Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have purified the 31-kDa precursor of human interleukin 1 beta (proIL1 beta) from recombinant Escherichia coli expressing the protein. The recombinant precursor was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, Western blot, and for biological and receptor binding activity. The protein migrates at the expected molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration columns. The specific activity of the recombinant precursor is less than 10(2) units/mg in the EL4 thymoma assay compared with 5 x 10(8) units/mg for the recombinant 17-kDa mature protein. The inactivity of the precursor is attributable to the inability of the protein to bind the IL1 receptor on EL4 cells as shown by receptor competition studies using 125I-labeled 17-kDa IL1 beta. Inactivity of the IL1 beta precursor is not due to degradation of the protein in either the bioactivity or receptor binding assays. The inactive IL1 beta precursor is converted to an active form following proteolysis with chymotrypsin which generates a carboxyl-terminal fragment of 17 kDa that is 6 orders of magnitude more active than the starting IL1 beta precursor. Removal of the first 114 amino acids from proIL1 beta generates a fully active molecule. In contrast, removal of the first 77 amino acids by treatment with trypsin only partially restores activity. The resultant 22-kDa protein exhibits a 600-fold increase in both biological and receptor binding activity, demonstrating a direct correlation between the ability of sequences within the pro-region to inhibit biological activity and inhibit binding to the IL1 receptor. Far-UV circular dichroism spectroscopy indicates that proIL1 beta is similar in secondary structure to mature IL1 beta; both proteins are nonhelical beta sheet proteins.
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PMID:Purification and characterization of human recombinant precursor interleukin 1 beta. 264 2

Although it is widely recognized that many proteins contain discrete functional domains, it is less certain whether smaller, less obviously discrete, units of structure will retain their specific function when transplanted into a different context. The observation that the potent inflammatory cytokine human interleukin 1 beta has the same overall structure as soybean trypsin inhibitor (STI) (Kunitz) prompted us to replace a tight turn in the cytokine sequence with the large loop in soybean trypsin inhibitor that binds to the active site of trypsin. Wild-type interleukin 1 beta (IL-1 beta) is highly resistant to proteolysis, but the chimeric STI/IL is specifically cleaved by trypsin, apparently in the inserted loop. Other chimeric interleukins have also been constructed, by replacing the same tight turn with inhibitory loops from other protein protease inhibitors: turkey ovomucoid inhibitor (TOI), a chymotrypsin inhibitor, and alpha 1-antitrypsin (AT), an elastase inhibitor. Although these loops come from proteins not related structurally to interleukin 1, they confer specific protease sensitivity or inhibition on the chimeric cytokine. The cytokine properties of these chimeric interleukins have also been evaluated. The chimeras formed from human IL-1 beta and all inhibitory loops tested bind to the interleukin 1 receptor with reasonable affinity. The typical cellular effects of IL-1, however, are not observed with all the recombinant proteins, thus confirming that receptor binding and signal transduction can be uncoupled. When these results are taken together with the results of site-directed mutagenesis of IL-1, reported in this paper and elsewhere, they allow the receptor and intracellular transduction sites on the protein to be mapped in detail.
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PMID:Modularity of protein function: chimeric interleukin 1 beta s containing specific protease inhibitor loops retain function of both molecules. 849 37

The major interleukin 1 beta (IL-1 beta) species from human plantar stratum corneum was purified and found to have an N-terminal amino acid sequence homologous to a stretch of the human IL-1 beta precursor, starting with His115. Whereas SDS-polyacrylamide gel electrophoresis followed by immunoblotting revealed only one component in plantar stratum corneum with IL-1 beta-like immunoreactivity, and with an apparent molecular mass around 18 kDa, isoelectric focusing under non-denaturing conditions showed one major component with isoelectric point around 6.1 and two minor components isoelectric at pH 6.3 and 6.9, respectively. Digestion of recombinant human IL-1 beta precursor with chymotrypsin, producing a C-terminal fragment with N-terminal Yal114, yielded a component with IL-1 beta-like immunoreactivity isoelectric at pH 6.3. Recombinant bacterial variants of human IL-1 beta with N-terminal amino acids corresponding to Val114, His115 and Ala117 were isoelectric at pH 6.3, 6.1 and 6.9, respectively. Cloning and subsequent nucleotide sequencing of IL-1 beta precursor cDNA from a human keratinocyte line showed total identify with the sequence previously published for the human monocyte IL-1 beta precursor. The authors conclude that the IL-1 beta species present in plantar stratum corneum have isoelectric points determined by their respective amino acid sequences, and that there is a mechanism for IL-1 beta activation in human epidermis not involving interleukin 1 beta convertase.
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PMID:Purification and characterization of interleukin 1 beta from human plantar stratum corneum. Evidence of interleukin 1 beta processing in vivo not involving interleukin 1 beta convertase. 970 14