Immobilized pH gradient two-dimensional gel electrophoresis and mass spectrometric identification of cytokine-regulated proteins in ME-180 cervical carcinoma cells.

Two-dimensional (2-D) polyacrylamide gel electrophoresis combined with mass spectrometry is a powerful combination of technologies that allows high resolution separation of proteins and their rapid identification. Immobilized pH gradient (IPG) first-dimensional gels have several advantages over carrier ampholyte isoelectric focusing, including a high degree of reproducibility, good protein spot resolution, and a selection of pH range. Here we demonstrate the utility and efficacy of combining IPG 2-D gel electrophoresis with mass spectrometry to identify interferon-gamma- (IFN) and tumor necrosis factor (TNF)-regulated proteins in ME-180 cervical carcinoma cells. Three cytokine-regulated proteins have been identified, using imidazole-zinc-stained preparative IPG 2-D gels and in-gel tryptic digestion followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry for determination of peptide masses and sequences: 1) triosephosphate isomerase, a glycolytic pathway enzyme, 2) proteasome subunit C3, which is important in protein degradation, and 3) Ran, a GTP-binding protein important in cell cycle regulation, protein import into the nucleus, and RNA export from the nucleus.

Functional expression of falcipain, a Plasmodium falciparum cysteine proteinase, supports its role as a malarial hemoglobinase.

Erythrocytic malaria parasites degrade hemoglobin as a principal source of amino acids for parasite protein synthesis. We have previously shown that a Plasmodium falciparum trophozoite cysteine proteinase, now termed falcipain, is required for hemoglobin degradation, and we have hypothesized that this proteinase is responsible for initial cleavages of hemoglobin. To further evaluate the biological role of falcipain, we expressed the enzyme in bacterial and viral expression systems. After expression in the baculovirus system, falcipain was enzymatically active and had biochemical properties very similar to those of the native proteinase. Recombinant falcipain rapidly hydrolyzed both denatured and native hemoglobin. Hemoglobin hydrolysis was blocked by cysteine proteinase inhibitors but not by inhibitors of other classes of proteinases. Our results support our hypothesis that falcipain is a critical malarial hemoglobinase that is responsible for both initial cleavages of hemoglobin and the subsequent hydrolysis of globin into small peptides.

The use of antisense mRNA to inhibit the tonoplast H+ ATPase in carrot.

Carrot root cells were transformed with the coding or 5' noncoding regions of the carrot vacuolar H+ ATPase A subunit cDNA cloned in the antisense orientation behind the cauliflower mosaic virus 35S promoter. Bafilomycin-sensitive ATPase, H(+)-pumping, and 14C-O-methyl-glucose uptake activities were specifically inhibited in the tonoplast fractions of mutant cell lines. Protein gel blotting confirmed that the expression of the A subunit was inhibited in the tonoplast fraction, but not in the Golgi fraction. Two-dimensional protein gel blots of total microsomes of wild-type and control transformant cell lines revealed two major immunoreactive polypeptides in the acidic pI range. In contrast, highly purified tonoplast membranes contained only the less acidic polypeptide. Because the less acidic polypeptide was preferentially diminished in the two antisense cell lines, we infer that the antisense constructs specifically blocked expression of a tonoplast-specific isoform of the V-ATPase A subunit in carrot. Regenerated plants containing the antisense constructs exhibited altered leaf morphologies and reduced cell expansion. The altered phenotype was correlated with the presence of the antisense construct.