Biochemical analysis of membrane proteins from an early maturation stage of phagosomes.
We used an improved technique for pulse-chase labeling of phagosomes using custom-made magnetic microparticles. With the help of a permanent magnet we purified both newly formed, nascent and early matured (i.e., 5-min-old) condensed phagosomes in high amounts. The protein patterns of membrane proteins of newly formed phagosomes and 5-min-old condensed ones were compared by two-dimensional (2-D) electrophoresis. The protein patterns allowed the detection of protein spots that changed in abundance between these two stages. Three protein spots abundant in condensed phagosomes only and one spot well-stained in both stages were collected from ten preparative Coomassie brilliant blue-stained 2-D gels. Following microdigestion, selected purified oligopeptides were sequenced by Edman degradation. While the oligopeptide sequences of proteins from two spots showed high homology to an already sequenced 25 kDa calcium binding protein, the other two showed no significant homology to protein sequences available in sequence databases. Presently polymerase chain reaction (PCR) and cloning experiments are set up to reveal the cDNAs of these proteins in order to study their function by knock-out and gene replacement experiments.
Maicher
M T
MT
Institute for General Zoology and Genetics, University of Münster, Germany.
Tiedtke
A
A
D000595:Amino Acid Sequence; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis*; D008969:Molecular Sequence Data; D010588:Phagosomes / Q000737:chemistry*; D015800:Protozoan Proteins / Q000032:analysis*; D016808:Tetrahymena thermophila / Q000737:chemistry*
D008565:Membrane Proteins; D015800:Protozoan Proteins