Identification of cDNA clones encoding secretory isoenzyme forms: sequence determination of canine pancreatic prechymotrypsinogen 2 mRNA
| Autor: | V Luc, G Scheele, S D Pinsky, K S LaForge |
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| Rok vydání: | 1983 |
| Předmět: |
Biology
Dogs Species Specificity Complementary DNA Animals Humans Amino Acid Sequence RNA Messenger Cloning Molecular Pancreas Peptide sequence Enzyme Precursors Multidisciplinary Base Sequence cDNA library Isoelectric focusing Endoplasmic reticulum Nucleic Acid Hybridization DNA Molecular biology Chymotrypsinogen Secretory protein Genes Biochemistry Zymogen activation Rough endoplasmic reticulum membrane Research Article |
| Zdroj: | Proceedings of the National Academy of Sciences. 80:7486-7490 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.80.24.7486 |
| Popis: | A cDNA library has been constructed from canine poly(A)+ mRNA. Clones containing cDNA inserts coding for prechymotrypsinogen 2 (isoelectric point = 7.1; Mr = 27,500), one of three canine pancreatic isoenzyme forms, were selected by colony hybridization using a cDNA probe synthesized from immunoselected prechymotrypsinogen 2 mRNA. To verify that cDNA clones code for prechymotrypsinogen 2 forms that translocate across rough endoplasmic reticulum membranes and fold into stable and identifiable secretory proteins, we conducted in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and optimal concentrations of glutathione and analyzed nascent translation products in their nonreduced state by two-dimensional isoelectric focusing/NaDodSO4 gel electrophoresis and fluorography. A near full-length chymotrypsinogen 2 cDNA and its primed extension were used to determine the nucleotide sequence for the entire coding region of prechymotrypsinogen 2 mRNA and 87 residues, including a poly(A) addition signal, in the 3' nontranslated region. The deduced amino acid sequence shows a 263-residue presecretory protein containing an 18-residue amino-terminal transport peptide (Met-Ala-Phe-Leu-Trp-Leu-Leu-Ser-Cys-Phe-Ala-Leu-Leu-Gly-Thr-Ala-Phe-Gly ), which we have previously shown to mediate the translocation of chymotrypsinogen 2 across the rough endoplasmic reticulum membrane. Following the transport peptide is a 245-residue proenzyme, which shows 82% and 80% sequence identity with bovine chymotrypsinogens A and B, respectively. Conserved among the three zymogens are 10 Cys residues that form five disulfide bonds in bovine chymotrypsinogens A and B and the residues that are required for zymogen activation, substrate binding, and catalytic activity. |
| Databáze: | OpenAIRE |
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