Differential activation of nitric-oxide synthase isozymes by calmodulin-troponin C chimeras.
The interactions of neuronal nitric-oxide synthase (nNOS) with calmodulin (CaM) and mutant forms of CaM, including CaM-troponin C chimeras, have been previously reported, but there has been no comparable investigation of CaM interactions with the other constitutively expressed NOS (cNOS), endothelial NOS (eNOS), or the inducible isoform (iNOS). The present study was designed to evaluate the role of the four CaM EF hands in the activation of eNOS and iNOS. To assess the role of CaM regions on aspects of enzymatic function, three distinct activities associated with NOS were measured: NADPH oxidation, cytochrome c reduction, and nitric oxide (*NO) generation as assessed by the oxyhemoglobin capture assay. CaM activates the cNOS enzymes by a mechanism other than stimulating electron transfer into the oxygenase domain. Interactions with the reductase moiety are dominant in cNOS activation, and EF hand 1 is critical for activation of both nNOS and eNOS. Although the activation patterns for nNOS and eNOS are clearly related, effects of the chimeras on all the reactions are not equivalent. We propose that cytochrome c reduction is a measure of the release of the FMN domain from the reductase complex. In contrast, cytochrome c reduction by iNOS is readily activated by each of the chimeras examined here and may be constitutive. Each of the chimeras were co-expressed with the human iNOS enzyme in Escherichia coli and subsequently purified. Domains 2 and 3 of CaM contain important elements required for the Ca2+/CaM independence of *NO production by the iNOS enzyme. The disparity between cytochrome c reduction and *NO production at low calcium can be attributed to poor association of heme and FMN domains when the bound CaM constructs are depleted of Ca2+. In general cNOSs are much more difficult to activate than iNOS, which can be attributed to their extra sequence elements, which are adjacent to the CaM-binding site and associated with CaM control.
Newman
Elena
E
Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Spratt
Donald E
DE
Mosher
Jennifer
J
Cheyne
Bo
B
Montgomery
Heather J
HJ
Wilson
Denney L
DL
Weinberg
J Brice
JB
Smith
Susan M E
SM
Salerno
John C
JC
Ghosh
Dipak K
DK
Guillemette
J Guy
JG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D002417:Cattle; D045304:Cytochromes c / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D019209:Troponin C / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*
D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019209:Troponin C; D009569:Nitric Oxide; D045304:Cytochromes c; C496320:NOS1 protein, human; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496322:Nos1 protein, rat; C496318:Nos2 protein, rat; C496316:Nos3 protein, rat