Commercially obtained cystine binding protein (CBP) of E. coli has been purified by HPLC, yielding a pure cystine binding protein not similar to any protein in the data banks. This protein has been partially sequenced, an N-terminal peptide made and an antibody to this peptide obtained which reacts with the whole protein. The commercial preparation of CBP also contains a histidine binding protein which can be used to assay for histidine in the same way that CBP is used to assay tissues for cystine. CBP of E. coli is being cloned by two methods. One, by using an E. coli expression library and identifying the correct CBP producing colony with our antibody to CBP. Two, by making oligonucleotides of sections of the known N-terminal sequence of CBP and using them as primers to identify CBP in the E. coli library. By this later method, we have obtained a partial sequence of the DNA of CBP and are are continuing the work to obtain the complete sequence. This DNA sequence will then be cloned in a suitable bacterium for large production of cystine binding protein. With this larger amount of material, the protein will be studied as to its metabolic function, specifically its cystine binding capability. Niemann-Pick C disease (NPC) was shown to be reversibly induced in normal fibroblasts by hydrophobic amines and certain steroids, thus creating a model system for study of this disease. We are identifying the trafficking patterns and metabolism of intracellular cholesterol using various inhibitors and stimulators. We have shown that normal and cystinotic fibroblasts take up ascorbic acid by two mechanisms, a high affinity Na+ dependent transporter and a low affinity Na+ independent transporter, neither of which can be accounted for by diffusion. Transport of ascorbic acid by the low affinity transporter is not different from normal in the two types of cells, however transport in cystinotic cells by the high affinity transporter is about 40% of that seen in normal cells. The Km's for both transporters were the same for both types of cells while the Vmax of the high affinity transporter was statistically lower than normal for cystinotic fibroblasts. This data indicates that cystinotic fibroblasts have a selective impairment in ascorbate transport.
