Search

Your search keyword '"Garty, Haim"' showing total 7 results
Start Over Author "Garty, Haim" Journal biochemistry
7 results on '"Garty, Haim"'

1. Purification of the human [alpha]2 isoform of Na, K-ATPase expressed in Pichia pastoris: stabilization by lipids and FXYD1

Author

Lifshitz, Yael, Petrovich, Ekaterina, Haviv, Haim, Goldshleger, Rivka, Tal, Daniel M., Garty, Haim, and Karlish, Steven J.D.

Subjects
Adenosine triphosphatase -- Research, Adenosine triphosphatase -- Physiological aspects, Phospholipids -- Research, Phospholipids -- Chemical properties, Cell metabolism -- Research, Biological sciences, Chemistry
Abstract

The physiological implications of interaction of human [alpha]1 and [alpha]2 isoforms of Na,K-ATPase with phospholipids and FXYD1 are discussed.

Published
2007

7. Purification of the human alpha2 Isoform of Na,K-ATPase expressed in Pichia pastoris. Stabilization by lipids and FXYD1.

Author

Lifshitz Y, Petrovich E, Haviv H, Goldshleger R, Tal DM, Garty H, and Karlish SJ

Subjects
Cell Membrane drug effects, Cell Membrane metabolism, Humans, Isoenzymes chemistry, Isoenzymes genetics, Isoenzymes isolation & purification, Isoenzymes metabolism, Kinetics, Membrane Proteins genetics, Models, Molecular, Phosphoproteins genetics, Pichia genetics, Protein Binding, Protein Denaturation, Protein Structure, Quaternary, Sodium-Potassium-Exchanging ATPase chemistry, Sodium-Potassium-Exchanging ATPase genetics, Temperature, Cholesterol pharmacology, Gene Expression, Membrane Proteins metabolism, Phosphatidylserines pharmacology, Phosphoproteins metabolism, Pichia metabolism, Sodium-Potassium-Exchanging ATPase isolation & purification, Sodium-Potassium-Exchanging ATPase metabolism
Abstract

Human alpha1 and alpha2 isoforms of Na,K-ATPase have been expressed with porcine 10*Histidine-tagged beta1 subunit in Pichia pastoris. Methanol-induced expression of alpha2 is optimal at 20 degrees C, whereas at 25 degrees C, which is optimal for expression of alpha1, alpha2 is not expressed. Detergent-soluble alpha2beta1 and alpha1beta1 complexes have been purified in a stable and functional state. alpha2beta1 shows a somewhat lower Na,K-ATPase activity and higher K0.5K compared to alpha1beta1, while values of K0.5Na and KmATP are similar. Ouabain inhibits both alpha1beta1 (K0.5 24.6 +/- 6 nM) and alpha2beta1 (K0.5 102 +/- 14 nM) with high affinity. A striking difference between the isoforms is that alpha2beta1 is unstable. Both alpha1beta1 and alpha2beta1 complexes, prepared in C12E8 with an added phosphatidyl serine, are active, but alpha2beta1 is rapidly inactivated at 0 degrees C. Addition of low concentrations of cholesterol with 1-stearoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (SOPS) stabilizes strongly, maintaining alpha2beta1 active up to two weeks at 0 degrees C. By contrast, alpha1beta1 is stable at 0 degrees C without added cholesterol. Both alpha1beta1 and alpha2beta1 complexes are stabilized by cholesterol at 37 degrees C. Human FXYD1 spontaneously associates in vitro with either alpha1beta1 or alpha2beta1, to form alpha1beta1/FXYD1 and alpha2beta1/FXYD1 complexes. The reconstituted FXYD1 protects both alpha1beta1 and alpha2beta1 very strongly against thermal inactivation. Instability of alpha2 is attributable to suboptimal phophatidylserine-protein interactions. Residues within TM8, TM9 and TM10, near the alphabeta subunit interface, may play an important role in differential interactions of lipid with alpha1 and alpha2, and affect isoform stability. Possible physiological implications of isoform interactions with phospholipids and FXYD1 are discussed.

Published
2007
Full Text
View/download PDF

Catalog

Books, media, physical & digital resources
See catalog results