ISOLATION AND CHARACTERIZATION OF 26-KDA AND 30-KDA RAT-LIVER PROTEINS IMMUNOREACTIVE TO ANTI-STEROL CARRIER PROTEIN-2 ANTIBODIES

Although the existing literature suggests that the sterol carrier prot ein-2 (SCP-S) gene has only two initiation sites encoding for a 58- an d a 15-kDa protein, respectively, this does not explain the profusion of other putative SCP-2-related proteins detectable on Western blottin g. Two of these additional anti-SCP-a immunoreactive proteins, 13.2 an d 46 kDa, appear due to proteolytic processing of the two gene transcr ipts. However, the origin of additional immunoreactive rat liver prote ins near 26 and 30 kDa is unclear. The latter proteins were consistent ly detected on Western blotting by three independent types of polyclon al antisera: anti-13.2-kDa SCP-2, anti-synthetic peptide from the amin o-terminus of the 13.2-kDa SCP-2, and Protein A affinity-purified anti -synthetic peptide to the amino-terminus of 13.2-kDa SCP-2. To resolve whether the 26- and 30-kDa proteins are SCP-2 gene products, each pro tein was isolated from rat liver and purified to homogeneity as indica ted by Tricine-SDS polyacrylamide gel electrophoresis, isoelectric foc using, and/or mass spectroscopy. Their masses, determined by MALDI-TOF mass spectroscopy, were 25.7 and 29.8 kDa, respectively. However, the mass spectral data were not consistent with either protein being an S CP-2 gene product. Peptide mass mapping of the 25.7-kDa protein reveal ed identity to the rat 25,784.79-Da glutathione-S-transferase. Further more, neither the mass nor the amino acid composition of the 29.8-kDa protein correlated with any SCP-S gene product or dimerized SCP-2 gene product. A database search of the amino acid composition identified t he protein as rat carbonic anhydrase. In summary, although the 26- and 29.8-kDa proteins may share some common epitopes with the 13.2-kDa SC P-2, they were not SCP-2 gene products. (C) 1998 Academic Press.