Studies on cystic fibrosis using isoelectric focusing. IV. Distinction between ciliary dyskinesia activity in cystic fibrosis and asthmatic sera and association of cystic fibrosis protein with the activity in cystic fibrosis serum
Wilson, G.B.; Fudenberg, H.H.
Pediatric Research 11(4): 317-324
ISSN/ISBN: 0031-3998 PMID: 846785 DOI: 10.1203/00006450-197704000-00011
The cystic fibrosis protein (CFP) and ciliary dyskinesia activities (CDA) in sera from CF homozygotes, heterozygote carriers, and individuals with bronchial asthma have been partially purified. Concurrently, the CDA's in sera from patients with cystic fibrosis (CF) or bronchial asthma were shown to be different substances by ion-exchange or gel permeation chromatographic procedures. Sephadex G-200 chromatography indicated that the CF-CDA eluted with a protein fraction of molecular weight (MW) 68,000-150,000 and that the asthma CDA was found in a protein fraction of MV greater than 150,000. The two activities could also be separated by DEAE-cellulose chromatography. Prior acidification of whole normal, CF homozygote, obligate heterozygote, or asthmatic sera to pH 3.7 using EDTA, followed by fractionation of Sephadex G-200 removed all the CDA's from fractions of highermolecular weight and shifted the activities to a protein fraction of MW 1,100-13,700. This procedure afforded a 200-fold purification of the CDA's in sera from patients with asthma or CF. EDTA treatment, however, also generated a CDA in previously nonreactive normal sera. Subsequent fractionation of the various active G-200 fractions on Bio-Gel P10 allowed for the separation of three separate activities (Bio-Gel Fractions I, II-IV, and V). Fraction I was shown to represent the activity in sera from patients with asthma and was determined to be C3a (MW 9,000). Fraction I was also found in normal, CF, and carrier sera and therefore is not a specific CDA. Fraction II-IV is thought to represent a CF-specific CDA (MW 5,000) since it could not be demonstrated in either normal or asthmatic sera but was found in sera of obligate heterozygotes. Fraction III-IV also did not react with antisera to human C3a. Fraction V was generated from all serum types upon acidification of the serum with EDTA and is thought to be a nonspecific CDA. Bio-Gel P10 filtration of Sephadex G-200 fractions provided 823-fold and 650-fold purification of the asthmatic and CF CDA's, respectively. Concurrent analysis of column fractions for CDA's by bioassay and for CFP by electrofocusing showed CFP only in fractions that contained the CF-CDA. Combined analyses employing acid disc gel electrophoresis, isoelectric focusing, and EDTA treatment of active CF-immunoglobulin (Ig) G and Sephadex G-200 column fractions, followed by Bio-Gel P10 chromatography, provided evidence that CFP was associated with the CF-CDA. It is unknown as yet whether CFP itself is responsible for the CF-CDA activity.