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High affinity binding of divalent cation to actin monomer is much stronger than previously reported 总被引:2,自引:0,他引:2
L C Gershman L A Selden J E Estes 《Biochemical and biophysical research communications》1986,135(2):607-614
Monomeric actin is known to bind tightly one divalent cation per molecule. We have quantitatively reinvestigated the affinity of actin for Ca++ and Mg++ using the fluorescent Ca++ chelator Quin2 to induce and measure the dissociation of Ca++ from Ca-actin, supporting these studies with measurements using 45Ca. We found that the KD for Ca-actin is actually 1.9 +/- 0.7 nM. Kinetic analysis supported this result and demonstrated a dissociation rate constant (k-) of 0.013 s-1 and an association rate constant (k+) of 6.8 X 10(6)M-1 s-1 for Ca-actin. Competitive binding studies indicated that the binding affinity of actin for Ca++ is 5.4 times that for Mg++, yielding a calculated KD for Mg-actin of about 10 nM. Thus, the tight-binding of divalent cations to actin is 3-4 orders of magnitude stronger than previously thought. 相似文献
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Isolation and characterization of a large,neurite-associated glycoconjugate from neuroblastoma cells
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A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species. 相似文献
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Richard F. Selden Andre Steinmetz Lee McIntosh Lawrence Bogorad Gerard Burkard Mfika Mubumbila Marcel Kuntz Edwin J. Crouse Jacques H. Weil 《Plant molecular biology》1983,2(3):141-153
A minimum of 37 genes corresponding to tRNAs for 17 different amino acids have been localized on the restriction endonuclease cleavage site map of theZea mays chloroplast DNA molecule. Of these, 14 genes corresponding to tRNAs for 11 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. One tRNA gene is in the smaller single-copy region. Each copy of the large repeated sequence contains, in addition to the ribosomal RNA genes, 11 tRNA genes corresponding to tRNAs for 8 amino acids. The genes for tRNA2 Ile and tRNAAla map in the ribosomal spacer sequence separating the 16S and 23S ribosomal RNA genes. The three isoaccepting species for the tRNAsLeu and the three for tRNAsSer, as well as the two isoaccepting species for tRNAAsn, tRNAGly, tRNAsIle, tRNAsMet, tRNAsThr, are shown to be encoded at different loci. Two independent methods have been used for the localization of tRNA genes on the physical map of the maize chloroplast DNA molecule: (a) cloned chloroplast DNA fragments were hybridized with radioactively-labelled total 4S RNAs, the hybridized RNAs were then eluted, and identified by two-dimensional polyacrylamide gel electrophoresis, and (b) individual tRNAs were32P-labelledin vitro and hybridized to DNA fragments generated by digestion of maize chloroplast DNA with various restriction endonucleases. 相似文献
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Deep-level diagnostic value of the rDNA-ITS region 总被引:14,自引:0,他引:14
The similarity of certain reported angiosperm rDNA internal transcribed
spacer (ITS) region sequences to those of green algae prompted our analysis
of the deep-level phylogenetic signal in the highly conserved but short
5.8S and hypervariable ITS2 sequences. We found that 5.8S sequences yield
phylogenetic trees similar to but less well supported than those generated
by a ca. 10-fold longer alignment from rDNA-18S sequences, as well as
independent evidence. We attribute this result to our finding that,
compared to 18S, the 5.8S has a higher proportion of sites subject to vary
and greater among-site substitution rate homogeneity. We also determined
that our phylogenetic results are not likely affected by intramolecular
compensatory mutation to maintain RNA secondary structure nor by evident
systematic biases in base composition. Despite historical homology, there
appears to be no ITS2 primary sequence similarity shared sufficient
similarity to cluster correctly on the basis of alignability. Our results
indicate that groups, however, share sufficient similarity to cluster
correctly on the basis of alignability. Our results indicate that ITS
region sequences can diagnose organismal origins and phylogenetic
relationships at many phylogenetic levels and provide a useful paradigm for
molecular evolutionary study.
相似文献
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Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro 总被引:1,自引:1,他引:0
JEROME F. LA PEYRE DORIS Y. SCHAFHAUSER ESAM H. RIZKALLA MOHAMED FAISAL 《The Journal of eukaryotic microbiology》1995,42(5):544-551
ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases. 相似文献
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