ISSN:
0173-0835
Keywords:
Immobilized pH gradient two-dimensional electrophoresis
;
Microsequencing
;
Human liver proteins
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Simplified methodology has been developed for the direct N-terminal amino acid microsequencing of human liver and hepatoma derived polypeptides, following micropreparative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Utilization of immobilized pH gradient (IPG) gel strips in the first dimension permitted protein loading of 0.5-2.0 mg with negligible diminution of polypeptide resolution. Following 2-D separation and electrotransfer to polyvinylidene difluoride (PVDF) membranes nearly 100 well resolved Ponceau S stained polypeptides were readily visualized, from which, 32 adult liver S-9 and 72 HepG2 nuclear cytosolic polypeptides were subjected to N-terminal microsequencing. Twenty normal adult liver and 54 HepG2 polypeptides yielded N-terminal sequence information, of which 17 and 19 polypeptides, respectively, exhibited high sequence homology to previously identified proteins. The initial yields of the proteins sequenced ranged from 2-14 pmols and yielded sequences of 14-26 amino acid residues. Many of the adult liver and HepG2 proteins contained inferred leader sequences since the first sequenced residue was several (20-30) residues from the methionine initiation site predicted by the cDNA of the adult liver. Quantitative comparison of 60 well characterized hepatic proteins between normal adult liver and two nontrans-formed, Chang and WRL-68, and four human hepatoma derived cell lines, HepG2, Huh-7, FOCUS, and SK-Hep, revealed a high homogeneity of protein expression both qualitatively and quantitatively in both whole cell lysate and purified nuclear preparations. Most notable differences include the previously characterized polypeptides: carbamoyl phosphate synthase, MER5 homologous protein, cytidylate kinase, phosphatidylethanolamine-binding protein and mitochondrial enoyl-CoA hydratase as well as three N-terminally blocked polypeptides: 11 (63 kDa/pI 7.00), 56 (26/6.45) and 59 (22/6.00) all of which were expressed at similar levels in normal adult liver tissue and each of the nontransformed, Chang and WRL-68, cell lines but not expressed or expressed at greatly decreased levels in each of tumor derived liver cell lines. Pyruvate carboxylase, superoxide dismutase, serotransferrin, liver fatty acid binding protein, 1-hydroxyprostaglandin dehydrogenase, NADH dehydrogenase (ubiqui-none) as well as three N-terminally blocked polypeptides: 9 (57/6.00), 53 (24/4.90) and 63 (16/4.70) were detected only in whole adult liver tissue and not in any of the cultured cell lines. Two additional polypeptides: U35, (27/6.05) and 58 (22/5.70) yielded N-terminal partial amino acid sequences but were not identified in established protein databases. We have shown that micropreparative IPG 2-D PAGE in combination with protein microsequencing provides a convenient one step procedure to rapidly obtain partial amino acid sequence information for nearly 100 individual polypeptides directly from a single 2-D PAGE gel with numerous applications to a wide variety of biological model systems.
Additional Material:
7 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.11501601321
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