Effect of limited trypsin digestion on the renal Na+−H+ exchanger and its regulation by cAMP-Dependent protein kinase
Tóm tắt
The Na+−H+ exchanger from solubilized rabbit renal brush border membranes is inhibited by cAMP-dependent protein kinase (PKA) mediated protein phosphorylation. To characterize this inhibitory response and its sensitivity to limited proteolysis, the activity of the transporter was assayed after reconstitution of the proteins into artificial lipid vesicles. Limited trypsin digestion increased the basal rate of proton gradient-stimulated, amiloride-inhibitable sodium uptake in reconstituted proteoliposomes and blocked the inhibitory response to PKA-mediated protein phosphorylation. To determine if the inhibitory response to PKA-mediated protein phosphorylation could be restored to the trypsin-treated solubilized proteins, nontrypsinized solubilized brush border membrane proteins were separated by column chromatography. The addition of small molecular weight polypeptides, fractionated on Superose-12 FPLC (V
e=0.7), to trypsinized solubilized brush border membrane proteins restored the inhibitory response to PKA-mediated protein phosphorylation. Similarly, the addition of the 0.1m NaCl fraction from an anion exchange column, Mono Q-FPLC, also restored the inhibitory response to PKA. Both protein fractions contained a common 42–43 kDa protein which was preferentially phosphorylated by PKA. These results indicate that limited trypsin digestion dissociates the activity of the renal Na+−H+ exchanger from its regulation by PKA. It is suggested that trypsin cleaves an inhibitory component of the transporter and that this component is the site of PKA-mediated regulation. Phosphoprotein analysis of fractions that restored PKA regulation raises the possibility that a polypeptide of 42–43 kDa is involved in the inhibition of the renal Na+−H+ exchanger by PKA-mediated, protein phosphorylation.
Tài liệu tham khảo
Beavo, J.A., Bechtel, P.J., Krebs, E.G. 1974. Preparation of homogenous cyclic AMP-dependent protein kinase(s) and its subunits from rabbit skeletal muscle.Methods Enzymol 38:299–308
Hoffman, J.F., Laris, P. 1974. Determination of membrane potentials in human andAmphiuma red cells by means of a fluorescent probe.J. Physiol. (London) 293:519–552
Kahn, A.M., Dolson, G.M., Hise, M.K., Bennett, S.C., Weinman, E.J. 1985. Parathyroid hormone and dibutyryl cyclic AMP inhibit Na+−H+ countertransport in brush border membrane vesicles from a suspension of rabbit proximal tubule.Am. j. Physiol. 248:F212-F218
Kinsella, J.L., Aronson, P.S. 1980. Properties of the Na+−H+ exchanger in renal microvillus membrane vesicles.Am. J. Physiol. 238:F461-F469
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurements with the Folin phenol reagent.J. Biol. Chem. 193:265–275
Shenolikar, S., Cohen, P. 1978 the substrate, specificity of cyclic AMP dependent protein kinase: Amino-acid sequences at the phosphorylation sites of Herring protamine (Clupeine).FEBS Lett. 86:92–98
Weinman, E.J., W. Dubinsky, S. Shenolikar 1988. Reconstitution of cAMP-dependent protein kinase, regulated renal Na+−H+ exchanger.J. Membrane Biol. 101:11–18
Weinman, E.J., Shenolikar, S., Kahn, A.M. 1987. cAMP-associated inhibition of Na+−H+ exchanger in rabbit kidney.Am. J. Physiol. 352:F19-F25