Abstract:
:Progress of mutagenesis studies on the relation of the structure of Na+/K+-ATPase to its reaction mechanism has been impeded by the paucity of information on the properties of small amounts of impure recombinant enzyme obtained in the currently available expression systems, and the uncertainty of whether expression in a new environment alters the various catalytic activities of this membrane enzyme. Hence, our aim was to make a detailed comparison of the properties of the extensively studied canine kidney Na+/K+-ATPase with those of its alpha1,beta1 subunits expressed in the baculovirus-infected Sf-9 cells. The active fraction of the recombinant enzyme, containing 10-20% of the expressed a subunits, was found to have normal molar activity, all the partial reactions, and the ability to catalyze ATP-dependent Na+/K+ exchange after reconstitution into proteoliposomes. Comparison of steady-state kinetics of the hydrolytic activities of recombinant and native enzymes showed that (a) ATP and Na+ plots of Na+-ATPase were the same in the two preparations; (b) apparent K+ affinity of K+-phosphatase of recombinant enzyme was lower than that of kidney enzyme; and (c) for Na+/K+- ATPase activity, apparent K+ affinity of recombinant enzyme was lower, and its apparent Na+ and ATP affinities were higher than those of kidney enzyme. The two enzymes had similar ADP- and K+-sensitive phosphointermediates, identical affinities for ouabain, and similar ligand sensitivities of dissociation rates of ouabain-enzyme complexes. Evidently, the recombinant enzyme has reduced affinity at cytoplasmic K+ sites, but no changes at multiple Na+, ATP, and ouabain binding sites. Likely causes of this selective change include altered glycosylation state of beta and interactions among active and inactive recombinant enzymes. The present results provide the necessary database for the appropriate use of an expression system in structure-function studies on canine alpha1,beta1 isoform of Na+/K+-ATPase, and indicate the need for similar studies on recombinant Na+/K+-ATPases obtained in other expression systems.
journal_name
Arch Biochem Biophysjournal_title
Archives of biochemistry and biophysicsauthors
Xie Z,Wang Y,Liu G,Zolotarjova N,Periyasamy SM,Askari Adoi
10.1006/abbi.1996.0237subject
Has Abstractpub_date
1996-06-01 00:00:00pages
153-62issue
1eissn
0003-9861issn
1096-0384pii
S0003-9861(96)90237-4journal_volume
330pub_type
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