A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry.

The growing availability of genomic sequence information, together with improvements in analytical methodology, have enabled high throughput, high sensitivity protein identification. Silver staining remains the most sensitive method for visualization of proteins separated by two-dimensional gel electrophoresis (2-D PAGE). Several silver staining protocols have been developed which offer improved compatibility with subsequent mass spectrometric analysis. We describe a modified silver staining method that is available as a commercial kit (Silver Stain PlusOne; Amersham Pharmacia Biotech, Amersham, UK). The 2-D patterns abtained with this modified protocol are comparable to those from other silver staining methods. Omitting the sensitizing reagent allows higher loading without saturation, which facilitates protein identification and quantitation. We show that tryptic digests of proteins visualized by the modified stain afford excellent mass spectra by both matrix-assisted laser desorption/ionization and tandem electrospray ionization. We conclude that the modified silver staining protocol is highly compatible with subsequent mass spectrometric analysis.

The pacL gene of Synechococcus sp. strain PCC 7942 encodes a Ca(2+)-transporting ATPase.

An ATP-dependent Ca2+ uptake activity was identified in plasma membrane vesicles prepared from Synechococcus sp. strain PCC 7942. This activity was insensitive to agents which collapse pH gradients and membrane potentials but sensitive to vanadate, indicating that the activity is catalyzed by a P-type Ca(2+)-ATPase. A gene was cloned from Synechococcus sp. strain PCC 7942 by using a degenerate oligonucleotide based on a sequence conserved among P-type ATPases. This gene (pacL) encodes a product similar in structure to eukaryotic Ca(2+)-ATPases. We have shown that pacL encodes a Ca(2+)-ATPase by demonstrating that a strain in which pacL is disrupted has no Ca(2+)-ATPase activity associated with its plasma membrane. In addition, Ca(2+)-ATPase activity was restored to the delta pacL strain by introducing pacL into a second site in the Synechococcus sp. strain PCC 7942 chromosome.

Characterization of a gene encoding a Ca(2+)-ATPase-like protein in the plastid envelope.

By screening an Arabidopsis expression library with an antiserum against chloroplast envelope proteins, we have isolated a partial cDNA with an open reading frame that encodes a polypeptide similar to P-type cation-transporting ATPases. The corresponding genomic clone was isolated and the complete coding sequence was deduced after identification and mapping of introns. The gene has been designated PEA1 (plastid envelope ATPase) and the predicted polypeptide PEA1p. PEA1p has 946 amino acids and a molecular mass of 104 kDa. This protein is 40-44% identical to various mammalian plasma membrane Ca(2+)-ATPases but lacks the C-terminal calmodulin binding domain present in the mammalian polypeptides. When aligned with mammalian plasma membrane Ca(2+)-ATPases, PEA1p has a 70- to 80-amino acid N-terminal region that extends beyond the N terminus of these enzymes. This extension has some similarity to the transit peptide of the plastid envelope phosphate translocator and may function to target the protein to the plastid. Antibodies raised against a portion of PEA1p recognize a single 90- to 95-kDa polypeptide in chloroplast inner envelope preparations. Transcript abundance as determined by RNase protection was found to be 7- to 9-fold higher in roots than in leaves. Possible roles for a plastid envelope calcium pump are suggested.

Calcium Transport in the Green Alga Mesotaenium caldariorum: Preliminary Characterization and Subcellular Distribution.

The subcellular localization and biochemical characterization of calcium transport were studied in the unicellular green alga Mesotaenium caldariorum. Membrane fractions prepared by osmotic lysis of Mesotaenium protoplasts exhibit high rates of ATP-dependent calcium uptake. Sucrose gradient centrifugation separates two pools of activity, which display specific activities for calcium transport as high as 15 nanomoles Ca(2+) per minute per milligram of protein. Marker enzyme analysis shows that this dual distribution of calcium transport activity is similar to that of vanadate-insensitive ATPase and pyrophosphatase, activities considered to be associated with the tonoplast. Plasma membranes, endoplasmic reticulum vesicles, mitochondrial membranes, and thylakoids band at higher densities than either calcium transport fraction. Both pools of ATP-dependent calcium uptake contain two components which are not separable on sucrose gradients but can be distinguished on the basis of inhibitor sensitivity. One component is inhibited by nigericin or trimethyltin chloride (I(50) values of 3 nanomolar and 4 micromolar, respectively), while the other component is vanadate sensitive (I(50) of 25 micromolar). These results suggest that direct Ca(2+) transport and Ca(2+)/H(+) antiport activities are present in both sucrose gradient fractions.

Visualization of bilin-linked peptides and proteins in polyacrylamide gels.

Biliproteins and bilipeptides subjected to discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of zinc acetate form a complex which fluoresces an orange color when viewed under ultraviolet light. The complex between the bilin chromophore and the zinc ion fluoresces at wavelengths which can be selectively visualized in gels by using a red filter. For the biliproteins phytochrome and C-phycocyanin the minimum detectable quantities are 100 and 50 ng, respectively. This is comparable to the sensitivity of Coomassie blue staining. The technique has been used for selective detection of phytochrome in plant extracts and to distinguish chromophore-bearing peptides from those not containing chromophore in proteolytic digests of phytochrome.

Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria.

We constructed the broad-host-range plasmid pUCD800 containing the sacB gene of Bacillus subtilis for use in the positive selection and isolation of insertion sequence (IS) elements in gram-negative bacteria. Cells containing pUCD800 do not grow on medium containing 5% sucrose unless the sacB gene is inactivated. By using pUCD800, we isolated a 1.4-kilobase putative IS element from Agrobacterium tumefaciens NT1RE by selection for growth on sucrose medium. This putative IS element appears to be unique to Agrobacterium strains.