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The activity of citrate synthase (CS), a mitochondrial enzyme in the energy metabolism pathway, was studied in Euplotes focardii (Hypotrichia) and Uronema marinum (Scuticociliatia), isolated from samples of Antarctic seawater and, for comparison in E. vannus and U. nigricans , related ciliates from the Adriatic. The temperature-dependent characteristics of CS were investigated in the range 0–55°C, to evaluate mechanisms of thermal regulation, such as optimal temperature levels, activation energy, and kinetic parameters. CS activity was up to double that recorded in temperate organisms at reaction temperatures between 0 and 10°C. The optimal temperature for enzymatic activity was independent of T amb . Activation energy for CS was significantly lower in Antarctic ciliates than in temperate ones, indicating a significant increase in the reaction rate. K M at various temperatures of the reaction substrates indicated the higher affinity of CS for acetyl-CoA in both Antarctic organisms at lower temperatures. The data for thermal profiles for K M , showing greater differences between E. focardii and E. vannus than between U. marinum and U. nigricans , support the distinctive physiological characteristics of each species. E. focardii is an endemic and psychrophilic species, whereas U. marinum is a typically ubiquitous species, more adaptable to environmental changes. 相似文献
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EDTA-insoluble organic structures of the hypercalcified peritubular matrix (PM) in horse dentine were observed by scanning electron microscopy. The PM was enveloped in double cylindrical structures composed of fibrillar sheaths in the inner and outer peripheries. Between the outer fibrillar sheath and intrinsic fibrils of the intertubular matrix, a calcified cementing membrane existed. Within the PM, warped cone-shaped structures of fibrillar sheaths, overlapping at intervals of 4-6 microns and semiconcentrically surrounding the dentinal tubule, extended from the inner fibrillar towards the outer fibrillar sheath. The cone-shaped fibrillar sheaths following the inner and outer fibrillar sheaths were identified as the incremental lines of the PM. Most of these fibrils may be collagen although it could not be confirmed, whereas non-collagenous organic materials in the lateral branches of the dentinal tubule are radially arranged in the PM. These EDTA-insoluble structures were three-dimensionally illustrated using an image-analysing system. 相似文献
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The complete primary structure of MSP-1, a major water-soluble glycoprotein in the foliated calcite shell layer of the scallop
Patinopecten yessoensis, is reported. The full-length complementary DNA for MSP-1 isolated by polymerase chain reaction contained a sequence for a
signal peptide of 20 amino acids followed by a polypeptide of 820 amino acids with calculated molecular mass of 74.5 kDa.
The deduced amino acid sequence of MSP-1 includes a high proportion of Ser (32%), Gly (25%), and Asp (20%), and the predicted
isoelectric point is 3.2; in these respects, MSP-1 is a typical acidic glycoprotein of mineralized tissues. A repeated modular
structure characterizes MSP-1, with a sequence unit between 158 and 177 amino acids in length being repeated 4 times in tandem
in the middle part of the protein. The repeated unit comprises 3 modules (SG, D, and K domains), each having a distinct amino
acid composition and sequence. The SG domain is almost exclusively composed of Ser and Gly residues. The D domain is rich
in Asp residues, potential N-glycosylation and phosphorylation sites. The K domain is rich in Gly residues and has a core of basic residues. The Asp residues
are arranged more or less regularly in the D domains, exhibiting some repeated motifs such as Asp-Gly-Ser-Asp and Asp-Ser-Asp.
Further, the 4 D domains indicate remarkable overall sequence similarities to each other. These observations suggest that
the regular arrangements of COO− groups in the D domain side chains may be important for specific control of crystal growth.
Received September 19, 2000; accepted February 9, 2001 相似文献
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W.-Q. Zhuang J.-H. Tay A. Maszenan S. Tay 《Applied microbiology and biotechnology》2002,58(4):547-554
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A novel matrix protein family participating in the prismatic layer framework formation of pearl oyster, Pinctada fucata 总被引:1,自引:0,他引:1
Zhang C Xie L Huang J Liu X Zhang R 《Biochemical and biophysical research communications》2006,344(3):735-740
Understanding the molecular composition and the formation mechanism of shell matrix framework is of great interest for biomineralization in mollusk shell. The cDNAs encoding a novel matrix protein family (KRMP) were cloned from the mantle of pearl oyster, Pinctada fucata. Analysis of the deduced amino acid sequences revealed that KRMP have a high proportion of lysine, glycine, and tyrosine, and their predict isoelectric points are higher than any other identified shell matrix protein to our knowledge. The deduced amino acid sequences of KRMP can be divided into three regions, including an N-terminal signal peptide, a lysine-rich basic region interacting with acidic proteins or CO(3)(2-), and a Gly/Tyr-rich region involved in the protein cross-link via quinone-tanning process. RT-PCR and in situ hybridization demonstrated that KRMP mRNA was specifically expressed in the mantle edge, involved in the prismatic layer formation. Taken together, it seems that KRMP is a matrix protein family participating in the framework formation of prismatic layer. 相似文献
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Supungul P Sootanan P Klinbunga S Kamonrat W Jarayabhand P Tassanakajon A 《Marine biotechnology (New York, N.Y.)》2000,2(4):339-347
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Identification of organic phosphorus covalently bound to collagen and non-collagenous proteins of chicken-bone matrix. The presence of O-phosphoserine and O-phosphothreonine in non-collagenous proteins, and their absence from phosphorylated collagen
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Lola Cohen-Solal Jane B. Lian Dora Kossiva Melvin J. Glimcher 《The Biochemical journal》1979,177(1):81-98
Non-collagenous phosphoproteins, almost all of which can be extracted in EDTA at neutral pH in the presence of proteinase inhibitors, are identified in the matrix of chicken bone, and are therefore not covalently bound to collagen. Similarly, all the peptides containing gamma-carboxyglutamic acid are present in the EDTA extract and none in the insoluble residue, confirming that none is covalently linked to chicken bone collagen. However, organic phosphorus is also found to be present in chicken bone collagen, principally in the alpha2-chains. Of the total protein-bound organic phosphorus present in chicken bone matrix, approx. 80% is associated with the non-collagenous proteins and 20% with collagen. The soluble non-collagenous proteins contain both O-phosphoserine and O-phosphothreonine and these account for essentially of their organic phosphorus content. In contrast, collagen contains neither O-phosphoserine nor O-phosphothreonine. Indeed, no phosphorylated hydroxy amino acid, phosphoamidated amino acid or phosphorylated sugar could be identified in purified components of collagen, which contain approximately four to five atoms of organic phosphorus per molecule of collagen. Peptides containing organic phosphorus were isolated from partial acid hydrolysates and enzymic digests of purified collagen components, which contain an as-yet-unidentified cationic amino acid. These data, the very high concentrations of glutamic acid in the phosphorylated peptides, and the pH-stability of the organic phosphorus moiety in intact collagen chains strongly suggest that at least part of the organic phosphorus in collagen is present as phosphorylated glutamic acid. This would indicate that the two major chemically different protein fractions in chicken bone matrix that contain organic phosphorus may represent two distinct metabolic pools of organic phosphorus under separate biological control. 相似文献
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The effect of manganese chloride (10 mg Mn l-1), EDTA (18 mg l-1) and a mixture of these compounds on the nitrogen balance in maize xylem exudate was investigated. The compounds were applied
as experimental solutions to the roots of 20 day old plants 24 h before excision. Application of Mn resulting in a lowered
nitrogen level in the xylem exudate increased the relative content of the organic N-compounds in the exudate, particularly
that of free amino acids. EDTA appreciably enhanced the content of total nitrogen in xylem exudate, however no significant
changes were found in the proportion of inorganic and organic N-compounds in comparison with the water control. The significant
features of the free amino acid exudate fraction of all experimental variants were, among others, the relatively high lysine
content and the absence of proline arid sulphur containing amino acids. By gel filtration on Sephadex G-25 of xylem exudates
UV (254 nm) absorbing fractions, four in the water control, five in Mn and six in EDTA variants were isolated. The UV absorbing
fractions with the exception of one in each experimental variant (Kav 0.73 in water control, Kav 0.61 in Mn-variant, Kav 0.75 in EDTA and Mn + EDTA variant) were of peptide character, proline and sulphur-containing amino acids were missing in
them. In the exceptional UV absorbing fractions (Kav 0.61–0.75) in spite of their high N-content (10.23%) after hydrolysis practically no amino acid could be detected. 相似文献
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Soluble protein (MPSP, myostracal prism soluble protein) obtained from
myostracum in oyster shell (Crassostrea gigas) was characterized using biochemical and molecular biological techniques. From an analysis of secondary protein structure, it was shown that β-structure was predominant in MPSP. And via in vitro assays, the relation of MPSP to biomineral phase and morphology was studied. SDS-PAGE revealed one major protein band of 20 kDa. An amino acid sequence of 160 amino acids was deduced for myostracum by characterization of the complementary DNA encoding the protein. The deduced protein was composed of a high proportion of Gly and Asp, typifying a calcium-binding protein for shell formation, and a relatively high proportion of Val, Ala and Ile, typifying an adhesive protein. In contrast to prevailing expectations, (Gly–Asp)n-type sequence motifs exist in MPSP, demanding a revision of previous theories of protein–mineral interactions. The cDNA sequence of myostracum is elucidated for the first time. 相似文献
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Xylem exudates were collected from 20-day-old maize seedlings grown for 24 h in labelled manganese chloride solution (10 ppm)
with or without EDTA (18 mgl-1).
The distribution of radioactivity on paper ohromatograms of exudates did not show the existence of free Mn. Fractionation
of exudate constituents by gel filtration on Sephadex G-26 showed that54Mn is associated with three UV (254 nm) -absorbing fractions. Two fractions contain amino acids (M. wt.:≧ 10 000 and ∼ 900).
The third fraction (M. wt. ∼ 600) however, appears to be a carrier of a different type, which is practically amino acid free,
although it contains 10.23% N (by elementary analysis). The properties of this carrier are discussed. Application of EDTA
has favoured the; transport of Mn in xylem sap with fractions of relatively lower molecular weights. 相似文献
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Thomas Latire Florence Legendre Mouloud Bouyoucef Frédéric Marin Franck Carreiras Muriel Rigot-Jolivet Jean-Marc Lebel Philippe Galéra Antoine Serpentini 《Cytotechnology》2017,69(5):815-829
Mollusc shells are composed of more than 95% calcium carbonate and less than 5% organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. In this study, we investigated the effects of matrix macromolecular components extracted from the shells of two edible molluscs of economic interest, i.e., the blue mussel Mytilus edulis and the Pacific oyster Crassostrea gigas. The potential biological activities of these organic molecules were analysed on human dermal fibroblasts in primary culture. Our results demonstrate that shell extracts of the two studied molluscs modulate the metabolic activities of the cells. In addition, the extracts caused a decrease of type I collagen and a concomitant increase of active MMP-1, both at the mRNA and the protein levels. Therefore, our results suggest that shell extracts from M. edulis and C. gigas contain molecules that promote the catabolic pathway of human dermal fibroblasts. This work emphasises the potential use of these shell matrices in the context of anti-fibrotic strategies, particularly against scleroderma. More generally, it stresses the usefulness to valorise bivalve shells that are coproducts of shellfish farming activity. 相似文献
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Bédouet L Marie A Berland S Marie B Auzoux-Bordenave S Marin F Milet C 《Marine biotechnology (New York, N.Y.)》2012,14(4):446-458
A successful strategy for the identification of shell proteins is based on proteomic analyses where soluble and insoluble fractions isolated from organic shell matrix are digested with trypsin with the aim of generating peptides, which are used to identify novel shell proteins contained in databases. However, using trypsin as a sole degradative agent is limited by the enzyme's cleavage specificity and is dependent upon the occurrence of lysine and arginine in the shell protein sequence. To bypass this limitation, we investigated the ability of trifluoroacetic acid (TFA), a low-specificity chemical degradative agent, to generate clusters of analyzable peptides from organic shell matrix, suitable for database annotation. Acetic acid-insoluble fractions from Haliotis tuberculata shell were processed by trypsin followed by TFA digestion. The hydrolysates were used to annotate an expressed sequence tag library constructed from the mantle tissue of Haliotis asinina, a tropical abalone species. The characterization of sequences with repeat motifs featured in some of the shell matrix proteins benefited from TFA-induced serial cutting, which can result in peptide ladder series. Using the degradative specificities of TFA and trypsin, we were able to identify five novel shell proteins. This pilot study indicates that a mild chemical digestion of organic shell matrix combined with trypsin generates peptides suitable for proteomic analysis for better characterization of mollusc shell matrix proteins. 相似文献
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Puverel S Houlbrèque F Tambutté E Zoccola D Payan P Caminiti N Tambutté S Allemand D 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2007,147(4):850-856
Biominerals contain both inorganic and organic components. Organic components are collectively termed the organic matrix, and this matrix has been reported to play a crucial role in mineralization. Several matrix proteins have been characterized in vertebrates, but only a few in invertebrates, primarily in Molluscs and Echinoderms. Methods classically used to extract organic matrix proteins eliminate potential low molecular weight matrix components, since cut-offs ranging from 3.5 to 10 kDa are used to desalt matrix extracts. Consequently, the presence of such components remains unknown and these are never subjected to further analyses. In the present study, we have used microcolonies from the Scleractinian coral Stylophora pistillata to study newly synthesized matrix components by labelling them with 14C-labelled amino acids. Radioactive matrix components were investigated by a method in which both total organic matrix and fractions of matrix below and above 5 kDa were analyzed. Using this method and SDS-PAGE analyses, we were able to detect the presence of low molecular mass matrix components (<3.5 kDa), but no free amino acids in the skeletal organic matrix. Since more than 98% of the 14C-labelled amino acids were incorporated into low molecular weight molecules, these probably form the bulk of newly synthesized organic matrix components. Our results suggest that these low molecular weight components may be peptides, which can be involved in the regulation of coral skeleton mineralization. 相似文献
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Importance of minor-groove contacts for recognition of DNA by the binding domain of Hin recombinase 总被引:6,自引:0,他引:6
Incorporation of the DNA-cleaving moiety EDTA.Fe at discrete amino acid residues along a DNA-binding protein allows the positions of these residues relative to DNA bases, and hence the organization of the folded protein, to be mapped by high-resolution gel electrophoresis. A 52-residue protein, based on the sequence-specific DNA-binding domain of Hin recombinase (139-190), with EDTA at the NH2 terminus cleaves DNA at Hin recombination sites. The cleavage data for EDTA-Hin(139-190) reveal that the NH2 terminus of Hin(139-190) is bound in the minor groove of DNA near the symmetry axis of Hin-binding sites [Sluka, J. P., Horvath, S. J., Bruist, M. F., Simon, M. I., & Dervan, P. B. (1987) Science 238, 1129]. Six proteins, varying in length from 49 to 60 residues and corresponding to the DNA-binding domain of Hin recombinase, were synthesized by solid-phase methods: Hin(142-190), Hin(141-190), Hin(140-190), Hin(139-190), Hin(135-190), and Hin(131-190) were prepared with and without EDTA at the NH2 termini in order to test the relative importance of the residues Gly139-Arg140-Pro141-Arg142, located near the minor groove, for sequence-specific recognition at five imperfectly conserved 12-base-pair binding sites. Footprinting and affinity cleaving reveal that deletion of Gly139 results in a protein with affinity and specificity similar to those of Hin(139-190) but that deletion of Gly139-Arg140 affords a protein with altered affinities and sequence specificities for the five binding sites. It appears that Arg140 in the DNA-binding domain of Hin is important for recognition of the 5'-AAA-3' sequence in the minor groove of DNA. Our results indicate modular DNA and protein interactions with two adjacent DNA sites (major and minor grooves, respectively) bound on the same face of the helix by two separate parts of the protein. 相似文献
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JOHN-ERIK HAUGEN HANS PETTER SEJRUP 《Lethaia: An International Journal of Palaeontology and Stratigraphy》1990,23(2):133-141
Haugen, J. E. & Sejrup, H. P. 1990 04 15: Amino acid composition of aragonitic conchiolin in the shell of Arctica islandica. Lethaia , Vol. 23, pp. 133–141. Oslo. ISSN 0024–1164.
The distribution of amino acids within the two aragonitic shell layers of modern specimens of the mollusc Arctica islandica (Linné) has been studied in detail. The mean total hydrolyzed amino acid content was 19 nmol*mg in the inner layer and 15 nmol/mg in the outer layer. No significant difference in amino acid composition could be found between the two layers. The layers contained minor amounts of free amino acids which made up 0.3–0.7% of the total hydrolyzed amino acid content. The composition of the free amino acid fraction was very similar in each of the two layers, but differed somewhat from the total hydrolyzed fraction. The total hydrolyzed fraction was dominated by aspartic acid, glycine. alanine and glutamic acid, which together made up 62% of the total amount of amino acids, whereas the free fraction was dominated by Asp. Ser, Gly and Tyr. Amino acid content and composition within a single layer showed little variation from umbo to the ventral margin. The amino acid composition is in accordance with previously reported data on similar mineral structures which support the theory of structure specific rather than species specific amino acid composition. * Arctica islandica. amino acids. shell structure . 相似文献
The distribution of amino acids within the two aragonitic shell layers of modern specimens of the mollusc Arctica islandica (Linné) has been studied in detail. The mean total hydrolyzed amino acid content was 19 nmol*mg in the inner layer and 15 nmol/mg in the outer layer. No significant difference in amino acid composition could be found between the two layers. The layers contained minor amounts of free amino acids which made up 0.3–0.7% of the total hydrolyzed amino acid content. The composition of the free amino acid fraction was very similar in each of the two layers, but differed somewhat from the total hydrolyzed fraction. The total hydrolyzed fraction was dominated by aspartic acid, glycine. alanine and glutamic acid, which together made up 62% of the total amount of amino acids, whereas the free fraction was dominated by Asp. Ser, Gly and Tyr. Amino acid content and composition within a single layer showed little variation from umbo to the ventral margin. The amino acid composition is in accordance with previously reported data on similar mineral structures which support the theory of structure specific rather than species specific amino acid composition. * Arctica islandica. amino acids. shell structure . 相似文献