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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
1
|
| pubmed:dateCreated |
1997-7-14
|
| pubmed:abstractText |
Prohormone substrates are required for investigation of the proteolytic processing of prohormones and proproteins into active peptide hormones and neurotransmitters. However, the lack of prohormone proteins has been a limiting factor in elucidating proteolytic mechanisms for conversion of prohormones into active peptides. Therefore, in this study, cloned cDNAs encoding the prohormones proenkephalin (PE), pro-neuropeptide Y (pro-NPY), pro-opiomelanocortin (POMC), and beta-protachykinin (beta-PT) were utilized to express recombinant prohormones in Escherichia coli. High-level expression of milligrams of prohormones was achieved with the pET3c expression vector utilizing the T7 promoter for production of PE, pro-NPY, and POMC, as demonstrated by SDS-PAGE gel electrophoresis, Western blots, and 35S-methionine labeling. In addition, beta-PT was expressed at high levels as fusion proteins with the maltose-binding protein and glutathione S-transferase by the pMAL-c and pGEX-2T expression vectors, respectively. Relative rates of processing by the established processing proteases "prohormone thiol protease" (PTP), 70-kDa aspartyl protease, and PC1/ 3 and PC2 (PC, prohormone convertase) were examined with purified PE, pro-NPY, and POMC. Distinct preferences of processing enzymes for different prohormones was demonstrated. PTP preferred PE and pro-NPY substrates, whereas little processing of POMC was detected. In contrast, the 70-kDa aspartyl protease cleaved POMC more readily than pro-NPY or PE. However, PC1/3 and PC2 prefer POMC as substrate. Demonstration of selectivity of processing enzymes for prohormone substrates illustrates the importance of expressing recombinant prohormones for in vitro processing studies.
|
| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/DNA, Complementary,
http://linkedlifedata.com/resource/pubmed/chemical/Endopeptidases,
http://linkedlifedata.com/resource/pubmed/chemical/Enkephalins,
http://linkedlifedata.com/resource/pubmed/chemical/Neuropeptide Y,
http://linkedlifedata.com/resource/pubmed/chemical/Pro-Opiomelanocortin,
http://linkedlifedata.com/resource/pubmed/chemical/Protein Precursors,
http://linkedlifedata.com/resource/pubmed/chemical/Protein Sorting Signals,
http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Fusion Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Tachykinins,
http://linkedlifedata.com/resource/pubmed/chemical/proenkephalin,
http://linkedlifedata.com/resource/pubmed/chemical/proneuropeptide Y,
http://linkedlifedata.com/resource/pubmed/chemical/protachykinin
|
| pubmed:status |
MEDLINE
|
| pubmed:month |
Jun
|
| pubmed:issn |
1046-5928
|
| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:volume |
10
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
80-8
|
| pubmed:dateRevised |
2008-11-21
|
| pubmed:meshHeading |
pubmed-meshheading:9179294-Animals,
pubmed-meshheading:9179294-Bacteriophage T7,
pubmed-meshheading:9179294-Blotting, Western,
pubmed-meshheading:9179294-Cloning, Molecular,
pubmed-meshheading:9179294-DNA, Complementary,
pubmed-meshheading:9179294-Electrophoresis, Polyacrylamide Gel,
pubmed-meshheading:9179294-Endopeptidases,
pubmed-meshheading:9179294-Enkephalins,
pubmed-meshheading:9179294-Escherichia coli,
pubmed-meshheading:9179294-Genes, Viral,
pubmed-meshheading:9179294-Neuropeptide Y,
pubmed-meshheading:9179294-Pro-Opiomelanocortin,
pubmed-meshheading:9179294-Promoter Regions, Genetic,
pubmed-meshheading:9179294-Protein Precursors,
pubmed-meshheading:9179294-Protein Processing, Post-Translational,
pubmed-meshheading:9179294-Protein Sorting Signals,
pubmed-meshheading:9179294-Rats,
pubmed-meshheading:9179294-Recombinant Fusion Proteins,
pubmed-meshheading:9179294-Swine,
pubmed-meshheading:9179294-Tachykinins
|
| pubmed:year |
1997
|
| pubmed:articleTitle |
High-level expression of the prohormones proenkephalin, pro-neuropeptide Y, proopiomelanocortin, and beta-protachykinin for in vitro prohormone processing.
|
| pubmed:affiliation |
Department of Medicine, University of California, San Diego, La Jolla 92093-0822, USA. vhook@ucsd.edu
|
| pubmed:publicationType |
Journal Article,
Comparative Study,
Research Support, U.S. Gov't, P.H.S.,
Research Support, U.S. Gov't, Non-P.H.S.
|