ABSTRACT
The conditioned medium of the murine macrophage PU5.1.8 was analyzed by two-dimensional gel electrophoresis in order to detect LPS-induced proteins. Spots of interest were identified by microsequencing of internal peptides generated by limited in situ acid hydrolysis. In total conditioned medium, several monokines (TNF-alpha and macrophage inflammatory protein-1 alpha and 1 beta) were identified as LPS-induced spots. Because minor spots could be masked by the complexity of the 2-D pattern, conditioned medium was successively fractionated by zinc precipitation and affinity chromatography (Procion red and Con A agarose). Zinc supernatant fraction, Procion red flow-through, and Con A eluate fractions were further analyzed by 2-D gel electrophoresis for the presence of LPS-induced spots. In these fractions serum amyloid A3, lipocalin 24p3, cathepsin B, and plasminogen activator inhibitor-I were characterized as LPS-induced proteins secreted by macrophages. Lipocalin 24p3 protein was retrieved for the first time. In addition to these proteins that follow a classical secretory pathway, several cellular proteins (mainly ribosomal proteins) were retrieved as LPS-induced proteins in the conditioned medium. Control experiments argue against the obvious explanation that the latter observation is caused solely by cellular leakage.
Subject(s)
Carrier Proteins/metabolism , Lipopolysaccharides/pharmacology , Macrophage Activation , Macrophages/metabolism , Serum Amyloid A Protein/metabolism , Amino Acid Sequence , Animals , Base Sequence , Carrier Proteins/chemistry , Cell Line , Electrophoresis, Gel, Two-Dimensional , Gene Expression , In Vitro Techniques , Macrophages/chemistry , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , Peptide Fragments/chemistry , RNA, Messenger/genetics , Serum Amyloid A Protein/chemistryABSTRACT
A method is described for the isolation of peptide fragments from proteins separated by polyacrylamide gel electrophoresis. After completion of the electrophoresis step, gels are stained with Ponceau S or Coomassie Blue. Gel portions containing protein stained with Ponceau S are excised and transferred to borosilicate glass digestion tubes containing 0.9 ml of 1 mM NaOH or 5 mM Na2HPO4. After complete dissociation of the dye from the protein, 0.1 ml of 20% formic acid is added and the protein is hydrolyzed in situ at 112 degrees C for four hours. Subsequently the acid solution is made 10% in acetonitrile and chromatographed as such on a C18 (C4) reversed-phase column using an appropriate large-volume sample loading syringe and injection loop. Proteins stained with Coomassie Blue can be hydrolyzed in situ after complete removal of the dye with an aqueous solution containing 40% acetone, 10% triethylamine and 5% acetic acid. The gel slices are next washed with HPLC-grade water and protein is hydrolyzed in 2% formic acid under standard conditions. Gel-related contaminants do not interfere with the peptide separation under the proper conditions of HPLC analysis.
Subject(s)
Peptide Fragments/chemistry , Peptide Mapping/methods , Amino Acid Sequence , Apoproteins/chemistry , Azo Compounds , Carbonic Anhydrases/chemistry , Chromatography, High Pressure Liquid , Cytochrome c Group/chemistry , Cytochromes c , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Hydrolysis , Molecular Sequence Data , Proteins/isolation & purification , Rosaniline Dyes , Transferrin/chemistryABSTRACT
The nematode worm Caenorhabditis elegans is known to undergo characteristic morphological as well as physiological signs of senescence. Two-dimensional gel electrophoresis shows that alterations also occur in the pattern of the nuclear proteins as a function of age. Non-histone proteins whose level exhibits a steep fall with age are egg-specific and not involved in senescence. However, a distinct set of non-histones accumulates with age and can be considered as senescence markers. Some of these are glycoproteins, as shown by their concanavalin A-binding properties. One age-specific polypeptide, called 'protein S-28', was further characterized by peptide mapping and determination of its N-terminal amino acid sequence.
