Three specific antipeptide antibodies and oligonucleotide probes synthesized to internal sequences of parafusin have been used to search for mammalian counterpart(s) of this protein. Parafusin is an exocytic-sensitive phosphoglycoprotein from a unicellular eukaryote Paramecium that was recently cloned and sequenced. Western and Southern blot analyses, polymerase chain reaction (PCR) and reverse transcriptase coupled PCR (RT-PCR) techniques have been used to examine rat liver and pancreas, human pancreas and a murine pancreatic β-cell line (βTC3) arising in transgenic mice. The parafusin-specific antibodies showed cross-reaction with a protein at ~ 63 kDa in 4 tissues, whereas a phosphoglucomutase-specific antibody also detected a second band of similar molecular weight in the βTC3 cells. The presence of two bands shows that parafusin homologue(s) and phosphoglucomutase are separate entities. βTC3 cells were shown to incorporate [β35]UDPGlc into the parafusin homologue in a Ca++ sensitive manner characterisitic of parafusin. Southern blot analysis revealed that the parafusin-specific probe hybridized with restriction enzyme digests of rat DNA in distinct patterns different from those observed with a phosphoglucomutase-specific probe. Rat genomic DNA and mRNA from the βTC3 cells were used as the templates for PCR and RT-PCR using internal parafusin primers. In both cases similarly sized products were obtained which hybridized in Southern analysis with a specific parafusin probe located within the amplified region. These results indicate that a parafusin homologue exists in mammalian cells.
|Original language||English (US)|
|Number of pages||8|
|Journal||European Journal of Cell Biology|
|State||Published - 1995|
ASJC Scopus subject areas
- Pathology and Forensic Medicine
- Cell Biology