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.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002845:Chromatography / Q000379:methods; D002923:Cilia / Q000201:enzymology*; D003550:Cystic Fibrosis / Q000097:blood / Q000201:enzymology*; D004573:Electrolytes / Q000494:pharmacology; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology*; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D010125:Ostreidae / Q000032:analysis; D011817:Rabbits
D004573:Electrolytes; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases