Isolation and characterization of gliadins from Aegilops squarrosa seeds

The major gliadin components were isolated from the seeds of the diploid species Aegilops squarrosa, a putative source of polyploid wheat D-genome. The isolation procedure included gel-filtration and reversed-phase high-performance liquid chromatography (HPLC). The purified proteins were characterized by electrophoretic mobility in polyacrylamide gel using acid Al-lactate system and a system containing sodium dodecyl sulfate. The amino acid composition of isolated omega-gliadins was determined. Using covalent chromatography on thiopropyl-Sepharose 6B it was found that omega-gliadins of A. squarrosa contain no SH-groups and/or S-S-bonds. The N-terminal amino acid sequences of A. squarrosa gliadins were determined. omega-Gliadins were found to contain three types of N-terminal amino acid sequences, one of which, SRQ, in hexaploid wheat is encoded by 1B chromosome. It was shown that some omega-gliadins of A. squarrosa have blocked N-terminal amino acids. The major component of the gamma-fraction was found to contain an N-terminal sequence of gamma 2 type encoded in polyploid wheat by 1D chromosome. Gliadins with electrophoretic mobility in the beta-zone of the spectrum possess the N-terminal sequence of alpha-type. The results obtained are discussed in terms of the origin of polyploid wheat genomes.     

The distribution and partial characterization of the serum apolipoproteins in the guinea pig.

1. Very-low-density (VLD), low-density (LD) and high-density (HD) lipoproteins were isolated by sequential ultracentrifugation from the serum of male guinea pigs fed on a diet containing 3--4% fat. The apoproteins of these lipoproteins (apo-VLD, apo-LD and apo-HD lipoproteins) were studied after delipidation with organic solvents or extraction with tetramethylurea. 2. The major apolipoprotein of LD lipoprotein isolated by gel filtration was found to closely resemble apolipoprotein B of human serum in its chemical and physical properties. Electrophoresis in sodium dodecyl sulphate-polyacrylamide gel showed that this apoprotein consisted of a number of polypeptides. 3. Tetramethylurea precipitated an apoprotein from guinea-pig serum lipoproteins that is probably the apolipoprotein B-like component. This apoprotein accounted for about 80% of the apo-LD lipoprotein, about 55% of the apo-VLD lipoprotein and about 50% of the apo-HD lipoprotein. 4. The distribution of apolipoproteins soluble in tetramethylurea was determined by densitometric scanning of stained polyacrylamide disc gels. 5. A glycine-rich component of high electrophoretic mobility (band I) and a triplet of soluble apolipoproteins (bands II-IV) were present in both VLD and LD lipoprotein classes. These components constituted a higher proportion of the tetramethylurea-soluble apoproteins of VLD lipoprotein (60--80%) than of LD lipoprotein (40--55%). 6. Small amounts (10--15%) of a component of intermediate mobility, which contained traces of half-cystine, were also present in both VLD and LD lipoproteins. 7. A group of soluble components of basic character (bands VI-X), present as minor components of VLD lipoprotein (10--20%), constituted a major proportion (30--45%) of the soluble apoproteins of LD lipoprotein. Two of these apoproteins were rich in lysine, and two of lower electrophoretic mobility were rich in arginine. 8. The pattern of tetramethylurea-soluble apoproteins in HD lipoprotein was distinguished by the presence of two polypeptides of low electrophoretic mobility as its predominant components. One of these components, band VI, resembled the A-I apolipoprotein of man in both its amino acid profile and in its electrophoretic mobility. The second major component, band VI-B, was rich in lysine and resembled the C-I apolipoprotein of man in amino acid composition. 9. The soluble components of bands I and IX were analogous in physicochemical properties to the R-X1 and R-X2 (high-arginine polypeptide) peptides of human serum lipoproteins respectively.     

The stepwise mutation model: an experimental evaluation utilizing hemoglobin variants

The stepwise mutation model of Ohta and Kimura (1973) was proposed to explain patterns of genetic variability revealed by means of electrophoresis. The assumption that electrophoretic mobility was principally determined by unit changes in net molecular charge has been criticized by Johnson (1974, 1977). This assumption has been tested directly using hemoglobin. Twenty-seven human hemoglobin variants with known amino acid substitutions, and 26 nonhuman hemoglobins with known sequences were studied by starch gel electrophoresis. Of these hemoglobins, 60 to 70% had electrophoretic mobilities that could be predicted solely on the basis of net charge calculated from the amino acid composition alone, ignoring tertiary structure. Only four hemoglobins showed a mobility that was clearly different from an expected mobility calculated using only the net charge of the molecule. For the remaining 30% of hemoglobins studied, mobility was determined by a combination of net charge and other unidentified components, probably reflecting changes in ionization of some amino acid residues as a result of small alterations in tertiary structure due to the amino acid substitution in the variant. For the nonhuman hemoglobins, the deviation of a sample from its expected mobility increased with increasing amino acid divergence from human hemoglobin A.-It is concluded that the net electrostatic charge of a molecule is the principal determinant of electrophoretic mobility under the conditions studied. However, because of the significant deviation from strict stepwise mobility detected for 30 to 40% of the variants studied, it is further concluded that the infinite-allele model of Kimura and Crow (1964) or a "mixed model" such as that proposed by Li (1976) may be more appropriate than the stepwise mutation model for the analysis of much of the available electrophoretic data from natural populations.     

The influence of site-specificity of single amino acid substitutions on electrophoretic separation of yeast iso-1-cytochromec

Summary This study dealt with the ability of non-denaturing gel electrophoresis to separate iso-1-cytochromec with single amino acid replacements isolated from revertants of variouscyc1 nonsense mutants of the yeastSaccharomyces cerevisiae. A total of 28 different iso-1-cytochromesc with single amino acid substitutions of one of seven amino acids at six positions were examined on nondenaturing polyacrylamide gels at pH 4.8. Each of these iso-1-cytochromesc exhibited 1 of 16 distinct electrophoretic mobilities. We could distinguish the majority of iso-1-cytochromesc, even those having the same replacement at different sites and those having different replacements that resulted in the same net charge. These results provide confirmation of the importance of site-specific effects on the electrophoretic mobility, and presumably other properties, of proteins differing in sequence by as little as one amino acid. They demonstrate that nondenaturing electrophoresis is able to separate the majority of, but not all, proteins differing by single amino acids.     

Mapping and characterization of antigenic epitopes and the nucleic acid-binding domains of the VP6 protein of bluetongue viruses.

Heterologously expressed VP6 and truncated VP6 proteins of bluetongue virus (BTV) serotype 11 purified to near homogeneity were used for structure and function analyses. The yield of the expressed VP6 was host cell dependent. Six antigenic epitopes of VP6 of BTV were identified and mapped by immunoblot analyses and enzyme-linked immunosorbent assay with oligoclonal antibodies. These determinants were surface accessible and conserved among the cognate VP6 proteins of five U.S. BTV serotypes. The amino acid sequences and sizes of these six antigenic epitopes were determined, and their precise locations were also mapped and confirmed by deletion analyses. The nucleic acid binding activities of VP6, confirmed by electrophoretic mobility shift assay, were concentration dependent. The binding activities and affinities of the purified expressed VP6 protein towards double-stranded RNA and double-stranded DNA were similar. Two domains of VP6, corresponding to three of the six antigenic epitopes, were responsible for the nucleic acid binding activities and have been mapped within 28 amino acids near the middle and 11 residues near the carboxyl terminus of VP6 by electrophoretic mobility shift assay and deletion mutant analyses. Synthetic oligopeptides corresponding to these three regions also exhibited similar concentration-dependent nucleic acid binding activities.     

Difference of proteins from inclusion bodies formed in the nucleus and cytoplasm of the cytoplasmic polyhedrosis virus-infected midgut in the silkworm,Bombyx mori

Strains of cytoplasmic polyhedrosis virus (CPV) of the silkworm Bombyx mori typically form proteinaceous inclusion bodies (IBs) which occlude many virions and are formed in the cytoplasm of the midgut epithelium. In contrast, an unusual strain of CPV termed “A” strain produces IBs containing no virions in the nuclei of the epithelial cells. In this case although the viruses multiply in the cytoplasm, few IBs are formed in the cytoplasm. To clarify why the A strain forms IBs in the nucleus, the structural differences on the IB proteins (IBPs) from A and a typical (H) strain were investigated. Analyses by SDS-PAGE showed A strain IBP had slightly lower electrophoretic mobility than these of H strain. When these IBPs were partially digested with Staphylococcus aureus V8 protease, one of the digested products between the two strains showed different electrophoretic mobility. Amino acid sequence analyses of peptides produced with lysylendopeptidase from IBP of A strain indicated that a histidine residue of H strain was replaced by a tyrosine residue. The carboxyl terminal regions of the two IBPs were also different; IBP of A strain was -Leu-Leu-Val-COOH, but the terminal residue of H strain could not be determined by the same method. These differences of amino acid sequence of IBPs between A and H strains may be responsible for the partitioning of them; i.e., in the case of A strain IBP may be transportable into the nucleus by the specific signal of amino acid sequence.     

Carbohydrate composition of normal and variant human alpha 1-protease inhibitors.

Carbohydrate composition of normal human alpha 1-protease inhibitor (PiM₁) and several variant inhibitors (PiM₂, PiM₃, PiA, PiS, and PiZ) was determined by methanolysis of the samples followed by quantitative analysis of both neutral and amino sugars using gas-liquid chromatography. All normal and variant inhibitors contained nine mannose, seven galactose, ten N-acetylglucosamine, and eight N-acetylneuraminic acid residues per molecule, and no significant difference was found in their carbohydrate compositions. PiA is a variant with the fastest anodal electrophoretic mobility, and PiZ is a variant with the slowest mobility thus far reported. The differences in electrophoretic mobility of these Pi variants are entirely due to their amino acid substitutions determined previously. These amino acid substitutions have no effect on the carbohydrate structure of the protease inhibitor.     

Characterization of protamines from four avian species

No data are available on the protamines of birds, with the exception of galline. We have characterized the protamines from four species of birds belonging to four different orders. All of them have very similar properties. They have been purified by carboxymethylcellulose chromatography and analyzed with respect to amino acid composition and electrophoretic behaviour. They are very arginine-rich proteins (63.4-67.3%) but do not contain lysine. Serine (12.0-18.2%), tyrosine (5.8-9.0%) and glycine (4.5-7.1%), along with arginine, make up the bulk of the amino acid residues in these molecules. The electrophoretic mobility of bird protamines in acetic acid-urea-polyacrylamide gels is intermediate between that of somatic histones and salmine. The molecular size, estimated from amino acid analysis and electrophoretic migration, is 65 +/- 5 amino acid residues.     

Studies on Nuclei of Paramecium aurelia. II. Amino Acid Composition and Electrophoretic Properties of the Chromosomal Basic Proteins*

SYNOPSIS. The basic proteins of Paramecium aurelia nucleus were extracted from isolated nuclei and deoxyribonucleoprotein (DNP) of such nuclei. About 35–40% of the nuclear protein, predominantly a lysine-rich histone, is acid soluble. Five major components of the histone can be distinguished by polyacrylamide gel electrophoresis. Some components of Paramecium histone are similar to those of mammalian histones in their electrophoretic mobility, but they differ from the latter in the electrophoretic velocity and relative levels. The basic to acidic amino acid ratio of the histone from the ciliate is ～1.1–1.5, and its amino acid composition resembles closely that of yeast histone. Through the use of Sephadex G-200 gel filtration for purification of the histones extracted directly from isolated nuclei 2 basic proteins were resolved—component I, with an elution volume of 1.4, constitutes ～20% and component II, with an elution volume of 1.9, ～80%.     

Chemistry and end-group analysis on purified M protein of type 12 group A streptococcal cell walls

M protein was extracted from the cell walls of streptococci by use of both acidic and alkaline buffers. These extracts were further purified by ammonium sulfate fractionation and column chromatography. Both diethylaminoethyl and carboxymethyl celluloses were employed to cover the pH range of 3.0 to 9.0. All of the M proteins isolated were immunologically related, but their physical and chemical properties varied dependent upon the pH range of isolation. Each isolate appeared to be homogeneous on the basis of immunodiffusion analysis, electrophoretic mobility, and ultracentrifugal analysis, but their amino acid analyses differed slightly. Two factors were shared by all isolates: (i) they all reacted with type-specific antisera and (ii) each seemed to have l-lysine as a single N-terminal amino acid.     

Multiple forms of hypophyseal prolactin. A new glycosylated form of prolactin with increased biological activity

Different forms of prolactin obtained from porcine hypophysis and differing in terms of molecular weight, electrophoretic mobility and biological activity were studied by polyacrylamide gel electrophoresis. A glycosylated form of prolactin with Mr 25 000 and possessing an increased biological activity towards pigeon crop was revealed. It was found that the carbohydrate component of this prolactin form is attached to asparagine at position 31; no differences were revealed between the amino acid composition of the major and glycosylated forms of the hormone. In the hypophysis, the glycosylated form content makes up to 30-40% of the total prolactin monomer content. A disulfide dimeric form of prolactin with Mr of about 50 000 was isolated and characterized.     

Type II collagen of lamprey

The major collagen in lamprey notochord is type II, as determined by its amino acid composition and solubility properties. This collagen has a distribution of charged residues indistinguishable from higher vertebrate Type II collagens as judged by its SLS banding pattern. Lamprey type II collagen has a higher thermal stability than lamprey skin collagen, in contrast to the identical melting temperatures for these types in mammals. A minor collagen in lamprey notochord has solubility properties, amino acid composition, and electrophoretic mobility similar to that of 1 alpha, 2 alpha, 3 alpha collagen in human cartilage.     

Biochemical characterization of the human carbonic anhydrase variant CA ih Hiroshima

Summary Some biochemical properties of a new red cell human carbonic anhydrase variant, CA Ih Hiroshima, have been determined. Evidence is presented that the amino acid substitution in the Japanese variant is not the same as the previously characterized CA Ic variant from Guam of similar electrophoretic mobility. Based on a comparison with the normal CA I isoenzyme, a proposal for the site of the amino acid substitution is presented.     

The structural gene for the ribosomal protein S18 in Escherichia coli. II. Chemical studies on the protein S18 having an altered electrophoretic mobility

A mutant of Escherichia coli strain CR341 has an altered 30 S ribosomal protein S18. The alteration involves a change in the electrophoretic mobility of S18. S18 proteins were purified from the mutant and the parent strain, respectively, and their amino acid composition and tryptic peptides were compared. The results have shown that the mutational alteration involves substitution of cysteine for arginine. In addition, we determined the electrophoretic mobility of S18 proteins modified by ethyleneimine. The modification, which involves conversion of cysteine residues to S-(2-aminoethyl)cysteine, causes a greater electrophoretic mobility increase in the mutant protein than in the wild type protein, resulting in identical mobilities for the aminoethylated proteins. This experiment gives further support to the conclusion that the original mobility difference between mutant and wild type proteins is due to the mutational substitution of cysteine for arginine. The S18 obtained from a recombinant was also studied. The recombinant protein was found to have the mobility of the wild type protein and the wild type primary structure, as judged by amino acid composition and tryptic peptide analysis. This recombinant was obtained from the mutant by introducing Hfr strain G10 chromosome segments in the region between 70 and 10 minutes, and not in the str-spc region at 64 minutes, as described in the preceding paper. These results, together with those in the preceding paper, show that the mutation studied here is in the structural gene for S18, and that it maps outside the str-spc region.     

Fractionation of Subunits in Xylanases from Trichoderma viride with a New Simple Preparative Polyacrylamide Gel Electrophoresis Apparatus

Formic acid was identified as the acidic component of antibiotic K-52B by gas-liquid chromatography, whereas amino sugar I hydrochloride was isolated from the acid hydrolysate as the basic sugar component. On the basis of infrared analyses of constituent oligosaccharides, formic acid was assumed to be linked to the hydroxyl group of galactose in oligosaccharide III. From the results of physicochemical properties and ninhydrin degradation, periodate oxidation and peracetylation studies of amino sugar I hydrochloride, C₈H₁₈N₂O₅. 2HC1 was considered to be a new diaminohexose with a substituent group.     

Amino acid sequence of chicken gizzard gamma-tropomyosin

Chicken gizzard muscle tropomyosin has been fractionated into its two major components, beta and gamma and the amino acid sequence of the gamma component established by the isolation and sequence analysis of fragments derived from cyanogen bromide cleavage and tryptic digestions. Despite its much slower mobility on sodium dodecyl sulfate-polyacrylamide electrophoretic gels, it has the same polypeptide chain length (284 residues) as the alpha and beta components of rabbit skeletal muscle. Evidence for microheterogeneity of the chicken gizzard component was detected both on electrophoretic gels and in the sequence analysis. The gamma component is more closely related to rabbit skeletal alpha-tropomyosin than to the beta component. While the protein is highly homologous to the rabbit skeletal tropomyosins, significant sequence differences are observed in two regions; between residues 42-83 and 258-284. In the latter region (COOH-terminal) the alterations in sequence are very similar to those seen in platelet tropomyosin when compared with the skeletal proteins.     

The histones of rainbow trout erythrocytes include an erythrocyte-specific histone.

The erythrocyte histones of rainbow trout were compared with those of goose by polyacrylamide gel electrophoresis. A band analogous to goose erythrocyte-specific histone V, but not identical in relative mobility or quantity, was found to be a component of trout erythrocyte histone. A similar component was also found in carp erythrocyte histone, but it was absent from trout liver histone. To reveal this band clearly, it was advantageous to displace the histone III monomer by oxidation. To verify the character of this protein, each of the main erythrocyte histones of trout were purified by chromatography on Amberlite CG-50, eluted with guanidinium chloride, and then further purified by exclusion chromatography on Bio-Gel P-60. Amino acid compositions of corresponding trout and goose histones, including that of the erythrocyte-specific histone, were sufficiently similar to establish their analogous identities. In general, the chromatographic and electrophoretic properties of histones I, IIb1, IIb2, and V from trout differed more from those of goose, than did their gross amino acid compositions. Comprehensive fractionation and characterization is necessary to extablish identities of corresponding histone fractions, An extensive quantitative variability was found among erythrocyte-specific histones of fish. This must be reconciled with hypothetical roles for this histone in erythropoiesis.     

Characterization of serum lipoproteins of the shark Centrophorus squamous.

1. Blood serum from the shark Centrophorus squamosus (Bonnaterre) was shown to contain VLD (very-low-density), LD (low-density) and HD (high-density) lipoproteins. 2. In shape, size and general physical properties, these lipoproteins were very similar to those described for other animals. The VLD lipoproteins were the major components of the mixture, and HD lipoproteins were present at the lowest amount. 3. In addition to the usual lipid components, the shark lipoproteins also contain substantial amounts of hydrocarbon, probably mainly squalene, and monoalkyldiacylglycerols. Only trace amounts of wax ester were detected. 4. The protein moiety of the VLD and LD lipoproteins contained a component which, in its solubility and electrophoretic properties, molecular weight and amino acid composition, resembled the B apolipoprotein of man and other mammals. This accounted for a large part of the total shark apolipoprotein. 5. There were also present smaller amounts of proteins which were soluble in 8M-urea. In their electrophoretic mobility on basic polyacrylamide gel, some of these were like the A and C apoproteins of man. 6. The electrophoretic distribution of the soluble proteins from the VLD and LD lipoproteins resembled that in higher mammals, but in the HD lipoproteins the similarity was less.     

An Experimental Investigation of the Unit Charge Model of Protein Polymorphism and Its Relation to the Esterase-5 Locus of DROSOPHILA PSEUDOOBSCURA, DROSOPHILA PERSIMILIS, and DROSOPHILA MIRANDA

The relationship between charge changes and electrophoretic mobility changes is investigated experimentally. The charge of several proteins is altered by reaction with small molecules of known structure and the change in electrophoretic mobility is measured. The method of Ferguson plots is used to separate charge and shape components of mobility differences. The average effect of an amino acid charge change on the mobility of the esterase-5( 1.00) allele of Drosophila pseudoobscura is estimated to be 0.046. This estimate is then used to apply the step model of Ohta and Kimura (1973) to electrophoretic mobility data for the esterase-5 locus of D. pseudoobscura and D. miranda. The variation in electrophoretic mobility at this locus was found to be in agreement with the predictions of the step model.     

Cuticular proteins: The neglected component

This paper emphasizes the importance of the protein component of cuticles. Correlation of electrophoretic charge distribution of individual cuticular proteins and physical properties of the cuticles from which they were extracted, as well as interpopulation and interspecies conservation of electrophoretic patterns, are used to argue that individual proteins play precise roles in the cuticle. Glycosylation of cuticular proteins is described, but no function for these modifications is yet known. Analogy is drawn to analyses of chorion proteins and the case is made that analysis of amino acid sequence data is likely to provide insights into how cuticular proteins and chitin interact to construct the diverse types of cuticles.