![Kinetics behaviors of Na,K-ATPase: Comparison of solubilized and DPPC:DPPE-liposome reconstituted enzyme [An article from: Comparative Biochemistry and Physiology, Part C]](https://www.ebooknetworking.net/books/B00/0RR/bigB000RR6JKQ.jpg)
Kinetics behaviors of Na,K-ATPase: Comparison of solubilized and DPPC:DPPE-liposome reconstituted enzyme [An article from: Comparative Biochemistry and Physiology, Part C]
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This digital document is a journal article from Comparative Biochemistry and Physiology, Part C, published by Elsevier in . The article is delivered in HTML format and is available in your Amazon.com Media Library immediately after purchase. You can view it with any web browser.
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We describe and compare the main kinetic characteristics of rabbit kidney Na,K-ATPase incorporated inside-out in DPPC:DPPE-liposomes with the C"1"2E"8 solubilized and purified form. In proteoliposomes, we observed that the ATP hydrolysis of the enzyme is favored and also its affinity for Na^+-binding sites increases, keeping the negative cooperativity with two classes of hydrolysis sites: one of high affinity (K"0"."5=6 @mM and 4 @mM for reconstituted enzyme and purified form, respectively) and another of low affinity (K"0"."5=0.4 mM and 1.4 mM for reconstituted enzyme and purified form, respectively). Our data showed a biphasic curve for ATP hydrolysis, suggesting the presence of (@a@b)"2 oligomer in reconstituted Na,K-ATPase similar to the solubilized enzyme. The Mg^2^+ concentration dependence in the proteoliposomes stimulated the Na,K-ATPase activity up to 476 U/mg with a K"0"."5 value of 0.4 mM. The Na^+ ions also presented a single saturation curve with V"M=551 U/mg and K"0"."5=0.2 mM with cooperative effects. The activity was also stimulated by K^+ ions through a single curve of saturation sites (K"0"."5=2.8 mM), with cooperative effects and V"M=641 U/mg. The lipid microenvironment close to the proteic structure and the K^+ internal to the liposome has a key role in enzyme regulation, affecting its kinetic parameters while it can also modulate the enzyme's affinity for substrate and ions.
Description:
We describe and compare the main kinetic characteristics of rabbit kidney Na,K-ATPase incorporated inside-out in DPPC:DPPE-liposomes with the C"1"2E"8 solubilized and purified form. In proteoliposomes, we observed that the ATP hydrolysis of the enzyme is favored and also its affinity for Na^+-binding sites increases, keeping the negative cooperativity with two classes of hydrolysis sites: one of high affinity (K"0"."5=6 @mM and 4 @mM for reconstituted enzyme and purified form, respectively) and another of low affinity (K"0"."5=0.4 mM and 1.4 mM for reconstituted enzyme and purified form, respectively). Our data showed a biphasic curve for ATP hydrolysis, suggesting the presence of (@a@b)"2 oligomer in reconstituted Na,K-ATPase similar to the solubilized enzyme. The Mg^2^+ concentration dependence in the proteoliposomes stimulated the Na,K-ATPase activity up to 476 U/mg with a K"0"."5 value of 0.4 mM. The Na^+ ions also presented a single saturation curve with V"M=551 U/mg and K"0"."5=0.2 mM with cooperative effects. The activity was also stimulated by K^+ ions through a single curve of saturation sites (K"0"."5=2.8 mM), with cooperative effects and V"M=641 U/mg. The lipid microenvironment close to the proteic structure and the K^+ internal to the liposome has a key role in enzyme regulation, affecting its kinetic parameters while it can also modulate the enzyme's affinity for substrate and ions.
