The cerebroside sulfate activator from pig kidney: purification and molecular structure

The activator protein for hydrolysis of cerebroside sulfate by arylsulfatase A was purified from pig kidney in high yield. This protein, also known as sphingolipid activator protein-1 and saposin-B, was particularly rich in pig kidney. Purification was achieved by a simple procedure involving homogenation and heat treatment followed by affinity, ion exchange, and gel filtration chromatographies. The final product was better than 90% pure by gel electrophoresis and HPLC. It was possible to sequence more than 60 amino acids from the N-terminus with only a few uncertain residues. The sequence differed from that predicted for the human protein by about 10%, with most amino acid variations being conservative. There appeared to be a residual glycosyl substituent on asparagine 21, but the sugar content was low and the protein failed to bind to concanavalin A. The cerebroside sulfate activator proved to be exceptionally resistant to denaturation or protease digestion. The apparent molecular mass was approximately 20,000 Da on preparative gel-filtration columns, but was variable when estimated by HPLC gel filtration. Values ranging from 30,000 to over 100,000 Da were observed in neutral buffers, while values around 15,000-16,000 Da were seen in acidic buffers such as those used for assay of the biological activity. This was further decreased to a putative subunit of 7000-8000 Da under severe denaturing conditions. Pig kidney is a convenient source for the large-scale preparation of this interesting protein which has heretofore been obtained from human sources.