The Calmodulin‐binding Domain of the Inducible (Macrophage) Nitric Oxide Synthase

Abstract

was GI nM. The binding affinity was lower, but still remarkably high in the presence of EGTA. The peptides counteracted the stimulation by calmodulin of a classical calmodulin-target enzyme, the Ca2+ pump of the plasma membrane.

Expression and Immunoaffinity Purification of Human Inducible Nitric-oxide Synthase

“…This study confirms that rH-iNOS also binds CaM tightly, and it is the first to demonstrate stoichiometric binding of CaM to the inducible NOS isoform. These findings are in agreement with recently published studies showing that CaM binds peptides derived from the consensus CaM binding domain of murine iNOS with a stoichiometry of 1:1 (24,50). Our analyses also demonstrate that rH-iNOS, lysed in the presence of excess BH 4 , remained essentially replete with that cofactor after subsequent repurification by DEAE chromatography in BH 4 -free buffers, demonstrating the tight binding of this cofactor.…”

“…These values were in good agreement with those obtained by amino acid analysis, which ranged from 114 to 260 nmol/min/mg at 37°C with an average of 164 Ϯ 57 (S.D., n ϭ 5). The K m determined for L-Arg was 2.30 Ϯ 0.25 M. In addition, two preparations of rH-iNOS with specific activities that differed by 3.3-fold gave indistinguishable IC 50 values for N G -methyl-L-Arg: 2.24 Ϯ 0.22 and 2.38 Ϯ 0.17 M, using 1 M L-Arg as substrate. In summary, rH-iNOS prepared by the immunoaffinity purification protocol was suitable for direct use in all subsequent enzyme analyses.…”

Thermodynamics of apocalmodulin and nitric oxide synthase II peptide interaction

“…Change in K D from NOS-II M521K to NOS-II 3K was 25-fold. Interaction of ApoCaM with WT NOS-II peptides was of similar affinity as the interaction of NOS-II M521K with ApoCaM (K D % 40 nM as reported in [13,14] for WT NOS-II and K D ¼ 66:8 nM for NOS-II M521K). We also tried to measure binding of ApoCaM to WT NOS-II peptides (mouse macrophage and human hepatic types) by ITC.…”

Increased Mitogen-Activated Protein Kinase Activity and TNF-α Production Associated with Mycobacterium smegmatis- but Not Mycobacterium avium-Infected Macrophages Requires Prolonged Stimulation of the Calmodulin/Calmodulin Kinase and Cyclic AMP/Protein Kinase A Pathways

“…CaM is a calcium-sensing protein that undergoes a conformation change upon binding calcium and is an important cofactor for a number of enzymes including inducible NO synthase (21). CaM also functions in activating gene transcription, and this is mediated, at least in part, by the activation of CaMKs.…”