Identification and partial purification of a (Na-K)ATPase stimulating serine protease from plasma of insulin-dependent diabetics.
Plasma from insulin-dependent diabetics shows an increased ability to specifically activate the (Na-K)ATPase from different sources. Several protease inhibitors like phenyl methyl sulfonyl fluoride, trypsin inhibitor, antithrombin III and aprotinin, produced a significant dose-dependent inhibition of the stimulatory effect produced by a 1/100 final dilution of plasma on the beef heart (Na-K)ATPase activity. Serine proteases employed at scalar concentrations in the ATPase medium gave a dose-dependent stimulation of the enzyme activity as did diabetic plasma. The maximum percent stimulation of the (Na-K)ATPase activity (about 60%) was reached by 0.56 microgram/ml of thrombin, 0.50 microgram/ml of kallikrein and 0.55 microgram/ml of trypsin. The protease-induced ATPase stimulation was significantly reduced by antithrombin III, trypsin inhibitor and by aprotinin. A partial purification of the activating plasma factor was obtained by eluting plasma on a heparin-Sepharose column. Two (Na-K)ATPase stimulating fractions were found, which eluted with 1.0 and 3.0 mol/l NaCl, respectively. Half-maximal stimulation of the enzyme occurred with 3.4 micrograms/ml proteins of fraction 1.0 mol/l and with 45 ng/ml proteins of fraction 3.0 mol/l, this last representing the most purified plasma fraction (about 8890-fold purification). The proteolytic activity of both plasma and purified plasma fractions was tested on Tos-Arg-OMe substrate which was hydrolyzed to a much higher degree by the most purified plasma fraction. Like the (Na-K)ATPase stimulation, the esterolytic activity was inhibited by protease inhibitors, the most effective to this regard being antithrombin.
Finotti
P
P
Department of Pharmacology, University of Padova, Italy.
Verbaro
R
R
D000818:Animals; D002417:Cattle; D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000201:enzymology*; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D009206:Myocardium / Q000201:enzymology; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000097:blood*; D000254:Sodium-Potassium-Exchanging ATPase / Q000097:blood / Q000378:metabolism*; D014106:Tosylarginine Methyl Ester / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D011480:Protease Inhibitors; D014106:Tosylarginine Methyl Ester; D012697:Serine Endopeptidases; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase