E. Wyroba et al., MAMMALIAN HOMOLOG OF THE CALCIUM-SENSITIVE PHOSPHOGLYCOPROTEIN, PARAFUSIN, European journal of cell biology, 68(4), 1995, pp. 419-426
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.