Arachin polymorphism in groundnut (arachis hypogaea l.)

Arachin, the major seed storage protein of groundnut, showed polymorphism. The polymorphic forms were due to differences in molecular size, net charge and polypeptide composition of the native protein. Purified arachin at low ionic strength resolved into monomeric and dimeric forms both on sucrose density gradient centrifugation and cellulose acetate membrane (CAM) electrophoresis. The dimers had more net negative charge compared with the monomers. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of arachin from the cultivar Spanish improved (SP) under non-reducing conditions showed three major components of M, 70.7, 63.8 and 60.9 k. Arachin from Trombay Groundnut 1 (TG-1) showed three components of M, 70.7, 63.8 and 59.5 k while in TG-18, a derivative of a cross between SP and TG-1, there were only two components of M, 70.7 and 63.8 k. Data from two dimensional gel electrophoresis in the presence and absence of 2-mercaptoethanol shows that each of the above components in turn consists of subunit pairs that are held together by disulphide linkages. The M,s of the major polypeptides for the three arachins is as follows: SP, 47.5, 45.1, 42.6 and 21.4 k; TG-1, 47.5, 45.1, 41.2 and 21.4 k; TG-18, 47.5,45.1 and 21.4 k. Two dimensional gel electrophoresis (IEF and SDS-PAGE) indicates that the arachin subunits consist of two major groups—the acidic polypeptides (three in SP, TG-1 and two in TG-18) and the basic polypeptides (three in SP, TG-1 and two in TG-18). The acidic polypeptides did not show charge variation while the basic polypeptides were charge heterogeneous. Absence of both an acidic polypeptide along with a basic polypeptide in TG-18 suggests that the acidic and basic polypeptides are probably products of the same gene and arise as a result of post-translational cleavage. Antibodies raised against purified arachin from SP reacted with arachin from TG-1 and TG-18 showing similar antigenic determinants. The acidic polypeptides show considerable homology in their structure as revealed by peptide mapping patterns.     

Effect of structural modifications on the assembly of a glycinin subunit.

A Gy4 glycinin cDNA was modified and used to produce structurally altered 11S storage protein subunits. We evaluated these modified subunits for their ability to assemble into oligomers. Alterations made in the acidic polypeptide changed the subunit solubility characteristics but did not eliminate assembly. Modifications in the basic polypeptide usually eliminated assembly of subunits into trimers. A region exhibiting high natural variability located at the COOH terminus of the acidic polypeptide that we have designated the hypervariable region was also studied. Extensive deletions and insertions were tolerated in the hypervariable region without perturbing subunit assembly. Some of the insertions significantly increased the methionine content in the Gy4 glycinin subunit. Together, our results indicated that the structure of the basic polypeptide was more critical for assembly of trimers than that of the acidic polypeptide, an observation that implies that the basic polypeptides direct trimer formation. The assembly assays described here will be useful in efforts to improve seed quality. Using them, the effects of modifications to the storage protein subunits can be rapidly evaluated before introducing the mutated genes into plants.     

One large subunit of ribulose 1,5-bisphosphate carboxylase oxygenase in Medicago,Spinacia and Nicotiana

Summary Isoelectric focusing of subunits of ribulose 1,5-bisphosphate carboxylase oxygenase of Medicago, Spinacia and Nicotiana were investigated, using a rapid isolation technique, without S-carboxymethylation. RuBPC-ase and its subunits were isolated by gel electrophoresis. Isoelectric focusing of RuBPC-ase of M. sativa and M. falcata showed that this enzyme consists of one large subunit (LSU) polypeptide and two or three small subunits (SSU), depending on the genotype. The pl of the LSU's was identical, but the pl of SSU's of the two genotypes was different. Amino acid composition and tryptic peptide maps further supported the concept of a conserved nature of LSU and heterogeneity of SSU polypeptides in Medicago. It was also found that S. oleracea, N. tabacum, N. glutinosa and N. excelsior have a single LSU polypeptide, but they differ in respect of pl values. The SSU polypeptides appeared to be variable. S-carboxymethylation affected the number as well as the pl values of LSU and SSU polypeptides. It is suggested that one LSU polypeptide is probably the general rule in higher plants, rather than the three LSU polypeptides demonstrated by Chen et al. (1977) and Wildman (1979).     

Purification and reconstitution of the proton-pumping ATPase of fungal and plant plasma membranes

The 11S storage protein (glycinin) of soybean [Glycine max (L.) Merr., cv. Raiden] was studied by polyacrylamide gel electrophoresis and amino acid sequence analysis. It contained the following subunits composed of acidic (A) and basic (B) polypeptides: A1aB2, A1bB1b, A2B1a, and A3B4. However, it lacked polypeptides A4, A5, and B3 which are present in many other cultivars. A new acidic polypeptide called A6 was present in a low amount and was characterized by amino acid sequence analysis. It was homologous to A4, although of a smaller apparent molecular weight. Since Raiden has an average protein content of about 40% and its glycinin fraction can be purified as a 350,000 D complex which is typical of other cultivars, the results imply polymorphism with respect to glycinin subunit composition. Because there is a wide variation in the methionine content of the various subunits, these findings suggest the possibility of genetically manipulating the nutritional quality of soybean seed protein by altering glycinin subunit composition.     

Reconstitution of Arachin from Isolated Subunits

Six subunits of arachin were isolated in urea solution. They were then reassociated by removing urea by co-dialysis against 20 mM sodium phosphate buffer (pH 7.9), containing 30% sucrose, 0.1 M> sodium chloride and 7 mM β-mercaptoethanol, without agitation at 25°C. The reconstitution yield was greater than 90%. The reconstituted molecule was indistinguishable from intact arachin in disc electrophoretic mobility, subunit composition, sedimentation behavior depending upon ionic strength, circular dichroism, ultraviolet absorption and fluorescence emission spectra, and stabilities against heating, proteases and guanidine hydrochloride. The reconstituted arachin was, therefore, suggested to be in native state.

On the other hand, we found that co-dialysis of four or five subunits of arachin formed hexamer which contained the corresponding four or five subunits. These hexamers were more labile than intact arachin against heating. These facts suggest that the assembly of all six subunits to a hexamer will most advantage the quaternary structure of arachin.     

Reevaluation of the Subunit Molecular Weights of Soybean 11S Globulin

The acidic and basic subunits are the main constituents of soybean 11S globulin. Each of these two subunits consists of three major polypeptides of similar size. The molecular weights of the acidic and basic subunits have been previously estimated to be 37,000 and 22,000, respectively, by SDS-polyacrylamide gel electrophoresis (Catsimpoolas et al, J. Set Food. Agric., 22, 448 (1971)). Reevaluation of the molecular weights by 6 m guanidine gel chromatography gave the values of 28,000 and 18,000, respectively. These are supported by results of equilibrium sedimentation in the same solvent. The previously reported values seem to have been overestimated, especially for the acidic subunits. The overestimations seem to be related to the high percentage of acidic amino acids, which causes the conformation of the SDS-protein polypeptide complexes of these subunits to deviate from those of proteins usually employed as standards for molecular weight estimations.     

The Ricinus communis 2S albumin precursor: a single preproprotein may be processed into two different heterodimeric storage proteins

Summary TheRicinus communis (castor bean) 2S albumin is a heterodimer of glutamine-rich, disulphidelinked 4 and 7 kDa polypeptides. A cDNA library was constructed using mRNA from maturing castor bean endosperm as template. Clones containing sequences complementary to albumin mRNA were isolated by hybridization using as a probe a mixture of synthetic oligonucleotides representing sequences predicted for a peptide present in the 2 S albumin large subunit. The nucleotide sequence contained an open reading frame encoding a preproprotein of 258 amino acid residues. The preproprotein included both polypeptides of the previously sequenced 2S albumin. In addition, this precursor included two further glutamine-rich sequences which, in terms of their size and conserved cysteine residues typically found in seed proteins of the 2S albumin superfamily, possibly represent the small and large polypeptide subunits of a second heterodimeric storage protein. A post-translational processing scheme is proposed which would result in a single preproprotein generating two distinct heterodimeric 2S albumins. The generation of a second heterodimer seems likely since polypeptide candidates for its small and large subunits were found in theRicinus 2S albumin fraction, and N-terminal protein sequencing confirmed the existence of the putative small subunit.     

Unassembled polypeptides of the plastidic ribosomes in heat-treated 70S-ribosome-deficient rye leaves

The polypeptides of the subunits of 70S ribosomes isolated from rye (Secale cereale L.) leaf chloroplasts were analyzed by two-dimensional polyacrylamide gel electrophoresis. The 50S subunit contained approx. 33 polypeptides in the range of relative molecular mass (M_r) 13000–36000, the 30S subunit contained approx. 25 polypeptides in the range of M_r 13000–40500. Antisera raised against the individual isolated ribosomal subunits detected approx. 17 polypeptides of the 50S and 10 polypeptides of the 30S subunit in the immunoblotting assay. By immunoblotting with these antisera the major antigenic ribosomal polypeptides (r-proteins) of the chloroplasts were clearly and specifically visualized also in separations of leaf extracts or soluble chloroplast supernatants. In extracts from rye leaves grown at 32° C, a temperature which is non-permissive for 70S-ribosome formation, or in supernatants from ribosome-deficient isolated plastids, six plastidic r-proteins were visualized by immunoblotting with the anti-50S-serum and two to four plastidic r-proteins were detected by immunoblotting with the anti-30S-serum, while other r-proteins that reacted with our antisera were missing. Those plastidic r-proteins that were present in 70S-ribosome-deficient leaves must represent individual unassembled ribosomal polypeptides that were synthesized on cytoplasmic 80S ribosomes. For the biogenesis of chloroplast ribosomes the mechanism of coordinate regulation appear to be less strict than those known for the biogenesis of bacterial ribosomes, thus allowing a marked accumulation of several unassembled ribosomal polypeptides of cytoplasmic origin.Abbreviations L polypeptide of large ribosomal subunit - M_r relative molecular mass - r-protein ribosomal polypeptide - S polypeptide of small ribosomal subunit - SDS sodium dodecyl sulfate     

An analysis of the subunit structure of the crystalloid protein complex from castor bean endosperm

Chromatographic and electrophoretic studies have shown that the subunits of the crystalloid protein, isolated from mature castor bean (Ricinus communis L. cv Hale) seed endosperm protein bodies, are heterogeneous with molecular weights in the range 49 to 53.5 kilodaltons (kD), and are quantitatively in unequal amounts. Each subunit comprises an αβ polypeptide pair which are reduced by 2-mercaptoethanol in two subgroups with molecular weights in the 29 to 34 kD and 20.5 to 23.5 kD ranges. Subunits and corresponding polypeptide pairs are also seen to be heterogeneous in pI following isoelectric focusing. In general, large polypeptides are acidic (pI 4.8-6.2) and small polypeptides basic (pI 7.4-9.4), although overlap of some isoelectric isomers does occur, notably in polypeptides derived from subunits which are quantitatively present in smaller amounts.     

Immunological relationships among the major seed proteins of cereals

The structural relationships among the major seed proteins of cereals was evaluated by Western blot analyses using antibodies raised against the wheat gliadin, rice glutelin acidic and basic subunits, and rice prolamine polypeptide. Consitent with the conservation of the primary sequences of these proteins, antibodies to the acidic and basic glutelin subunits cross-reacted with homologous polypeptides from oat as well as pea. The rice glutelin antibodies did not react with the major seed proteins from barley, rye, maize and sorghum. Antibodies raised against the acidic glutelin subunit reacted with the wheat glutenins but antibodies to the basic glutelin subunit did not. A comparison of the published primary sequences of a high molecular weight glutenin and rice glutelin showed little similarity except for a conserved peptide with the motif arg-gln-leu-gln-cys. The possible significance of this conserved element shared by these widely different proteins is discussed. Similar studies with the wheat gliadin antibody showed immunologically related components in plants of the subfamily Festucoideae except for rice. Antibodies raised against the rice prolamine recognized only the rice prolamine, indicating that this polypeptide was structurally distinct from other cereal prolamines. Overall, these results support and help clarify the evolutionary relationship of the cereals.     

Urea-Elicited Changes in Relative Electrophoretic Mobility of Certain Glycinin and beta-Conglycinin Subunits

Six molar urea in sodium dodecyl sulfate-polyacrylamide gels altered the relative electrophoretic mobility of several soybean protein subunits. Glycinin acidic polypeptide components A₃ and A₄ could be resolved from the other acidic polypeptides. A variant of the δ′ subunit of β-conglycinin was identified.     

DNA-dependent RNA polymerases from Acanthamoeba castellanii. Comparative subunit structures of the homogeneous enzymes.

The constituent polypeptides of the three classes of DNA-dependent RNA polymerase from Acanthamoeba castellanii were compared by several electrophoretic methods. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) reveals that a number of polypeptide components of the isozymes have identical molecular weights. Two-dimensional electrophoresis (isoelectric focusing in 8 M urea:SDS-polyacrylamide gel electrophoresis) demonstrates that the polypeptides of identical molecular weights also have identical isoelectric pH values. These polypeptides were also coincident after electrophoresis in 8 M urea at acidic or basic pH values followed by a second electrophoretic separation in the presence of SDS. By these criteria, subunits of molecular weight 13,300, 15,500, 17,500, 22,500, 37,000, and 39,000 are indistinguishable in polymerase I and III. The 13,300, 15,500, and 22,500 subunits are also shared by the class II polymerase. In addition, electrophoresis in 8 M urea under basic conditions reveals microheterogeneity in the 17,500 molecular weight subunit. The strikingly similar pattern of common subunits between yeast and Acanthamoeba suggests that a universal arrangement of functional units may be an essential feature of the eukaryotic polymerases.     

The quaternary structure of bovine brain kinesin.

In the present work we have studied the subunit composition of kinesin, the microtubule-activated, mechanochemical ATPase, isolated from bovine brain. Polypeptides with mol. wts of 120 and 62 kd are the major components of the kinesin preparation. These polypeptides could not be separated by electrophoresis under nondenaturing conditions or by FPLC on a MonoQ column, and are therefore assumed to form a tight complex. As shown by immunoblotting with polyclonal and monoclonal antibodies to the 120-kd polypeptide and by one-dimensional peptide mapping, the 62-kd polypeptide does not appear to be a proteolytic product of the 120-kd component. Densitometric scanning of polyacrylamide-SDS gels shows that these polypeptides are present in a complex in a 1:1 molar ratio. The mol. wt of native kinesin was studied by sedimentation equilibrium and was found to be 386 +/- 14 kd. A comparison of the mol. wts of individual polypeptides with the mol. wt of the intact molecule indicates that the native molecule contains two 120-kd subunits and two 62-kd subunits.     

Specific subunit pairs of legumin from Vicia faba

Four pairs of disulphide-linked acidic (α) and basic (β) subunits were isolated from legumin of Vicia faba. Pairing between α- and β-subunits is nonrandom, supporting the view that each subunit pair arises from a common precursor polypeptide, already containing intramolecular disulphide bonds, when cleavage to the subunit pair takes place. The subunit pairs belong to two structural types: type A contains Met, whereas type B lacks Met. In addition to these four subunit pairs, at least two more pairs are present in legumin in minor amounts.     

Multiple molecular forms of peanut lectin: Classification of isolectins and isolectin distribution among genotypes of the genus Arachis

Peanut lectin (or an immunologically indistinguishable material) is present in seeds of 4556 genotypes of the peanut, Arachis hypogaea, and in 65 genotypes of related species of Arachis. Seeds of one line of A. villosa and three lines of unclassified Arachis spp. are devoid of the lectin. Peanut lectin from 116 A. hypogaea genotypes is resolved by isoelectric focusing into three related isolectin profiles, which are designated the V, S, and V₂ types. Each is composed of from six to eight separate isolectins. Peanut lectin from A. monticola, A. pusilla, and one genotype of Arachis spp. is of the V type; isolectin profiles from other wild Arachis genotypes are variable, but comprise several distinct groups. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate resolves peanut lectin preparations from 37 genotypes into the lectin subunit of M_r 30,000 and a second polypeptide of M_r 60,000. Lectin preparations from five genotypes lack the M_r 60,000 polypeptide band and have a subunit that migrates slightly faster (and therefore probably is of lower molecular weight) than the subunits of all other tested lines. Peanut lectin preparations from 62 lines have specific hemagglutinating activities ranging from 1024 to 4196 with desialyzed human Type O erythrocytes. The lectin from one genotype exhibits substantially less hemagglutinating activity and is hemolytic.     

Variability of the Low Molecular Weight Globulin,Conglutin δ, Within Lupin Species

Conglutin δ, a 2S globulin, was purified and compared in six species or varieties of lupin seeds. A common pattern is suggested, present in all species, corresponding to a protein which could exist as a monomer or a dimer. The first form contains one subunit, from 11 to 16.2 kDa, according to the species. It possesses a quaternary structure closely related to conglutin δ1 and was previously described in the narrow-leaved lupin. The second form contains two similar subunits (23 to 26 kDa) and could be the conglutin δ2. These two subunits are associated even when SDS is used and are probably disulfide-linked subunits. Each subunit is composed of two disulfide-linked polypeptides. One is acidic with molecular weight from 14 to 17.3 kDa and the second is acidic to neutral, from 2.4 to 4.5 kDa. Three species (L. luteus, L. arboreus and L. pilosus) present a supplementary subunit, with different molecular weight and p than that previously described and which never associates in a dimer form. It has been purified in L. luteus. When native, this protein is oligomeric. The subunit of 12 kDa in this species is composed of a polypeptide of 9 kDa (pl 4.5) disulfide-linked to one of 3 kDa (pl 6.5). This supplementary protein remains partly associated with the first in the yellow lupin (L. luteus). It probably corresponds to a new protein, different from conglutin δ.     

Biosynthesis and processing of the alpha subunit of the voltage-sensitive sodium channel in rat brain neurons

The sodium channel from rat brain is a complex of alpha (260 kd), beta 1 (36 kd), and beta 2 (33 kd) subunits. The alpha and beta 2 subunits are linked by disulfide bonds. The earliest biosynthetic precursor of the alpha subunit is a 203 kd core polypeptide with sufficient high-mannose carbohydrate chains to increase its apparent size to 224 kd. It is processed to 224 kd and 249 kd precursor forms containing complex carbohydrate chains before it achieves the mature size of 260 kd. Most newly synthesized alpha subunits are not disulfide-linked to beta 2 subunits, but remain as a metabolically stable pool of intracellular subunits. alpha subunits disulfide-linked to beta 2 are found preferentially at the cell surface. A possible role for this intracellular pool as a rate-limiting step in the regulation of the cell surface density and localization of sodium channels in developing neurons is proposed.     

Synthesis and assembly of the cytochrome b-f complex in higher plants

The cytochrome b-f complex is composed of four polypeptide subunits, three of which, cytochrome f, cytochrome b-563 and subunit IV, are encoded in chloroplast DNA and synthesised within the chloroplast, and the fourth, the Rieske FeS protein, is encoded in nuclear DNA and synthesised in the cytoplasm. The assembly of the cytochrome b-f complex therefore requires the interaction of subunits encoded by different genomes. A key role for the nuclear-encoded Rieske FeS protein in the assembly of the complex is suggested by a study of cytochrome b-f complex mutants. The assembly of individual subunits of the complex may be regulated by the availability of prosthetic groups. The genes for the chloroplast-encoded subunits and cDNA clones for the Rieske FeS protein have been isolated and characterised. Cytochrome f and the Rieske FeS protein are synthesised initially with N-terminal presequences required for their correct assembly within the chloroplast. The deduced amino acid sequences of the four subunits have been used to suggest models for the arrangement of the polypeptides in the thylakoid membrane.