Determination and characterization of ciliary ATPase in the presence of serum from cystic fibrosis patients.

The purpose of this investigation was twofold: (1) to identify and characterize 
the enzymatic ATP hydrolysis system of epithelial cilia, and (2) to 
develop a quantitative, biochemical test for the ciliotoxic cystic fibrosis (CF) 
factor based on inhibition of ATP utilization by ciliary preparations. Our 
rationale for selecting this system for CF factor analysis relates to 
the tight and essential mechanochemical coupling of functioning cilia. Using rabbit 
tracheal epithelium as the source, a high molecular weight (greater than 
200,000) ATPase was identified, partially purified, and extensively characterized. The properties 
of this protein were similar to those observed in previous studies 
of others with flagellar and ciliary dynein (the motility-associated ATPase) isolated 
from microorganisms. Analysis of the pH profile revealed a broad range 
of high enzymatic activity between 6.5 and 9. Studies with potential 
cation activators showed that the enzyme is activated equally by either 
Ca2+ or Mg2+ in equimolar concentrations. No activation occurred in the 
presence of Zn2+, Na+, H+, or Na+ plus K+ and the 
effect of Mg2+ or Ca2+ was not inhibited by Na+, K+, 
or Na+ plus K+. The enzyme hydrolyzed Mg2+- containing solutions of 
UTP, CTP, and ADP at 51-54% the rate of ATP dephosphorylation, 
whereas Mg-deoxy-ATP was hydrolyzed 79% as effectively as ATP. Using a 
newly devised, analytical technique with [gamma-32P]ATP as the substrate, the ATP 
hydrolysis of various ciliary preparations from rabbit trachea and oyster gill 
(including motile suspensions) was monitored in the presence of sera from 
CF homo- and heterozygotes. Reproducible rates of ATP dephosphorylation averaging 27 
nmol/min/mg protein were demonstrable with homogenates of ciliated epithelium. None of 
the test systems evaluated, however, were capable of demonstrating CF-related differences 
in ATPase activity or ATP utilization. Although these attemps have been 
unsuccessful thus far, the approach described in this report provides an 
example of an objective, quantitative, biochemical assessment of ciliary function. 