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. 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. Ca2+ important that to a of as and The calmodulin (CaM) CaM, nitric-oxide nitric constitutively expressed endothelial nitric-oxide synthase iNOS, inducible nitric-oxide synthase neuronal nitric-oxide synthase C is the Ca2+ that to the activation of is in the of the interactions and of C is a is to activate the The of is in to CaM in that both have domains by a each domain is of EF In the of three of the four EF hand regions in the are the of of the EF hands hand of from the in CaM in that the is a in the the has a sequence with The EF hand of not Ca2+ of a to a has more and three more than not activate enzymes The domain of CaM Ca2+ with a than the domain but the site of Ca2+ with 1 of more than the site in CaM of the domains of CaM can be for the domains of to the of regions of the the of domains or elements of the EF hand between CaM and to regions that enzyme activation of CaM including each of the four domains from have been CaM is domain 1 of and domains and of synthase enzymes are with each a reductase domain and FMN The oxygenase domain for and as as the and CaM-binding domain the oxygenase and reductase domains of The of CaM to nNOS and endothelial NOS electron transfer into the the reductase domain and the to transfer to the heme in the oxygenase domain Although the investigation of nNOS interactions with CaM and mutant forms of CaM including chimeras have been previously reported, there has been a investigation of the other constitutively expressed NOS or the inducible isoform (iNOS). enzyme and activation study both nNOS and eNOS has important in the that of CaM has constitutively expressed NOS enzymes is the of CaM and activation of the inducible NOS The present study was designed to evaluate the role of the four CaM EF hands in the activation of the eNOS and iNOS chimeras were in the To assess the role of CaM regions on aspects of NOS enzymatic function, three distinct activities associated with NOS were electron transfer from NADPH to the reductase by NADPH electron transfer from FMN to by cytochrome c and electron transfer to the heme by generation of with of the chimeras in the and activation of iNOS and cNOS enzymes by that domain 2 and the associated sequence iNOS domain and at low Ca2+ NOS and neuronal and endothelial NOS were expressed in Escherichia coli and as previously The human iNOS enzyme a of the and was co-expressed with the or mutant CaM in The iNOS enzymes were a of and by as previously C and for the C chimeras CaM CaM CaM and CaM were a from The CaM is to CaM that of CaM are by from The CaM is to CaM that 2 and are by 2 and of The required the of by a in the in CaM The CaM of the CaM that are by of The CaM the CaM that are by of the CaM of 1 and 2 and the from CaM and 3 and from were all and the of with and for with the iNOS enzyme. The regions for CaM CaM CaM CaM and CaM were into the the and at the and of each of the in the The were and The was to that no the of the of coli with the were to 1 of in a with of the The were at the at at which the were with and 3 of at The were and the was and at The were on and in of 1 1 and The was as previously in on and at was on the a the as previously of .NO was the oxyhemoglobin as previously The were at in a the iNOS, and nNOS were at and in a the of the were 1 1 and was in at and NADPH and were present in the with the enzyme the was The NADPH was to to with in the enzyme from the of the The was by the to the oxyhemoglobin at NADPH and and CaM or were to the NADPH of NADPH by NOS was at as previously reactions were by the of and on a at The of CaM was 2 The in a of NADPH FMN and iNOS, or eNOS. c reduction of cytochrome c was at as previously reactions were by the of NADPH and on a at The of CaM was 2 which .NO The in a of NADPH FMN cytochrome c and iNOS, or eNOS. for were the from sequence human human and human iNOS, CaM and with Ca2+ and EF hands from were from with the chimeras in which EF hand 1 or 2 was from of the EF hand was a of CaM to the to at the The no or and no and their to the of CaM, and 1 a sequence including the of CaM, and the chimeras in The CaM and the human CaM and from the (CaM) and are the from and are to which are in the The CaM CaM the in which the to the CaM EF hand by the sequence from The of the CaM are by the and the the of the EF hand regions are by the The sequence from in CaM from the to the of 3 and not in the (CaM) CaM the to the of 3 or in the Ca2+ the and the of the three extra in the CaM the for the of and to the of CaM has sequence elements to the from to the C CaM the sequence elements of CaM and CaM The of is important than be the regions including the of and are in sequence and between and The important was the of the in CaM have been in CaM or and expressed on their a of to the chimeras were by and were to be The chimeras as when are The were in the chimeras when in a Ca2+ not was as a to assess the of CaM and The for each of the was 1 of the not The iNOS enzyme was co-expressed with CaM and each of the The of the iNOS co-expressed with CaM the of iNOS enzyme The of iNOS when the enzyme was co-expressed with CaM or CaM were more than when the enzyme was co-expressed with CaM CaM or CaM to on the of iNOS with each of the chimeras, a that the chimeras the of CaM the CaM-binding of The of the iNOS enzymes co-expressed with each of the chimeras were of NOS not of heme and of the enzymes were of .NO the oxyhemoglobin assay. cNOS by in EF hands and of CaM are important for nNOS chimeras elements to of elements were to activate .NO production from nNOS by than 3 and In contrast, the EF hand 2 was to activate .NO production by The in the of .NO production by nNOS activated by the chimeras was CaM CaM CaM CaM CaM of the chimeras were to activate .NO production by nNOS in the of 1 are with of nNOS activation by chimeras by the and the oxyhemoglobin .NO capture activation of cNOS 3 for the of the no c c 3 for the of the no in a We present here the of the activation of eNOS by .NO production from eNOS activated by CaM or were as CaM CaM CaM CaM CaM Although the of activation for the eNOS was to that of the nNOS is that CaM was not to activate the eNOS enzyme. the of CaM all of the chimeras were to activate .NO production by eNOS to a than nNOS The activation of nNOS and eNOS by CaM in a NADPH to .NO production of more than three of the of for eNOS were previously the NADPH was associated with the of to the of the The of NADPH by nNOS activated by CaM or the chimeras the for .NO that by reduction of by the NOS reductase The of NADPH to .NO was the for nNOS activated by CaM and CaM that CaM and the chimeras by stimulating the nNOS enzyme to from the to the heme in the oxygenase domain. of NADPH to .NO for the CaM and CaM chimeras a of electron transfer from .NO NADPH by eNOS activated by CaM or the chimeras not the as for the production of .NO NOS activation by all of the chimeras of NADPH to .NO that eNOS may be more than nNOS to of NADPH from the production of .NO when activated by mutant CaM The cytochrome c reduction that all of the chimeras CaM activated electron transfer in the reductase domain of the nNOS enzyme. The reduction of cytochrome c were than the of .NO for the enzymes activated by the CaM and CaM The of cytochrome c reduction by nNOS activated by CaM was the as when activated by The EF hands and of are for the activation of electron transfer the reductase domain of In contrast, in EF hand 1 of CaM to be important to in the reductase domain by in or no electron transfer to cytochrome was when the role of the four CaM domains in the activation of electron transfer the reductase domain of eNOS enzyme. The of domain 3 or in CaM the of reduction of cytochrome In to the with the CaM production of cytochrome c reduction by eNOS. In to the nNOS the CaM cytochrome c reduction for eNOS. The of production of .NO by eNOS bound to CaM in with nNOS may be in by in the reductase domain as by the of cytochrome c iNOS by of iNOS activation by CaM have been by the between the enzyme and with the of iNOS to has the of iNOS. We have by the human iNOS enzyme with each of the chimeras in previously reported, the of iNOS with CaM in a that a in when with and The of iNOS with the chimeras to regions of CaM that are important for activation and of The of CaM domains or with their in of .NO production from to and In contrast, the of domain 2 of CaM with in a CaM that activated NOS to The CaM activated iNOS to The of CaM to each of co-expressed not in in the of the that the CaM-binding domains were with the mutant CaM not activation of for the of the c for the of the in a The of to the Ca2+ in a in .NO production by all of the chimeras was for the iNOS enzyme co-expressed with CaM CaM and CaM The of Ca2+ the of the calcium In contrast, the of to the iNOS co-expressed with CaM and CaM in a in enzyme that was comparable with the on iNOS co-expressed with The of a activation by chimeras is with the EF hand of is not to Ca2+ and the of Ca2+ with 1 of than the site in CaM that domains 2 and 3 of CaM contain important required for the of CaM to the iNOS enzyme. The of CaM in the of in for all of the enzymes co-expressed with the chimeras for the CaM that activation not The mutant CaM chimeras to be by CaM when in a for the that there are elements in CaM for the of CaM to iNOS. of the of the chimeras to activate NADPH to their activation of .NO by iNOS and The of CaM, CaM or CaM with iNOS in a of NADPH .NO in the of that in the in the iNOS mechanism is more than that in the cNOS the of NADPH have been associated with the of to the the between iNOS and cNOS enzymes may be to a for the FMN in the iNOS enzymes more to a but a much of .NO The iNOS co-expressed with CaM CaM or CaM in the but the of to a than in the of NADPH The CaM domains 2 and 3 contain elements to the of the iNOS enzyme. The cytochrome c were to electron transfer from the to electron The iNOS enzymes co-expressed with the chimeras all or more in the of both and low of Ca2+. are with study electron transfer from the reductase domain of iNOS enzymes to electron is CaM the of cytochrome c reduction to CaM are for iNOS enzymes co-expressed with CaM CaM and CaM may be to the of in CaM that are not for electron transfer but may the of electron transfer by as of the interactions between and FMN or of the reductase domain. of the sequence the oxygenase and reductase of and iNOS with the from the of The of in a is present in all as previously by and The is in that sequence elements that to be by the of the in the CaM in is from a of the that the of the CaM-binding site in eNOS is from the sequence elements as the in nNOS and iNOS. have been by the of the or elements in nNOS and be to both the of CaM with the site and interactions of CaM with the oxygenase domain. The of the in NOS enzymes the CaM-binding site and to the domain is of the the and present in the and the is the in the of the domain. In contrast, the between the CaM-binding site and the oxygenase domain is not and in from to the between the CaM-binding site and the reductase domain of CaM and from and have a general mechanism of activation of The of the the of CaM the In activation from the of domain. of on the the and the have for CaM to with The aspects of interactions between CaM and are in a the of the Ca2+ to the domain of with a of the and domains CaM The of CaM to the cNOS enzymes has been to electron transfer at the on iNOS is The present investigation of CaM interactions with all three NOS was to of the activation of which CaM regions EF hands and with their have effects on the of .NO production and NADPH of nNOS enzymes 3 and by the cytochrome c reduction regions of can domains 3 and with on electron transfer from NADPH into the reductase domain of that the of the CaM and CaM chimeras to activate .NO production is associated with the reduction of heme by In contrast, the of the domain of CaM and the by the domain in in a that can activate is a the much in sequence in the CaM and that the in CaM and CaM CaM important with sequence regions the CaM-binding investigation the eNOS enzyme a but with the nNOS the transfer of into the reductase domain of eNOS as by the reduction of the cytochrome c was to the of the CaM was the of the chimeras activated eNOS to of the with In the activation of eNOS by CaM and CaM was as a of with In with the nNOS CaM CaM and CaM all activated electron transfer to cytochrome c in eNOS to a than activated .NO that the activation of .NO production by from a of FMN to heme electron In contrast, CaM is in the activation of cytochrome c reduction in eNOS than in the activation of .NO that the of CaM to activate .NO production in eNOS from electron transfer to The investigation of the role of regions of CaM on the activation of iNOS required the of for each of the the from a in the role of CaM in the activation of iNOS when with the cNOS enzymes and with each of the chimeras in the or of a Ca2+ not electron transfer into the reductase domain as the cytochrome c assay. are with the reductase domains of both human and iNOS In to CaM activated .NO production by iNOS was than that by CaM Ca2+ Although CaM activates .NO production to the in iNOS and eNOS when expressed as a of with CaM, is more to that in the cNOS CaM is the in iNOS is the CaM is more than CaM in .NO production by iNOS, and CaM and CaM may be more CaM is more than CaM but than the for CaM that the domain of CaM is important for iNOS than for eNOS and but the of EF hand is and is to be of CaM is in all than CaM or CaM CaM and CaM are as of iNOS as CaM when in the of Ca2+. are for The of CaM and CaM when bound to Ca2+ may be the of in the of the CaM in the 3 and which are in that the CaM of iNOS is than the CaM of eNOS and nNOS and that the important interactions eNOS and nNOS activation by CaM are in iNOS. The of elements in the domain and the of CaM or the of of the domain the Ca2+ of The of to the in a in .NO production by iNOS co-expressed with CaM CaM or CaM in .NO production with a in NADPH but not cytochrome c reduction, which was by all In the of CaM CaM and CaM all activated NADPH to a than .NO that at low Ca2+ of chimeras to iNOS electron CaM a EF hand that is of Ca2+ and that is to be of interactions to the of the CaM the of CaM is that the EF hand of CaM and the associated regions are in iNOS the other the CaM and CaM that regions are important of iNOS of the domain chimeras were at as as CaM in electron transfer to cytochrome CaM CaM and CaM were more than the for iNOS, and nNOS in the for the activation of cytochrome c reduction are than the for the activation of .NO The iNOS that cytochrome c reduction can from a of the by CaM isoform are in the in and CaM is in cytochrome c reduction by and CaM is than the other chimeras at cytochrome c reduction in eNOS. for both cNOS enzymes the that CaM can activate reductase domain by a mechanism other than stimulating electron transfer into the oxygenase domain that a is for the eNOS effects of the chimeras on all the reactions are not between nNOS and eNOS with CaM in and between cNOS enzymes have been for the of CaM on enzyme and electron transfer The in the of all chimeras in investigation are in Although by the that more than of NOS enzymes with CaM the of CaM bound to the CaM-binding of eNOS for the of The between the CaM and CaM the of in CaM by the in and the of the Ca2+ with the that the EF hand of not Ca2+. of three for the of .NO production by the cNOS The of a on iNOS by the of CaM is with the that iNOS is at of Ca2+ when the of CaM may not be Ca2+ of are for the of and CaM, in with the eNOS in CaM to be in with eNOS CaM-binding in The of and is on the of CaM and in CaM, in the of in the of CaM, CaM, are for the of and CaM, in CaM to be in with eNOS CaM-binding in The of and is on the of CaM and in in the of in a for the CaM a in the in with the eNOS is the of the in CaM by 3 not in has been that the of the of CaM the to the of the domains with to each other and to the The three in the of the CaM the by and the of in the and C domains of CaM by as in are but are to be in the The effects of CaM on the Ca2+/CaM of .NO production by iNOS be to a in the of the of CaM with the enzyme or in the of the domains of CaM when bound to iNOS. The CaM has with the eNOS when with CaM of and the of of and have been to be a for the of to to CaM-binding may be important in the activation of cNOS enzymes as as the Ca2+/CaM of .NO production by iNOS bound to CaM study chimeras and mutant that the of the CaM to activate nNOS is on the of and to in CaM The domain of Ca2+ with 1 of more than the site in CaM The CaM has but 2 of are The .NO production by nNOS is than that by eNOS when activated by CaM 3 and effects on the of cNOS enzymes were previously when the in domain of CaM were In the of CaM the CaM is by a and the that is in with the eNOS In to the with the cNOS the activation for iNOS when co-expressed with CaM is to the The in the and activation of the three NOS by CaM are to elements in regions of the CaM the of a of of chimeras on the of CaM and from which were In each the of the the of CaM for of the of the of the EF hand is from a regions are by the the of CaM the was from The of CaM bound to a from the eNOS CaM-binding as by is by the is in to other CaM that in with and the CaM-binding of the cNOS are to with CaM in in the with by the of and the of CaM of four elements, each of a EF hand and associated four EF hands in Ca2+ and with the The interactions are with and the of 2 and are in the with the nNOS than in with other but not the in interactions with other regions of NOS are to be important The of the in with other CaM-binding EF hand to the oxygenase domain and EF hand 2 to the domain and the adjacent of the that the sequence of the domain. EF hands 1 and 3 are that and the adjacent sequence regions with from the oxygenase or the reductase The of the C with a is by a In EF hands 3 and with the and regions Ca2+. EF hand 1 of is of Ca2+ with a for in of the in Ca2+ are present in the other three EF Although the EF hand of the CaM the to readily a of the CaM is to that a to CaM but a a when into the CaM but that and The in was the for from the from the the C sequence elements between and were CaM the with the of the is the sequence regions are and in the of the which in CaM is by the bound Ca2+ and the of the other EF The no interactions or with and be readily to of to the The of the CaM is in In including all of EF hand were from the to the of were a CaM to the with the of EF hands 3 and on the the of three in the between four and The no interactions or with and be readily to a of of for CaM in a of the in the to all four EF hands on the The the of the forms EF hand the the of the CaM The extra a in the by the The CaM CaM and CaM were the of the for the sequence be readily into the CaM with of the the between and of the interactions or with the and each be readily to a of The of in with the are or are in CaM in CaM in CaM and in CaM each of the four were important in the of CaM to NOS and the activation of .NO the of EF hands 1 and 2 from to be more important than the of EF hands 3 and the of the regions in are more and their in Ca2+ and is or The here not and in the of CaM regions with the NOS elements in activation, is that interactions with the CaM-binding site are to for the The with to of the domain and the that the and domains are in or with the of bound regions contain that are associated with CaM and are to with the CaM The domain is and has of the in the has been to the of eNOS to a which was as for Although is that mutant that cNOS is that adjacent sequence regions as of the CaM and for The of which has been in of electron transfer is not to with CaM and is to be important adjacent associated with the oxygenase domain. We have that the the CaM-binding site with the oxygenase domain is not in NOS that the interactions between bound CaM and the oxygenase domain a may not be electron transfer from FMN to heme in a NOS is to the oxygenase domain of the other of bound CaM with adjacent to the of the oxygenase domain which electron transfer is We that electron transfer in NOS by a mechanism in which the domain from the to which by a and to the oxygenase for electron transfer to heme CaM in a of the the release of the FMN domain. from the between activation of cytochrome c reduction and .NO production can be readily of the domain is for the activation of cytochrome but .NO production of the FMN domain to the oxygenase domain. that are to release of the FMN domain are not to of the FMN and oxygenase domains and may the association of the FMN and oxygenase c reduction by iNOS is readily activated by each of the chimeras examined here and may be constitutive. The disparity between cytochrome c reduction and .NO production at low Ca2+ can be attributed to poor association of heme and FMN domains when the bound CaM constructs are depleted of Ca2+. Interactions with the reductase moiety are dominant in cNOS activation, and EF hand 1 is critical for activation of both nNOS and eNOS. In cNOS enzymes are much more difficult to activate than iNOS, which can be attributed to the extra sequence elements which are adjacent to the CaM-binding and associated with CaM control. We for the the for the in investigation and for

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.