Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.9 (ribonuclease)
 6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Structural isoforms of the insulin receptor that occur in various tissues have been postulated to be involved in certain actions of insulin in target cells. To determine whether these insulin-receptor subtypes are caused by alterations in the receptor primary structure, we used RNA heteroduplex mapping and amplification of cDNA to detect variation in the coding region of insulin-receptor mRNA from 5 rat tissues. A complete series of overlapping antisense [32P]RNA probes was prepared from plasmids containing segments of a full-length rat insulin-receptor cDNA, and probes were hybridized individually in solution with polyadenylated RNA from rat brain, kidney, liver, skeletal muscle, and spleen. After ribonuclease digestion, probe fragments were analyzed by denaturing gel electrophoresis. Tissue-specific cleavage of the mRNA:RNA probe heteroduplex, attributable to sequence mismatch, was detected only for a single probe covering the distal alpha-subunit, as expected for the known alternative splicing of rat insulin-receptor mRNA in this region. No evidence for additional heterogeneity of the receptor mRNA coding region was observed in the 5 tissues studied either by RNA heteroduplex mapping or, in some areas, by regional amplification of insulin-receptor cDNA. Cell-free translation of size-fractionated polyadenylated RNA was used to further demonstrate that each of the major insulin-receptor mRNA size classes in rat liver contained both forms of the alternatively spliced mRNA transcripts and produced two insulin-proreceptor polypeptides. These results suggest that heterogeneity of the insulin-receptor mRNA coding region affecting the receptor primary structure is limited to the distal alpha-subunit near the subunit cleavage site.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Heterogeneity of messenger RNA that encodes the rat insulin receptor is limited to the domain of exon 11. Analysis by RNA heteroduplex mapping, amplification of cDNA, and in vitro translation. 139 3

We used a ribonuclease cleavage assay to screen for insulin receptor mRNA sequence alterations in 12 patients with syndromes of severe insulin resistance. Uniformly labeled [32P]antisense RNA probes complementary to insulin receptor mRNA were prepared by an SP6 or T7 RNA polymerase transcription reaction. Four probes ranging in size from 670-1470 bases were used to examine the entire 4.2-kilobase receptor protein-coding region. Patient RNA samples were hybridized to individual probes in solution, and mismatched sequences were detected by susceptibility to cleavage by a mixture of RNAses A and T1. The method was validated with insulin receptor mRNAs from cells transfected with cDNA constructs bearing known point and deletion mutations. Alterations in the insulin receptor mRNA sequence of two patients were detected. A patient with the type A syndrome of severe insulin resistance (A2-Boston) had a mutation in the insulin receptor beta-subunit mRNA sequence that localized to the region coding for amino acid residues 1174-1211 near the tyrosine kinase domain. The second alteration was a sequence polymorphism in the insulin receptor alpha-subunit mRNA in a patient with lipoatropic diabetes (LA-2) that localized to a region within amino acids 268-272. Direct sequence analysis revealed that the ribonuclease cleavage sites in patients A2-Boston and LA-2 were due to distinct single base changes in the insulin receptor gene and mRNA. Additional insulin receptor mRNA sequence polymorphisms were also identified as mismatches between the labeled RNA probes used and mRNA from several cultured human cell types. This study demonstrates that ribonuclease cleavage can rapidly detect and localize insulin receptor mRNA sequence mutations and polymorphic variations as small as single base changes. Further analysis of insulin receptor mRNA sequence alterations identified in this way may elucidate a possible genetic basis for functional insulin receptor defects in patients with severe insulin resistance and can also reveal some insulin receptor sequence polymorphisms that occur in the population at large.
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PMID:Insulin receptor messenger ribonucleic acid sequence alterations detected by ribonuclease cleavage in patients with syndromes of insulin resistance. 273 94

Insulin-like growth factors (IGF-I and -II) bind with high affinity to IGF-binding proteins (IGFBPs). IGFBP-3 contains vicinal cysteines in sequence which is similar to the active sites in thioredoxin and protein disulfide isomerase. We tested if, in analogy with these redox enzymes, IGFBP-3 could catalyze the isomerization of intramolecular disulfide bridges in protein substrates. IGFBP-3 (30 microM) was able to reactivate reduced ribonuclease at a rate of 38% of that of thioredoxin. Also recombinant IGF-I induced the regeneration of ribonuclease activity. Thiol redox reactions are known to play a role in regulating conformational changes in the insulin receptor and possibly also in the IGF-I receptor. Therefore, the intrinsic isomerase activities of IGF-I may be important in the activation of its receptor. The observed effects of IGFBP-3 may help to elucidate the mechanism by which this binding protein can modulate the actions of IGF-I.
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PMID:Insulin-like growth factors (IGFs) and IGF binding protein-3 display disulfide isomerase activity. 750 99

The liver is the major site of insulin metabolism. Previous studies have suggested that hepatocytes were chiefly responsible for this activity, while contributions of Kupffer and other nonparenchymal liver cells remained controversial. In this study, we compared 125I-insulin binding and degradation by rat hepatocytes with insulin binding and degradation by sinusoidal Kupffer and endothelial cells. Kupffer cells were separated from endothelial cells by centrifugal elutriation. Hepatocytes had approximately 3.5 times more insulin binding sites than Kupffer cells and approximately eight times more binding sites than endothelial cells. In addition, wheat germ agglutinin (WGA)-purified solubilized receptors from all three cell types bound insulin in proportions similar to whole cells. Moreover, all three cell types were shown with a ribonuclease (RNase) protection assay to express insulin receptor mRNA. Hepatocytes degraded approximately four times more insulin than Kupffer cells, while endothelial cells degraded only negligible amounts of insulin. Based on morphometric data available in the literature, we estimated that nonparenchymal cells could account for approximately 10% to 15% of hepatic insulin degradation. We concluded that rat hepatocytes, Kupffer cells, and endothelial cells all have specific insulin receptors, and that nonparenchymal cells play a small but significant role in insulin degradation.
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PMID:Insulin binding and degradation by rat liver Kupffer and endothelial cells. 848 71