Proteins of the chloroplast and cytoplasmic ribosomes of Euglena

The proteins of chloroplast and cytoplasmic ribosomes, isolatedfrom Euglena gracilis, have been compared by electrophoresison SDS-polyacrylamide gels. The proteins of the cytoplasmicribosomes were more numerous and larger on the average thanthose of the chloroplast ribosomes. There were about 14 proteins detected in the small subunit ofthe chloroplast ribosome, ranging from 11,000 to 43,000 daltonsand 16 proteins of 10,000 to 36,000 daltons from the large subunit.The banding patterns of the proteins of the subunits were quitedistinct from each other. The subunits of the cytoplasmic ribosomes were obtained by dissociationof the monomer with EDTA, and in 100 mm and 500 mil KCl andthe effects of these conditions of dissociation on the proteinsof the subunits compared. Regardless of the means of dissociation,the small and large subunits each gave 20–21 proteinsranging from 10,000 to 49,000 daltons. However, a comparisonof scans of the subunits indicated a selective and partial strippingof ribosomal proteins by high salt and by EDTA; i.e. differentproteins were sensitive to the two treatments. Native subunits, presumed to occur free in the cytoplasm werealso isolated. In addition to the ribosomal proteins found insmall subunits obtained by dissociation, the native small subunitcontained substantial amounts of high-molecular-weight proteins. Small, variable amounts of high-molecular-weight proteins arealso associated with chloroplast ribosome subunits, but thequantities depend on the method of purification of the subunits.Because these components are virtually eliminated followingtwo cycles of density gradient centrifugation, we infer thatthey are adventitious. These observations reflect on the relative merit among severalreported methods of purification of chloroplast and cytoplasmicribosomes. ¹ Present address: Department of Biochemistry, College of Medicineand Dentistry of New Jersey, Piscataway, N.J. 08854, U.S.A. (Received May 13, 1975; )     

Role of the amino- and carboxy-terminal regions in the folding and oligomerization of wheat high molecular weight glutenin subunits

The high molecular weight glutenin subunits are considered one of the most important components of wheat (Triticum aestivum) gluten, but their structure and interactions with other gluten proteins are still unknown. Understanding the role of these proteins in gluten formation may be aided by analyses of the conformation and interactions of individual wild-type and modified subunits expressed in heterologous systems. In the present report, the bacterium Escherichia coli was used to synthesize four naturally occurring X- and Y-type wheat high molecular weight glutenin subunits of the Glu-1D locus, as well as four bipartite chimeras of these proteins. Naturally occurring subunits synthesized in the bacteria exhibited sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration properties identical to those of high molecular weight glutenin subunits extracted from wheat grains. Wild-type and chimeric subunits migrated in sodium dodecyl sulfate gels differently than expected based on their molecular weights due to conformational properties of their N- and C-terminal regions. Results from cycles of reductive cleavage and oxidative reformation were consistent with the formation of both inter- and intramolecular disulfide bonds in patterns and proportions that differed among specific high molecular weight glutenin species. Comparison of the chimeric and wild-type proteins indicated that the two C-terminal cysteines of the Y-type subunits are linked by intramolecular disulfide bonds, suggesting that the role of these cysteines in glutenin polymerization may be limited.     

cDNA cloning of the two subunits of phospholipase A2 inhibitor PLIgamma from blood plasma of the Chinese mamushi, Agkistrodon blomhoffii siniticus

Three phospholipase A2 (PLA2) inhibitors (PLI) have been purified from the blood plasma of the Chinese mamushi, Agkistrodon blomhoffii siniticus; 1 of these, PLIgamma, contains 2 homologous subunits, PLIgamma-A and PLIgamma-B. The cDNAs encoding these 2 subunits of PLIgamma were isolated from a liver cDNA library by using fragments from polymerase chain reaction amplifications as probes and sequenced. The respective nucleotide sequences encoded 19-residue signal sequences, followed by 181-residue proteins. The calculated molecular masses were 20123 and 20150 Da for the PLIgamma-A and PLIgamma-B subunits, respectively; and PLIgamma-A included a N-linked carbohydrate site at Asn-157. The sequences of these subunits contained 2 internal repeats of disulfide-bonding pattern characteristic to those of urokinase-type plasminogen activator receptor and members of the Ly-6 superfamily. A phylogenetic analysis comparing the amino acid sequences of PLIgamma-A and PLIgamma-B with those for other snakes revealed that the gene duplication leading to these 2 subunits occurred before the divergence of Viperidae and Elapidae.     

Characterization of antigenic sialoglycoprotein subunits of the placental brush border membranes: Comparison with liver and kidney membrane subunits by two-dimensional electrophoresis

The sialoglycoprotein subunits of human placental brush border membranes were labeled by sequential treatment with periodate and (³H)-sodium borohydride, which trititates sialic acid, and by lactoperoxidase-catalyzed (¹²⁵I) iodination of tyrosine residues. The labeled subunits were characterized with respect to their affinity for antisera raised against Triton X-100 extracts of placental brush border membranes. The immunochemically reactive components were analyzed by two-dimensional electrophoresis according to a modification of the O'Farrell technique [20] enabling the assignment of estimated M_r? and pI. Of the 33 ³H-labeled brush border subunits present in Triton X-100-solubilized membrane preparations, 18 subunits reacted with antiplacental brush border antisera insolubilized on CNBr-activated Sepharose or in immunoprecipitates. Fourteen of these tritiated subunits were also labeled with ¹²⁵I, confirming that these are glycoproteins. The plasma membranes of normal human liver and microsomes from kidney were examined for the placental brush border glycoprotein subunits by reaction with insolubilized antiplacental brush border antisera and two-dimensional electrophoresis of the reacting tritium-labeled subunits. Comparison of the two-dimensional electrophoretic maps of the immunochemically reacting glycoproteins from liver, kidney, and placenta resulted in the identification of seven placental subunits in common with liver and kidney on the basis of antigenic cross-reactivity, M_r?, and pI. Four placental glycoproteins were not found in the other tissues and are potentially specific to the placenta. Three of the placental subunits were only seen in placenta and kidney. Three of the subunits ran at the dye front and could not be assigned molecular weights. One of the subunits was poorly labeled by tritiation of sialic acid and was not considered.     

Structural and genetical studies on the high-molecular-weight subunits of wheat glutenin

Summary The high-molecular-weight (HMW) subunits of glutenin from about 185 varieties were fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). About 20 different, major subunits were distinguished by this technique although each variety contained, with only a few exceptions, between 3 and 5 subunits. Further inter-varietal substitution lines to those already described (Payne et al. 1980) were analysed and the results indicate that all the HMW subunits are controlled by the homoeologous group 1 chromosomes. All hexaploid varieties studied except ‘NapHal’ contained two major subunits controlled by chromosome 1D. Their genes were shown to be tightly linked genetically for only four different types of banding patterns were observed. The nominal molecular weights determined after fractionation in 10% polyacrylamide gels were between 110,000 and 115,000 for the larger of the two subunits and between 82,000 and 84,000 for the smaller. One quarter of the varieties contained only one major HMW subunit controlled by chromosome 1B whereas the rest had two. The chromosome 1B subunits were the most varied and nine different banding patterns were detected. All the subunits had mobilities which were intermediate between those of the two chromosome 1D-controlled subunits. Only two types of HMW subunit controlled by chromosome 1A were detected in all the varieties examined; a single variety never contained both of these subunits and 40% of varieties contained neither. The chromosome 1A-controlled subunits had slightly slower mobilities in 10% gels than the largest HMW subunit controlled by chromosome 1D. About 100 single grains were analysed from each of five different crosses of the type (F₁ of variety A × variety B) × variety C. The results indicate that the genes on chromosome 1B which control the synthesis of subunits 6, 7, 13, 14 and 17 are allelic, as are the genes of the chromosome 1A-controlled subunits, 1 and 2.     

Reassembly of the Regularly Arranged Subunits in the Cell Wall of Lactobacillus brevis and Their Reattachment to Cell Walls

The reassembly of tetragonally arranged subunits in the cell wall of Lactobacillus brevis and the reattachment of the subunits to cell wall fragments were investigated by electron microscopy. The subunits dissociated from the cell wall with guanidine hydrochloride (GHCl) reassembled into the same regular array as seen in the native cell wall after dialysis against neutral buffer even in the absence of specific cations. The subunits could also reattach to the cell wall fragments from which they had been removed by treatment with GHCl, sodium dodecyl sulfate or cold trichloroacetic acid but not to those treated with hot formamide. Heterologous reattachment of the subunits occurred on cell wall fragments obtained from L. fermentum but not on those from L. plantarum or L. casei subsp. casei. On the basis of these observations and chemical analyses of the cell wall fragments, the subunits of L. brevis appeared to be bound by hydrogen bonds to a neutral polysaccharide moiety in the cell wall but not to peptidoglycan or teichoic acid.     

Identification and localization of G-proteins in the clonal adipocyte cell lines HGFu and Ob17

HGFu and Ob17 are cell lines derived from adipose tissue of lean (+/?) and ob/ob mice, respectively. Neither adenylyl cyclase activity nor G protein abundance and subcellular distribution have been assessed previously in these cells. Cyclase activity was low and resistant to catecholamine stimulation in both cell lines. However, the enzyme could be stimulated to high levels by forskolin and Mn²⁺. G_sα (largely the long isoform), G_iα2, and Gβ were the major G protein subunits identified. The levels of G protein mRNA expression were similar in both cell lines and, unlike actin expression, did not change as a result of differentiation. Immunoblotting and ADP-ribosylation of the G peptides corroborated these results. Assessment of the subcellular localization of the subunits by indirect epifluorescence and scanning confocal microscopy showed that each of the subunits had a characteristic subcellular pattern. G_sα showed vesicular cytoplasmic and nuclear staining; G_iα2 colocalized with actin stress fibers and disruption of these structures altered the distribution of G_iα2; β subunits showed some colocalization with the stress fibers as well as a cytoplasmic vesicular and nuclear pattern. As a result of differentiation, there was reorganization of the actin, together with the G_iα2 and β fibrous patterns. Both cell lines showed similar modifications. The induction of differentiation in these cells is therefore not associated with changes in adenylyl cyclase activity nor of the abundance of G-protein subunits, although reorganization of some of these subunits does accompany actin reorganization.     

High-molecular-weight glutenin subunits in the Cylindropyrum and Vertebrata section of the Aegilops genus and identification of subunits related to those encoded by the Dx alleles of common wheat

The high-molecular-weight (HMW) glute-nin subunit composition of seven species from the Cylindropyrum and Vertebrata sections of the Aegilops genus was studied using SDS-PAGE and Western blot analysis. Two subunits were detected in Ae. caudata and three in Ae. cylindrica. In both species, subunits showing electrophoretic mobility similar to that of 1Dx2 were present. Western blot analysis using a monoclonal antibody (IFRN 1602) specific for the 1Ax and 1Dx subunits of bread wheat showed that the 1Dx-like subunit of Ae. caudata gave only a weak reaction. This indicates that Ae. caudata expresses subunits which are more distantly related to the 1Dx subunits. Two subunits were detected in each of the 60 accessions of Ae. tauschii, including several 1D^tx subunits showing different electrophoretic mobilities from those of the 1Dx subunits commonly found in bread wheat. All of the 1D^tx subunits reacted strongly with IFRN 1602, confirming their close relationship to the 1Dx subunits of bread wheat. Three subunits were found in Ae. crassa (6 x), four in Ae. ventricosa and Ae. juvenalis and five in Ae. vavilovii. In these four species, the subunits that showed electrophoretic mobility similar, or close, to that of 1Dx2 all reacted with IFRN 1602. In addition, Ae. ventricosa contained a subunit showing electrophoretic mobility slower than that of 1Dx2.2, which also reacted with IFRN 1602. These results suggest that the D-genome component in the multiploid Aegilops species express at least one HMW glutenin subunit that is structurally related to the 1Dx subunits of bread wheat. Received: 5 November 1999 / Accepted: 12 February 2000     

Organization and topology of photosystem I subunits

Intact spinach (Spinacia oleracea) thylakoid membranes were treated with various proteases and photosystem I (PSI) complexes were isolated from these membranes to define the membrane topology of specific PSI subunits. Trypsin treatment caused cleavage of the PSI-D and E subunits. Thermolysin treatment cleaved the PSI-D, E, H, and K subunits, and also caused limited degradation of the reaction center core PSI-A and B subunits. Pronase treatment produced the most dramatic results as the PSI-A and B subunits were cleaved to 47-, 45-, 26-, and 24-kilodalton products. In addition, pronase degraded the PSI-D, E, H, K, and L subunits. Proteolytic cleavage sites for several of the products were identified by amino acid sequencing. The results indicate that PSI-A, B, D, E, H, K, and L subunits all have stroma-exposed regions, and these findings are summarized in a model describing the subunit organization of PSI.     

Analysis of the evolution of the family of the Sig genes encoding plant sigma factors

Within the latter decade, sigma subunits of bacterial-type RNA polymerases were found in eukaryote cells. In the higher plants and algae, these subunits determine the promoter specificity of the chloroplast multisubunit RNA polymerase. In the higher plants, sigma subunits are encoded by the family of nuclear Sig genes comprising 5–6 genes. Several conclusions as to the evolution of this gene family were deduced from the comparison of the deduced amino acid sequences and the sites of intron location.     

Equal Expression of the Maternal and Paternal Alleles for the Polypeptide Subunits of the Major Storage Protein of the Bean Phaseolus vulgaris L

Discontinuous sodium dodecyl sulfate slab gel electrophoresis of G1 globulin from several strains of Phaseolus vulgaris L. seed permitted clear resolution of the constituent polypeptides. Three strains (Tendergreen, Canadian Wonder, and BBL 240) had subunits of molecular weight 53,000, 47,000 and 43,000 while two strains (Seafarer and PI 229,815) had 50,500, 47,000 and 43,000 molecular weight subunits. F₁ seed from the cross BBL 240 × PI 229,815 showed four polypeptides on dissociation of the G1 protein; however, the amount of each of the 53,000 and 50,500 subunits was half that of the 47,000 subunit. This is interpreted as evidence that both the maternal and paternal loci for these polypeptides are transcribed and translated with similar efficiency. All of the polypeptides were found to have associated sugar residues.     

Relationships among the subunits of the high molecular weight proteinase,macropain (proteasome)

An analysis of the subunits of the high molecular weight proteinase, macropain (multicatalytic proteinase or proteasome) from human erythrocytes has been conducted using N-terminal amino acid sequencing, gel electrophoresis and reverse-phase peptide mapping. This analysis provided evidence for the existence of 13 subunits of different primary structure. Five subunits were susceptible to the Edman degradation and yielded unique N-terminal sequences. Similarities among these sequences, however, indicated that the subunits are homologues. Two-dimensional gel electrophoresis discriminated 10 major components, which included two of the subunits for which N-terminal sequences had been determined and eight N-terminally blocked subunits. Tryptic peptide mapping indicated that all 10 of these components have a different amino acid sequence. Tryptic peptides from some of the subunits were subjected to amino acid sequence analysis, and the data indicated that all the subunits tested in this way are related by common ancestry. The data suggest that at least nine of the total of 13 subunits are encoded by members of the same gene family; the remaining four subunits have not yet been investigated in sufficient detail to establish their relationships. No evidence for a close relationship with any previously investigated proteinase family has been found. Finally, through a comparison of the ‘latent’ and ‘active’ forms of macropain, the study established a close similarity in the subunit composition of these catalytically very different species, although proteolytic degradation of selected subunits appears in the active form of the enzyme.     

Phycocyanin synthesis and degradation in the blue-green bacterium Anacystis nidulans.

Cellular content and rates of synthesis of the apoprotein subunits of phycocyanin in Anacystis nidulans cultures undergoing, and recovering from, nitrate starvation were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total and immunoprecipitable soluble proteins. Results indicated that (i) nitrate starvation provokes coordinate degradation of apoprotein subunits: (ii) de novo synthesis of these subunits is selectively depressed during starvation; (iii) nitrate restoration provokes coordinate increases in the rates of synthesis of these subunits, although maximal rates are not achieved for 6 to 10 h after readdition of nitrate; and (iv) illumination affects both relative and absolute rates of apoprotein formation.     

Identification of cytochrome c oxidase subunits in nuclear yeast mutants lacking the functional enzyme.

Yeast mutants specifically lacking cytochrome c oxidase activity were screened for cytochrome c oxidase subunits by one- and two-dimensional electrophoresis, electrophoresis in exponential gradient gels, and immunoprecipitation with antisera against one or more of the cytoplasmically made subunits of the enzyme. Two cytochrome c oxidase-less nuclear mutants previously described from this laboratory each lack one or more mitochondrially synthesized cytochrome c oxidase subunits while possessing all four cytoplasmically synthesized subunits of that enzyme. The subunits remaining in these mutants were not assembled with each other; the cytoplasmically made subunits IV and VI could be released from the mitochondria by sonic oscillation, in contrast to the situation in wild type cells. No electrophoretically detectable alterations were found in any of the cytochrome c oxidase subunits present in the mutants. Nuclear mutations may thus cause both a loss as well as a defective assembly of mitochondrially made cytochrome c oxidase subunits.     

Structural homology among the major 7s globulin subunits of soybean seed storage proteins

The soybean seed 7S globulin subunits, i.e. α, α′, β and γ-subunits of β-conglycinin, the γ-conglycinin subunit and the HI/HII and LII subunits of basic 7S globulin were purified and the NH₂-terminal amino acid sequences of all these subunits except the γ-subunit of β-conglycinin were determined. Only the NH₂-terminal regions of the α and α′-subunits showed high sequence homology. However, sequencing of tryptic peptides from the seven subunits revealed that internal region sequences were highly homologous among the four subunits of β-conglycinin. In contrast to the β-conglycinin subunits, no sequence homology was found among the other subunits. On the basis of these results, the major 7S globulin fraction is considered more heterogeneous in primary structure than another major globulin fraction, 11S globulin (glycinin), in soybean seeds.     

Relationship between the Kinetic Properties and the Small Subunit Composition of Nicotiana Ribulose-1, 5-bisphosphate Carboxylase

Genetic variability in the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in several Nicotiana species has been characterized by isoelectric focusing patterns. This heritable variation provides an opportunity to examine the functional role of each of these subunits. In this study, specifically designed RuBPCase enzymes composed of identical large subunits but different small subunits were constructed in vivo by interspecific hybridization between the species N. sylvestris, N. tabacum, N. glauca, N. glutinosa, N. plumbaginifolia, and N. tomentosiformis. Small subunit polypeptides were combined to form a sequence of one, two, three, and four polypeptides with the large subunit of N. sylvestris. Kinetic properties of these hybrid enzymes were compared. No differences in the specific activity of either carboxylation or oxygenation nor in K_m values for ribulose 1,5-bisphosphate, CO₂, or O₂ were detected among the RuBPCase enzymes from the various interspecific hybrids. Likewise, the ratio of carboxylation to oxygenation was constant.     

Subunits of Xanthine Dehydrogenase and Their Differential Appearance in Chick Tissues During Development

Xanthine dehydrogenase (XDH) from adult chick liver comprises two polypeptide chains of different size in a molar ratio of 1: 1. The molecular weights of these subunits were estimated to be 155K (α) and 135K (β) daltons (1). However, XDH isolated from the liver of newly hatched chick was not found to represent the equimolar ratio of these two subunits; that is, the amount of subunit β was lower than that of subunit α. While examamining electrophoretically the change in the amounts of these subunits in the liver, the subunit α was found to appear earlier in the embryonic stage, but β only after hatching. In the kidney, however, both subunits were detected before hatching, being consistent with the fact that XDH exists before hatching in the kidney. The two subunits also appeared differentially in the kidney; i.e., subunit α appeared earlier than subunit β. In either tissue, the rate of increase in XDH activity corresponded to that of subunit β. Thus, the synthesis of two subunits of XDH are separately regulated at least until just after hatching.     

Binding of the delta subunit to rod phosphodiesterase catalytic subunits requires methylated, prenylated C-termini of the catalytic subunits

PDE6 (type 6 phosphodiesterase) from rod outer segments consists of two types of catalytic subunits, alpha and beta; two inhibitory gamma subunits; and one or more delta subunits found only on the soluble form of the enzyme. About 70% of the phosphodiesterase activity found in rod outer segments is membrane-bound, and is thought to be anchored to the membrane through C-terminal prenyl groups. The recombinant delta subunit has been shown to solubilize the membrane-bound form of the enzyme. This paper describes the site and mechanism of this interaction in more detail. In isolated rod outer segments, the delta subunit was found exclusively in the soluble fraction, and about 30% of it did not coimmunoprecipitate with the catalytic subunits. The delta subunit that was bound to the catalytic subunits dissociated slowly, with a half-life of about 3.5 h. To determine whether the site of this strong binding was the C-termini of the phosphodiesterase catalytic subunits, peptides corresponding to the C-terminal ends of the alpha and beta subunits were synthesized. Micromolar concentrations of these peptides blocked the phosphodiesterase/delta subunit interaction. Interestingly, this blockade only occurred if the peptides were both prenylated and methylated. These results suggested that a major site of interaction of the delta subunit is the methylated, prenylated C-terminus of the phosphodiesterase catalytic subunits. To determine whether the catalytic subunits of the full-length enzyme are methylated in situ when bound to the delta subunit, we labeled rod outer segments with a tritiated methyl donor. Soluble phosphodiesterase from these rod outer segments was more highly methylated (4.5 +/- 0.3-fold) than the membrane-bound phosphodiesterase, suggesting that the delta subunit bound preferentially to the methylated enzyme in the outer segment. Together these results suggest that the delta subunit/phosphodiesterase catalytic subunit interaction may be regulated by the C-terminal methylation of the catalytic subunits.     

长穗偃麦草中E和E1基因组高分子量麦谷蛋白基因启动子部分序列的进化分析

通过PCR克隆的方法,获得了分别来自二倍体长穗偃麦草的E基因组和四倍体长穗偃麦草的E_1基因组的4个高分子量麦谷蛋白亚基(HMW-GS)基因启动子的部分序列。序列分析表明,它们之间的同源性较高,两个x型亚基启动子序列之间只有1个碱基的差异,而两个y型亚基启动子序列完全相同,x和y型亚基启动子序列之间的长度和部分碱基位点都有差异。推测四倍体长穗偃麦草中的E_1基因组可能起源于二倍体的E基因组。与来自小麦族的A、B、D和G基因组部分亚基基因的启动子序列比较表明,小麦族的这一区域在进化上是相当保守的,不同基因组来源的序列同源性都在90％以上。经过对这些序列的聚类分析,表明长穗偃麦草的y型HMW-GS基因与其他亚基基因的进化关系较远,而x型亚基基因与一个来自小麦1B染色体的亚基基因关系最近。