Major Polypeptide Components Research Articles

Isolation and Characterization of a Novel ATPase-Photosystem I Reaction Center Complex in the Chloroplast Thylakoid Membrane

A novel 1 MDa intra-membrane ‘super-complex’ dominated by the chloroplast ATP synthase and the photosystem I (PSI) reaction-center complex has been isolated from spinach thylakoid membranes. This super-complex has been characterized by assay of ATPase activity, negative stain electron microscopy, tandem mass spectrometry, and Western assay of subunits. High-resolution Orbitrap mass spectrometry was used to quantitatively analyze the presence and relative amount of the multiple major and minor polypeptide components in the super-complex. This is the first isolation of a higher order complex involving intact ATP synthase and Photosystem I to be identified and documented by negative stain electron microscopy. The importance of the ATPase-PSI super-complex in the thylakoid domain may imply efficient channeling of substrates and cofactors utilized for localized CO2 fixation. Studies supported by NIHGMS-038323 and the Henry Koffler Distinguished Professorship (WAC), and NIHDK-063491 (JPW).

Association between single nucleotide polymorphism of apoVLDL-II gene with growth and body composition traits in Iranian commercial broiler line

Very low density apolipoprotein-II (apoVLDL-II) is a major polypeptide component of avian VLDL. The function of apoVLDL-II is the transport of neutral lipids (triacylglycerol) in the form of VLDL in the plasma. The apoVLDLII gene is dormant in embryos, chicks and roosters but can be activated by estrogen. The current study was designed to investigate the association of an apoVLDL-II gene polymorphism on chicken growth and body composition traits. Genomic DNAs were extracted from 400 chickens from four different commercial broiler lines. Genotyping for the apoVLDL-II gene by using PCR-RFLP method and SfcI restriction endonuclease showed a mutation in 492-bp fragment located on the first intron. Polymorphism in apoVLDL-II gene was significantly (P < 0.05) associated with body weight at 6 week (BW6), carcass weight (CW), breast muscle weight (BMW), drumstick weight (DW) and wing weight (WINW). No significant difference was observed in the back weight (BAKW) and abdominal fat weight (AFW). This research suggests that apoVLDL-II gene could be a candidate gene that can affect some growth traits in chickens.

Protective effect of extracellular protein metabolite of Luteococcus japonicus subsp. casei on cells subjected to heating and UV irradiation

Extracellular protein metabolite isolated from the culture liquid of Luteococcus japonicus subsp. casei had reactivating and protective effects on UV-irradiated and heated cells. The extracellular metabolite, produced by cells in the logarithmic growth phase, was present in culture liquid in minuscule amounts. Mass spectral analysis showed that, along with the major component with a molecular weight of 7.6 kDa, the preparation contained low quantities of three minor proteins. Apparently, the biological activity of the exometabolite is determined by the major polypeptide component.

The identification of foam-forming soluble proteins from wheat (Triticum aestivum) dough

Proteomic methods have been used to identify foam-forming soluble proteins from dough that may play an important role in stabilising gas bubbles in dough, and hence influence the crumb structure of bread. Proteins from a soluble fraction of dough (dough liquor) or dough liquor foam have been separated by two-dimensional gel electrophoresis, and 42 identified using a combination of matrix-assisted laser desorption/ionization-time of flight and quadrupole-time of flight analyses. Major polypeptide components included beta-amylase, tritin and serpins, with members of the alpha-amylase/trypsin inhibitor family being particularly abundant. Neither prolamin seed storage proteins nor the surface-active protein puroindoline were found. Commonly used dough ingredients (NaCl, Na L-ascorbate) had only a minor effect on the 2-DE protein profiles of dough liquor, of which one of the more significant was the loss of 9 kDa nonspecific lipid transfer protein. Many proteins were lost in dough liquor foam, particularly tritin, whilst a number of alpha-amylase inhibitors were more dominant, suggesting that these are amongst the most strongly surface-active proteins in dough liquor. Such proteins may play a role determining the ability of the aqueous phase of doughs, as represented by dough liquor, to form an elastic interface lining the bubbles, and hence maintain their integrity during dough proving.

Protein Biosynthesis in the Embryo and Endosperm during Embryogenesis in Pinus sylvestris L.

Protein biosynthesis in the embryo and endosperm during embryogenesis in the Scots pine was studied using electrophoretic and immunochemical methods. Proteins of the albumin-globulin fraction were visualized already at the early embryonic stages. The major polypeptide components (48–60, 37–39, and 20–22 kDa) were gradually accumulated in the course of maturation. A high synchrony was noted between the stages of embryogenesis and molecular events related to protein biosynthesis and accumulation in the developing seed.

Cluster of genes encoding the major egg envelope protein of zebrafish

The proteins of fish egg envelopes are encoded by genes that are closely related to the genes for human zona pellucida proteins. A cluster of three genes coding for an egg envelope protein was isolated from the zebrafish, Danio rerio. The three genes, zp2a, zp2b, and zp2c, are located within an 11 kb region and are each comprised of eight exons spanning 1.85 kb. The exon-intron structures of the genes are nearly identical; however, their deduced amino acid sequences diverge at exon 7 (zp2b and zp2c from zp2a) and exon 8 (zp2c from zp2b). Exons 2-7 have a structural organization similar to exons in the carboxy-terminal half of the human zona pellucida ZP1, ZP2, and ZPB genes, suggesting they arose from a common ancestral gene. Sequence comparisons indicate that the deduced zebrafish proteins are most closely related to human ZPB. Zebrafish mRNAs coding for each of the three ZP2 variants have been found either as full-length cDNAs or expressed sequence tags. Distinct from the wf(female) gene of winter flounder which we first reported (Lyons et al., 1993: J Biol Chem 268:21351-21358), expression of the zebrafish zp2 genes was found to be ovary-specific, instead of liver-specific, and the promoter regions of zp2a and zp2b, while different, both contained E-box sequences (CANNTG) that have been demonstrated to be essential for coordination of zona pellucida gene expression in mammalian oocytes. Mixed peptide sequence analysis was used to identify the major polypeptide component of isolated zebrafish egg envelopes as the zp2 gene product.

Compositional changes during commercial processing of flaxseed

Compositional changes occurring during processing of flaxseed are of primary importance in adding value to flaxseed products. The physicochemical characteristics of flaxseed products at four stages of commercial processing, cleaning, flaking, pressing and solvent extraction, were determined to improve the functional properties of flaxseed products. The effect of industrial oil extraction on proximate composition, protein extractability, gel electrophoretic profiles, in vitro protein digestibility and viscosity of eight flaxseed products was assessed. The processes of flaking, heating, solvent extraction and solvent removal significantly increased the contents of protein, ash and soluble carbohydrate, and decreased oil content, total phenolics and protein solubility. Pepsin digestibility ranged from 11 to 72%, and generally increased with processing. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis of solubilized proteins showed the presence of four major polypeptide components, with only minimal changes in the protein profiles among the samples as a result of processing. The viscosity of the slurry was largely unaffected but shear thinning indices increased with processing. Multiple-regression analysis showed that protein content was related to carbohydrate content, cyanogenic content, and viscosity, but not associated with protein solubility and digestibility.

Accumulation of 19-kDa plasma membrane polypeptide during induction of freezing tolerance in wheat suspension-cultured cells by abscisic acid.

Suspension-cultured cells derived from immature embryos of winter wheat (Triticum aestivum L. cv. Chihoku) were used in experiments designed to obtain clues to the mechanism of the ABA-induced development of freezing tolerance. Cultured cells treated with 50 microM ABA for 5 d at 23 degrees C acquired the maximum level of freezing tolerance (LT50; -21.6 degrees C). The increased freezing tolerance of ABA-treated cells was closely associated with the remarkable accumulation of 19-kDa polypeptides in the plasma membrane. The 19-kDa polypeptide components were isolated by preparative gel electrophoresis and were further separated into one major (AWPM-19) and other minor polypeptide components by Tricine-SDS-PAGE. N-terminal amino acid sequence of AWPM-19 was determined, and a cDNA clone encoding AWPM-19 was isolated by PCR from the library prepared from the ABA-treated cultured cells. The cDNA clone (WPM-1) encoded a 18.9 kDa hydrophobic polypeptide with four putative membrane spanning domains and with a high pI value (10.2). Expression of WPM-1 mRNA was dramatically induced by 50 microM ABA within a few hours. These results suggest that the AWPM-19 might be closely associated with the ABA-induced increase in freezing tolerance in wheat cultured cells.

Synthesis of the Mammalian Synaptic Vesicle Protein Synaptophysin in Insect Cells: A Model for Vesicle Biogenesis

The N-glycosylated integral membrane protein synaptophysin is one of the major polypeptide components of small presynaptic transmitter-containing vesicles in neurons and of similar vesicles in neuroendocrine cells of mammals. Functional properties, including a possible participation in channel formation, have been investigated by integration of purified synaptophysin into planar lipid bilayers. To overcome some of the inherent limitations of such anin vitroapproach we have overexpressed the rat synaptophysin cDNA in nonneuronal, non-neuroendocrine insect cells with the help of recombinant baculovirus. The complete polypeptide was produced in infected ovarian Sf9 cells at levels exceeding those observed in rat brain. The partially N-glycosylated molecules could be extracted from membranes with non-ionic detergents, most effectively withn-octyl-β-d-glucopyranoside, and could be enriched on chromatofocusing columns. By immunoelectron microscopy synaptophysin was shown to be integrated in the correct orientation into the endoplasmic reticulum, various pleomorphic vesicles and the plasma membrane. Using cell fractionation, including density gradient centrifugation and immunoisolation, we characterized distinct synaptophysin-rich vesicles. These vesicles may help to understand molecular principles of vesicle biogenesis in general and the function of synaptophysin in particular.

Immunisation of sheep with an integral membrane glycoprotein complex of Haemonchus contortus and with its major polypeptide components

Sheep were immunised against Haemonchus contortus with an integral membrane glycoprotein complex isolated from the intestines of the parasite as antigen. This antigen has been termed Haemonchus galactose-containing glycoprotein complex. Sodium dodecyl sulphate ( SDS) polyacrylamide gel analysis has shown that it is composed of several polypeptides but so far these have proved refractory to separation when in the native state. However when dissociated by SDS, it was found to be as efficacious as in its native state, although it was less consistently protective when tested after being dissociated and reduced. An attempt was therefore made to identify the protective ingredient(s) of the dissociated complex by testing its major polypeptides individually after they had been separated on SDS polyacrylamide gels under non-reducing conditions. Partial protection was induced by protein bands with molecular weights of about 200 kD and less than 50 kD, but none of the individual fractions tested was as efficacious as the unseparated complex, suggesting that either more than one component was essential for a consistent effect or that the separation technique had damaged crucial protective epitopes.

Expression of the synaptophysin gene family is not restricted to neuronal and neuroendocrine differentiation in rat and human

Abstract The integral membrane protein synaptophysin is one of the major polypeptide components of the small, electron-translucent, transmitter-containing vesicles in neurons and of similar vesicles in neuroendocrine (NE) cells. In an attempt to identify synaptophysin-related molecules, such as synaptoporin, it was noticed in polymerase chain reaction (PCR) experiments that products having the expected size could be amplified not only from neuronal and NE cells, but also from non-NE cells. Northern blot hybridization analyses demonstrated that certain non-NE cells express low amounts of synaptophysin mRNA although the encoded polypeptide could not be detected. These observations, however, did not explain the consistent amplification of cDNA fragments regardless of cell type. PCR products were therefore cloned and a novel type of cDNA was identified in rat and human. The partial human cDNA was completed by isolation of phage cDNA clones constructed from a human keratinocyte cell line (HaCaT) and by PCR. When used in hybridization experiments with genomic DNA, this clone recognized a single gene. The 2106 bp cDNA contains an open reading frame coding for a polypeptide of calculated molecular weight 28,565 and having an isoelectric point of 8.45. This polypeptide is very similar to synaptophysin in the four transmembrane domains and the connecting loop regions but lacks the characteristic cytoplasmic tail. Extensive PCR analyses and Northern blot hybridization experiments demonstrated that the synaptophysin-related gene is ubiquitously expressed in vitro and in vivo. To stress the ubiquity of expression in contrast to the restricted distribution of synaptophysin and synaptoporin, I propose to refer to the encoded polypeptide as pantophysin.

Fucoxanthin-chlorophyll a/c light-harvesting complexes of Laminaria saccharina: partial amino acid sequences and arrangement in thylakoid membranes.

The N-terminus of the major polypeptide component of the light-harvesting complex (LHC) from the brown alga Laminaria saccharina is blocked. Two partial sequences, one near the N-terminus and the other near the C-terminus, have been obtained by chemical cleavage with acetic acid and N-chlorosuccinimide. Four peptides were separated after trypsin digestion of the thylakoid membranes. One fragment is not phosphorylated, is not blocked, and has been sequenced. Purification on a reversed-phase column showed two forms of the LHC protein: the more hydrophobic form appears to be bound to photosystem I. These results are compared with LHC from other Chromophytes and the CAB family of green plants.

Purification of immature cores of mouse mammary tumor virus and immunolocalization of protein domains.

The immature capsids of the mouse mammary tumor virus (MMTV), known as intracytoplasmic A particles, have been isolated from murine L1210 leukemia cells. The diameter of the isolated particles was 80 nm as determined by negative staining. Two polypeptides of 77 and 110 kDa were found to be their major polypeptide components, in agreement with the expected sizes of the Gag and Gag-Pro precursor polypeptides of the mature MMTV proteins. Both polypeptides were recognized by antibodies directed toward the matrix (p10) and capsid (p27) proteins of MMTV. Immunogold labeling of p10 on isolated A particles, visualized by negative staining, showed that this protein is located at the surface of the immature capsids, whereas p27 can be detected only in broken or disrupted particles, suggesting that it has an internal location. These observations were confirmed by immunolabeling of both proteins on thin sections of A particle-producing cells. In addition, the viral protease had a more internal position than p27. Since the sequential order of the viral proteins in the Gag precursor is p10-pp21-p27-p14 and that in Gag-Pro is p10-pp21-p27-p30-protease, our results demonstrate the radial organization of the polypeptide precursors forming the intracytoplasmic A particles.

Autoantibodies to major and minor nuclear lamins are not restricted to autoimmune diseases

Autoantibodies to lamins, the major polypeptide components of the nuclear lamina, have been reported in selected sera from patients with autoimmune diseases, including anti-lamin B in systemic lupus erythematosus (SLE) and anti-lamins AC in autoimmune chronic active hepatitis (CAH). We have studied the frequency, specificity and isotypy of autoantibodies to major and minor lamins by immunoblotting on purified rat liver lamins in 190 sera from normal controls ( n = 62), rheumatic disease controls ( n = 42), and autoimmune disease patients ( n = 86). The frequency of anti-lamin in normal controls was 85.5%, and ranged from 77 to 100% in the other groups. Anti-lamin frequency was not related to age, sex, or disease duration. Reactivity with lamin A or with minor lamins only was observed with 7 various sera and 2 normal sera, respectively. Between groups, the proportions of reactive sera were not different for lamins AC (18–47%) and for lamin B (22–36%). In particular, anti-lamin B and anti-lamins AC were not more common in SLE or CAH than in normal sera. The most frequent lamin specificity of SLE sera was anti-lamins ABC. Anti-lamin isotypes were IgG and/or IgM. Titers of IgM antibodies were not higher in any group. However, IgG antilamin titers were higher in CAH than in normal, ankylosing spondylitis, or SLE sera. The highest end point titers (≧1:3200) were observed with CAH, SLE, and rheumatoid arthritis (RA) sera with IgG anti-lamins AC, B, or ABC, or with IgM anti-lamins ABC. None of these SLE and RA patients had evidence of liver disease. Reactivity with minor lamins was more frequent in CAH. We conclude that antilamin autoantibodies are present in sera from most individuals and that the highest titers are found in sera from patients with autoimmune diseases.

Properties of subunits of the multicatalytic proteinase complex revealed by the use of subunit-specific antibodies

The multicatalytic proteinase (MCP) is a high-molecular-mass non-lysosomal proteinase that gives rise to a characteristic pattern of bands of molecular mass 22-34 kDa on SDS/PAGE gels. Isoelectric-focusing gels of the enzyme purified from rat liver show 16 bands with isoelectric points in the range of pH 5-8.5. Two-dimensional PAGE gels reveal that there are more than the previously reported 13 polypeptides associated with the MCP from rat liver and show a pattern of 15-20 major spots and several minor ones, similar to that of MCP isolated from some other sources. Possible relationships between the different polypeptides were investigated by immunoblot analysis of electrophoretically purified proteinase subunits with affinity-purified subunit-specific antibodies as well as antibodies raised against individual denatured subunits of the complex. The results demonstrate that many of the major polypeptide components of the MCP complex are antigenically distinct. Moreover comparison of immunoreactive material in crude cell extracts with that in purified MCP preparations has shown that the polypeptides are not derived from a smaller number of higher-molecular-mass subunits. Also, individual subunits have the same apparent molecular mass in a variety of rat tissues, suggesting close similarity between MCPs of different tissues. The highest concentrations of MCP subunits occur in liver and kidney. Gel-filtration analysis of crude extracts has demonstrated that MCP polypeptides are also associated with a higher-molecular-mass complex, which may be the 26 S proteinase that has been implicated in the degradation of ubiquitin-protein conjugates.

H +-ATPase, a primary pump for accumulation of neurotransmitters, is a major constituent of brain synaptic vesicles

Upon treatment with sodium carbonate, rat brain synaptic vesicles lost ATP-dependent H+ transport and released major polypeptide components (about 72, 57, 41, 34 and 33 kDa). These polypeptides, consisting about 15% of the total protein, were identified as subunits of H(+)-ATPase by immunoblotting with antibodies against H(+)-ATPase from chromaffin granules. The same treatment also abolished the ATP-dependent, bafilomycin-sensitive uptakes of glutamate, serotonin and gamma-aminobutyrate by the synaptic vesicles. These results indicated that H(+)-ATPase is a major constituent of the vesicles (consisting about 20% of their total protein) and is a primary pump for accumulation of neurotransmitters.

Subunit organization of PSI particles from brown algae and diatoms: polypeptide and pigment analysis

P700 enriched fractions were isolated from two brown algae and one diatom using sucrose density centrifugation after digitinin solubilization. They had a Chl a/P700 ratio of about 250 to 375 according to the species, they were enriched in long-wavelength absorbing Chl a and exhibited a fluorescence emission maximum at 77 K near 720 nm. They all presented a major polypeptide component at 66±2 kDa, but their polypeptide composition was rather complex and somewhat different from one species to another. Further solubilization with dodecylmaltoside of those 'native' PSI particles allowed the separation of two or three fractions. The lightest, xanthophyll-rich, fraction was identified to be a light-harvesting complex. It contained no P700 and had a major polypeptide of molecular weight near 20 kDa (at the same molecular weight than the respective LH 'native' fraction of each species) and exhibited a 77 K peak fluorescence emission at 685 nm. The other fractions were enriched in P700 and almost entirely depleted in xanthophylls. When two of them are present, they both exhibited a major polypeptide at 66±2 kDa and were totally devoid of the LH polypeptide, but the two fractions widely differed one from another in the abundance and molecular weight of the other polypeptide components. The most purified of these two fractions presented a composition similar to PSI core complex from green plants.

Identification of a major polypeptide component of the sea urchin fertilization envelope

A monoclonal antibody (MAb No. 25-16), raised against purified cortical secretory vesicles (CVs) from the eggs of Strongylocentrotus purpuratus, has been used to identify a previously uncharacterized CV-derived polypeptide component of the sea urchin fertilization envelope (FE). MAb No. 25-16, an IgG 1, bound to a group of proteins with M r ≈ 200,000 on immunoblots of CVs. This same group of proteins also was detected in fertilization product and in soft FEs prepared from early embryos, indicating that the antigen is released at fertilization by CV exocytosis and becomes incorporated into the FE. The multiple components recognized by MAb No. 25-16 apparently did not result from proteolysis during sample preparation or differential N-linked glycosylation. No simplification of the SDS-gel or immunoblot patterns was observed when samples of fertilization product or cell surface complex were prepared in the presence of a cocktail of protease inhibitors; nor was a change in mobility of any of the antigen forms detected following treatment with endoglycosidase F. Upon partial denaturation and reduction of the protein by incubation at room temperature in the presence of SDS and dithiothreitol, the antigen was shown to undergo a decrease in relative mobility on SDS-PAGE. Complete reduction and denaturation, by boiling in dithiothreitol-containing SDS sample buffer or by an on-blot reduction technique, resulted in loss of the epitope. The protein component recognized by MAb No. 25-16 underwent a striking increase in mobility on SDS-PAGE after chelation of calcium ions with EGTA. Immunogold labeling on thin sections of unfertilized eggs revealed that the antigen is located in the spiral lamellar cores of all CVs. In fertilized eggs, fixed 5 min after insemination, the antigen was detected in the FE. Based on these biochemical and immunological data, we suggest that the antigen recognized by MAb No. 25-16 is released exocytotically from the CVs into the perivitelline space at fertilization and becomes incorporated into the FE. The abundance of this antigen suggests that it may function as a structural component of the FE.

Characterization of matrix domains of the hamster acrosome.

In this study we describe the purification and the structural and biochemical properties of a detergent-stable complex of the hamster sperm acrosome. This complex consists of two distinct acrosomal matrix domains and a layer of electron-dense material, termed the acrosomal lamina, derived from the luminal surface of the outer acrosomal membrane. This complex has been isolated by centrifugation of detergent-extracted sperm suspensions on Percoll density gradients. The complex contains two major polypeptides of Mr 29,000 and Mr 22,000 and minor polypeptides of Mr 64,000-62,000, 56,000 and 35,000. Gelatin-containing sodium dodecyl sulfate-polyacrylamide gels demonstrate that bands of proteinase activity are not the major polypeptide components of the complex. These data demonstrate that the matrix of the acrosome is compartmentalized into domains of differing structural properties that occupy specific locations in the intact acrosome and that matrix components are physically associated with the outer acrosomal membrane. These data indicate that a structural framework is present within the acrosome and we speculate that it may be involved in sequestering hydrolases into specific spatial domains and could affect the temporal release of activity of selected hydrolases during the acrosome reaction.

Characterization of the postacrosomal sheath of bovine spermatozoa.

A purified head fraction was prepared from bovine epididymal spermatozoa and was utilized to identify the solubility characteristics and major polypeptide components of the postacrosomal sheath. Sperm heads extracted in nonionic-detergent-containing or high-salt-containing solutions retained an intact postacrosomal sheath, but it was readily solubilized by high pH extraction solutions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major polypeptide of 58,000 daltons (58-kD) in the high pH extract solution. Antibodies to the 58-kD polypeptide specifically reacted with the postacrosomal segment by immunofluorescence and by electron microscopic immunohistochemistry were shown to bind the postacrosomal sheath. We conclude that this 58-kD polypeptide is a constituent of the postacrosomal sheath and that its distribution is restricted to the postacrosomal segment.