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 course of studies on sheep renal morphology. This protein has subsequently been identified as the GM2 activator protein (GM2AP). 2. The 'novel' protein, with an apparent molecular weight of 18-22 kDa and a pI between 4.7 and 4.9, was isolated from enriched granular fractions of sheep kidney cortex using two-dimensional (2-D) polyacrylamide gel electrophoresis. Following electroelution, the N-terminal amino acid sequence was determined and, applying the preferred codon usage formula, an oligodeoxyribonucleotide probe was constructed for examination of sites of expression of this novel protein using northern analyses and hybridization histochemistry. 3. Western blots of the 2-D gels onto nitrocellulose membranes permitted us to select the appropriate spots for injection into rabbits for production of polyclonal antibodies. The antibodies were used to confirm the sites of protein production using immunohistochemistry. 4. Northern analyses revealed that GM2AP mRNA has a widespread distribution in ovine tissues. In the kidney, GM2 was expressed in all major renal arteries and arterioles. In the liver, the expression of the gene was prominent in the hepatic vein and ducts. Antibodies 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.
Butkus
A
A
Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria, Australia.
Coghlan
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015180:Electrophoresis, Gel, Two-Dimensional; D049289:G(M2) Activator Protein; D005678:G(M2) Ganglioside / Q000235:genetics; D007668:Kidney / Q000098:blood supply / Q000378:metabolism; D008099:Liver / Q000098:blood supply / Q000378:metabolism; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D012756:Sheep / Q000235:genetics*
D049289:G(M2) Activator Protein; D011506:Proteins; D012333:RNA, Messenger; D005678:G(M2) Ganglioside