Preparation of active enzyme samples for IR studies of Na+/K+-ATPase.
In the case of the integral membrane protein Na+/K+-ATPase, preparation of highly concentrated samples for IR difference spectroscopy often leads to inactivation of the enzyme. Therefore, we compared the activity of Na+/K+-ATPase using different techniques of sample preparation. The loss of activity can be minimized by cooling the sample to 10 degrees C and by the addition of glycerol and dithiothreitol. The activity of Na+/K+-ATPase isolated from pig kidney is independent of the protein concentration whereas the enzyme from shark rectal gland is inactivated at concentrations above 1 microg/microL and is thus unsuitable for IR experiments.
Thoenges
Detlef
D
Institut für Biophysik, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7/Haus 74, 60590 Frankfurt/Main, Germany.
Zscherp
Christian
C
Grell
Ernst
E
Barth
Andreas
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005990:Glycerol / Q000494:pharmacology; D006868:Hydrolysis; D007668:Kidney; D012754:Sharks; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D013055:Spectrophotometry, Infrared / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared; D013552:Swine; D013696:Temperature; D013997:Time Factors; D014867:Water / Q000737:chemistry
D014867:Water; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D005990:Glycerol; D004229:Dithiothreitol