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PubMed2003

New strategy for the determination of gliadins in maize- or rice-based foods matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: fractionation of gliadins from maize or rice prolamins by acidic treatment.

A procedure for determining small quantities of gliadins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) in gluten-free foods containing relatively large amounts of prolamin proteins from maize or rice is described. We report for the first time that gliadins, the ethanol-soluble wheat prolamin fraction, can be quantitatively solubilized in 1.0 M acetic acid, while the corresponding ethanol-soluble maize or rice prolamin fraction remains insoluble in acetic acid. We describe a methodology for the detection of gliadins in maize and rice foods based on a two-step procedure of extraction (60% aqueous ethanol followed by 1 M acetic acid). Subsequent MALDI-TOFMS analysis of the resulting acidic extract from these gluten-free foods clearly confirms the presence of a typical mass pattern corresponding to gliadin components, ranging from 30 to 45 kDa. Depending on the percentages of maize or rice flours employed in the elaboration of these foods, the combined procedure enables levels of gliadins from 100 to 400 ppm to be detected. The efficiency of this combined procedure corroborates enzyme-linked immunosorbent assay data for a large number of maize/rice gluten-free foods by means of direct visualization of the characteristic gliadin mass pattern in maize or rice foods.

Food Analysis↗

PubMed1997

A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2.

The prolamin box (P-box) is a highly conserved 7-bp sequence element (5'-TGTAAAG-3') found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolamin-box binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys2-Cys2 zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts in vitro with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression.

Amino Acid Sequence↗

PubMed1995

Morphometric Analysis of Rice Seed Protein Bodies (Implication for a Significant Contribution of Prolamine to the Total Protein Content of Rice Endosperm).

Electron microscopic observation of thin sections of rice (Oryza sativa L.) endosperm revealed two types of protein bodies (PBs): spherical and irregular-shaped ones. Immunocytochemical localization studies using antibodies raised against purified glutelins, prolamines, and globulins indicated that the prolamines were localized in the spherical PB, whereas the irregular-shaped PB contained glutelins and globulins. We counted and measured the surface area and the relative volume of 2303 PBs randomly selected from two different developmental stages and from different locations within the endosperm. The ratio of spherical to irregular-shaped PBs was 1:1.6. Double-label immunogold electron microscopic localization indicated that the globulins represented about 18% of the surface area of the irregular-shaped PBs. Based on our morphometric analysis, we estimate the relative contribution of glutelin as 53%, that of prolamine as 35%, and that of globulin as 12% of the total seed protein.

Journal Article↗

PubMedDate not supplied

[Free and bound amino acids during germination of maize seeds with implemented foreign prolamines].

Wheat prolamines were preincorporated into the endosperm of maize seed, and concentrations of free and bound amino acids were measured in the endosperm and seedlings during seed germination. Incorporation of foreign prolamines lowered the decrease rate of free proline concentration in both the endosperm (sprouting and shoots) and the embryo (sprouting). The seedlings at the stage of shoots were characterized by higher concentrations of aspartic acid, glutamic acid, and tyrosine. At the stage of transition to autotrophic nutrition, the pool of amino acids that are physiologically important for the plant growth was greater in maize seedlings with incorporated wheat prolamines.

Amino Acids↗

PubMed1984

Microsurgical approach to pancreatic duct occlusion in the pig by means of a prolamine solution.

Prolamine is an alcoholic solution of zein, with the property of polymerizing within 15 minutes in a humid medium. Such a substance has been injected in the pancreatic duct of 10 pigs with a microsurgical technique, causing complete occlusion. The prolamine gel produces a progressive fibrosclerosis of the exocrine pancreatic parenchyma, sparing the endocrine portion. The resulting pancreatic atrophy avoids the occurrence of inflammatory complications that often hamper surgery on such gland. The 10 pigs under study showed post-operative normal values of blood glucose and insulin, and a decrease in serum alpha-amylase levels. Complications occurred in only one case, and the microscopic pattern was similar in all cases. Microsurgical approach and use of prolamine are considered effective both in pancreatic transplantation and in chronic pancreatitis.

Animals↗

PubMed1995

The involvement of Opaque 2 on beta-prolamin gene regulation in maize and Coix suggests a more general role for this transcriptional activator.

The maize opaque 2 (o2) mutation is known to have numerous pleiotropic effects. Some polypeptides have their expression depressed while others are enhanced. The best characterized effects of the o2 mutation are those exerted on endosperm genes encoding the storage protein class of the 22 kDa alpha-zeins and the ribosome inactivating protein b-32. The Opaque 2 (O2) locus encodes a basic domain-leucine zipper DNA-binding factor, O2, which transcriptionally regulates these genes. In the maize-related grass Coix lacryma-jobi, an O2-homologous protein regulates the 25 kDa alpha-coixin family. We show in this paper that O2 transcriptionally regulates the structurally and developmentally different class of the beta-prolamins. A new O2-binding box was identified in beta-prolamin genes from maize and Coix that, together with the boxes previously identified in other endosperm expressed genes, forms a curious collection of O2 cis elements. This may have regulatory implications on the role of O2 in the mechanism that controls coordinated gene expression in the developing endosperm. Considering that the O2 locus controls at least three distinct classes of genes in maize endosperm, we propose that the O2 protein may play a more general role in maize endosperm development than previously conceived.

Base Sequence↗

PubMed1995

Homologies between prolamins of different minor millets.

Minor millets, viz. Barnyard millet, Proso millet, Little millet, Foxtail millet and Kodo millet, one variety in each grown in Tamil Nadu Agricultural University (TNAU), Coimbatore, Tamil Nadu were selected for the study. The protein contents of the selected decorticated millets were found to be 11.0, 12.3, 12.9, 10.5 and 10.6% respectively. Fractionation of these proteins revealed that prolamin forms major storage protein in Foxtail millet whereas, glutelin forms major storage protein in all the other millets. The extractability was studied using different solvents, viz. isopropyl alcohol, t-butyl alcohol and ethyl alcohol with varying concentration of 2-mercapto ethanol. Electrophoretic pattern of the extracted prolamins from these millets were compared and found that a protein band at the molecular weight range of 20 kD was found homologous in all except Proso millet. The extractability of the 20 kD protein in 90% isopropyl alcohol showed its strong hydrophobic nature.

Amino Acids↗

PubMed1988

Comparative investigations of partial amino acid sequences of prolamins and glutelins from cereals. VIII. Amino acid sequences of glutelin peptides.

The peptide fractions isolated from chymotryptic hydrolysates of wheat, rye and barley glutelins were separated by high-performance liquid chromatography on octadecyl silica gel. The peptides obtained were analysed for their amino acid composition and some also for their amino acid sequence. Characteristic sequences of peptides from wheat glutelin can be grouped into three types. The first type contains a high amount of Gly and frequently Tyr in the N-terminal positions. A typical partial sequence, which occurs repeated in two peptides, is QGQQPGQGQ. Sequences of this type are found in high-molecular-weight subunits. The second type is characterized by the sequence SQn (n = 3-5) followed by a hydrophobic tripeptide e.g., PPF, PVL; the most frequent sequence is SQQQQPPF. Low-molecular-weight subunits contain sequences of this type. The third type, which has partial sequences such as QQPQQPFP, corresponds to typical peptides from prolamin. Most sequences of peptides from rye and barley glutelins can be divided into two groups. The predominant type shows prolamin-like sequences, e.g., PQQPXPQQ with X being F or I. The second type is similar to glycine-rich peptides from wheat glutelin, except that repeating sequences are less frequent.

Amino Acid Sequence↗

PubMed2003

Spectroscopic characterization and structural modeling of prolamin from maize and pearl millet.

Biophysical methods and structural modeling techniques have been used to characterize the prolamins from maize ( Zea mays) and pearl millet ( Pennisetum americanum). The alcohol-soluble prolamin from maize, called zein, was extracted using a simple protocol and purified by gel filtration in a 70% ethanol solution. Two protein fractions were purified from seed extracts of pearl millet with molecular weights of 25.5 and 7 kDa, as estimated by SDS-PAGE. The high molecular weight protein corresponds to pennisetin, which has a high alpha-helical content both in solution and the solid state, as demonstrated by circular dichroism and Fourier transform infrared spectra. Fluorescence spectroscopy of both fractions indicated changes in the tryptophan microenvironments with increasing water content of the buffer. Low-resolution envelopes of both fractions were retrieved by ab initio procedures from small-angle X-ray scattering data, which yielded maximum molecular dimensions of about 14 nm and 1 nm for pennisetin and the low molecular weight protein, respectively, and similar values were observed by dynamic light scattering experiments. Furthermore, (1)H nuclear magnetic resonance spectra of zein and pennisetin do not show any signal below 0.9 ppm, which is compatible with more extended solution structures. The molecular models for zein and pennisetin in solution suggest that both proteins have an elongated molecular structure which is approximately a prolate ellipsoid composed of ribbons of folded alpha-helical segments with a length of about 14 nm, resulting in a structure that permits efficient packing within the seed endosperm.

Alcohols↗

PubMed2000

Inhibitory serpins from wheat grain with reactive centers resembling glutamine-rich repeats of prolamin storage proteins. Cloning and characterization of five major molecular forms.

Genes encoding proteins of the serpin superfamily are widespread in the plant kingdom, but the properties of very few plant serpins have been studied, and physiological functions have not been elucidated. Six distinct serpins have been identified in grains of hexaploid bread wheat (Triticum aestivum L.) by partial purification and amino acid sequencing. The reactive centers of all but one of the serpins resemble the glutamine-rich repetitive sequences in prolamin storage proteins of wheat grain. Five of the serpins, classified into two protein Z subfamilies, WSZ1 and WSZ2, have been cloned, expressed in Escherichia coli, and purified. Inhibitory specificity toward 17 proteinases of mammalian, plant, and microbial origin was studied. All five serpins were suicide substrate inhibitors of chymotrypsin and cathepsin G. WSZ1a and WSZ1b inhibited at the unusual reactive center P(1)-P(1)' Gln-Gln, and WSZ2b at P(2)-P(1) Leu-Arg-one of two overlapping reactive centers. WSZ1c with P(1)-P(1)' Leu-Gln was the fastest inhibitor of chymotrypsin (k(a) = 1.3 x 10(6) m(-1) s(-1)). WSZ1a was as efficient an inhibitor of chymotrypsin as WSZ2a (k(a) approximately 10(5) m(-1) s(-1)), which has P(1)-P(1)' Leu-Ser-a reactive center common in animal serpins. WSZ2b inhibited plasmin at P(1)-P(1)' Arg-Gln (k(a) approximately 10(3) m(-1) s(-1)). None of the five serpins inhibited Bacillus subtilisin A, Fusarium trypsin, or two subtilisin-like plant serine proteinases, hordolisin from barley green malt and cucumisin D from honeydew melon. Possible functions involving interactions with endogenous or exogenous proteinases adapted to prolamin degradation are discussed.

Amino Acid Sequence↗

PubMed2003

Antibodies to oat prolamines (avenins) in children with coeliac disease.

BACKGROUND: The use of oats in a gluten-free diet for children with coeliac disease is presently under investigation. In this study we measured the content of antibodies to oat prolamines (avenin) in sera from coeliac children and reference children. METHODS: Crude avenin was prepared by extraction with ethanol and salt-solution and used as antigen in a three-step ELISA. Sera from 81 children, including 34 children with verified coeliac disease, were analysed for both IgA and IgG antibodies to avenin and gliadin. Sera were also incubated with gliadin before exposure to avenin, and vice versa, to assess a possible cross-reaction between the species. Keyhole limpet hemocyanin (KLH) was used as a negative control. RESULTS: Children with coeliac disease on a normal diet had significantly higher levels of antibodies to avenin, both IgG and IgA, than reference children (P < 0.001) and the levels correlated positively with gliadin antibodies, especially of IgA-type (r = 0.798). Both anti-avenin and anti-gliadin antibodies were only absorbed by the corresponding protein. CONCLUSIONS: Children with coeliac disease have antibodies to oat proteins at significantly higher levels than reference children. The absorption test did not indicate a cross-reactivity between the prolamines of wheat and oats. The method will be employed for repeated sampling of anti-avenin antibodies during a prospective interventional study with a gluten-free diet supplemented with oats.

Adolescent↗

PubMed1999

Genes controlling prolamin biosynthesis, Pro1 and Pro2, in foxtail millet, Setaria italica (L.) Beauv.

Variation and genetic control of seed protein in foxtail millet (Setaria italica) were studied using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Variation in the electrophoregram of the total seed protein were detected in the range between 20 and 30 kDa which is derived from the polymorphism of five prolamin bands. The segregation for each of the bands in F2 seeds showed that these bands are governed by seven alleles at two loci, Pro1 and Pro2, which are not linked to one another. Among 271 local cultivars examined, eight out of ten possible genotypes were observed. With its level of diversity comparable to that of isozymes, the alleles conferring prolamin polymorphism are useful genetic markers.

Alleles↗

PubMed1992

[Extraction and characterization of grain prolamins of 6 sorghum Sorghum bicolor L. Moench cultivars].

The purpose of this work was to isolate and characterize prolamines from grains of sorghum, by their solubility and molecular properties. One variety, Zaraza-1 (SV-V51), and five hybrids (Dekalb-55, Dekalb-64, Wac-5005, Pioneer-815B and Savanna-5) were used. The prolamines, called kafirins, were extracted with 70% isopropanol at 60 degrees C, and then fractionated by two methods: 95% ethanol and gel filtration with Sephadex G-200. The molecular weight was determined by electrophoresis in a discontinuous polyacrylamide gel with sodium dodecyl sulfate (PAGE-SDS). The alpha kafirin (soluble fraction in 95% ethanol), and 7.13% of the grain's total protein, and the beta kafirin represents 21.64% of kafirin (the insoluble fraction), represents 78.36% of the kafirin and 25.39% of total protein. Three fractions were obtained from the column fractionation, two important ones (A and C), and one (B) in a lesser amount. The proportion of these fractions varied between cultivars, so that in SV-V51, D-64 and Wac-5005, A is predominant, and in high tannin sorghum, C is the predominant one. Kafirins reduced with 2-mercaptoethanol were separated in four subunits with molecular weights of: 15,000, 21,300, 24,300 and 51,500 daltons. Unreduced kafirin showed a larger number of subunits (six) whose molecular weights were: 15,000, 24,300, 48,500, 69,000, 80,000 and 93,500 daltons. Reduced and kafirin showed the same number of subunits as those of nonfractioned kafirin reduced with 2-mercaptoethanol.

Chemical Fractionation↗

PubMed1978

Heterogeneity of avenin, the oat prolamin. Fractionation, molecular weight and amino acid composition.

The prolamin, avenin, was extracted from oat seeds and shown to be maximally extractable in 45% (w/w) ethanol. A purification procedure is described and some properties of avenin are examined. As with wheat and barley prolamins, salt soluble and glutelin fractions were simultaneously extracted. Starch gel electrophoresis revealed two novel fractions, which were isolated by ion-exchange chromatography. These fractions have similar amino acid composition, threonine as the N-terminal amino acid and both have a 22 500 molecular weight. It is suggested that the avenin constituents have a common ancestral gene.

Amino Acids↗

PubMed1989

Angioscope-assisted occlusion of venous tributaries with prolamine in in situ femoropopliteal bypass: preliminary results of canine experiments.

Ten mongrel dogs underwent left lower extremity in situ femoropopliteal bypass with femoral vein. A 20 to 25 cm myocutaneous bridge was left between femoral and popliteal anastomoses. A 2.8 mm angioscope was introduced intraluminally to visualize venous tributaries (VT). A balloon occlusion catheter was placed alongside the angioscope and directed in each VT. Prolamine was injected into each VT to effect occlusion. Seven dogs were followed up for 1 week and three dogs for 1 month. A total of 34 VT (range one to five per dog) were available for attempted occlusion. Twenty-nine of 84 (85%) VT were able to be occluded based on comparison of pre-VT and post-VT occlusion angiograms. Poor visualization of VT or VT too small to admit the 5F catheter were reasons for failure. We conclude that (1) in the canine model studied angioscope-assisted occlusion of femoropopliteal during bypass is technically feasible, (2) this technical detail makes unnecessary medial thigh dissection for exposure of the vein graft, and (3) during short-term observation prolamine appeared to be a suitable occluding substance.

Anastomosis, Surgical↗

PubMed1988

A continuous acetic acid system for polyacrylamide gel electrophoresis of gliadins and other prolamines.

A polyacrylamide gel electrophoresis system buffered by acetic acid alone was developed for electrophoresis of prolamines. When applied to gliadin electrophoresis, the acetic acid system produces more bands than does a conventional aluminum lactate-lactic acid system (using 12% acrylamide gels). The acetic acid system is relatively simple, requiring a single buffer component that is universally available in high purity.

Acetates↗

PubMed2004

Proteomic analysis of the effect of heat stress on hexaploid wheat grain: characterization of heat-responsive proteins from non-prolamins fraction.

The effect of heat stress on hexaploid wheat grain proteome was recently analyzed in our previous works. Proteomic tools allowed the characterization of heat-responsive proteins of total endosperm, composed mainly of prolamins. The present work completes this study; our aim was to analyze the effect of heat stress on the water-soluble fraction, composed essentially of albumins and globulins. These proteins were separated by two-dimensional electrophoresis (2-DE), visualized by Coomassie Brilliant Blue (CBB) staining and analyzed by Melanie-3 software. Of the 43 heat-changed proteins, 24 were found to be up-regulated whereas 19 spot proteins were down-regulated. All of these proteins were subjected to matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by database searching which allowed the identification of 42 spots. Of these, some were enzymes involved in different metabolic pathways of plants, such as granule-bound starch synthase and glucose-1-phosphate adenyltransferase, involved in the starch synthesis pathway; beta-amylase, involved in carbohydrate metabolism, and the ATP synthase beta-chain that was related to four heat-decreased proteins. Moreover, five heat up-regulated proteins showed similarities with small heat shock proteins while three other spots were related to elongation factors or eucaryotic translation initiation factors. Proteins involved in abiotic stresses or in plant defense mechanism were also identified and are discussed.

Databases, Protein↗

PubMed1983

A simple and rapid dot-immunobinding assay for zein and other prolamins.

A simple method for the immunochemical assay of zein and other prolamins is described. Alcohol-solubilized zein is spotted onto chromatography paper disks or strips, incubated with antiserum (30-60 min), washed with several changes of Tris-buffered saline (10 min), incubated with protein A-peroxidase (30 min), and washed with several changes of Tris-buffered saline (10 min). the binding of zein-specific antibody is visualized by peroxidase-catalyzed color production from 4-chloro-1-naphthol. The method is simple, inexpensive, and rapid (2 h total time) and has potential application to other insoluble antigens.

Densitometry↗