Sodium and potassium ion-dependent change in oligomerization of Na,K-ATPase in C12E8 detected by low-angle laser light scattering technique in combination with high performance porous silica-gel chromatography.

The research article discusses an investigation of the molecular weights and subunit structures of Na,K-ATPase, an enzyme from horse kidney, using a combination of tests. Notably, the study identified how sodium and potassium ions influenced the enzyme’s behavior and structure.

Method of Investigation

• The researchers used a combination of porous silica gel chromatography, laser light scattering (LS), and refractive index (RI) measurements in an organic compound called C12E8
• The enzyme was eluted (separated out from a mixture) using either NaCl or KCl. Each method yielded different protein peaks in the resulting filtrate—three for the NaCl solution and four for the KCl solution
• The peaks were labeled: Na-1, Na-2, Na-2′, Na-3 (NaCl-contained eluents), and K-1, K-2, K-3 (KCl-contained eluents)

Findings and Analysis

• Enzyme activities for Na,K-ATPase and K-p-nitrophenylphosphatase were found across all peaks. These activities were completely halted by the addition of a toxin called ouabain
• The research team compared the ratios from laser light scattering (LS) to that of differential refractive index (RI) for standard proteins and peaks derived in the study
• The study measured relative refractive index increments for three substances, BSA, thyroglobulin, and C12E8, obtaining values of 0.144, 0.141, and 0.135 respectively
• The research found that the size of the Na,K-ATPase enzyme in the primary peak from KCl eluents was twice as large as that from NaCl eluents
• Other peaks, named Na-3 and K-3, appeared at the same time during the process and demonstrated the same LS/RI values. This suggests that the sizes of Na-3 and K-3 are one-third of Na-2′ and one-sixth of K-2

The research provided a refined method of investigating sodium and potassium ion-dependent changes in Na,K-ATPase and demonstrated how different ions influence the enzyme’s structure and activity.