, and the regulation of these channels is now known to be defective in cystic fibrosis. Indanyl-oxyacetic acid 94 (IAA-94) is a high-affinity ligand for the chloride channel, and an affinity resin based on that structure was developed. Solubilized proteins from kidney and trachea membranes were applied to the affinity matrix, and four proteins with apparent molecular masses of 97, 64, 40, and 27 kilodaltons were eluted from the column by excess IAA-94. A potential-dependent 36Cl- uptake was observed after reconstituting these proteins into liposomes. Three types of chloride channels with single-channel conductances of 26, 100, and 400 picosiemens were observed after fusion of these liposomes with planar lipid bilayers. Similar types of chloride channels have been observed in epithelia.     

Increasing pre‐acclimation temperature reduces the freezing tolerance of winter‐type faba bean (Vicia faba L.)

Autumn‐sown winter‐type faba bean (Vicia faba L.) has been shown to have a yield advantage over spring sowing. Still, adoption of this overwintered pulse crop remains limited in temperate locations, due to inadequate winter hardiness. This research sought to understand how the prevailing temperature during emergence and seedling development, that is pre‐acclimation, influences freezing tolerance. Seedlings grown under a controlled “warm” 17/12°C (day/night) pre‐acclimation environment were initially less freezing tolerant than those grown under a “cold” 12/5°C temperature treatment. Stem and particularly root tissues were primarily responsible for slower cold acclimation, and there was a genotype specific response of above‐ground tissues to pre‐acclimation treatment. Both above and below‐ground tissues should be tested across a range of pre‐acclimation temperatures when screening faba bean germplasm for freezing tolerance.     

The effect of energy reserves and cryoprotectants on overwintering mortality in Mercenaria mercenaria notata (Say 1822) at two tidal levels

The objective of this study is to identify possible causes of the high winter mortality noted in juvenile Mercenaria mercenaria notata in eastern Canada. The percentage mortality, shell growth, concentration of energy reserves, and production of cryoprotectant molecules were compared between notata and native quahogs kept at intertidal and subtidal levels. Overwintering mortality of the notata strain reached 47.2% and was 3–9 times higher than that of the native strain. Shell increment was higher in the native than in the notata strain and also at the intertidal than at the subtidal level. The quahogs from the subtidal zone had a higher concentration of protein than those from the intertidal zone in August and April but a lower concentration in February. The notata strain had a lower concentration of lipids and glucose (34.9 and 0.21 mg g⁻¹ dry weight) than the native strain (42.2 and 0.28 mg g⁻¹ dry weight). Thermal hysteresis was detected in none of the quahog groups. High winter mortality in the notata strain seems to be caused, in part, by a lower capacity to stock lipid compared with the native strain. The higher concentration of glucose in the native strain may favour survival in cold water by helping to reduce the freezing point of the animals’ fluids.     

Protein Distribution in Gluten Products Isolated During and After Wet-Milling of Maize Grains

The protein distribution in five gluten samples isolated during and after wet-milling of maize grains (slurry before and after filtration, total industrial gluten meal, and coarse and fine fractions obtained after sieving) was investigated by sequential extraction. Six fractions (FI-FVI), including residue, were isolated. Heating filtered slurry to draw water away did not alter protein distribution. Compared with values reported in the literature for endosperm protein, we found a decrease in FI and FIV, respectively, extracted with salt alone and with reductant, due to proteolysis and partial elimination of nonprotein nitrogen during slurry filtration, and an increase in FII and decrease in FIII, alcohol-soluble proteins extracted without and with reductant, respectively, due to the presence of SO₂ in the steeping liquor. Gluten, with respect to the endosperm from which it originated, was richer in zeins (FII + FIII) and glutelins (FV + FVI) due to partial removal of salt-soluble proteins (FI + FIV) during the isolation process.     

Quantitative estimation of accumulation of protein fractions in unripe and ripe maize grain

The changes in amounts per grain of six protein fractions isolated by selective extraction, which had been determined during the development of INRA 260 and opaque 2 maize grains, were again studied by taking as development variable the mount of total protein per grain (N_p), defined as the difference between total and non protein nitrogen content. The accumulation of each protein fraction in grain is depicted with one straight line or aseries of linear segments whose slopes represent the relative rates of accumulation of each fraction with reference to that of total protein. According to changes in the slopes, four phases of accumulation can be distinguished for the INRA 260 variety. Phase I is characterized by the presence of basic proteins (salt-soluble proteins, G₃ glutclins and residue) alone. During phase II, which begins with the synthesis of endosperm specific proteins (zeins, G₁ and G₂ glutelins), all protein fractions are accumulated. Phases III and IV are concerned with the changes in accumulation of fractions belonging to basic proteins. With opaque 2 variety, phases II, III and IV cannot be differentiated. For a given variety v and from the second phase, the amount N_i,v of basic proteins, zeins, G₁ and G₂ glutelins is related to N_p by the equation N_i,v=a_p,i,v+b_p,i,vN_p where a_p,i,v and b_p,i,v remain constant throughout development and are independent on cultural pratices. This equation also represents the phenotypic of fraction i as function of total protein of mature grain. Similar equations for expressing the intra- and intervarietal changes of protein fractions as function of total nitrogen content of ripe grain can be found.