MAMMALIAN HOMOLOG OF THE CALCIUM-SENSITIVE PHOSPHOGLYCOPROTEIN, PARAFUSIN

Three specific antipeptide antibodies and oligonucleotide probes synth esized to internal sequences of parafusin have been used to search for mammalian counterpart(s) of this protein. Parafusin is an exocytic-se nsitive phosphoglycoprotein from a unicellular eukaryote Paramecium th at was recently cloned and sequenced (Subramanian et al., Proc. Natl. Acad. Sci, USA 91, 9832 9836 (1994)). Western and Southern blot analys es, polymerase chain reaction (PCR) and reverse transcriptase coupled PCR (RT-PCR) techniques have been used to examine rat liver and pancre as, human pancreas and a murine pancreatic beta-cell line (beta TC3) a rising in transgenic mice. The parafusin-specific antibodies showed cr oss-reaction with a protein at similar to 63 kDa in 4 tissues, whereas a phosphoglucomutase-specific antibody also detected a second band of similar molecular weight in the beta TC3 cells. The presence of two b ands shows that parafusin homologue(s) and phosphoglucomutase are sepa rate entities. beta TC3 cells were shown to incorporate [beta(35)]UDPG lc into the parafusin homologue in a Ca++-sensitive manner characteris itic of parafusin. Southern blot analysis revealed that the parafusin- specific probe hybridized with restriction enzyme digests of rat DNA i n distinct patterns different from those observed with a phosphoglucom utase-specific probe. Rat genomic DNA and mRNA from the beta TC3 cells were used as the templates for PCR and RT-PGR using internal parafusi n primers. In both cases similarly sized products were obtained which hybridized in Southern analysis with a specific parafusin probe locate d within the amplified region. These results indicate that a parafusin homologue exists in mammalian cells.