Continuation of a project is proposed to isolate, characterize and reverse the action of the primary gene product in the inherited disease, cystic fibrosis. Isolation and characterization of the Cystic Fibrosis Mucociliary Inhibitor (CFMI) will be carried out through conventional protein isolation methods including isoelectric focusing, affinity chromatography, gel filtration and ion exchange chromatography. Ultimately, the primary structure analysis will be completed on the CFMI and its normal counterpart. Preparative steps will be monitored by the oyster gill ciliary assay which is capable of detecting the CFMI in relative concentrations, which have been established for each step in the preliminary purification procedures. Indication that the CFMI is specific for cystic fibrosis genotypes is provided by the fact that preparations from heterozygotes contain approximately one-half (0.4 to 0.7) of the CFMI concentration found in preparations of homozygotes. Analogous fractions from non-CF individuals failed to inhibit mucociliary activity, even when tested in concentrations from 2 to almost 100 fold greater. Sera of individuals with bronchial asthma, chronic rhinitis and pancreatic insufficiency do not inhibit mucociliary activity. Progress in purification methods is evidenced by a 38,000 fold purification of the serum CFMI to two isoelectrically focused zones, pI 7.5-8.0 and pI 8.9-10.4, which inhibit mucociliary activity. The urinary CFMI has been purified 370 fold to a single isoelectrically focused peak, at pI 6.0-7.2. Analogous fractions from non-CF (normal) urine and sera do not inhibit mucociliary activity. When purified to homogeneity the CFMI will serve as an antigen to prepared monospecific antibodies. The development of an immunological or biochemical test for a factor specific for cystic fibrosis will significantly aid in genetic counseling and prenatal diagnosis. The aims of this project are to: (1) purify the CFMI, (2) characterize the CFMI biochemically, (3) characterize the normal counterpart of the CFMI, and (4) detect the malfunction exerted by the CFMI, and (5) develop a test routinely capable of quantitating a specific factor in cystic fibrosis genotypes.
NIH ExPorter
NIH ExPorter