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Query: UNIPROT:P02794 (ferritin)
 17,525 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ferritin messenger RNA has been shown to be translationally inactivated by the binding of a cytosolic protein to a 28-nucleotide iron-responsive element (IRE) located in the 5'-untranslated region of the mRNA. This interaction has been studied using quantitative receptor-ligand binding methods with gel retardation and nitrocellulose filter binding assays for the separation of bound complex from free RNA. In competition assays the entire 5'-untranslated region and the isolated IRE bound identically. The specificity of the RNA binding was studied using IRE variants. Two IREs from transferrin receptor mRNA and several variants with single base substitutions in the stem or loop had similar affinities. RNAs which could not form a stem-loop structure bound 1000-fold less well. These studies demonstrate the importance of the RNA conformation and the relative insensitivity of binding to much of the primary sequence. Saturation assays with increasing concentrations of 32P-IRE resulted in a binding hyperbola characteristic of mass action binding to a single class of sites with a KD = 0.09 nM. At 37 degrees C the dissociation rate is 0.04 min-1 (t 1/2 = 17 min). This rate is fast enough to account for the shift of ferritin RNA from the ribonucleoprotein pool to polysomes after rats are injected with iron. Determination of the concentration of the repressor requires accounting for three interconverting pools: free active repressor, mRNA-bound protein, and inactive (low affinity) repressor. Rat liver cytosol has a concentration of free active repressor of about 1 pmol/mg protein. Protein bound to endogenous mRNA can be measured by pretreatment with micrococcal nuclease or by separation with DEAE-Sepharose chromatography; it is present at a level similar to that of the free active protein. Inclusion of high levels of thiol reductants in the binding incubations reduces the inactive or low affinity repressor, forming unstably activated protein which has the same KD as the endogenous active protein; this inactive or low affinity protein is 2-4 times more abundant. A mechanism for iron regulation is proposed which accounts for the kinetics, the multiple protein pools, and the characteristics of the protein in these pools.
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PMID:Determinants of the interaction between the iron-responsive element-binding protein and its binding site in rat L-ferritin mRNA. 232 9

The filamentous bacteriophage f1 can be transformed into a spherical particle (spheroid) or an intermediate shortened filament with a flared end (I-forms) by exposure to a chloroform-water interface at 22 or 4 degrees C, respectively. The protein composition of bacteriophage f1 spheroids and I-forms was examined by separating the proteins from the purified. [35S]cysteine-labeled particles by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. Quantitation of the radioactivity on the gels showed that I-forms and spheroids contain the same complement of minor coat proteins as do untreated f1 phage. This composition is unchanged after removal of the DNA, either by digestion with micrococcal nuclease or by centrifugation of the particles through CsCl density gradients, indicating that none of the minor coat proteins is held in the particles solely through an interaction with the DNA. We also examined the location of the A protein in I-forms by decoration with ferritin-conjugated antibodies and examination under the electron microscope and found that the A protein is located specifically at the flared end of the I-form particle, through which the DNA is extruded and at which contraction into spheroids begins. The implications of these results with regard to the orientation of the DNA within the capsid and the process of infection are discussed.
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PMID:Minor coat protein composition and location of the A protein in bacteriophage f1 spheroids and I-forms. 709 58

A previous report from this laboratory described an estrogen-regulated endoribonuclease activity on Xenopus liver polysomes which had properties one might expect for a messenger ribonuclease involved in the regulated destabilization of albumin mRNA (Pastori, R. L., Moskaitis, J. E., and Schoenberg, D. R. (1991) Biochemistry 30, 10490-10498). This report describes the purification and properties of this ribonuclease. The purified nuclease fraction contained a doublet of 62 and 64 kDa and a small amount of a 40-kDa peptide. In situ analysis on both denaturing and nondenaturing gels using an albumin transcript as substrate showed all three proteins possess nuclease activity. Peptide mapping and Western blot with a polyclonal antiserum showed the 62- and 64-kDa peptides to be isoforms, and the 40-kDa peptide to be a degradation product of the larger species. Two-dimensional gel electrophoresis further separated the 62- and 64-kDa species into three pairs of proteins, with isoelectric points of 9.6, 9.8, and 9.8. The purified ribonuclease rapidly degraded a full-length albumin transcript, yet had no effect on either a full-length albumin antisense transcript or full-length ferritin transcript. A number of properties of the purified nuclease were characterized, including the effects of salt, divalent cations, EDTA, sulfhydryl reagents, and temperature. Treatment of the polysomal nuclease with micrococcal nuclease had no effect, indicating that this enzyme does not require an RNA cofactor for activity. Finally, primer extension mapped the major cleavage site to an overlapping repeated sequence APyrUGA, with cleavage between and adjacent to the two pyrimidine residues generating fragments with 5'-hydroxyls.
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PMID:Purification and characterization of an estrogen-regulated Xenopus liver polysomal nuclease involved in the selective destabilization of albumin mRNA. 789 Jul 44