Purification and properties of insulin-activated nitric oxide synthase from human erythrocyte membranes.
A membrane bound form of nitric oxide synthase of human erythrocytes that could be activated by insulin was purified to homogeneity by detergent solubilization of the purified membrane preparation of these cells. The purified enzyme (M(r) 230 KD) was found to be composed of one heavy chain (M(r) 135 KD) and one light chain (Mr 95 KD) held together by disulphide bond(s). Scatchard plot analysis of insulin binding to the purified enzyme showed the presence of 2 different populations of the binding sites and the activation were directly related to the hormone binding to the protein. Line weaver Burk plot of the purified enzyme showed that the stimulation of the enzymic activity by insulin was related to the decrease of K(m) with simultaneous increase of V(max). Treatment of the purified enzyme with anti insulin receptor antibody inhibited the activation of the enzyme and the binding of the hormone to the protein. Furthermore NO itself, at low concentration (<0.4 microM) activated the enzyme, but at higher concentration (>0.8 microM) had no effect on the activation. Incubation of the purified enzyme with insulin simultaneously stimulated the tyrosine kinase and nitric oxide synthase activities of the preparations, that could be inhibited by genistein (an inhibitor of tyrosine kinase). These results indicated that the insulin activated nitric oxide synthase could be the insulin receptor itself.
Bhattacharya
S
S
Sinha Institute of Medical Science and Technology, West Bengal, Tamluk, India.
Chakraborty Patra
S
S
Basu Roy
S
S
Kahn
N N
NN
Sinha
A K
AK
D002118:Calcium / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000201:enzymology*; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D008274:Magnesium / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011972:Receptor, Insulin / Q000276:immunology / Q000378:metabolism
D004791:Enzyme Inhibitors; D007328:Insulin; D009569:Nitric Oxide; D019833:Genistein; D019001:Nitric Oxide Synthase; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D008274:Magnesium; D002118:Calcium