Purification and properties of insulin-activated nitric oxide synthase from human erythrocyte membranes

Sujoy Bhattacharya, S. Chakraborty Patra, S. Basu Roy, N. N. Kahn, Asru K. Sinha

Research output: Contribution to journalArticle

32 Citations (Scopus)

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 (Mr 230 KD) was found to be composed of one heavy chain (Mr 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 Km with simultaneous increase of Vmax. 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 μM) activated the enzyme, but at higher concentration (>0.80 μM) 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 oftyrosine kinase). These results indicated that the insulin activated nitric oxide synthase could be the insulin receptor itself.

Original languageEnglish (US)
Pages (from-to)441-449
Number of pages9
JournalArchives of Physiology and Biochemistry
Volume109
Issue number5
DOIs
StatePublished - Dec 1 2001

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Erythrocyte Membrane
Nitric Oxide Synthase
Insulin
Enzymes
Insulin Receptor
Hormones
Insulin Antibodies
Enzyme Activation
Genistein
Disulfides
Detergents
Protein-Tyrosine Kinases
Anti-Idiotypic Antibodies
Carrier Proteins
Phosphotransferases
Erythrocytes
Binding Sites
Cell Membrane
Light
Membranes

All Science Journal Classification (ASJC) codes

  • Physiology
  • Physiology (medical)

Cite this

Purification and properties of insulin-activated nitric oxide synthase from human erythrocyte membranes. / Bhattacharya, Sujoy; Chakraborty Patra, S.; Basu Roy, S.; Kahn, N. N.; Sinha, Asru K.

In: Archives of Physiology and Biochemistry, Vol. 109, No. 5, 01.12.2001, p. 441-449.

Research output: Contribution to journalArticle

Bhattacharya, Sujoy ; Chakraborty Patra, S. ; Basu Roy, S. ; Kahn, N. N. ; Sinha, Asru K. / Purification and properties of insulin-activated nitric oxide synthase from human erythrocyte membranes. In: Archives of Physiology and Biochemistry. 2001 ; Vol. 109, No. 5. pp. 441-449.
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