Wheat Germ Protein Research Articles

Characterization of wheat germ protein synthesis initiation factor eIF-4C and comparison of eIF-4C from wheat germ and rabbit reticulocytes.

Eukaryotic protein synthesis initiation factor (eIF)-4C was purified from wheat germ and the molecular weight was calculated to be approximately 19,000 by SDS-polyacrylamide gel electrophoresis. A similar molecular weight was determined by gel filtration chromatography indicating that wheat germ eIF-4C is functional as a single polypeptide chain. An efficient in vitro translation system dependent upon the addition of eIF-4C was developed. This system was used to determine the concentrations of eIF-4C required for the half-maximal rate of translation of satellite tobacco necrosis virus RNA, alfalfa mosaic virus RNA 4, and barley alpha-amylase mRNA. No significant differences in the concentrations of eIF-4C required for the translation of these mRNAs were observed, although differences were noted for eIF-4A and eIF-4F. This finding suggests that eIF-4C is not involved in the binding of mRNA to 40 S ribosomal subunits. In heterologous assays, rabbit reticulocyte eIF-4C was as active as wheat germ eIF-4C in the wheat germ eIF-4C-dependent system. In addition, wheat germ eIF-4C substituted for rabbit reticulocyte eIF-4C in in vitro assay systems from rabbit reticulocytes. These results indicate that eIF-4C from wheat and rabbit contain conserved functional domains.

Characterization of the ATP-dependent binding of wheat germ protein synthesis initiation factors eIF-(iso)4F and eIF-4A to mRNA.

The ATP-dependent binding of wheat germ protein synthesis initiation factors eIF-(iso)4F and eIF-4A to an oligoribonucleotide has been investigated by direct fluorescence titration techniques. In addition, the effect of ATP on the interaction between another cap-binding initiation factor, eIF-4F, and eIF-4A was studied using the same methods. Comparison of the equilibrium association constants (K(eq)) indicate that 1) hydrolyzable ATP affects the affinity of eIF-(iso)4F for eIF-4A, regardless of whether or not mRNA was previously bound to the eIF-(iso)4F; in contrast, ATP had no effect on the eIF-(iso)4F/oligoribonucleotide interaction; 2) in the presence of ATP, the binding of the binary eIF-(iso)4F.eIF-4A complex to the oligoribonucleotide is of similar affinity as the binding of the oligoribonucleotide to the eIF-(iso)4F alone; the stoichiometry of this ternary eIF-(iso)4F.eIF-4A.mRNA complex was found to be 1:1:1; and 3) a similar ATP effect is observed for the eIF-4F/eIF-4A interaction as for the eIF-(iso)4F.eIF-4A complex.

Wheat acetyl-CoA carboxylase.

The acetyl-CoA carboxylase present in both wheat germ and total wheat leaf protein contains ca. 220 kDa subunits. It is the major biotin-dependent carboxylase present in wheat chloroplasts. Active acetyl-CoA carboxylase purified from wheat germ is a homodimer with an apparent molecular mass of ca. 500 kDa. The enzyme from wheat germ or from wheat chloroplasts is sensitive to the herbicide haloxyfop at micromolar levels. The incorporation of 14C-acetate into fatty acids in freshly cut wheat seedling leaves provides a convenient in vivo assay for both acetyl-CoA carboxylase and haloxyfop.

Effect of thermal processing on the nutritive value of wheat germ protein

Wheat germs are used as industrial products for nourishment so that it was necessary to determine the nutritive value of proteins in raw and in roasted wheat germs (temperature: 130-150 degrees C for 20 min). Protein quality evaluation has been determined by a biological method--feeding young growing rats. The rats were fed 10% level protein diets, based on raw and roasted wheat germs. The results show that protein of roasted wheat germs has higher digestibility (D) and protein efficiency ratio (PER) than the raw wheat germs which proves that roasting destroyed digestion enzymes inhibitors. Furthermore, the net protein utilization (NPU) has also been improved by roasting. Biological value (BV) of raw germs approximates to the value of roasted germs. The results lead us to the conclusion that roasting saves and improves protein parameters in wheat germs.

Functionality of Wheat Germ Protein in Comminuted Meat Products as Compared with Corn Germ and Soy Proteins

ABSTRACTWheat germ protein flour (WGPF), corn germ protein flour (CGPF), and soy flour (SF) were used as additives at a level of 3.5% in comminuted meat products (CMP). Frankfurters with protein additives showed increased water‐holding capacity and batter stability and decreased cooking loss. Improved viscosity and adhesiveness were observed with protein additives when the level of added water was constant. Protein additives also influenced textural and sensory properties of frankfurters. WGPF at a level of 3.5% was found similar to effects of SF and CGPF. WGPF is a potential nonmeat protein additive that can be utilized as an extender in CMP such as frankfurters and bologna.

Characterization of the interaction of wheat germ protein synthesis initiation factor eIF-3 with mRNA oligonucleotide and cap analogs

Direct fluorescence titration experiments of wheat germ protein synthesis initiation factor eIF-3 with mRNA cap and oligoribonucleotide analogues were performed in order to determine the equilibrium association constants (Keq) for the eIF-3.mRNA interaction as a function of pH and temperature. These data suggest that (i) the eIF-3.mRNA interaction is not cap-specific (i.e., m7G-specific), (ii) ATP hydrolysis is not involved in the interaction, and (iii) the interaction is primarily ionic in nature. Competition experiments between a rabbit alpha-globin mRNA oligoribonucleotide analogue and either mRNA cap analogues or nucleoside triphosphates (NTPs) are also reported; these experiments indicate that NTPs act as both activators and competitive inhibitors of the mRNA.eIF-3 association. The results are consistent with a partially uncompetitive binding mechanism, whereby at low NTP concentrations (less than or equal to 10 microM) the bound NTP enhances subsequent mRNA binding to eIF-3, perhaps by inducing a conformational change, and at higher NTP concentrations, the NTP acts as a competitive inhibitor for the mRNA binding site on eIF-3.

Interaction of wheat germ protein synthesis initiation factors eIF-3, eIF-(iso)4F, and eIF-4F with mRNA analogs

The interaction of wheat germ eIF-3 with the wheat germ cap-binding proteins eIF-(iso)4F and eIF-4F as a function of pH and ionic strength is described. Direct fluorescence titration experiments are used to measure the equilibrium association constants (Keq) for the binary protein/protein complexes as well as for the interaction of eIF-3 with methylated cap analogues and rabbit alpha-globin mRNA oligonucleotide analogues. The Keq values for ternary eIF-3/eIF-(iso)4F/analogue and eIF-3/eIF-4F/analogue interactions were also measured. The equilibrium binding constants were used to calculate coupling free energies, which provide an estimate of the cooperativity for the interaction of the mRNA analogues, eIF-3, and either eIF-4F or eIF-(iso)4F. These data suggest a mechanism in which the binding of eIF-(iso)4F or eIF-4F to mRNA enhances the subsequent binding of eIF-3 to the message. This may lead to favorable positioning of the complex on the ribosome and thereby enhance translation.

Crosslinking of hemin to a specific site on the 90-kDa ferritin repressor protein.

Incubation of a 90-kDa ferritin repressor protein (FRP) with small amounts of radiolabeled hemin resulted in the formation of a strong interaction between the two that was stable to SDS/PAGE. (We refer to this interaction as a "crosslink," without intending to imply knowledge as to its chemical nature.) Of seven other proteins tested individually, only apohemopexin and bovine serum albumin showed similar crosslinking ability, albeit to a much lower extent. [14C]Hemin specifically crosslinked to FRP in the presence of a 50-fold excess of total wheat germ proteins. Inclusion of catalase did not prevent the reaction of hemin with FRP, suggesting that H2O2 is not involved. The subsequent addition of a stoichiometric amount of apohemopexin did not reverse the reaction. Exhaustive digestion of the complex with Staphylococcus aureus V8 protease produced a major labeled peptide of 17 kDa. These results show the existence of a highly specific, uniquely reactive hemin binding site on FRP.

Dietary Plant Materials and Development of Diabetes in the BB Rat

The present study was designed to examine further the impact of individual plant protein sources found in a diabetogenic, cereal-based, rodent laboratory diet, NIH-07 [open formula, nonpurified rat and mouse diet (positive control)], on the development of diabetes. Diabetes-prone BB rats that were pan-T(OX19+)-lymphopenic were fed a low diabetogenic diet during gestation and lactation. Progeny of these rats were fed a normal or autoclaved NIH-07 diet, or one of eight other diets based on the AIN-76A formulation, with modified protein sources as follows: hydrolyzed casein (HC), soybean meal, HC+ trypsin inhibitor (TI) in water (2 mg/mL, wheat germ, alfalfa seeds, Brewer's yeast, red lentils and a plant protein mixture. Feeding soybean meal increased the incidence of diabetes compared with the negative control, HC diet (47% vs. 12% incidence, P = 0.02). Wheat germ, alfalfa seeds and plant protein mixture resulted in an intermediate incidence of diabetes of 33%; the incidence was lower for Brewer's yeast and lentils (20% and 13%). Autoclaving (121 degrees C, 10 min) the NIH-07 diet or the presence of TI in drinking water had a minimal effect on diabetes frequency, suggesting heat-labile plant toxicants were not directly involved. Thus, certain dietary plant protein sources or associated agents may influence the development of spontaneous diabetes in the BB rat.

Wheat germ initiation factors 4F and (iso) 4F interact differently with oligoribonucleotide analogs of rabbit .alpha.-globin mRNA

The binding of capped oligoribonucleotide analogues of the 5' terminus of rabbit alpha-globin mRNA to wheat germ protein synthesis initiation factors eIF-4F and eIF-(iso)4F was measured by direct fluorescence techniques. An analysis of the equilibrium association constants (Keq) indicates that both eIF-4F and eIF-(iso)4F recognize primarily the m7G cap structure but differ in the recognition of other structural features. eIF-4F is sensitive to the position and sequence of hairpin structures within the oligoribonucleotide, while eIF-(iso)4F shows a preference for linear sequences. These differences suggest that wheat germ eIF-4F and eIF-(iso)4F may have discriminatory activity for mRNA recognition.

In Vitro Binding of Wheat-Germ Proteins to the 5′-Upstream Region of the rolC Gene of Ri Plasmid

In Vitro Binding of Wheat-Germ Proteins to the 5′-Upstream Region of the rolC Gene of Ri Plasmid

A comparison of the binding of methylated cap analogs to wheat germ protein synthesis initiation factors 4F and (iso) 4F

The binding of the 5'-terminal cap analogues m7GpppG and m7GTP to wheat germ protein synthesis initiation factors eIF-4F and eIF-(iso)4F as a function of pH, ionic strength, and temperature is described. Equilibrium binding data indicate that eIF-4F and eIF-(iso)4F have different mechanisms for interacting with the 5'-cap structure, but the complexes formed between m7GpppG and wheat germ factor eIF-(iso)4F more closely resemble complexes formed between this cap analogue and either mammalian eIF-4E or eIF-4F. The binding of these initiation factors to the hypermethylated cap analogues m2,7GMP, m2,7GpppG, and m2,2,7GpppG is also investigated. The differences in affinity of eIF-4F and eIF-(iso)4F for the hypermethylated 5'-terminal cap structures suggest that these factors may have discriminatory activity.

Crystal structure of a protein‐toxin α1‐purothionin at 2.5Å and a comparison with predicted models

Alpha 1-Purothionin (alpha 1-P), a wheatgerm protein and lytic toxin, has a secondary and tertiary structure similar to that of crambin as revealed by CD and NMR studies. alpha 1-P crystallizes in the tetragonal space group 1422 with unit cell dimensions: a = b = 53.59 and c = 69.79 A. X-ray diffraction data have been measured to 2.5 A Bragg spacing. The crystal structure has been determined by molecular replacement methods, using an energy-minimized alpha 1-P model structure derived from crambin (Whitlow and Teeter: Journal of Biomolecular Structure and Dynamics 2:831-848, 1985, Journal of the American Chemical Society 108:7163-7172, 1986). The energy-minimized model gives a slightly cleaner rotation solution and better refinement against the x-ray data than do the crambin or unminimized alpha 1-P structures. The final crystallographic residual with the data in the 10-2.5 A resolution range is 0.216. The refined alpha 1-P structure has a backbone rms difference of 0.74 A from crambin and 0.55 A from the energy-minimized alpha 1-P model. A low resolution NMR model of alpha 1-P calculated from metric matrix distance geometry and restrained molecular dynamics differs from crambin's backbone by 2.3 A rms deviation (Clore et al.: EMBO Journal 5:2729-2735, 1986). Backbone dihedral angles for our predicted model differ from the refined alpha 1-P structure in only one region (at a turn where there is a deletion relative to crambin). The NMR model had differences in four regions.

Isolation and properties of lipolysis inhibitory proteins from wheat germ and wheat bran

Proteins inhibiting pancreatic lipase in vitro have been isolated from wheat germ and wheat bran, with relative molecular mass ranging from 24,400 to 27,500. Inhibition of pancreatic lipase by the wheat germ proteins is related to their ability to interact with the emulsified substrate and to hinder the adsorption of the enzyme on the interface. The extent of inhibition depends on the amount of substrate and is independent of the enzyme concentration. Bile salts forming micelles in the concentration range used are able to partially reverse the inhibition of pancreatic lipase by the wheat germ proteins. The nutritional significance of the data obtained is discussed.

Immunocytochemical Localization of a Wheat Germ Lysozyme in Wheat Embryo and Coleoptile Cells and Cytochemical Study of Its Interaction with the Cell Wall

Among several wheat (Triticum aestivum L.) germ proteins able to lyse Micrococcus lysodeikticus, one lysozyme (W1A) was purified by ion-exchange chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis. Polyclonal antibodies against this lysozyme were raised in rabbits. The in situ localization of W1A lysozyme was achieved by the indirect protein A-gold technique. Large amounts of W1A lysozyme were found in cell walls whereas intercellular spaces, cytoplasm, and organelles were nearly free of labeling. Specificity of labeling was assessed with several controls. In an attempt to detect the presence of binding sites, W1A lysozyme was complexed to colloidal gold. Particles were specifically distributed in large amounts over wheat embryo and coleoptile cell walls. The absence of labeling over isolated coleoptile cell walls treated with 0.1 and 0.4 molar potassium hydroxide for hemicellulose extraction indicated that W1A lysozyme binding sites were probably of hemicellulosic nature.

A kinetic light-scattering study of the binding of wheat germ protein synthesis initiation factor 3 to 40S ribosomal subunits and 80S ribosomes.

The rate constants for eucaryotic initiation factor 3 (eIF3) association and dissociation with 40S ribosomal subunits and 80S monosomes have been determined. These rate constants were determined by laser light scattering with unmodified eIF3. The affinity of eIF3 for 40S subunits is about 30-fold greater than for 80S ribosomes. This difference in affinity resides mainly in the association rate constants. Rate constants of 8.8 X 10(7) and 7.3 X 10(6) M-1 s-1 were obtained for eIF3 binding to 40S subunits and 80S ribosomes, respectively. From thermodynamic cycles, the affinity of eIF3-40S subunits for 60S subunits is about 30-fold lower than free 40S subunits for 60S subunits. A calculation shows that under these conditions and assuming simple equilibria, approximately 12% of ribosomal subunits would associate via a reaction of 40S-eIF3 with 60S subunits as opposed to a path where eIF3 dissociates from the 40S subunits prior to association with 60S subunits.

Protein translocation across wheat germ microsomal membranes requires an SRP-like component

Different wheat germ extracts were tested for the presence of membranes capable of translocating and processing nascent secretory proteins. One lysate was found in which nascent prehuman-placental lactogen (phPL) was translocated and processed to mature human placental lactogen (hPL). Processing was found to occur concomitant with translocation across membranes. Translocation across the wheat germ membrane required a component which is similar to the mammalian signal recognition particle (SRP). It bound to DEAE-Sepharose, had a sedimentation coefficient of 11S and contained a 7S RNA. In addition to hPL, the plant protein zein and the bacterial protein beta-lactamase were translocated across and processed by wheat germ membranes. Transport was found to occur only co-translationally. Our results show that the wheat germ protein translocation system is similar to the mammalian one. Unlike the mammalian SRP, the particle purified from wheat germ did not arrest elongation of nascent secretory proteins.

Mathematical treatment of data for calculating activities of alpha-amylase isoenzymes.

Methods for determining alpha-amylase isoenzymes by selective inhibition with a wheat-germ protein are practical and easy, and give accurate, precise results. The incomplete specificity of the inhibitory action is not a major drawback but does necessitate mathematical treatment of the data (i.e., enzymic activities measured before and after preincubation with the inhibitor) to ascertain the amount of the different isoamylases. Such an algorithm is quite simple and straightforward, because the isoenzymes can be calculated either arithmetically or geometrically, by using a linear standard curve, empirically obtained, that relates the fraction of activity remaining after inhibition (R/T) to the pancreatic isoenzyme fraction or to the percentage of total alpha-amylase (P/T or 100 X P/T). An alternative method, plotting R/T against the ratio of pancreatic to salivary isoenzyme (P/S), is inconvenient, necessarily yields a nonlinear curve, needlessly complicates the calculations, and has been a persistent source of confusion in many articles dealing with the differentiation of isoamylases.

The mode of inhibition of the biosynthesis of phenylalanine ammonia lyase by its product cinnamic acid in aging potato parenchyma tissue

Excised potato parenchyma tissue upon aging in air and light, showed synthesis of RNA, as assessed by the incorporation of [3H]-uridine, reaching a maximum in 18 h. The increase in RNA synthesis was in parallel with the increase in phenylalanine ammonia lyase synthesis. The treatment of the tissue, initially with actinomycin D at 15 μg/g tissue, or with trans-cinnamate at 3·5 mM, caused 65% and 50% inhibition of RNA synthesis, respectively. The inhibition was reduced to 20% by delaying the treatment of trans-cinnamate to the ninth hour. A comparative study on the nuclear fractions, isolated from trans-cinnamate treated and untreated aged parenchyma tissues showed that trans-cinnamate treatment inhibited thein vitro RNA synthesis by 38%. Studies on thein vivo synthesis of PAL and other proteins showed that trans-cinnamate treatment mainly impaired thede novo synthesis of PAL. The total RNA, isolated from trans-cinnamate treated parenchyma, was 66% less efficient in the translation of PAL, in cell free wheat germ protein synthesizing system. The translation product, purified by affinity chroma tography on phenylalanine conjugated sepharose-4B was found to be homogeneous and showed a single radioactive peak corresponding to protein band on polyacrylamide gel electrophoresis.

Structural Relationship among the Rice Glutelin Polypeptides

When the glutelin protein fraction of rice (Oryza sativa L.) seeds was fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, three size classes of proteins, 51 kilodaltons (kD), 34 to 37 kD, and 21 to 22 kD, as well as a contaminating prolamine polypeptide of 14 kD were detected. Antibodies were raised against these proteins and employed in studies to determine whether a precursor-product relationship existed among the glutelin components. Antibodies of the 34 to 37 kD and 21 to 22 kD polypeptides strongly reacted with the 51 kD protein, and conversely, anti-51 kD protein cross reacted with both of the putative subunits. Immunoprecipitation of in vitro translated products resulted in the synthesis of only the precursor form, indicating that the alpha and beta subunits are proteolytic products of the 51 kD precursor protein. The poly(A)(+) RNA directed in vitro translated product was about 2000 daltons larger than both the authentic glutelin precursor and the in vitro translated product from polysome run-off synthesis. Western blot analysis of the 34 to 37 kD and 21 to 22 kD polypeptides partially digested with Staphylococcus aureus V8 protease revealed distinct patterns indicating that these proteins are structurally unrelated. As observed for the glutelins, the rice prolamines are also synthesized as a precursor of 16 kD, 2000 daltons larger than the mature polypeptide. Addition of dog pancreatic microsomal membranes to a wheat germ protein translation system resulted in the processing of the prolamine preprotein but not the preproglutelin to the mature form.