Showing papers in "Methods of Molecular Biology in 1999"

Protein identification and analysis tools in the ExPASy server

Abstract: Protein identification and analysis software performs a central role in the investigation of proteins from two-dimensional (2-D) gels and mass spectrometry. For protein identification, the user matches certain empirically acquired information against a protein database to define a protein as already known or as novel. For protein analysis, information in protein databases can be used to predict certain properties about a protein, which can be useful for its empirical investigation. The two processes are thus complementary. Although there are numerous programs available for those applications, we have developed a set of original tools with a few main goals in mind. Specifically, these are: 1. To utilize the extensive annotation available in the Swiss-Prot database wherever possible, in particular the position-specific annotation in the Swiss-Prot feature tables to take into account posttranslational modifications and protein processing. 2. To develop tools specifically, but not exclusively, applicable to proteins prepared by two dimensional gel electrophoresis and peptide mass fingerprinting experiments. 3. To make all tools available on the World-Wide Web (WWW), and freely usable by the scientific community. In this chapter we give details about protein identification and analysis software that is available through the ExPASy World Wide Web server.

Human plasma platelet-activating factor acetylhydrolase.

Abstract: Human plasma platelet-activating factor (PAF) acetylhydrolase hydrolyzes the sn-2 acetyl residue of PAF, but not phospholipids with long chain sn-2 residues. It is associated with low density lipoprotein (LDL) particles, and is the LDL-associated phospholipase A, activity that specifically degrades oxidatively damaged phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1989) J. Biol. Chem. 264, 5331-5334). To identify potential substrates, we synthesized phosphatidylcholines with sn-2 residues from two to nine carbon atoms long, and found the V/k ratio decreased as the sn-2 residue was lengthened: the C5 homolog was 50%, the C6 207’0, while the C9 homolog was only 2% as efficient as PAF. However, the presence of an W-oxo function radically affected hydrolysis: the half-life of the sn-2 9-aldehydic homolog was identical to that of PAF. We oxidized [2-arachidonoyl]phosphatidylcholine and isolated a number of more polar phosphatidylcholines. We treated these with phospholipase C, derivatized the resulting diglycerides for gas chromatographic/mass spectroscopic analysis, and found a number of diglycerides where the m/z ratio was consistent with a series of short to medium length sn-2 residues. We treated the polar phosphatidylcholines with acetylhydrolase and derivatized the products for analysis by gas chromatography/mass spectroscopy. The liberated residues were more polar than straight chain standards and had mlz ratios from 129 to 296, consistent with short to medium chain residues. Therefore, oxidation fragments the sn-2 residue of phospholipids, and the acetylhydrolase specifically degrades such oxidatively fragmented phospholipids.

Preparation of HeLa cell nuclear and cytosolic S100 extracts for in vitro splicing.

Abstract: Following the initial discovery of split genes in 1977, it took several years before in vitro systems were successfully developed to study the biochemistry of pre-mRNA splicing. The first systems relied on coupling of transcription and splicing in whole-cell extracts and were fairly inefficient, because of the different optima for these two reactions (1,2). It was later shown that these reactions could be uncoupled (3,4), but obtaining discrete pre-mRNAs in useful amounts remained an obstacle until in vitro transcription with bacteriophage RNA polymerases (3) was adopted for this purpose. Another useful development was a nuclear extract preparation procedure that was initially developed for in vitro transcription studies (5). No splicing was detected in this study. However, the same extract preparation procedure, in conjunction with pre-mRNAs transcribed from cloned genes by SP6 RNA polymerase, was used to define optimal conditions for in vitro splicing (6). This system results in relatively efficient and accurate splicing, and is now in wide use, with slight variations from laboratory to laboratory. Variations in extract preparation include primarily the use of slightly different buffers and salts for nuclear extraction or dialysis.

Mammalian in vitro splicing assays.

Abstract: Splicing reactions are typically carried out using nuclear extracts, S100 extracts complemented with SR proteins, or partially purified fractions derived from the crude extracts. The extract preparation procedures are described in Chapter 24. Extracts derived from HeLa cells are used most commonly (see Note 1). The pre-mRNA substrates are usually prepared by in vitro runoff transcription with a bacteriophage polymerase (see Chapter 1). The intermediates and products of splicing are most conveniently visualized by urea/polyacrylamide gel electrophoresis (urea-PAGE) and autoradiography, which requires the use of labeled pre-mRNA substrate. The protocols provided here are based on ref. 1, with modifications introduced in refs. 2,3, and have been routinely used in our laboratory for many years.

The Avidin–Biotin Complex (ABC) Method and Other Avidin–Biotin Binding Methods

Abstract: Immunoenzyme methods can be enhanced by the use of the high affinity molecules, avidin and biotin. The binding of avidin to biotin is almost irreversible. By labeling a detection enzyme such as horseradish peroxidase with biotin, and a secondary antibody (reactive against the antigen detecting primary antibody) with biotin as well, these two compounds can then be linked irreversibly with avidin. For this process, the biotinylated enzyme is complexed with avidin in solution and this avidin-biotin complex (ABC) is then introduced to the biotinylated secondary antibody, where it binds to primary antibody-antigen sites. Also, enzyme-labeled avidin molecules can be used to bind biotinylated secondary antibodies with greater resolution. Finally, biotinylated tyramide used in conjunction with peroxidase precipitates even greater amounts of biotin molecules for detection by enzyme-labeled avidin molecules.

Transcellular regulation of eicosanoid biosynthesis.

Abstract: Models for in vivo scenarios of transcellular biosynthesis provide invaluable information about the regulation of eicosanoid biosynthesis that is likely to occur during multicellular events in vivo. The experimental approach of studying eicosanoid generation during cell-cell interactions and receptor-mediated cell activation represents a significant advancement beyond initial observations of eicosanoid formation and bioaction in isolated cell types that were activated under less physiologically relevant conditions. The experimental models reviewed in this chapter should be viewed as specific examples or as approaches to the study of cell-cell interactions. These examples may serve as guidelines to investigate novel cell-cell scenarios (see Fig. 3) and advance the emerging area of transcellular biosynthesis of bioactive lipid mediators.