LOCATION OF GANGLIOSIDE GM2 ACTIVATOR PROTEIN GENE-EXPRESSION IN SHEEP

1. The present study was aimed at characterizing and establishing the site of production of a 'novel' protein isolated in 1988 during the co urse of studies on sheep renal morphology, This protein has subsequent ly been identified as the GM2 activator protein (GM2AP). 2. The 'novel ' protein, with an apparent molecular weight of 18-22kDa and a pi betw een 4.7 and 4.9, was isolated from enriched granular fractions of shee p kidney cortex using two-dimensional (2-D) polyacrylamide gel electro phoresis, Following electroelution, the N-terminal amino add sequence was determined and, applying the preferred codon usage formula, an oli godeoxyribonucleotide probe was constructed for examination of sites o f expression of this novel protein using northern analyses and hybridi zation histochemistry, 3. Western blots of the 2-D gels onto nitrocell ulose membranes permitted us to select the appropriate spots for injec tion into rabbits for production of polyclonal antibodies. The antibod ies were used to confirm the sites of protein production using immunoh istochemistry. 4. Northern analyses revealed that GM2AP mRNA has a wid espread distribution in ovine tissues. In the kidney, GM2 was expresse d in all major renal arteries and arterioles, In the liver, the expres sion of the gene was prominent in the hepatic vein and duets. Antibodi es raised against the GM2AP confirmed that the protein was present at the same sites as the mRNA, 5. These are the first studies showing the location of GM2 activator gene expression in normal mammalian tissues . The arterial site of production has implications for local action or an important role in membrane integrity throughout the kidney.