Secondary structure of the Neurospora crassa plasma membrane H+-ATPase as estimated by circular dichroism.

In a previous communication, a water-soluble, hexameric form of the Neurospora crassa plasma membrane H+-ATPase was described (Chadwick, C. C., Goormaghtigh, E., and Scarborough, G. A. (1987) Arch. Biochem. Biophys. 252, 348-356). To facilitate physical studies of the hexamers, the H+-ATPase isolation procedure has been improved, resulting in a structurally and functionally stable hexamer preparation that contains only 5 to 10% non-ATPase protein, approximately 12 mol of enzyme-bound lysophosphatidylcholine/mol of H+-ATPase monomer, and little or no residual plasma membrane phospholipid. Importantly, when activated by lysophosphatidylglycerol, which satisfies the acidic phospholipid requirement of the enzyme, the hexameric quaternary structure of the enzyme is retained, indicating that the functional properties of the water-soluble hexamers are relevant to those of the native, membrane-bound enzyme. The circular dichroism (CD) spectrum of this H+-ATPase preparation has been measured from 184 to 260 nm and used to estimate the secondary structure of the enzyme. The H+-ATPase is estimated to consist of approximately 36% helix, 12% antiparallel beta-sheet, 8% parallel beta-sheet, 11% beta-turn, and 26% other (irregular) structure. There is no change in the CD spectrum when known enzyme ligands are added to the H+-ATPase solution, suggesting that any changes in secondary structure that might occur during ligand binding and/or catalytic cycling are either minor or result in compensatory changes in secondary structure. The CD spectrum of the H+-ATPase is also compared to published spectra of the animal cell Na+/K+- and Ca2+-ATPases and is shown to be quite similar in shape and intensity, suggesting that all of these ATPases, which have significant sequence homology and are mechanistically similar, may have similar secondary structure composition as well.