Purification and Properties of Insulin-Activated Nitric Oxide Synthase from Human Erythrocyte Membranes

Abstract

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.

Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease

“…on May 10, 2018. by guest www.bloodjournal.org From protein with eNOS epitopes. Two groups hypothesized that RBCs express a novel NOS isoform, 23,25 but no sequence confirmation analysis was provided.…”

“…6,15,16,20,[23][24][25] However, positive staining with anti-iNOS antibodies has also been reported, 20,25 and others suggested that RBCs might express a novel NOS isoform. 23,25 Thus, doubts about the functional significance of the NOS-like activity in RBCs remain, and little is known about NOS isoform identity and disease relevance.We therefore sought to definitively identify and characterize the activity of human red cell NOS using an advanced multilevel analytical approach. Using HPLC, LC-MS/MS, flow cytometry, laser scanning microscopy, and enzymatic assay together with functional studies we here demonstrate that human RBCs contain an active eNOS that gives rise to constitutive intracellular NO formation under normoxic conditions.…”

“…Two earlier studies suggested that RBCs also contain iNOS. 20,25 We are unable to confirm these reports as we failed to isolate any iNOS from crude human RBC lysates of healthy donors using specific anti-NOS2 antibodies for immunoprecipitation.Although a L-NNA/L-NAME-inhibitable NOS activity was demonstrated by measuring NO metabolites or citrulline in the supernatant of intact erythrocytes 2,15,16,19 and RBC membranes,16,23 2 reports suggested RBCs harbor an inactive NOS. 20,22 It was important to confirm, therefore, that the protein identified as eNOS in our study was catalytically active, L-arginine:citrulline conversion was dependent on the presence of Ca 2ϩ /calmodulin, and activity was inhibited by L-NAME.…”

“…on May 10, 2018. by guest www.bloodjournal.org From protein with eNOS epitopes. Two groups hypothesized that RBCs express a novel NOS isoform, 23,25 but no sequence confirmation analysis was provided.The main technical hurdle in the biochemical characterization of proteins in RBCs is the presence of overwhelmingly high level of hemoglobin, which represents a formidable obstacle to purification, activity determination, immunostaining, and colorimetric/ fluorimetric assay. In this study, we chose to isolate NOS from hemolysates under native conditions to permit combined structural and functional analysis.…”

Restoration by aspirin of impaired plasma maspin level in human breast cancer

“…Aspirin activated nitric oxide synthase activity was determined by the formation of NO in reaction mixture by the conversion of oxyhemoglobin to methemoglobin [13] as described before [14]. The formation of NO was independently verified by chemiluminescence's method [15].…”