BIOCHEMICAL ASSESSMENT OF CILIARY ACTIVITY IN THE PRESENCE OF CYSTIC FIBROSIS (CF) SERUM

Abstract

Since Spock, et al. (Pediat. Res. 1:173, 1967) first observed that serum from CF patients and heterozygotes induced ciliary dysrhythmia, several “CF factor” bioassays have been described. Such methods, however, suffer from several disadvantages in that they are: a) subjective, b) qualitative, and c) difficult to reproduce. We ourselves had consistently negative experiences with both rabbit tracheal (RT) and oyster gill (OG) assays. In an effort to develop a quantitative chemical assay. we have tried a fresh approach by measuring motility-coupled ciliary ATP hydrolysis in the presence of serum. In addition, to characterize the ATPase activity several basic studies were performed with OG and RT cilia. Homogeneous, motile cilia were prepared from the former by mincing in 0.05 M Tris HCl (pH 8.0), 0.3 mM EDTA; this preparation, which on electron microscopy showed intact cilia, hydrolyzed ATP in proportion to calculated beat frequency. A high molecular wt. ATPase was purified from RT cilia and shown to have properties similar to flagellar dynein. The effect of various factors (time, temp., pH, cations, and substrate concentration) on ciliary ATP hydrolysis was determined. Explants, homogenates, and minces of RT and OG were preincubated for 30-45 min with either normal, CF,or CF heterozygote sera; subsequent addition of ATP-γ-³²P and measurement of ³²Pi production revealed that the latter two groups could not be distinguished from normal serum. Studies are continuing but this objective, quantitative assay has thus far failed to demonstrate the “CF factor.”

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