Purification and characterization of a DnaK-like and a GroEL-like protein from Porphyromonas gingivalis

Heat-shock proteins of Porphyromonas gingivalis were demonstrated and two of them were purified and further characterized. The amplified de novo synthesis of two different proteins, with apparent molecular weights of 75 kDa and 68 kDa, was observed by autofluorography when a P. gingivalis culture incubated in a 14C-labeled amino acid mixture was shifted from 37 degrees C to 44 degrees C. Both proteins possessed ATP-binding abilities and were purified to almost homogeneity employing affinity chromatography on ATP-agarose followed by preparative SDS-PAGE. Purified 75 kDa and 68 kDa proteins had isoelectric points of 4.4 and 4.6, respectively. They were shown to be immunoreactive with commercial anti-DnaK and anti-GroEL polyclonal antibodies, respectively. Immunoblotting analysis of whole cells using antiserum raised against each purified protein from P. gingivalis, confirmed elevated synthesis of both proteins during thermal shock. A GroEL protein reacted strongly with antiserum against the 68 kDa protein. However, a DnaK protein reacted weakly with antiserum to the 75 kDa protein. Analysis of the N-terminal amino acid sequence of the DnaK-like protein (75 kDa) showed a high degree of homology with those of the HSP70 family including both prokaryotic and eukaryotic cells. The N-terminal amino acid analysis of the GroEL-like protein (68 kDa) indicated that it was identical to those of cloned GroEL homologues from P. gingivalis.     

Purification and Characterization of a DnaK-like and a GroEL-like Protein from Porphyromonas gingivalis

Heat-shock proteins of Porphyromonas gingivalis were demonstrated and two of them were purified and further characterized. The amplified de novo synthesis of two different proteins, with apparent molecular weights of 75 kDa and 68 kDa, was observed by autofluorography when a P. gingivalis culture incubated in a ¹⁴C-labeled amino acid mixture was shifted from 37°C to 44°C. Both proteins possessed ATP-binding abilities and were purified to almost homogeneity employing affinity chromatography on ATP-agarose followed by preparative SDS-PAGE. Purified 75 kDa and 68 kDa proteins had isoelectric points of 4.4 and 4.6, respectively. They were shown to be immunoreactive with commercial anti-DnaK and anti-GroEL polyclonal antibodies, respectively. Immunoblotting analysis of whole cells using antiserum raised against each purified protein from P. gingivalis, confirmed elevated synthesis of both proteins during thermal shock. A GroEL protein reacted strongly with antiserum against the 68 kDa protein. However, a DnaK protein reacted weakly with antiserum to the 75 kDa protein. Analysis of the N-terminal amino acid sequence of the DnaK-like protein (75 kDa) showed a high degree of homology with those of the HSP70 family including both prokaryotic and eukaryotic cells. The N-terminal amino acid analysis of the GroEL-like protein (68 kDa) indicated that it was identical to those of cloned GroEL homologues from P. gingivalis.     

Presclerotized eggshell protein from the liver fluke Fasciola hepatica

Trematode parasites protect their eggs with a tough tanned eggshell. Eggshell precursor proteins are synthesized and stockpiled within the extensive vitellaria of the animal. A major eggshell precursor protein with an apparent molecular weight of 31,000 and pI of 7.4 was isolated from the vitellaria of Fasciola hepatica. This protein, which represents 6-7% of the total protein in mature Fasciola, is unique in containing rather high levels of the amino acid 3,4-dihydroxyphenylalanine (DOPA), i.e., 110 residues per 1000. Other prominent amino acids are glycine, aspartic acid, and lysine. A prominent DOPA-containing tryptic peptide derived from eggshell precursor protein has the sequence Gly-Gly-Gly-DOPA-Gly-Gly-DOPA-Gly-Lys. DOPA residues disappear during the maturation of the eggshell and by treatment in vitro with mushroom polyphenol oxidase. This disappearance may be related to the formation of cross-links in the eggshell protein.     

Identification of a putative eggshell precursor protein of the female Schistosoma japonicum

In adult worms of Schistosoma japonicum, a prominent radiolabelled female-specific protein (34 kDa) was demonstrated on fluorography of SDS gels with the pulse incorporation of 14C-tyrosine in vitro, though it was difficult to detect major female-specific proteins by direct staining methods. This female-specific protein was demonstrated to localize exclusively in the vitelline cells by indirect immunofluorescence using the rabbit anti-34 kDa female protein antiserum. It was shown that 14C-tyrosine was selectively incorporated into the vitelline cells by the pulse labelled autoradiographs. Two days after the exposure of worms to radio-tyrosine, the shells of eggs in the uterus were demonstrated to have become radioactive, indicating that 14C-tyrosine-labelled protein was used as a material for the eggshell. In the fluorograph of proteins extracted from newly laid eggs in vitro, the prominent band was not found at the 34 kDa region, but a lot of radioactivity appeared at higher than 100 kDa. The results suggested that a 34 kDa female protein was a precursor of the eggshell and became a much larger protein molecule as a result of cross-linking during eggshell hardening.     

Eggshell zona radiata-proteins from cod (Gadus morhua): extra-ovarian origin and induction by estradiol-17 beta.

Using Atlantic cod (Gadus morhua) as a model organism, the aim of this report was to delineate whether teleostean eggshell zona radiata proteins have their origin, i.e., site of synthesis, in gonadal or somatic tissues. Estradiol-17 beta was administered intraperitoneally to one-year-old cod (Gadus morhua) with either undeveloped gonads or with differentiated gonads. By immunoblotting procedures estradiol-dependent protein induction was investigated using specific rabbit antisera directed against cod eggshell proteins and brown trout vitellogenin. No immunological cross-reactions were observed between the two antisera, and eggshell proteins and vitellogenin were detected in blood plasma and somatic tissues only in estradiol-treated cod. Three plasma-components were immunoreactive to antiserum directed against eggshell proteins, and these proteins possessed molecular weights of 78, 54 and 47 kDa, identical to the molecular weights of the cod eggshell alpha, beta and gamma zona radiata-proteins. These three immunoreactive plasma-components were observed after administration of estradiol-17 beta to both sexes, also in males having reached spermiation, and in juveniles of either sex without developed gonads. The data are interpreted to signify that cod eggshell zona radiata-proteins originate in an extra-ovarian tissue and are transported in the blood for deposition in the ovaries. We propose that oogenesis involves estradiol-17 beta regulation of both eggshell zona radiata-proteins and vitellogenin synthesis.     

The proteome of the insoluble Schistosoma mansoni eggshell skeleton

In schistosomiasis, the majority of symptoms of the disease is caused by the eggs that are trapped in the liver. These eggs elicit an immune reaction that leads to the formation of granulomas. The eggshell, which is a rigid insoluble structure built from cross-linked proteins, is the site of direct interaction between the egg and the immune system. However, the exact protein composition of the insoluble eggshell was previously unknown. To identify the proteins of the eggshell of Schistosoma mansoni we performed LC-MS/MS analysis, immunostaining and amino acid analysis on eggshell fragments. For this, eggshell protein skeleton was prepared by thoroughly cleaning eggshells in a four-step stripping procedure of increasing strength including urea and SDS to remove all material that is not covalently linked to the eggshell itself, but is part of the inside of the egg, such as Reynold’s layer, von Lichtenberg’s envelope and the miracidium. We identified 45 proteins of which the majority are non-structural proteins and non-specific for eggs, but are house-keeping proteins that are present in large quantities in worms and miracidia. Some of these proteins are known to be immunogenic, such as HSP70, GST and enolase. In addition, a number of schistosome-specific proteins with unknown function and no homology to any known annotated protein were found to be incorporated in the eggshell. Schistosome-specific glycoconjugates were also shown to be present on the eggshell protein skeleton. This study also confirmed that the putative eggshell protein p14 contributes largely to the eggshell. Together, these results give new insights into eggshell composition as well as eggshell formation. Those proteins that are present at the site and time of eggshell formation are incorporated in the cross-linked eggshell and this cross-linking does no longer occur when the miracidium starts secreting proteins.     

Immunological and chemical characterization of rat liver cytochrome c oxidase

Rat liver cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase; EC 1.9.3.1) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into 12 different polypeptide chains. Specific antisera against the holoenzyme and against purified subunits IV and VIII were used to characterize the enzyme complex. The antiserum against subunit IV precipitates from sodium dodecyl sulfate-dissociated mitochondria only subunit IV and from Triton X-100-dissolved mitochondria all 12 polypeptide chains, indicating their integral location within the enzyme complex. Different antisera against the holoenzyme only precipitate subunits IV, V and VIb from sodium dodecyl sulfate-dissociated mitochondria, suggesting the location of these subunits on the surface layer of the complex. Subunit VIII is thought to be located within the complex, since a specific antiserum does not precipitate the complex. The amino acid composition of all 12 protein subunits is different, thus excluding their origin from proteolytic degradation. The proteolytic degradation of subunit IV into IV during isolation of the enzyme was corroborated by the very similar amino acid composition of both proteins.     

Ovocalyxin-32, a novel chicken eggshell matrix protein. isolation, amino acid sequencing, cloning, and immunocytochemical localization

The eggshell is a highly ordered structure resulting from the deposition of calcium carbonate concomitantly with an organic matrix upon the eggshell membranes. Mineralization takes place in an acellular uterine fluid, which contains the ionic and matrix precursors of the eggshell. We have identified a novel 32-kDa protein, ovocalyxin-32, which is expressed at high levels in the uterine and isthmus regions of the oviduct, and concentrated in the eggshell. Sequencing of peptides derived from the purified protein allowed expressed sequence tag sequences to be identified that were assembled to yield a full-length composite sequence whose conceptual translation product contained the complete amino acid sequence of ovocalyxin-32. Data base searches revealed that ovocalyxin-32 has limited identity (32%) to two unrelated proteins: latexin, a carboxypeptidase inhibitor expressed in the rat cerebral cortex and mast cells, and a skin protein, which is encoded by a retinoic acid receptor-responsive gene, TIG1. High level expression of ovocalyxin-32 was limited to the isthmus and uterus tissue, where immunocytochemistry at the light and electron microscope levels demonstrated that ovocalyxin-32 is secreted by surface epithelial cells. In the eggshell, ovocalyxin-32 localizes to the outer palisade layer, the vertical crystal layer, and the cuticle of the eggshell, in agreement with its demonstration by Western blotting at high levels in the uterine fluid during the termination phase of eggshell formation. Ovocalyxin-32 is therefore identified as a novel protein synthesized in the distal oviduct where hen eggshell formation occurs.     

Common amino acid domain among endopolygalacturonases of ascomycete fungi

The endopolygalacturonase (EC 3.2.1.15) enzymes produced in vitro by three ascomycete fungi, Aspergillus niger, Sclerotinia sclerotiorum, and Colletotrichum lindemuthianum were studied by using thin-layer isoelectric focusing and activity stain overlay techniques. The polygalacturonases from A. niger and S. sclerotiorum consisted of numerous isoforms, whereas the endopolygalacturonase from C. lindemuthianum consisted of a single protein species. The most abundant endopolygalacturonase isoform produced by each of these organisms was purified and characterized. Biochemical parameters, including molecular weight, isoelectric point, kinetic parameters, temperature and pH optima, and thermal stability, were determined. Considerable differences in physical and chemical properties were demonstrated among these fungal polygalacturonases. Antibodies raised against individual proteins exhibited little cross-reaction, suggesting that these enzymes differ structurally as well as biochemically. In contrast, the analysis of the N-terminal amino acid sequences of the three proteins showed extensive homology, particularly in a region labeled domain 1 in which 84% of the amino acids were conserved.     

Common amino acid domain among endopolygalacturonases of ascomycete fungi.

The endopolygalacturonase (EC 3.2.1.15) enzymes produced in vitro by three ascomycete fungi, Aspergillus niger, Sclerotinia sclerotiorum, and Colletotrichum lindemuthianum were studied by using thin-layer isoelectric focusing and activity stain overlay techniques. The polygalacturonases from A. niger and S. sclerotiorum consisted of numerous isoforms, whereas the endopolygalacturonase from C. lindemuthianum consisted of a single protein species. The most abundant endopolygalacturonase isoform produced by each of these organisms was purified and characterized. Biochemical parameters, including molecular weight, isoelectric point, kinetic parameters, temperature and pH optima, and thermal stability, were determined. Considerable differences in physical and chemical properties were demonstrated among these fungal polygalacturonases. Antibodies raised against individual proteins exhibited little cross-reaction, suggesting that these enzymes differ structurally as well as biochemically. In contrast, the analysis of the N-terminal amino acid sequences of the three proteins showed extensive homology, particularly in a region labeled domain 1 in which 84% of the amino acids were conserved.     

Protein import into yeast mitochondria is inhibited by antibodies raised against 45-kd proteins of the outer membrane.

Import of several precursor proteins into isolated yeast mitochondria is inhibited by rabbit antiserum raised against the total mitochondrial outer membrane or against electrophoretically purified 45-kd outer membrane proteins. Antisera against other outer membrane proteins are only marginally active or inactive. Inhibition by the antiserum against 45-kd proteins is only weak with untreated mitochondria, but reaches 80-90% with mitochondria that had been pretreated with 0.1 mg/ml trypsin. This trypsin pretreatment by itself inhibits precursor import only slightly (30-50%). Selective inhibition of import does not correlate with binding of the various IgGs to the mitochondrial surface and is also observed with the corresponding Fab fragments. Inhibition by antibodies against 45-kd outer membrane proteins strongly suggests the existence of a mitochondrial surface protein mediating protein import and offers a means of isolating this protein.     

An immunologic probe for a defined region of the myelin proteolipid

Antiserum has been prepared against an isolated polypeptide fragment, designated BPS4, which comprises residues 181-211 of the bovine myelin proteolipid. The antiserum recognizes the intact bovine proteolipid protein but not several other polypeptide fragments within the molecule, nor the myelin basic protein, thus demonstrating specificity of the antiserum. In a competitive enzyme-linked immunosorbent assay, both the major proteolipid and the DM 20 bands observed on sodium dodecyl sulfate-polyacrylamide gels reacted equally well with the antiserum, indicating that the BPS4 segment is present in both molecular species. The rat myelin proteolipid protein cross-reacted with antiserum against the intact bovine protein but showed minimal cross-reactivity with the antiserum against the bovine BPS4 fragment. This was demonstrated in parallel experiments using three types of preparations, namely, sodium dodecyl sulfate-solubilized myelin, delipidated myelin, and isolated proteolipid apoprotein. The difference between the bovine and rat proteins, which presumably reflects amino acid sequence differences, is thus detectable by the antiserum against the polypeptide fragment but not by the antiserum against the intact protein. Isolated bovine myelin membranes did not bind the antiserum in the absence of detergent or without delipidation. On the other hand, in vesicles reconstituted with the intact bovine apoprotein, the BPS4 segment was oriented on the exterior face of the liposome where it was capable of binding antibody and was susceptible to Pronase digestion.     

滨藜（Atriplex patens）核糖体失活蛋白基因的克隆和分析

为开发利用新疆野生植物滨藜,采用反转录-多聚酶链式反应(RT-PCR),从滨藜（Atriplex patens）叶中克隆核糖体失活蛋白（ribosome inactivating proteins ,RIPs）基因的完整读码框ORF片段.序列分析表明,该片段大小为843 bp,编码1 条长280个氨基酸肽链, 其中N 端的26个氨基酸是信号肽.滨藜核糖体失活蛋白（ApRIP）属Ⅰ型核糖体失活蛋白.同源性比较分析显示,滨藜RIP与藜科植物藜的核糖体失活蛋白,天花粉蛋白（TCS）和美洲商陆蛋白（PAP）基因序列的同源性分别为82%,41%和55%.氨基酸序列比对和SWISS-MODEL同源模建分析表明,滨藜RIP的3′端氨基酸肽链具有与其它RIP相同的活性中心位点"EAARXKXI".     

Mass spectrometric sequencing of endotoxin proteins of Bacillus thuringiensis ssp. konkukian extracted from polyacrylamide gels

The amino acid sequences of the crystal proteins of Bacillus thuringiensis ssp. konkukian strain HL-47 are unknown. We used 1-D denaturing polyacrylamide electrophoresis, nano-ESI-Q-TOF-MS, and protein database searching to analyze these proteins. On SDS-PAGE gels, a preparation of purified crystal proteins exhibited 110, 102, 76, 55, 37, and 30 kDa protein bands. Immunoblotting of the gel with antiserum raised to this preparation revealed that four crystal proteins, of 110, 102, 55, and 37 kDa, reacted with the specific antiserum. The 102-kDa major protein reacted strongly. The other crystal proteins showed weak immunoreactivity. The 102 and 55 kDa proteins were analyzed by ESI-MS. The internal amino acid sequence of the 102-kDa major protein has similarity to the sequences of the surface layer protein of B. thuringiensis ssp. finitimus and B. anthracis. However, the internal amino acid sequences of the 55 kDa protein did not show any homology to proteins in the databases. Proteomic analysis of these proteins leads to the conclusion that the sequence data provided the protein databases of the crystal proteins of the konkukian ssp.     

Use of a novel strategy for the preparation and characterization of an antipeptide antibody capable of recognizing members of the annexin family

Annexins are structurally-related proteins which bind phospholipids in a Ca2+-dependent manner. We have used a novel coupling strategy to prepare an antiserum directed against a 17-amino acid synthetic peptide that resembles the sequence of a highly-conserved portion of these proteins. This antipeptide serum specifically recognizes 5 of 6 human annexins on Western blots, despite differences between the protein and peptide sequences of 3 or 4 amino acids. The antiserum does not recognize endonexin II, whose sequence differs from that of the peptide by 6 amino acids. The availability of multiple proteins with known amino acid sequence has allowed analysis of structural requirements for recognition by this antibody. In some situations, use of such an antibody may allow the identification of a protein as a member of a family.     

Purification,characterization, and in vitro mineralization studies of a novel goose eggshell matrix protein,ansocalcin

Biomineralization is an important process in which hard tissues are generated through mineral deposition, often assisted by biomacromolecules. Eggshells, because of their rapid formation via mineralization, are chosen as a model for understanding the fundamentals of biomineralization. This report discusses purification and characterization of various proteins and peptides from goose eggshell matrix. A novel 15-kDa protein (ansocalcin) was extracted from the eggshell matrix, purified, and identified and its role in mineralization evaluated using in vitro crystal growth experiments. The complete amino acid sequence of ansocalcin showed high homology to ovocleidin-17, a chicken eggshell protein, and to C-type lectins from snake venom. The amino acid sequence of ansocalcin was characterized by the presence of acidic and basic amino acid multiplets. In vitro crystallization experiments showed that ansocalcin induced pits on the rhombohedral faces at lower concentrations (<50 microg/ml). At higher concentrations, the nucleation of calcite crystal aggregates was observed. Molecular weight determinations by size exclusion chromatography and sodium dodecyl sulfate -polyacrylamide gel electrophoresis showed reversible concentration-dependent aggregation of ansocalcin in solution. We propose that such aggregated structures may act as a template for the nucleation of calcite crystal aggregates. Similar aggregation of calcite crystals was also observed when crystallizations were performed in the presence of whole goose eggshell extract. These results show that ansocalcin plays a significant role in goose eggshell calcification.     

Role of Abscisic Acid in Drought-Induced Freezing Tolerance,Cold Acclimation,and Accumulation of LT178 and RAB18 Proteins in Arabidopsis thaliana

Embryogenic tissues of Pinus caribaea Morelet var hondurensis produce extracellular proteins; among them germins have been identified. Two-dimensional electrophoresis followed by electroblotting onto a polyvinylidene difluoride membrane allowed isolation and N-terminal amino acid sequencing of extracellular GP111, which is present within the five embryogenic cell lines studied. The amino acid sequence showed strong homologies with the sequences of germins deduced from cDNA sequencing, starting at the same amino acid position but one, compared with other sequences of mature germins deduced from protein sequencing. Immunoblots of embryogenic and nonembryogenic extracellular proteins indicated that the polypeptide GP111 plus two others with similar relative molecular mass values are present in embryogenic cell lines but not in nonembryogenic ones. They were recognized by an antiserum raised against the nonglycosylated monomer of wheat germin. The cross-reaction between pine and wheat apoproteins was highly specific. An antiserum against the glycosylated pentameric germin-like protein (an oxalate oxidase) of barley cross-reacted with all three, as well as with several other glycosylated polypeptides.     

Identification and characterization of a human cytomegalovirus gene coding for a membrane protein that is conserved among human herpesviruses.

A rabbit antiserum was raised against envelope material from purified human cytomegalovirus strain AD169. The serum recognized polypeptides 200, 170, 160, 75, 58, and 45 kilodaltons in size. It was used to screen a cDNA library constructed from poly(A)+ RNA from human cytomegalovirus-infected cells in the expression vector lambda gt11. A recombinant bacteriophage expressing cytomegalovirus-specific sequences was identified, and the corresponding gene was mapped to the HindIII R fragment. The gene is transcribed into a late 1.5-kilobase RNA. The nucleotide sequence of the coding region was determined. Computer analysis of the gene product revealed a polypeptide containing multiple potential membrane-spanning domains, representing a type of protein not identified in the envelope of herpesviruses before. The protein shows homology on the amino acid level to hypothetical proteins from reading frames BBRF3 of Epstein-Barr virus, UL10 of herpes simplex virus type 1, and ORF50 of varicella-zoster virus. By using an antiserum raised against procaryote-expressed parts of the cytomegalovirus membrane protein, a 45-kilodalton structural component of the virus was identified as the gene product.