Blanco G, Melton R J, Sánchez G, Mercer R W
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biochemistry. 1999 Oct 12;38(41):13661-9. doi: 10.1021/bi991207b.
Different isoforms of the sodium/potassium adenosinetriphosphatase (Na,K-ATPase) alpha and beta subunits have been identified in mammals. The association of the various alpha and beta polypeptides results in distinct Na,K-ATPase isozymes with unique enzymatic properties. We studied the function of the Na,K-ATPase alpha4 isoform in Sf-9 cells using recombinant baculoviruses. When alpha4 and the Na pump beta1 subunit are coexpressed in the cells, Na, K-ATPase activity is induced. This activity is reflected by a ouabain-sensitive hydrolysis of ATP, by a Na(+)-dependent, K(+)-sensitive, and ouabain-inhibitable phosphorylation from ATP, and by the ouabain-inhibitable transport of K(+). Furthermore, the activity of alpha4 is inhibited by the P-type ATPase blocker vanadate but not by compounds that inhibit the sarcoplasmic reticulum Ca-ATPase or the gastric H,K-ATPase. The Na,K-ATPase alpha4 isoform is specifically expressed in the testis of the rat. The gonad also expresses the beta1 and beta3 subunits. In insect cells, the alpha4 polypeptide is able to form active complexes with either of these subunits. Characterization of the enzymatic properties of the alpha4beta1 and alpha4beta3 isozymes indicates that both Na,K-ATPases have similar kinetics to Na(+), K(+), ATP, and ouabain. The enzymatic properties of alpha4beta1 and alpha4beta3 are, however, distinct from the other Na pump isozymes. A Na, K-ATPase activity with similar properties as the alpha4-containing enzymes was found in rat testis. This Na,K-ATPase activity represents approximately 55% of the total enzyme of the gonad. These results show that the alpha4 polypeptide is a functional isoform of the Na,K-ATPase both in vitro and in the native tissue.
在哺乳动物中已鉴定出钠/钾腺苷三磷酸酶（Na，K - ATPase）α和β亚基的不同同工型。各种α和β多肽的组合产生具有独特酶特性的不同Na，K - ATPase同工酶。我们使用重组杆状病毒研究了Na，K - ATPaseα4同工型在Sf - 9细胞中的功能。当α4和钠泵β1亚基在细胞中共表达时，会诱导Na，K - ATPase活性。这种活性表现为哇巴因敏感的ATP水解、由ATP进行的Na（+）依赖性、K（+）敏感且哇巴因可抑制的磷酸化，以及哇巴因可抑制的K（+）转运。此外，α4的活性受到P型ATP酶阻滞剂钒酸盐的抑制，但不受抑制肌浆网Ca - ATP酶或胃H，K - ATP酶的化合物的抑制。Na，K - ATPaseα4同工型在大鼠睾丸中特异性表达。性腺也表达β1和β3亚基。在昆虫细胞中，α4多肽能够与这些亚基中的任何一个形成活性复合物。α4β1和α4β3同工酶的酶特性表征表明，两种Na，K - ATPase对Na（+）、K（+）、ATP和哇巴因具有相似的动力学。然而，α4β1和α4β3的酶特性与其他钠泵同工酶不同。在大鼠睾丸中发现了一种具有与含α4的酶相似特性的Na，K - ATPase活性。这种Na，K - ATPase活性约占性腺总酶的55％。这些结果表明，α4多肽在体外和天然组织中都是Na，K - ATPase的功能同工型。
