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1.
Calcium has been suggested to be an important element in the regulation of microtubule dynamics 'in vivo'. In this report we have analyzed the possibility that microtubule-associated protein 2 (MAP2) binds calcium. MAP2 was blue-stained with the cationic carbocyanine dye 'stains-all' in a similar way to that of calcium-binding proteins and bound 45Ca as estimated from dot-blotting experiments. The calcium-binding characteristics of MAP2, determined by equilibrium dialysis, indicated that MAP2 bound about 3 mol (n = 2.9 +/- 0.4) of calcium per mol of protein (Kd = (0.9 +/- 0.2).10(-5) M). Analysis of the Scatchard plots from equilibrium dialysis and dot-blot assays indicated that MAP2 also presented low-affinity calcium-binding sites (Kd = (0.3 +/- 0.2).10(-4) M). Incubation of nitrocellulose blots of proteolytically digested MAP2 with 45Ca indicated that the calcium-binding sites were located in the region that is not involved in the interaction with tubulin (projection region). 相似文献
2.
Larval-specific protein (LSP) is the most abundant protein in the hemolymph of cockroaches shortly before molting, but is rapidly cleared from the hemolymph during the molt (Kunkel, J. G., and Lawler, D. M. (1974) Comp. Biochem. Physiol. 47B, 697-710). Blatta orientalis LSP was purified by sedimentation in preparative sucrose gradients followed by 2-hydroxypropylamino-cellulose anion-exchange chromatography and gel filtration on a column of Bio-Gel A-1.5m. The amino acid composition of LSP includes 16.3 mol % tyrosine and 4.9 mol % phenylalanine, but virtually no cysteine and little methionine. The following physical properties were determined for LSP: R8 = 68.3 A, 8(20),w = 17.8, and V = 0.723. From these values an Mr = 507,900 was calculated. In electron micrographs, LSP appears as rectangular particles of 121 by 134 A. In disc polyacrylamide gel electrophoresis, native LSP exhibits a single band, but in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, LSP is resolved into a doublet of closely spaced bands of Mr = 88,100 and 84,400 present in a ratio of 1.38:1. These data indicate that native B. orientalis LSP is a hexamer of subunits averaging approximately Mr = 86,000. Crossed immunoelectrophoresis of Blattella germanica larval serum indicates that LSP in that species is a hexamer composed of a random assortment of two subunits of different charge in the ratio 1.25:1. The amino acid composition and physical properties of LSP suggest that LSP may be the hemimetabolous analogue of the tyrosine- and phenylalanine-rich storage proteins of holometabolous insects. 相似文献
3.
Summary Evidence is presented for a carbohydrate-binding property of aprotinin, which is preserved both in a fluorescein isothiocyanate (FITC) conjugate and a cyanogen bromidelinked Sepharose conjugate of the protein. Both conjugates similarly retain their tryptic and chymotryptic inhibitory properties. The FITC conjugate is shown to be a single species with respect to charge and to molecular weight and shows a specific binding of normal materials containing sialosyl or uronosyl groups, which accords with its histochemical behaviour. The Sepharose-conjugate showed a similar specificity.R.W.S. holds a grant from the Cancer Research Campaign. We thank Dr. G. M. W. Cook for discussion and advice. 相似文献
4.
Biondi RM 《Trends in biochemical sciences》2004,29(3):136-142
Phosphoinositide-dependent protein kinase 1 (PDK1) is a protein kinase that phosphorylates and activates several other protein kinases from the AGC group (which includes PKA, PKG and PKC), to which PDK1 also belongs. Recent data suggests that PDK1 specificity is achieved by regulation of its interaction with substrates and supports a rather simple model explaining how PDK1 interacts with different substrates. The data further suggests that PDK1 interacts with its substrates when they are in a particular conformation (inactive). PDK1 has the ability to recognize, interact with and phosphorylate specific substrate conformations and thus sets PDK1 at the centre of a protein conformation sensor mechanism. The PDK1-substrate interaction model describes, at a molecular level, the mechanism used by PDK1 to sense the conformation of its substrates. 相似文献
5.
PACT, a protein activator of the interferon-induced protein kinase, PKR. 总被引:23,自引:2,他引:21
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PKR, a latent protein kinase, mediates the antiviral actions of interferon. It is also involved in cellular signal transduction, apoptosis, growth regulation and differentiation. Although in virus-infected cells, viral double-stranded (ds) RNA can serve as a PKR activator, cellular activators have remained obscure. Here, we report the cloning of PACT, a cellular protein activator of PKR. PACT heterodimerized with PKR and activated it in vitro in the absence of dsRNA. In mammalian cells, overexpression of PACT caused PKR activation and, in yeast, co-expression of PACT enhanced the anti-growth effect of PKR. Thus, PACT has the hallmarks of a direct activator of PKR. 相似文献
6.
From a user's point-of-view we are in the Golden Age of protein crystallographic software. In the past few decades, solving protein structures has gone from a task requiring man-months of effort to a process requiring minutes on an ordinary laptop with no human intervention required. The birth of XtalView coincided with the mainstream use of synchrotron radiation, seleno-Met phasing and it continues to be used in this age of robotic crystallization, Fed-Ex data collection and fully automated structure solution "pipelines". This article is a retrospective history of protein crystallographic computing and a discussion of the current state of the art. 相似文献
7.
MKP-7, a novel mitogen-activated protein kinase phosphatase, functions as a shuttle protein 总被引:1,自引:0,他引:1
Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) negatively regulate MAPK activity. In the present study, we have identified a novel MKP, designated MKP-7, and mapped it to human chromosome 12p12. MKP-7 possesses a long C-terminal stretch containing both a nuclear export signal and a nuclear localization signal, in addition to the rhodanese-like domain and the dual specificity phosphatase catalytic domain, both of which are conserved among MKP family members. When expressed in mammalian cells MKP-7 protein was localized exclusively in the cytoplasm, but this localization became exclusively nuclear following leptomycin B treatment or introduction of a mutation in the nuclear export signal. These findings indicate that MKP-7 is the first identified leptomycin B-sensitive shuttle MKP. Forced expression of MKP-7 suppressed activation of MAPKs in COS-7 cells in the order of selectivity, JNK p38 > ERK. Furthermore, a mutant form MKP-7 functioned as a dominant negative particularly against the dephosphorylation of JNK, suggesting that MKP-7 works as a JNK-specific phosphatase in vivo. Co-immunoprecipitation experiments and histological analysis suggested that MKP-7 determines the localization of MAPKs in the cytoplasm. 相似文献
8.
DrrC, a daunorubicin resistance protein with a strong sequence similarity to the UvrA protein involved in excision repair of DNA, is induced by daunorubicin in Streptomyces peucetius and behaves like an ATP-dependent, DNA binding protein in vitro. The refolded protein obtained from expression of the drrC gene in Escherichia coli was used to conduct gel retardation assays. DrrC bound a DNA segment containing the promoter region of a daunorubicin production gene only in the presence of ATP and daunorubicin. This result suggests that DrrC is a novel type of drug self-resistance protein with DNA binding properties like those of UvrA. Western blotting analysis with a polyclonal antiserum generated against His-tagged DrrC showed that the appearance of DrrC in S. peucetius is coincident with the onset of daunorubicin production and that the drrC gene is induced by daunorubicin. These data also showed that the DnrN and DnrI regulatory proteins are required for drrC expression. The level of DrrA, another daunorubicin resistance protein that resembles ATP-dependent bacterial antiporters, was regulated in the same way as that of DrrC. 相似文献
9.
Differential IgG-binding characteristics of staphylococcal protein A, streptococcal protein G, and a chimeric protein AG 总被引:4,自引:0,他引:4
M Eliasson R Andersson A Olsson H Wigzell M Uhlén 《Journal of immunology (Baltimore, Md. : 1950)》1989,142(2):575-581
Various Gram-positive bacteria express different types of IgG-binding receptors, each of which displaying certain unique binding properties. To evaluate specificity and avidity aspects of the differential binding pattern, a set of competitive binding assays was employed, by using staphylococcal protein A (SPA), streptococcal protein G (SPG), and a chimeric protein AG. These receptors were analyzed, in a reciprocal fashion, for binding and inhibition of binding to a selected panel of polyclonal and monoclonal Ig. Results of the study reveal that a majority of the determinants on human and bovine IgG, recognized by SPA and SPG, are either coextensive or closely overlapping. Accordingly, a minor portion of the determinants appear to be unique in the sense that a particular determinant(s) is selectively identified by one of the two receptors. Binding assays involving purified Fc fragments from human IgG, suggest that SPG shows exclusive specificity for an Fab region determinant(s) not recognized by SPA, whereas the Fc determinants for SPA and SPG are identical or overlapping. Furthermore, one of the IgG subclasses of bovine origin appears to be seen by the SPG receptor only. The competition study also demonstrates that the novel chimeric protein AG receptor shows higher or equal avidity for variants of human IgG molecules compared to the best of its parental constituents. It can thus be deduced that chimeric receptors might be useful as optimized tools for immunologic applications. 相似文献
10.
A multifunctional masquerade-like protein has been isolated, purified, and characterized from hemocytes of the freshwater crayfish, Pacifastacus leniusculus. It was isolated by its Escherichia coli binding property, and it binds to formaldehyde-treated Gram-negative bacteria as well as to yeast, Saccharomyces cerevisiae, whereas it does not bind to formaldehyde-fixed Gram-positive bacteria. The intact masquerade (mas)-like protein is present in crayfish hemocytes as a heterodimer composed of two subunits with molecular masses of 134 and 129 kDa. Under reducing conditions the molecular masses of the intact proteins are not changed. After binding to bacteria or yeast cell walls, the mas-like protein is processed by a proteolytic enzyme. The 134 kDa of the processed protein yields four subunits of 65, 47, 33, and 29 kDa, and the 129-kDa protein results in four subunits of 63, 47, 33, and 29 kDa in 10% SDS-PAGE under reducing conditions. The 33-kDa protein could be purified by immunoaffinity chromatography using an Ab to the C-terminal part of the mas-like protein. This subunit of the mas-like protein has cell adhesion activity, whereas the two intact proteins, 134 and 129 kDa, have binding activity to LPSs, glucans, Gram-negative bacteria, and yeast. E. coli coated with the mas-like protein were more rapidly cleared in crayfish than only E. coli, suggesting this protein is an opsonin. Therefore, the cell adhesion and opsonic activities of the mas-like protein suggest that it plays a role as an innate immune protein. 相似文献
11.
Protein U, a late-developmental spore coat protein of Myxococcus xanthus, is a secretory protein. 总被引:3,自引:2,他引:1
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Protein U is a spore coat protein produced at the late stage of development of Myxococcus xanthus. This protein was isolated from developmental cells, and its amino-terminal sequence was determined. On the basis of this sequence, the gene for protein U (pru) was cloned and its DNA sequence was determined, revealing an open reading frame of 179 codons. The product from this open reading frame has a typical signal peptide of 25 amino acid residues at the amino terminal end, followed by protein U of 154 residues. This result indicates that protein U is produced as a secretory precursor, pro-protein U, which is then secreted across the membrane to assemble on the spore surface. This is in sharp contrast to protein S, a major spore coat protein produced early in development, which has no signal peptide, indicating that there are two distinct pathways for trafficking of spore coat proteins during the differentiation of M. xanthus. 相似文献
12.
We report here a novel case of amyloid-like aggregation of a plant protein. A sweet-tasting protein, monellin, experiences an irreversible heat denaturation at pH 2.5 and 85 degrees C. Addition of 100 mM NaCl couples this process with protein aggregation. The aggregates were structured as regular fibers with approximately 10 nm width and capable of binding to Congo red, similarly to well-known amyloid fibrils. The amyloid-like aggregation process was also successfully monitored with a calorimetric method. This work supports the universality of the amyloid-like aggregation, not restricted to some special categories of protein. 相似文献
13.
B. Ketterer 《The Biochemical journal》1965,96(2):372-376
14.
Metallothioneins, a diverse protein family 总被引:1,自引:0,他引:1
Grennan AK 《Plant physiology》2011,155(4):1750-1751
15.
T Furukawa T Uchiumi R Tokunaga S Taketani 《Archives of biochemistry and biophysics》1992,298(1):182-186
We examined the properties of a new iron-binding protein purified previously from rat liver (T. Furukawa, S. Taketani, H. Kohno, and R. Tokunaga, 1991, Biochem. Biophys. Res. Commun. 181, 409-415). The protein was digested with trypsin and the peptides were analyzed by reverse-phase high-performance liquid chromatography. The partial amino acid sequences of the tryptic peptides coincided with that of rat ribosomal protein P2. Immunoblot analysis and iron-binding assay confirmed that the iron-binding protein and ribosomal protein P2 are identical. Then the iron binding ability of ribosomal protein P2 was examined in rat hepatoma H4IIEC3 cells incubated with radioactive iron. When immunoprecipitation with anti-iron-binding protein serum was performed using cells incubated with 59Fe-citrate, about 4% of the 59Fe radioactivity in cells was associated with the iron-binding protein through 30 to 90 min of incubation. About 1.5% of radioactive iron in cells incubated with 59Fe-transferrin was found in immunoprecipitates with anti-iron-binding protein serum during 1 to 5 h of incubation, and 4 to 7% of the radioactivity was found in immunoprecipitates with a monoclonal antibody against ribosomal P proteins in the same incubation. These results demonstrate that ribosomal proteins P2 binds iron taken up by the cells. 相似文献
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18.
Sphingomyelin plays complex structural and signaling functions in the plasma membrane. Of special interest is that hydrolysis of sphingomyelin to ceramide can modulate dynamics of membrane rafts, which serve as signaling platforms for various receptors. This review is focused on a recently discovered sphingomyelin-binding protein, lysenin, which can be used as a unique probe to trace distribution and turnover of sphingomyelin in cellular membranes. We analyze the primary and secondary structures of lysenin with respect to its interaction with the plasma membrane. The specificity of lysenin binding to sphingomyelin, revealed by both biochemical and cytochemical approaches, is discussed. 相似文献
19.
Endo-(1,4)-beta-xylanases of plant and fungal origin play an important role in the degradation of arabinoxylans. Two distinct classes of proteinaceous endoxylanase inhibitors, the Triticum aestivum xylanase inhibitor (TAXI) and the xylanase inhibitor protein (XIP), have been identified in cereals. Engineering of proteins in conjunction with enzyme kinetics, thermodynamic, real-time interaction, and X-ray crystallographic studies has provided knowledge on the mechanism of inhibition of XIP-I towards endoxylanases. XIP-I is a 30 kDa protein which belongs to glycoside hydrolase family 18, and folds as a typical (beta/alpha)8 barrel. Although the inhibitor shows highest homology with plant chitinases, XIP-I does not hydrolyse chitin; probably due to structural differences in the XIP-I binding cleft. The inhibitor is specific for fungal xylanases from glycoside hydrolases families 10 and 11, but does not inhibit bacterial enzymes. The inhibition is competitive and, depending on the xylanase, the Ki value can be as low as 3.4 nM. Site-directed mutagenesis of a xylanase from Aspergillus niger suggested that the XIP-I binding site was the conserved hairpin loop "thumb" region of family 11 xylanases. Furthermore, XIP-I shows the ability to inhibit barley alpha-amylases of glycoside hydrolase family 13, providing the first example of a protein able to inhibit members of different glycoside hydrolase families (10, 11, and 13), and additionally a novel function for a protein of glycoside hydrolase family 18. 相似文献
20.
Phosphorylation of a bacterial activator protein, OmpR, by a protein kinase, EnvZ, results in stimulation of its DNA-binding ability 总被引:27,自引:0,他引:27
The Escherichia coli OmpR protein is an activator protein specific for the ompF and ompC genes, which respectively encode the outer membrane proteins, OmpF and OmpC. The EnvZ protein is a protein kinase specific for the OmpR protein. In this study, we compared the in vitro DNA-binding ability of the phosphorylated form of the OmpR protein with that of the non-phosphorylated form by means of non-denaturing gel retardation analysis and DNase I footprinting analysis. The results indicate that the phosphorylation of the OmpR protein results in stimulation of its in vitro DNA-binding ability as to both the ompF and ompC promoter DNAs. 相似文献