1. Feeding 2-aminoanthraquinone (2-AAQ) in the diet to Fischer rats led to nephrotoxicity in females, caused by deposits of crystalline material in the kidney tubules. 2. This material consisted of 2-AAQ, N-acetyl-2-AAQ and N-formyl-2-AAQ. N-Formyl-2-AAQ was also identified in the ether extract of urine of rats fed 2-AAQ. 3. This represents the first case of identification of the N-formyl derivative of a primary aromatic amine as a metabolite in vivo.

The deduced amino acid sequences of antifreeze proteins (AFPs) from larvae of the beetle Dendroides canadensis were determined from both complementary DNAs (cDNAs) and from peptide sequencing. These consisted of proteins with a 25-residue signal peptide and mature proteins 83 (Dendroides antifreeze protein; DAFP-1) or 84 (DAFP-2) amino acids in length which differed at only two positions. Peptide sequencing yielded sequences which overlapped exactly with those of the deduced cDNA sequences of DAFP-1 and DAFP-2, while the partial sequence of another AFP (DAFP-3) matched 21 of 28 residues. Seven 12- or 13-mer repeating units are present in these antifreeze proteins with a consensus sequence consisting of: Cys-Thr-X3-Ser-X5-X6-Cys-X8-X9-Ala-X11-Thr-X1 3, where X3 and X11 tend toward charged residues, X5 tends toward threonine or serine, X6 toward asparagine or aspartate, X9 toward asparagine or lysine, and X13 toward alanine in the 13-mers. The most interesting feature of these proteins is that throughout the length of the mature antifreeze proteins every sixth residue is a cysteine. These sequences are not similar to any of the known fish AFPs, but they are similar to AFPs from the beetle Tenebrio molitor.

No abstract

Two-dimensional gel electrophoresis with subsequent analysis by mass spectrometry was applied to study differences in protein expression between benign and malignant solid tumors from human beast, lung and ovary cells. Cells from freshly resected clinical material were lysed and the extracts were subjected to isoelectric focusing with immobilized pH gradients followed by second-dimensional separation on 10-13% sodium dodecyl sulfate (SDS)/polyacrylamide gels. Polypeptides were identified using matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry after in-gel protein digestion. Some of the upregulated polypeptides in malignant cells are of potential importance as markers of tumor proliferation. Twenty such proteins were identified, ten constituting novel identifications and ten sequence verifications of previously gel-matched proteins. The proteins identified span a wide range of functions, but several cases of protein truncation were found. Truncated forms of cytokeratins 6D and 8, and of cathepsin D were identified. Truncated froms of these over-expressed proteins support the presence of proteolytic processing steps in tumor material. The protein processing and the difference between protein and mRNA abundancies in tumors of different malignancy and origin suggest that studies at the protein level are important for an understanding of tumor phenotypes.

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 nontransformed, 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 (ubiquinone) 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.