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Modifications Induced by Plasma from Insulin-Dependent Diabetic Patients and by Lysophosphatidylcholine on Human Na⁺,K⁺-Adenosine Triphosphatase¹

Department of Diabetology (R.A.R., P.F.), INRCA Ancona, and the Institutes of Biochemistry (G.Z., E.S., R.S., L.M.) and Obstetrics and Gynecology (N.C.), University of Ancona, Ancona, Italy; and the Institute of Physiology, Czech Academy of Sciences (E.A.), Prague, Czech Republic

Address all correspondence and requests for reprints to: Prof. L. Mazzanti, Istituto di Biochimica, Universitià di Ancona, Via P. Ranieri 65, 60131 Ancona, Italy.

To investigate the molecular mechanisms of the inhibition of Na⁺,K⁺-adenosine triphosphatase (Na⁺,K⁺-ATPase) in diabetes mellitus, we incubated Na⁺,K⁺-ATPase purified from human placenta of six healthy nondiabetic women with plasma from six insulin-dependent diabetic (IDDM) men and six healthy controls and with different concentrations of lysophosphatidylcholine (LPC). We determined the enzyme activity, anthroyl ouabain-binding capacity, dissociation constant (K_d), and average lifetime values () by the static and dynamic fluorescence of anthroyl ouabain. The lipid annulus of the enzyme was studied by static and dynamic fluorescence of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). Moreover, we studied the lipid microenvironment surrounding the Na⁺,K⁺-ATPase purified from the placentas of six healthy women and six insulin-dependent diabetic women, determining the percent composition of phospholipids of the lipid annulus. The addition of total and protein-free IDDM plasma to normal Na⁺,K⁺-ATPase significantly inhibited the enzymatic activity even at the lowest concentration studied (1:100), whereas the ouabain-binding capacity, K_d, and were not affected by IDDM plasma. The fluorescence polarization and lifetime values of TMA-DPH were significantly decreased by diabetic plasma. The incubation of Na⁺,K⁺-ATPase with LPC caused an inhibition of the enzymatic activity without modifications of the anthroyl ouabain-binding capacity and dissociation constant. The fluorescence polarization and lifetime values of TMA-DPH were significantly decreased by 5 µmol/L LPC. The study of the phospholipids surrounding Na⁺,K⁺-ATPase demonstrated a significant increase in the percent LPC content in IDDM patients compared with controls together with a concomitant decrease in phosphatidylcholine. These observations indicate that the inhibition caused by diabetic plasma on Na⁺,K⁺-ATPase is not dependent on a modification of the ouabain-binding site and that it seems to mimic the effect of LPC addition. A link between modification of the lipid moiety of the enzyme and Na⁺,K⁺-ATPase inhibition might be hypothesized.