Specific expression of annexin III in rat-small-hepatocytes

Small hepatocytes are cells that express characteristic phenotypes such as a high growth potential and differentiation capacity. In order to identify rat-small-hepatocyte specific proteins, we separated the cellular proteins of isolated small and parenchymal hepatocytes by 2D polyacrylamide gel electrophoresis. Comparison of their profiles revealed a protein with a molecular mass of 37 kDa in the small hepatocytes that was not present in the parenchymal hepatocytes. Proteolytic peptide mass fingerprinting was used to identify the protein and it was found to be annexin III. The validity of the identification was confirmed by Western blot analysis with anti-annexin III antibody.     

Protein kinases CKI and CKII are implicated in modification of ribosomal proteins of the yeast Trichosporon cutaneum

Phosphorylation of acidic ribosomal proteins P1/P2-P0 is a common phenomenon in eukaryotic organisms. It was found previously that in Trichosporon cutaneum, unlike in other yeast species, in addition to the two acidic ribosomal proteins, two other proteins of 15 kDa and 19 kDa of the small ribosomal subunit were phosphorylated. Here we describe two protein kinases: CKI and CKII, which are engaged in the modification of T. cutaneum ribosomal proteins. The acidic ribosomal proteins and the protein of 19 kDa were modified by CKII associated with ribosomes, while the protein of 15 kDa was modified by CKI. Protein kinase CKI was purified from cell-free extract (CKIC) and from ribosomal fraction (CKIR). The molecular mass of CKIC was established at 33 kDa while that of CKIR at 35-37 kDa. A protein of 40 kDa copurified with CKIR but not CKIC. Heparin significantly increased 40 kDa protein phosphorylation level by CKIR. Microsequencing analysis revealed the presence of CKI recognition motifs in the N-terminal fragment of the 40 kDa protein.     

Co-purification of A1 adenosine receptors and guanine nucleotide-binding proteins from bovine brain

A1 adenosine receptors and guanine nucleotide-binding proteins (G proteins) solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate have been co-purified from bovine cerebral cortex. A portion of solubilized receptors which displays high affinity GTP-sensitive agonist binding (40-50%) adheres tightly to agonist affinity columns composed of N6-aminobenzyladenosine-agarose. A1 adenosine receptors and G proteins are rapidly and selectively coeluted from agonist columns by the addition of 8-p-sulfophenyltheophylline, but only in combination with Mg2+-GTP or N-ethylmaleimide, agents which lower the affinity of receptors for agonists. Purified receptors and G protein alpha-subunits can be detected with the potent A1-selective antagonist radioligand, [125I]3-(4-amino-3-iodo)phenethyl-1-propyl-8-cyclopentylxanthine (125I-BW-A844U) and [35S]guanosine 5'-3-O-(thio)triphosphate [( 35S]GTP gamma S), respectively. Pretreatment of solubilized receptors with 0.1 mM N-ethylmaleimide or 0.1 mM R-phenylisopropyladenosine abolishes adsorption of receptors and G proteins to affinity columns. Following removal of 8-p-sulfophenyltheophylline and GTP, purified receptors bind agonists (2 sites) and antagonists (1 site) with affinities similar to crude soluble receptors and typical of A1 receptors. Some receptors may be denatured as a result of purification since only 23% of the radioligand binding sites which adhere to the affinity column can be detected in the eluate. The Bmax of purified receptors, 820 +/- 100 pmol/mg protein (n = 3) is 1800-fold higher than crude soluble receptors. The specific activity of [35S]GTP gamma S binding sites in affinity column eluates is 4640 pmol/mg protein. Assuming a 1:1 stoichiometry, this specific activity indicates that receptor-G protein complexes are greater than 50% pure following affinity chromatography. The photoaffinity labeled purified receptor was identified by polyacrylamide gel electrophoresis as a single band with a molecular mass of 35 kDa which when deglycosylated undergoes a characteristic shift in molecular mass to a sharp band at 32 kDa. In addition to the receptor, silver staining revealed polypeptides with molecular masses of 39 and 41 kDa, which are ADP-ribosylated by pertussis toxin, and 36 kDa corresponding to G protein beta-subunits.     

胡杨甜菜碱醛脱氢酶基因的功能分化

甜菜碱醛脱氢酶(BADH)在植物抗逆反应中发挥着重要作用。文中从胡杨cDNA克隆到2个甜菜碱醛脱氢酶基因,分别命名为PeBADH1和PeBADH2。PeBADH1和PeBADH2均编码503个氨基酸的蛋白质,预测分子量分别是54.93 kDa和54.90 kDa。组织表达模式分析发现这2个基因在正常生长、盐和H2O2胁迫下,在不同组织中的表达模式有较大差异。在大肠杆菌中表达并纯化了2个基因的重组蛋白。酶活性分析显示PeBADH1和PeBADH2蛋白对底物的活性分别是0.073μmol/(min.mg)和0.107μmol/(min.mg)。热力学稳定性分析显示这2个蛋白的热力学稳定性具有明显差异。因此,基因表达模式差异与蛋白质酶学性质的不同预示着这2个基因可能存在功能上的分化。     

Proteomic analysis of human bronchoalveolar lavage fluid: expression profiling of surfactant-associated protein A isomers derived from human pulmonary alveolar proteinosis using immunoaffinity detection

Human bronchoalveolar lavage fluid (BALF) proteins from pulmonary alveolar proteinosis (PAP) obtained by washing the epithelial lining of the lung with phosphate-buffered saline, were separated using high resolution two-dimensional gel electrophoresis (2-DE) under denaturing and reducing conditions. By Western blotting, the proteins were transferred from polyacrylamide gel onto a chemical resilient membrane. The surfactant-associated protein A (SP-A) isomers were then identified with enhanced chemiluminescence detection (ECL) using antibody-antigen reaction. Some of the gels were treated with silver staining after 2-DE. The molecular masses of SP-A isomers in BALF from PAP ranged from 20.5 to 26, 26 to 32, and 32 to 42 kDa, respectively; and isoelectric points (pI) were in pH range of 4.5-5.4 under denaturing and reducing conditions. In the mass range of 20.5-26 kDa and pI of 4.5-5.4, there were five isomers, and in mass range of 26-32 kDa and pI of 4.5 to 5.4, there were at least eight isomers on the ECL detection film. However, in the mass range of 32-42 kDa and pI of 4.5-5.4, there were three isomers separated one from another but there was also a cluster of overlapping spots on the ECL detection film. Thus, this communication describes a characteristic 2-DE pattern of SP-A isomers in BALF from PAP as follows. (1) The five isomers of mass 20.5-26 kDa and pI of 4.5-5.4; (2) the eight isomers of mass 26-32 kDa and pI of 4.5-5.4; and (3) the three isomers of mass 32-42 kDa and pI of 4.5-5.4.     

Variation in the surface layer proteins of Clostridium difficile

Surface layers (S-layers) form regular crystalline structures on the outermost surface of many bacteria. Clostridium difficile possesses such an S-layer consisting of two protein subunits. Treatment of whole cells of C. difficile with 5 M guanidine hydrochloride revealed two major proteins of different molecular masses characteristic of the S-layer on SDS-PAGE. In this study 25 isolates were investigated. A high degree of variability in the molecular mass of the two S-layer proteins was evident. Molecular masses ranged from 48 to 56 kDa for the heavier protein and from 37 to 45 kDa for the lighter protein. A further protein component of 70 kDa was detectable in all isolates. No cross-reaction was seen between the two major proteins from isolates that produced different S-layer patterns, and most S-layer proteins from isolates with the same or similar banding patterns did not cross-react. The S-layer proteins, when detected by a combination of Coomassie blue staining and immunoblotting, are a useful marker for phenotyping.     

江南卷柏脱氢抗坏血酸还原酶的分子特性

脱氢抗坏血酸还原酶 (DHAR) 在植物抗坏血酸?谷胱甘肽循环中发挥着重要作用。利用同源克隆技术从江南卷柏中克隆到2个脱氢抗坏血酸还原酶基因,分别命名为SmDHAR1和SmDHAR2。SmDHAR1和SmDHAR2分别编码218和241个氨基酸,预测分子量分别是23.97 kDa和27.33 kDa。基因组序列分析显示这2个基因分别含有5和6个内含子。器官表达模式分析发现这2个基因在根、茎、叶中均有表达,是组成型表达基因。在大肠杆菌中表达并纯化了2个基因的重组蛋白。酶活性分析显示SmDHAR1和SmDHAR2蛋白对底物DHA的活性有显著差异,分别是19.76和0.17 μmol/(min·mg)。热力学稳定性分析显示这2个重组蛋白的热力学稳定性具有明显差异。因此,基因结构与酶学性质的差异预示着这2个基因可能存在功能上的分化。     

Molecular cloning of an Arabidopsis cDNA encoding a dynamin-like protein that is localized to plastids

Dynamin-related proteins are high molecular weight GTPase proteins found in a variety of eukaryotic cells from yeast to human. They are involved in diverse biological processes that include endocytosis in animal cells and vacuolar protein sorting in yeast. We isolated a new gene, ADL2, that encodes a dynamin-like protein in Arabidopsis. The ADL2 cDNA is 2.68 kb in size and has an open reading frame for 809 amino acid residues with a calculated molecular mass of 90 kDa. Sequence analysis of ADL2 revealed a high degree of amino acid sequence similarity to other members of the dynamin superfamily. Among those members ADL2 was most closely related to Dnm1p of yeast and thus appears to be a member of the Vps1p subfamily. Expression studies showed that the ADL2 gene is widely expressed in various tissues with highest expression in flower tissues. In vivo targeting experiments showed that ADL2:smGFP fusion protein is localized to chloroplasts in soybean photoautroph cells. In addition experiments with deletion constructs revealed that the N-terminal 35 amino acid residues were sufficient to direct the smGFP into chloroplasts in tobacco protoplasts when expressed as a fusion protein.     

Fluorimetric coupled enzyme assay for lysoplasmalogenase activity in liver.

Two new proteins with apparent molecular masses of 53 kDa and 190 kDa have been identified in both sarcoplasmic reticulum and human blood platelets using a monoclonal antibody, FII1b5. The sarcoplasmic reticulum FII1b5 antigens were present in the terminal cisternae fraction, but were absent from light sarcoplasmic reticulum. The platelet and skeletal muscle proteins were not sensitive to digestion with endoglycosidase H under conditions that removed carbohydrate from the 53 kDa glycoprotein in sarcoplasmic reticulum or GPIIIa in platelet microsomes and did not bind 45Ca in a nitrocellulose overlay calcium-binding assay. These results distinguished the FII1b5 antigens from the 53 kDa glycoprotein and calsequestrin of sarcoplasmic reticulum. The 190 kDa platelet and sarcoplasmic reticulum proteins were extracted from membranes with high concentrations of NaCl, indicating that the high molecular mass FII1b5 antigens are peripherally associated with the bilayers. In contrast, the platelet and muscle 53 kDa proteins remained membrane-bound in the presence of high salt concentrations, suggesting that they are integral proteins.     

On the size and the role of a free cytosolic pool of acidic ribosomal proteins in yeast Saccharomyces cerevisiae

A small but distinct amount of yeast acidic ribosomal proteins A1/A2 was detected in cytosol by immunoblotting on a two-dimensional gel electrophoretogram, while 38 kDa acidic protein A0 was not detected. The free forms of A1/A2 in the cytosol were eluted in gel filtration at the molecular mass of about 30 kDa under non-denaturation conditions, suggesting that they exist as a dimer or timer without association with A0. The amount of free A1/A2 was determined by immunoblotting to be 0.3% of the ribosome-bound A1/A2 in yeast. The time course of incorporation of radioactive amino acid showed that the cytosolic free A1/A2 are labeled more rapidly with high specific radioactivity than the ribosome-bound A1/A2. This result suggested that some of the cytosolic A1/A2, if not all, are newly-synthesized proteins which are ready for incorporation into cytoplasmic ribosomes.     

Human and mouse cementum proteins immunologically related to enamel proteins

SDS-polyacrylamide gel electrophoresis, immunoblot and amino acid composition analyses were applied to human and mouse acellular cementum proteins immunologically related to enamelins and amelogenins. In this analysis, anti-mouse amelogenin, anti-human enamelin and synthetic peptide (e.g., -LPPHPGHPGYIC-) antibodies were shown to cross-react with tooth crown-derived enamelin with a molecular mass of 72,000 Da (72 kDa), amelogenins (26 kDa), and also to four human cementum proteins (72, 58, 50 and 26 kDa) and two mouse cementum proteins (72 and 26 kDa). Each of the antibodies recognized tooth root-derived cementum polypeptides which share one or more epitopes with tooth crown-derived enamel proteins. The molecular mass and isoelectric points for crown-derived and root-derived enamel-related proteins were similar. Analysis of human and mouse cementum proteins revealed a characteristic amino acid composition enriched in glutamyl, serine, glycine, alanine, proline, valine and leucine residues; compared to the major enamel protein amelogenin, cementum proteins were low in proline, histidine and methionine. The human and mouse putative intermediate cementum proteins appear to represent a distinct class of enamel-related proteins. Moreover, these results support the hypothesis that epithelial root sheath epithelia express several cementum proteins immunologically related to canonical enamel proteins.     

Pregnancy associated glycoprotein-1, -6, -7, and -17 are major products of bovine binucleate trophoblast giant cells at midpregnancy

Pregnancy associated glycoproteins (PAGs) are extensively glycosylated secretory proteins of ruminant trophoblast cells. In cattle placenta several PAG cDNAs are expressed, but the variety of correspondent proteins and their degree of glycosylation are not well characterized. Thus, we purified PAGs by using a protocol which included a lectin (Vicia villosa agglutinin) affinity chromatography. Due to their specific glycosylation pattern, PAGs derived from binucleate trophoblast giant cells were highly enriched by this protocol. PAGs were purified from cotyledons of 2 day 100 placentas and from a single placenta at day 155 and 180. In all samples three major bands (75; 66; 56 kDa) were detected by one-dimensional SDS-PAGE. Mass-spectrometric analysis identified the 75 kDa band as a mixture of PAG-7 and PAG-6, the 66 kDa band as PAG-1 and the 56 kDa band as PAG-17. N-terminal sequencing of the day 100 sample confirmed the mass spectrometric identifications. Enzymatic release of N-glycans with peptide-N-glycanase-F from PAGs reduced the molecular weight to approximately 37 kDa which corresponds to the theoretical molecular mass of PAGs. Limited peptide-N-glycanase-F treatment revealed that all four N-glycosylation sites are quantitatively occupied in PAG-1. Compared to PAG-1 the number of potential N-glycosylation sites is lower in PAG-17 (three sites) and higher in PAG-6 and -7 (five and six sites, respectively). This suggests that the number of attached N-glycans is the main determinant of molecular mass of bovine PAGs. The degree of glycosylation may be a major factor regulating the plasma half life of PAGs.     

Primary structure, genomic organization and expression of the major secretory protein of murine epididymis, ME1

The mouse cDNA and its genomic clones encoding the epididymal secretory glycoprotein ME1 were identified. The Me1 gene spans 15kb with four exons and three introns. The deduced amino-acid sequence of the ME1 cDNA revealed that it consists of 149 amino acid residues, which contain a signal peptide characteristic of secretory proteins, six cysteine residues and a proline-rich region conserved in the orthologous proteins. Northern blot analysis revealed that 1.3kb ME1 mRNA is highly expressed in the mouse epididymis. The polyclonal antibodies generated against human HE1 (ME1 orthologous protein) expressed in bacteria reacted with approximately 17 to 25kDa components in mouse epididymis crude extract. The reduction of the molecular mass of the recombinant ME1 protein with the digestion of glycopeptidase A indicated that it is modified by Asn-linked glycosylation.     

Changes of soluble proteins in leaf and thylakoid exposed in high saline condition of a mangrove taxa Bruguiera gymnorrhiza

One-year-old seedlings of Bruguiera gymnorrhiza (L) Savingay were exposed to 500 mM NaCl for 6d under hydroponic culture condition to characterize the changes in leaf and thylakoid protein profiles in response to short-term salt exposures. Significant changes in leaf dry mass, chlorophylls and soluble leaf proteins were observed in short term of salt exposures, as it happens under tidal situations in nature. Chlorophyll a/b ratio showed decrease of light harvesting efficiency in salt treatment. Total soluble proteins in leaves were extracted from control and NaCl-treated plants at 2d intervals and were analyzed by SDS-PAGE. Intensity of several protein bands of different molecular mass of leaf protein profile ranging from 10 to 86 kDa (10, 16, 23, 33, 37, 42, 44, 50 and 86 kDa) were decreased due to high salt treatment. Out of these, 16, 23 and 33 kDa protein bands decreased dramatically from 1–3 fold but recovered in 7d growth, except the 33 kDa band. SDSPAGE profile of thylakoid protein revealed that both number and the intensity of several protein bands got altered by salt concentration. However, 33 kDa protein band of thylakoid reappeared in recovery that might not be of the same characteristics with same molecular mass as shown in total leaf protein profile. The numbers of major bands found in SDS-PAGE were reduced when analyzed in urea-SDS-PAGE to minimize protein aggregations by high salt. It was noted that 47 kDa disappeared while some proteins of apparent molecular mass like 23 kDa, 33 kDa, 37 kDa and 50 kDa degraded to minor bands. Partial restoration of protein bands occurred when the salt-treated plants were brought back to initial growth condition. These results clearly demonstrate that short term high salt concentration could cause major alterations to photosynthetic apparatus of a true non salt-secreting tree mangrove Bruguiera gymnorrhiza and adapted against fluctuation of salinity by altering leaf protein pool relatively more than the thylakoid proteins.Key words: Bruguiera gymnorrhiza, Mangrove, Polypeptides, Salt shock, Sodium chloride, Thylakoid     

Low-molecular-mass proteins in cyanobacterial photosystem II: identification of psbH and psbK gene products by N-terminal sequencing

The O2-evolving photosystem II core complex was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus Copeland. Analysis by SDS-polyacrylamide gel electrophoresis revealed that the complex contained at least seven low-molecular-mass proteins in addition to the well characterized CP47 apoprotein, CP43 apoprotein, 33 kDa extrinsic protein, D1 protein, D2 protein and large subunit of cytochrome b-559. The separation of these low-molecular-mass proteins were very similar between cyanobacterial and higher plant PS II. N-terminal sequences of the 6.5 kDa and 3.9 kDa proteins of cyanobacterial core complex were determined after blotting to a polyvinylidene difluoride membrane. The sequence of the 6.5 kDa protein showed high homology with an internal sequence of plant psbH gene product, so-called 10 kDa phosphoprotein, but did not conserve the Thr residue which is specifically phosphorylated in plants. The sequence of the 3.9 kDa protein corresponded to the K protein of higher plants (mature form of psbK gene product). These results indicate that the products of both psbH and psbK genes are present in cyanobacterial PS II as well as being associated with the O2-evolving core complex.     

Purification of the spliced leader ribonucleoprotein particle from Leptomonas collosoma revealed the existence of an Sm protein in trypanosomes. Cloning the SmE homologue

Trans-splicing in trypanosomes involves the addition of a common spliced leader (SL) sequence, which is derived from a small RNA, the SL RNA, to all mRNA precursors. The SL RNA is present in the cell in the form of a ribonucleoprotein, the SL RNP. Using conventional chromatography and affinity selection with 2'-O-methylated RNA oligonucleotides at high ionic strength, five proteins of 70, 16, 13, 12, and 8 kDa were co-selected with the SL RNA from Leptomonas collosoma, representing the SL RNP core particle. Under conditions of lower ionic strength, additional proteins of 28 and 20 kDa were revealed. On the basis of peptide sequences, the gene coding for a protein with a predicted molecular weight of 11.9 kDa was cloned and identified as homologue of the cis-spliceosomal SmE. The protein carries the Sm motifs 1 and 2 characteristic of Sm antigens that bind to all known cis-spliceosomal uridylic acid-rich small nuclear RNAs (U snRNAs), suggesting the existence of Sm proteins in trypanosomes. This finding is of special interest because trypanosome snRNPs are the only snRNPs examined to date that are not recognized by anti-Sm antibodies. Because of the early divergence of trypanosomes from the eukaryotic lineage, the trypanosome SmE protein represents one of the primordial Sm proteins in nature.     

Immunological characterization of microtubule-associated proteins specific for the immature brain

Immunoblotting analysis was used to detect the microtubule-associated proteins present at different stages of rat brain development. Polyclonal antibodies were raised against the two main adult brain microtubule-associated proteins: MAP-2 (300 kDa) and TAU (60-70 kDa). Whatever the stage of development, anti-MAP-2 serum detected high molecular mass proteins and at immature stages a protein of 62 kDa. This protein which has previously been referred to as 'young TAU slow' is, therefore, immunologically related to MAP-2. The anti-TAU serum (but not the anti-MAP-2 serum) detected at immature stages of development a 48 kDa protein which also disappears at adulthood. This 48 kDa entity which has been referred to as 'young TAU fast' is progressively replaced by the closely spaced bands (60-70 kDa) of adult TAU proteins. The 62 and 48 kDa proteins appear therefore to be immunologically distinct and represent two microtubule-associated proteins specific to the immature brain.     

The early light-inducible proteins of barley. Characterization of two families of 2-h-specific nuclear-coded chloroplast proteins

Within 1-2 h of illumination of etiolated barley plants the mRNAs of seven nuclear-coded proteins are transiently induced. It is proposed that at least some of these proteins are precursors to chloroplast membrane proteins since after posttranslational transport 2-h-specific bands of 18.5 kDa, 18 kDa and 13.5 kDa have been found bound to thylakoid membranes. cDNA clones for these early light-inducible proteins (ELIPs) have been isolated. Hybrid-release translation shows that part of their information must be homologous since the complete set of early light-inducible translation products is obtained with all investigated clones although the proportions of the translated bands vary for individual clones. From hybridization data it is concluded that two ELIP families of high (24-27 kDa) and of low (16-18 kDa) molecular mass exist which are induced in parallel. Induction of ELIPs occurs even at very low light intensities and is saturated at about 1000 lx. Therefore, ELIPs are not considered to represent light stress proteins but to play a regulatory role during development.