Neutral Endopeptidase Neprilysin Is Copurified with Na,K-ATPase from Rabbit Outer Medulla and Hydrolyzes Its α-Subunit

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M. A. Groubman¹, Y. V. Kamanina¹, I. Yu. Petrushanko², A. M. Rubtsov¹, and O. D. Lopina^1*

¹Department of Biochemistry, Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 939-3955; E-mail: od_lopina@mail.ru

²Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia

^* To whom correspondence should be addressed.

Received June 29, 2010

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Preparations of Na,K-ATPase from outer medulla of rabbit kidney purified in accordance with the method of P. L. Jorgensen were shown to contain as admixture a protease that moves with α-subunit (~100 kDa) as a single protein band during one-dimensional SDS-PAGE. The electro-elution of proteins of this band from polyacrylamide gel results in the appearance of two protein fragments (~67 and 55 kDa) that are stained with polyclonal antibodies against Na,K-ATPase α-subunit. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis showed that the neutral membrane-bound endopeptidase neprilysin is located in one protein band together with the Na,K-ATPase α-subunit. Addition of thiorphan, a specific inhibitor of neutral endopeptidase, eliminates proteolysis of the α-subunit. The data demonstrate that Na,K-ATPase α-subunit may be a natural target for neprilysin.

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KEY WORDS: Na,K-ATPase α-subunit, neprilysin, proteolysis, mammalian kidney

DOI: 10.1134/S000629791010010X

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