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Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.  相似文献
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We have isolated cDNA clones encoding the entire sequence of the bovine 46 kd cation-dependent mannose 6-phosphate (CD Man-6-P) receptor. Translation of CD Man-6-P receptor mRNA in Xenopus laevis oocytes results in a protein that binds specifically to phosphomannan-Sepharose, thus demonstrating that our cDNA clones encode a functional receptor. The deduced 279 amino acid sequence reveals a single polypeptide chain that contains a putative signal sequence and a transmembrane domain. Trypsin digestion of microsomal membranes containing the receptor and the location of the five potential N-linked glycosylation sites indicate that the receptor is a transmembrane protein with an extracytoplasmic amino terminus. This extracytoplasmic domain is homologous to the approximately 145 amino acid long repeating domains present in the 215 kd cation-independent Man-6-P receptor.  相似文献
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In order to study the mechanisms for the generation of length diversity within the 5' flanking region of the human insulin gene, we have isolated and sequenced a previously uncharacterized allele. This allele, of a size intermediate between those three already described in the literature, encompasses 1,156 base pairs (bp) and contains 81 reiterated tandem oligonucleotides of 14-15 bp each. Population analysis on 298 independently sampled individuals by Southern blotting of genomic DNA demonstrates that the polymorphic portion of the insulin 5' flanking region varies from 400 to more than 8,000 nucleotides, being encoded by from 30 to over 540 oligomeric repeats. Length variability 5' to the insulin gene is a result primarily of unequal crossing over, which generates an expansion or contraction in the number of tandem repeat units per chromosome. A similar mechanism probably accounts for nondispersed reiterated sequences at other loci in the human genome.  相似文献
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Previous studies by Southern blot analysis of human X mouse somatic cell hybrids localized the renin gene to region p21----qter of human chromosome 1. Using a DNA insert encoding exons 2-5, the renin gene was mapped to human chromosome bands 1q25----q32 by in situ hybridization. The sublocalization of the renin gene will facilitate subsequent detailed linkage analysis of human chromosome 1.  相似文献
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Expression of cloned bovine adrenal rhodanese   总被引:2,自引:0,他引:2  
A cDNA for the enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) has been cloned from a bovine adrenal library. An initiator methionine codon precedes the amino-terminal amino acid found in the isolated protein. Rhodanese is synthesized in the cytoplasm and transferred to the mitochondrial matrix. Thus, any amino-terminal sequence required for organelle import is retained in the mature protein. Furthermore, the DNA sequence shows that there are three additional amino acids, Gly-Lys-Ala, at the carboxyl terminus that are not found by protein sequencing. Additionally, comparison of the published amino acid sequence with that encoded by the open reading frame revealed three differences in the amino acid sequence. Comparison of the bovine and chicken liver sequences shows an overall level of 70% sequence homology, but there is complete identity of all residues that have been implicated in the function of the enzyme. When two mammalian cells, cos-7 and 293 cells, were transiently transfected with a plasmid containing the rhodanese coding region, rhodanese activity in lysates increased approximately 20-fold. Fluorograms of denaturing polyacrylamide gels detected a large increase in a polypeptide that co-migrated with the native protein and reacted with anti-rhodanese antibodies. Nondenaturing gels showed two active species that co-migrated with the two major electrophoretic forms purified by current procedures. Escherichia coli, transformed with a plasmid containing the rhodanese coding region, showed a 15-fold increase in rhodanese activity over baseline values. When the E. coli recombinant protein was analyzed on a nondenaturing gel, only one species was observed that co-electrophoresed with the more electropositive variant seen in purified bovine liver rhodanese. This single variant could be converted by carboxypeptidase B digestion to a form of the enzyme that co-migrated with the more electronegative species isolated from bovine liver. Thus, two major, enzymatically active electrophoretic variants, commonly observed in mammalian cells, can be accounted for by carboxyl-terminal processing without recourse to other post-translational modifications.  相似文献
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The NH2-terminal sequence of rhodanese influences many of its properties, ranging from mitochondrial import to folding. Rhodanese truncated by >9 residues is degraded in Escherichia coli. Mutant enzymes with lesser truncations are recoverable and active, but they show altered active site reactivities (Trevino, R. J., Tsalkova, T., Dramer, G., Hardesty, B., Chirgwin, J. M., and Horowitz, P. M. (1998) J. Biol. Chem. 273, 27841-27847), suggesting that the NH2-terminal sequence stabilizes the overall structure. We tested aspects of the conformations of these shortened species. Intrinsic and probe fluorescence showed that truncation decreased stability and increased hydrophobic exposure, while near UV CD suggested altered tertiary structure. Under native conditions, truncated rhodanese bound to GroEL and was released and reactivated by adding ATP and GroES, suggesting equilibrium between native and non-native conformers. Furthermore, GroEL assisted folding of denatured mutants to the same extent as wild type, although at a reduced rate. X-ray crystallography showed that Delta1-7 crystallized isomorphously with wild type in polyethyleneglycol, and the structure was highly conserved. Thus, the missing NH2-terminal residues that contribute to global stability of the native structure in solution do not significantly alter contacts at the atomic level of the crystallized protein. The two-domain structure of rhodanese was not significantly altered by drastically different crystallization conditions or crystal packing suggesting rigidity of the native rhodanese domains and the stabilization of the interdomain interactions by the crystal environment. The results support a model in which loss of interactions near the rhodanese NH2 terminus does not distort the folded native structure but does facilitate the transition in solution to a molten globule state, which among other things, can interact with molecular chaperones.  相似文献
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A stable analogue has been prepared of the enediolate anion believed to occur transiently in the reaction of phosphoglucose isomerase. This compound, 5-phosphoarabinonate, is the strongest known competitive inhibitor of the enzyme (Ki = 3 times 10(-7) M below pH 7). A distinctive pH dependence of binding, also found for two other aldonic acid omega-phosphates, 6-phosphogluconate and 4-phosphoerythronate, involves pertubation of a pKa from 7.0 in the free enzyme to 9.0 in the enzyme-inhibitor complex. This perturbation may reflect a catalytically advantageous increase in basicity which occurs around the transition state of the normal enzymatic reaction.  相似文献
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In contrast to the strongly pH-dependent inhibition of phosphoglucose isomerase by substrate analogues with a free carboxyl group, inhibition of this enzyme by neutral sugar phosphates is essentially invariant between pH 7 and 9. Competitive inhibition constants for glucitol 6-phosphate (40 muM), arabinose 5-phosphate (50 muM), and erythritol 4-phosphate (100 muM) were found to be of the same order of magnitude as that reported previously for substrate binding constants (50 to 240 muM). The unique exception is erythrose 4-phosphate whose Ki (0.7 muM, independent of pH) reflects a tightness of binding similar to that found at pH values near or below neutrality for the transition state analogue 5-phosphorarabinonate. The pH independence of inhibition by erythrose 4-phosphate and other neutral sugar phosphates may reflect a mode and locus of binding to phosphoglucose isomerase different from that of the aldonate inhibitors.  相似文献
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