Patent Abstract:
a method of screening a substance of interest for heme independent modulation of enzymatic activity of soluble guanylyl cyclase is disclosed , comprising obtaining αβ cys105 mutant sgc enzyme ; determining activity of the mutant enzyme for forming cgmp from gtp in the presence of the substance of interest in a reaction medium ; determining activity as in step , except in the absence of the substance of interest ; optionally , including an activator other than the substance of interest in steps b ) and c ); e ) comparing results of – to yield a comparison result ; and f ) from that value of that result , assessing activity of the substance of interest for modulating cgmp production by the mutant enzyme . increased or decreased formation of cgmp in the presence of the substance of interest indicates activity of the substance for modulating heme independent cgmp production .

Detailed Description:
a heme - deficient mutant sgc with a substituted hist105 residue is described herein , which has a high basal specific activity and displays properties similar to no - stimulated wild type sgc . the role of the heme - coordinating bond and of the coordinating his105 residue in governing the function of the sgc regulatory domain is discussed . the heme - coordinating his105 residue of the β subunit of soluble guanylyl cyclase was substituted with cysteine , resulting in a heme - deficient enzyme . analysis of this enzyme indicated that the mutant sgc has a high basal activity comparable with the activity of the wild type sgc activated by nitric oxide . the mutant was significantly inhibited by dtt , but not glutathione , and was heme - insensitive without dtt . the mutant can be partially reconstituted with heme after treatment with dtt and is activated by no , although both heme and no activation are lost after gel filtration . the mutant is only partially stimulated by no - independent activators such as protoporphyrin ix , fatty acids and allosteric activators , but the dtt - inhibited mutant shows activation by these reagents . intracellular mutant sgc displays a remarkably high level of cgmp synthesis , which is also not affected by nitric oxide . based on the properties of this constitutively active αβ cys105 enzyme a revision of the functional role of the his105 residue is proposed . the mechanism of sgc activation by no is discussed . reagents . hemin , grace media , fbs and imidazole were purchased from sigma . the no donor 3 -( 2 - hydroxyl - 1 - methyl - 2nitrosohydrazino )- n - methyl - 1 - propanamine ( noc - 7 ) was from calbiochem . the 3 -( 5 ′- hydroxymethyl - 2 ′ furyl )- 1 - benzyl - indazole ( yc - 1 ) activator was from alexis co . [ α 32 p ] gtp was from nen . cdna &# 39 ; s and expression vectors . the design and generation of baculoviruses expressing sgc α and β subunits and the generation of the site - directed substitution of βhis105 were described previously ( 26 ) ( appended hereto ). sgc expression and purification . wild type and αβ cys105 mutant sgc were expressed in sf9 cells as described previously ( 26 ). purification of sgc was performed as described earlier ( 26 ), the disclosure of which is hereby incorporated herein by reference , with the following modifications . cells were harvested 72 h post infection , resuspended in loading buffer ( 25 mm triethanolamine , ph 7 . 5 . 10 glycerol , 4 mm mgcl 2 and 1 mm phenylmethylsulfonyl fluoride , and 5 μg / ml each of pepstatin a , leupeptin , aprotinin , and chymostatin ,) and lysed by sonication . the lysate was subjected to 100 , 000 × g centrifugation for 1 h . the high - speed supernate was loaded on a 60 ml deae - sepharose ( amersham pharmacia biotech ) column and washed with 60 ml of loading buffer without protease inhibitors . the proteins were eluted with loading buffer containing 250 mm nacl and the eluate was directly loaded on a 30 ml his - bind resin ( novagen ) column . the column was washed with 60 ml of loading buffer followed by 60 ml of loading buffer with 45 mm imidazole . the enzyme was eluted with 175 mm imidazole and 2 ml fractions were collected . the absorbance spectra ( wild type enzyme ) and activity ( both wild type and αβ cys105 mutant ) in the elution fractions were determined and the positive fractions were pooled for further studies . the enzyme at this stage was approximately 90 – 95 % pure . to remove imidazole , sgc - containing fractions were diluted 3 fold with 25 mm triethanolamine ph 7 . 5 , loaded on a 2 ml hi - trap deae - sepharose column ( amersham pharmacia biotech ), washed with 10 ml of loading buffer and eluted with 25 mm triethanolamine ph 7 . 5 , 250 mm nacl , 10 glycerol , 4 mm mgcl 2 , 0 . 5 mm edta , 0 . 5 mm egta . the enzyme obtained at this stage ( 95 % or greater purity ) was used for experiments . assay of sgc activity . enzyme activity was assayed by formation of [ 32 p ] cgmp from α [ 32 p ] gtp at 37 ° c . in a final volume of 100 μl . incubation medium contained 50 mm triethanolamine - hcl buffer ( ph 7 . 4 ), 1 mm 3 - isobutyl - 1 - methylxanthine ( ibmx ), 1 mg / ml bsa . 1 mm cgmp , 3 mm mgcl 2 , 0 . 05 mg / ml creatine phosphokinase , 5 mm creatine phosphate , 0 . 1 mm egta . 200 μm gtp ( about 10 . 000 cpm / pmol ). the reaction was started by addition of the substrate ( gtp ) with or without activators . 1 mm dtt , gsh or cysteine were added to the incubation medium before the addition of gtp . thiol concentration varied in some experiments as indicated . samples containing 0 . 2 μg sgc were incubated for 10 or 15 min and the reaction was stopped by addition of 500 μl 150 mm zinc acetate and 500 μl 180 mm sodium carbonate . the pellet of zinc carbonate containing most of the unreacted gtp was removed by centrifugation and the supernatant fractions were loaded onto columns filled with 1 g of neutral alumina . columns were then eluted with 10 ml 0 . 1 m tris - hcl buffer ( ph 7 . 5 ) and cherenkov radiation in flow through plus eluate was counted in a lkb liquid scintillation spectrometer . samples incubated in the absence of sgc were used as a negative control . the concentration of dimethylsulfoxide ( dmso ) used as a vehicle for yc - 1 was not higher than 0 . 1 and had no effect on sgc activity as determined previously . uv - vis spectroscopy . all absorbance measurements were recorded with a dual - beam cecil 9500 spectrophotometer at 25 ° c . to monitor the heme reconstitution of the αβ cys105 mutant , 2 μm αβ cys105 sgc in 25 mm triethanolamine ph 7 . 5 , 10 % glycerol , 250 mm nacl , 4 mm mgcl 2 , 0 . 5 mm edta and 0 . 5 mm egta were treated with 2 mm dtt for 15 min at room temperature . hemin stock solution ( 5 mm ) was prepared in dmso and was reduced by dilution in 25 mm tea , ph 7 . 5 containing 5 mm dtt to a working solution of 500 μm . the reduced heme was kept under argon in a gas - tight vial during the reconstitution . identical amounts of reduced hemin ( between 0 . 1 μm to 15 μm were added with a gas - tight syringe to both sample and reference cuvettes , which contained 25 mm triethanolamine ph 7 . 5 , 10 % glycerol , 250 mm nacl , 4 mm mgcl 2 , 0 . 5 mm edta , 0 . 5 mm egta and 2 mm dtt . difference spectra were recorded between 370 and 600 nm with a speed of 200 nm / min . to measure the effects of no on the spectra of reconstituted αβ cys105 enzyme , 50 μm noc - 7 was added to both sample and reference cuvettes and spectra recorded 15 min later . assay of cgmp accumulation in intact cells . 48 hours postinfection sf9 cells expressing either wild type or αβ cys105 mutant sgc were washed twice with dulbecco &# 39 ; s pbs and preincubated for 10 min in pbs with 0 . 5 mm ibmx in 50 μl final volume of 107 cell / ml cell suspension . after this , 1 mm snp or vehicle was added and the cells are incubated for an additional 5 min at 37 ° c . the reaction was terminated by addition of 50 μl of 1 m perchloric acid and cgmp was extracted on ice for 1 h . the extract was centrifuged , neutralized with 2m k 2 co 3 and used for cgmp determination by radioimmunoassay ( 9 , 27 ). the pellet was dissolved in 0 . 1 m naoh and used for protein assay by the method of lowry ( 28 ). sds - page and western blot . purified sgc was separated on 7 . 5 sds - page as described previously ( 29 ) and transferred to immobilon ™- p membrane ( millipore corp . bedford , mass .) according to manufacturer &# 39 ; s protocol . immunodetection of non - tagged ( 3 subunit was performed using a 1 : 1000 dilution of polyclonal rabbit antibodies raised against β subunit of human sgc ( 12 ). hexahistidine - tagged a subunit was detected by using 1 : 2000 dilution of monoclonal anti - hexahistidine antibodies ( quiagen ). blots were developed using the ecl detection system ( amersham pharmacia biotech ) according to the manufacturer &# 39 ; s protocol . coomassie blue r250 staining of sds - page gels was performed as described previously ( 29 ). sensitivity of the mutant αβ cys105 enzyme to dtt was determined , and the results are shown in fig1 a , together with results for the wild type enzyme . the activity of purified αβ cys105 or wild type enzymes was measured with 1 mm dtt ( open bars ) or in the absence of any thiols ( solid bars ). the activity of enzyme was measured with 100 μm snp or without snp ( basal ) as described in materials and methods to test the effects of cystamine on sgc , the enzyme was first preincubated for 5 min at room temperature with 1 mm cystamine in the presence or absence of 1 mm dtt and then the activity was measured in the presence of 1 mm cystamine . referring now to the graph shown in fig1 b , the effects of dtt and gsh on the αβ cys105 were compared . purified αβ cys105 mutant enzyme was preincubated with reaction buffer ( see material and methods ) containing indicated concentrations of gsh ( opened squares , dotted line ) or dtt ( solid diamonds , solid line ) for 10 min at room temperature and then tested for activity . one sample ( opened triangle ) was treated with 10 mm gsh , then supplemented with 1 mm dtt before the activity was tested . the data are normalized to the sample without any thiols ( specific activity 1 ± 0 . 1 pmol / min / mg ) which is defined as 100 . arrow indicates the decrease in activity after the addition of dtt to gsh - treated enzyme . data representative of 5 independent experiments with similar results performed in triplicates are shown . values are shown as means ± s . d . the results of these tests , demonstrating that the mutant αβ cys105 enzyme has a high activity inhibited by dtt , but not gsh , are discussed in the results , below . studies were carried out to determine the effects of the allosteric activator yc - 1 , and the results are shown in fig2 a – b . the activity of αβ cys105 ( solid bars ) and wild type enzyme ( opened bars ) preincubated with ( b ) or without ( a ) 1 mm dtt was tested in the presence of vehicle ( dmso ), 100 μm yc - 1 , 100 μm snp , or both yc - 1 and snp as described in material and methods . the numbers above bars indicate the fold stimulation versus vehicle - treated enzyme . data representative of 3 independent experiments with similar results performed in triplicates are shown . values are shown as means ± s . d . the experimental data shows that yc - 1 activates the αβ cys105 mutant more effectively in the presence of dtt ( fig2 b ) than without ( fig2 a ), also discussed below in the results . the activity of the purified αβ cys105 enzyme was measured in the presence of increasing concentrations of aa with ( opened triangles , dotted line ) or without ( solid diamonds , solid line ) 1 mm dtt . the data in each treatment group are normalized to sample without aa , which is defined as 100 . data representative of 3 independent experiments with similar results performed in triplicates are shown in fig3 . values are shown as means ± s . d . specific activity of the dtt - treated enzyme without aa in this experiment was 0 . 13 ± 0 . 03 μmol / min / mg , and the specific activity of non - treated enzyme was 1 . 14 ± 0 . 05 μmol / min / mg . these results , which are discussed below , reveal that aa activates αβ cys105 mutant sgc more effectively in the presence of dtt than in its absence . the activity of the purified αβ cys105 enzyme was measured in the absence ( opened bars ) or presence of ( solid bars ) 1 μm protoporphyrin . before addition of ppix the enzymes were treated with 1 mm dtt or treated with vehicle . the data in each treatment pair are normalized to sample without ppix assumed as 100 . the numbers above bars indicate the specific activity of the sample in μmol / min / mg . data representative of 3 independent experiments with similar results performed in triplicates are shown in fig4 . values are shown as means ± s . d . ppix - dependent activation of wild type and αβ cys105 mutant enzymes is discussed below in results . heme reconstitution of αβ cys105 enzyme and snp or yc - 1 stimulation activation by 100 μm yc - 1 and / or 100 μm snp was tested on a dtt - treated heme reconstituted αβ cys105 enzyme . snp stimulation : mutant αβ cys105 enzyme was incubated with ( fig5 a , open bars ) or without ( fig5 a , solid bars ) 1 mm dtt for 15 min at room temperature and reconstituted with 1 μm hemin reduced as described in materials and methods . the activity of the reconstituted enzyme was tested in the presence or absence of 100 μm snp . one sample was reconstituted with 10 μm hemin , then subjected to gel filtration through a hitrap desalting column ( amersham pharmacia biotech ) and activity was tested in the presence of 100 μm snp ( after gf ). allosteric activation : the numbers above bars indicate the specific activity ( fig5 a pmole / mg / min ) or fold stimulation versus the basal activity of heme - deficient αβ cys105 enzyme ( fig5 b ). data representative of 4 ( fig5 a ) or 3 ( fig5 b ) independent experiments with similar results performed in triplicates are shown . values are shown as means ± s . d . absorption spectra of reconstituted enzyme are shown in fig5 c , d . difference spectra of 2 μm heme - deficient dtt - treated αβ cys105 enzyme ( no hemin ) and enzyme reconstituted with increasing concentrations of reduced hemin was recorded as described in materials and methods . spectra of αβ cys105 enzyme without hemin or reconstituted with 1 μm and 10 μm hemin are shown in fig5 c . the enzyme reconstituted with 10 μm hemin was subjected to gel filtration ( after gf ) or treated for 15 min with 50 μm noc - 7 ( with no ) and the spectra recorded , as shown in fig5 d . purified mutant αβ cys105 enzyme ( 5 μg ) was incubated with 2 mm dtt for 1 hour at room temperature or treated directly with non - reducing (− β - me , lane 1 ) or reducing (+ β - me , lanes 2 and 3 ) loading sds - page buffer . western blotting with anti - 6his and anti - β - sgc antibodies was performed to determine the position of hexahistidine - tagged α ( fig6 a , top panel ) and β subunits ( fig6 a , bottom panel ), respectively . in a separate experiment the mobility of the α and β subunits of αβ cys105 mutant ( fig6 b , lane 4 ) and wild type enzymes ( fig6 b , lane 5 ) was visualized by coomassie staining of 5 μg purified sgc separated by sds - page . as discussed in results , below , these data show that no disulfide bonds between mutant sgc subunits are formed . sf9 cells expressing wild type sgc ( fig7 , open bars ) or αβ cys105 sgc ( fig7 , solid bars ) were harvested , washed with pbs , and challenged with vehicle or 1 mm snp for 5 min in the presence of 0 . 5 mm ibmx . accumulated cgmp was extracted and quantified as described in materials and methods . data representative of 3 independent experiments with similar results performed in sextuplicates are shown in fig7 . values are shown as means ± s . d . the differences in intracellular accumulation of cgmp in sf9 cells expressing the wild type enzyme or the mutant form are discussed below . heme deficient αβ cys105 mutant sgc has different specific activity in the presence or absence of dtt . baculoviruses expressing the sgc α subunit with a c - terminal histidine tag and the β subunit carrying a his105 → cys substitution were used to generate the mutant αβ cys105 enzyme as described previously ( 26 ). the enzyme was heme deficient and did not respond to nitric oxide stimulation ( fig1 a ), which corroborates previous findings ( 26 ). typically , sgc activity in vitro is measured in the presence of at least 1 mm dtt . specific activity of the αβ cys105 enzyme in the presence of 1 mm dtt was 0 . 15 μmol / min / mg . with or without addition of sodium nitroprusside ( fig1 , white bars ). under similar conditions the wild type enzyme had a specific activity of 0 . 02 μmol / min / mg without no and 1 . 4 μmol / min / mg in the presence of sodium nitroprusside ( snp ). these values are in agreement with previous measurements ( 26 ). however , when the activity was measured in the absence of dtt , we observed a significant change in the catalytic properties of the αβ cys105 mutant . while the activity of the wild type enzyme did not change significantly without dtt ( fig1 , compare white and black bars ), the mutant αβ cys105 enzyme showed a significantly higher activity ( 0 . 95 μmol / min / mg , fig1 , compare white and black bars ) even in the absence of any sgc activators . the addition of snp did not change the activity of the αβ cys105 mutant , but significantly increased the activity of the wild type enzyme ( fig1 a ). to test whether the mutation affected the properties of the catalytic center of sgc the km for mg ++ - gtp substrate was measured . we found that the gtp - km for the αβ cys105 mutant was about 150 μm both in the presence or absence of dtt ( table 1 ). these values are well within the 65 – 450 μm range of gtp - km measured for the recombinant sgc ( 12 , 30 – 32 ), suggesting that the mutation did not affect the catalytic center . treatment of wild type sgc with thiol modifying agents including cystamine and cystine ( 33 , 34 ) have been shown to inhibit sgc , presumably by modification of some thiol groups essential for catalysis . we compared the inhibitory effect of cystamine on the αβ cys105 mutant and wild type enzymes ( fig1 a ). in corroboration with previous reports ( 33 ), wild type sgc was inhibited by cystamine and this inhibition was reversed in the presence of dtt . the mutant αβ cys105 enzyme exhibited a similar sensitivity to cystamine , but was less sensitive in the presence of dtt , supporting the conclusion that the catalytic sites of the wild type and αβ cys105 enzymes have similar properties . thus , the his105 → cys substitution significantly increased the activity of the αβ cys105 mutant not through changes in the catalytic center , but rather by affecting the mechanism of sgc regulation . analysis of various reducing agents indicated that the catalytic properties of the mutant αβ cys105 enzyme depend on the structure of the thiol used . concentration - response measurements indicate that an inhibitory effect of dtt was prominent at concentrations higher than 1 mm ( fig1 b ). on the contrary , when glutathione was used as the reducing agent , a slight stimulatory effect of gsh at a concentration above 1 mm was observed . in the presence of gsh , the mutant αβ cys105 enzyme had a specific activity of up to 2 μmol / min / mg ( fig1 b ). this stimulatory effect was abolished when 1 mm dtt was administered to the gsh - treated enzyme . the αβ cys105 mutant exhibited a higher stimulation by allosteric regulators in the presence of dtt . a group of structurally related compounds are known to activate the heme competent sgc without changing the absorbance spectrum of the heme moiety . the allosteric regulator yc - 1 and structurally related pyrazolopyridine bay - 41 - 2272 ( 35 , 36 ) are members of this family . we previously demonstrated that removal of the heme moiety from sgc due to his105 → cys substitution only partially affected the activation of the enzyme by allosteric activators ( 26 ). this suggested that activation of sgc by allosteric regulators is both heme - dependent and heme - independent . we found that yc - 1 activation of the αβ cys105 mutant is affected by dtt . the αβ cys105 enzyme preserved some activation by yc - 1 and was activated up to 3 - fold in the presence of dtt ( fig2 , solid bars ). however , in dtt - free conditions the activity of the αβ cys105 mutant increased only by 30 with yc - 1 activation . the extent of yc - 1 stimulation of the mutant αβ cys105 enzyme was not affected by snp . the wild type enzyme was more efficiently stimulated by yc - 1 , displaying 11 - fold stimulation and a specific activity of 0 . 15 μmol / min / mg ( fig2 ). snp enhanced yc - 1 dependent stimulation of the wild type enzyme , but not of the mutant αβ cys105 enzyme . identical effects on the mutant and wild type enzyme were observed when 2 μm bay - 41 - 2272 was used instead of 100 μm yc - 1 ( data not shown ), concentrations that are maximally effective ( 35 , 37 ). activation of the mutant αβ cys105 enzyme by arachidonic acids . some unsaturated fatty acids , such as arachidonic acid , activate wild type sgc independent of nitric oxide and heme ( 38 ). the mutant αβ cys105 enzyme was tested to determine if it preserved these properties . in the absence of dtt , the high activity of the αβ cys105 enzyme was only modestly stimulated by arachidonic acid ( fig3 ). however , in the presence of dtt , arachidonic acid stimulated the mutant αβ cys105 enzyme and at 1 mm concentration stimulation was 3 . 5 fold with a 0 . 5 ( μmol / min / mg activity . wild type enzyme was also stimulated by arachidonic acid with a maximum 3 fold activation ( data not shown ), which was not affected by dtt . protoporphyrin ix activates the αβ cys105 mutant more efficiently in the presence of dtt . as the αβ cys105 mutant is heme - deficient , it was decided to test whether the replacement of histidine 105 disrupted only the coordinating bond or induced more profound changes in the heme - binding domain . protoporphyrin ix ( ppix ) has been shown to effectively stimulate sgc ( 39 ), especially the heme - deficient sgc ( 40 ). in the present investigation , the effect of ppix on the αβ cys105 enzyme was tested . in the absence of dtt , 1 μm ppix stimulated basal activity of the αβ cys105 mutant only by 80 to a specific activity of 2 . 2 μmol cgmp / min / mg ( fig4 ). however , in the presence of dtt , the inhibited αβ cys105 enzyme was more receptive to activation by ppix and exhibited a 5 . 4 - fold stimulation from 0 . 13 μmol / min / mg to 0 . 70 μmol / min / mg activity at 1 μm ppix . for comparison , ppix - dependent activation of the heme - containing wild type enzyme resulted in similar activity in the presence or absence of dtt , 0 . 36 and 0 . 31 μmol / min / mg , respectively ( fig4 ). wild type enzyme also showed a higher fold stimulation by ppix in the presence of dtt , due to a slight decrease in the basal activity in the presence of dtt ( 0 . 02 umol / min / mg with dtt vs . 0 . 05 μmol / min / mg without dtt ). thus , the αβ cys105 mutant could be activated by ppix in the presence of dtt , suggesting that the mutation did not irreversibly disturb the conformation of the heme - binding domain . since ppix activation of the αβ cys105 mutant suggested that the heme - binding domain retained its properties to bind the protoporphyrin moiety , the αβ cys105 mutant was tested to determine whether it can be reconstituted with heme . in the absence of dtt , even 1 μm hemin did not confer any sensitivity to snp ( fig5 a ). higher concentrations of hemin ( 10 μm ) also did not have any significant effect ( data not shown ). however , in the presence of 1 mm dtt , the αβ cys105 mutant partially restored its no - activation upon addition of hemin . in view of these data , it is suggested that the heme domain of the αβ cys105 mutant is capable of accepting heme , which is properly oriented and can bind nitric oxide to stimulate the enzyme . however , the complex of the αβ cys105 enzyme and heme is not stable . the reconstituted enzyme lost its heme and no activation after the reconstitution buffer was removed by gel filtration ( fig5 a ). no stimulation was not detected even when the activity was measured immediately after chromatography . the reconstitution of the αβ cys105 mutant with heme did not change the extent of activation by yc - 1 ( fig5 b ). however , the heme - reconstituted αβ cys105 mutant demonstrated a greater effect of yc - 1 plus no stimulation ( fig5 b ), exhibiting at least partial restoration of this catalytic property of sgc . similar data were obtained , when bay41 - 2272 was used as the allosteric activator ( data not shown ). catalytic data correlated well with spectroscopic studies of the αβ cys105 mutant . addition of reduced hemin to the dtt - treated αβ cys105 enzyme resulted in the appearance of a soret peak ( fig5 c ). however , it should be noted that the maximum of the observed soret peak of the reconstituted αβ cys105 mutant was at 417 nm vs . 431 nm , characteristic for wild type sgc . hemin binding to the αβ cys105 mutant was saturated at 13 - fold molar excess of reduced hemin ( data not shown ), suggesting that interaction between the heme prosthetic group and the enzyme heme pocket is weak . in complete agreement with the activity data ( fig5 a ), the soret peak of the bound heme disappeared after the reconstituted enzyme was passed through a gel filtration column ( fig5 c ). no disulfide bonds between α and β subunits of the αβ cys105 enzyme . as the activity of the αβ cys105 mutant is strongly affected by dtt , a reduction of a putative disulfide bond or a mixed thiol is implicated . a disulfide bond between two subunits of the enzyme , or between two distant cysteine residues of the same subunit will affect the mobility of these subunits on sds - page under non - reducing conditions . the mobililty of the α and β subunits of the αβ cys105 mutant and wild type enzyme in both reducing and non - reducing sds - page was compared ( fig6 ). mobility of both the α and β subunits of the αβ cys105 enzyme was not affected by the treatment with dtt or presence of β - mercaptoethanol in the sds - page loading buffer and was no different from the wild type subunits . thus , no disulfide bonds between the subunits , or distal intramolecular bonds were apparent . in view of these results , it is suggested that sensitivity to dtt is due to the formation of either some close range disulfide bonds , or direct modification of sgc thiols by oxidation or formation of mixed thiols with small molecular thiols , such as cysteine or glutathione . intracellular αβ cys105 enzyme is heme - deficient and more active than no - stimulated wild type enzyme . as the properties of the αβ cys105 mutant are affected differently by various thiols , it was decided to test the activity of the αβ cys105 enzyme in intact cells . moreover , since the αβ cys105 mutant could be transiently reconstituted in vitro with heme ( fig5 ), it was also tested whether intracellular conditions are more favorable for the formation of heme competent enzyme than conditions in vitro . intracellular cgmp accumulation was examined in sf9 cells expressing the αβ cys105 mutant or the wild type enzymes ( fig7 ). addition of 1 mm snp to the wild type sf9 / αβ cells increased the rate of cgmp accumulation almost four - fold from 0 . 9 nmol / mg / 5 min to 3 . 6 nmol / mg / 5 min . it was observed that snp did not affect the accumulation of cgmp in the sf9 cells expressing the αβ cys105 mutant ( fig7 ), thus it is proposed that the intracellular αβ cys105 mutant is also heme - deficient . however , the basal level of cgmp accumulation in the sf9 / αβ cys105 cells was about ten times higher ( 33 nmol / mg / 5 min ) than the rate in no - activated sf9 cells overexpressing wild type enzyme , although the level of expression was the same ( data not shown ). binding of nitric oxide to fe ++ in the heme moiety of soluble guanylyl cyclase is the central event leading to the stimulation of sgc . no induced changes in the interaction between heme and the coordinating histidine 105 residue result in conformational changes which significantly increase the enzyme &# 39 ; s specific activity . in previous studies of recombinant wild type human sgc , we ( 12 , 26 ) and others ( 41 ) found that the wild type enzyme displayed a specific activity of ˜ 1 . 5 μmol / min / mg after exposure to no . in the present disclosure , an αβ cys105 mutant variant of the human sgc is described that lacks both the heme moiety and the heme coordinating histidine 105 residue , and which exhibits high constitutive activity similar to activity no - stimulated wild type enzyme . the substitution of the histidine 105 residue by a cysteine resulted in a heme deficient sgc , as demonstrated by the lack of a soret peak characteristic for sgc ( fig5 c ). in the absence of thiols , the αβ cys105 enzyme has a high specific activity of 1 . 2 ± 0 . 3 nmol / mg / min ( n = 4 independent purifications ). the specific activity of the αβ cys105 mutant varied somewhat with preparation , but this activity was always comparable to the activity of no - induced wild type enzyme as shown in fig1 . high activity of the αβ cys105 enzyme may be explained by changes in the catalytic center or in the regulatory domain . however , the αβ cys105 mutant has a gtp - km similar to previously measured gtp - km for the wild type enzymes ( table 1 ) and displays identical to wild type enzyme susceptibility to cystamine inhibition , which is attributed to inhibition of the catalytic function of sgc . these findings suggest that the function of the catalytic center was not affected by the mutation and cannot account for the marked constitutive increase in αβ cys105 enzyme activity . since the substituted his 105 is not part of the catalytic domain , but has a demonstrated role in heme coordination , we postulate that the observed αβ cys105 mutant phenotype reflects changes in the function of the regulatory domain . wild type enzyme can be stimulated in a no - independent manner by protoporphyrin ix , arachidonic acid or with allosteric activators like yc - 1 and bay41 - like compounds . without dtt the activity of αβ cys105 enzyme was enhanced modestly by no - independent activators of sgc , such as protoporphyrin ix , arachidonic acid or yc - 1 ( fig2 , 3 and 4 ). the mutation did not affect the binding sites of any of the tested no - independent activators . the exposure of highly active αβ cys105 enzyme to millimolar concentrations of dtt not only resulted in a 5 – 7 fold decrease in enzymatic activity , but also restored the mutant &# 39 ; s sensitivity to all no - independent stimulators . this restoration indicates that the mutation did not directly affect the structural elements necessary for the binding or activation by these no - independent agents . it appears that the αβ cys105 mutant is purified in a constitutively activated state that is not sensitive to additional stimulation . such properties are similar to the properties of the no - stimulated wild type enzyme , which exhibits a blunted response to allosteric regulators at maximal no activation ( 37 , 42 ). thus , it is proposed that the obtained mutant , although heme deficient and without a histidine 105 residue , achieved a similar conformation as the wild type enzyme stimulated by nitric oxide . measurements of cgmp accumulation in sf9 cells ( fig7 ) also demonstrate that the αβ cys105 enzyme is constitutively highly active . an earlier report describing the substitution of the histidine 105 residue of the bovine β subunit with phenylalanine ( 21 , 40 ) clearly demonstrated the heme - coordinating role of the his105 residue . we substituted the heme - coordinating histidine with a cysteine residue , which is also known to coordinate heme , e . g . nitric oxide synthase ( 43 ). as summarized in table 1 , the human sgc mutant carrying the βhis105 → cys substitution shares many properties with the bovine sgc with ( βhis105 → phe substitution . both αβ phe105 and αβ cys105 enzymes are heme - deficient and do not respond to no stimulation . however , after heme reconstitution both αβ phe105 and αβ cys105 mutants are activated by no 2 . 7 - ( 40 ) and 3 . 6 - fola ( fig5 a and b ), respectively . the soret bands for both heme reconstituted mutants were shifted in comparison to the wild type enzyme ( λ max = 431 nm ). αβ cys105 enzyme had a soret peak at 417 nm ( fig5 c ), while the αβ phe105 sgc had a maximum at 400 nm ( 40 ). spectral differences of these mutants and wild type enzyme are probably due to different heme coordinating residues or lack of such coordination . although there are many similarities between these two mutants , there are also some significant differences . αβ cys105 displayed a high basal activity of 1 . 2 μmol / min / mg , while the αβ phe105 had a substantially lower activity of only about 40 nmol / min / mg ( 40 ). even the dtt - inhibited αβ cys105 had a higher activity than the αβ phe105 enzyme ( table 1 ). another important difference between these mutants is activation by protoporphyrin ix . αβ phe105 enzyme was able to bind ppix , as demonstrated by spectral studies , but was not stimulated by ppix ( 40 ). on the contrary , the αβ cys105 mutant , presented in this report , was stimulated by ppix , especially after addition of dtt ( fig4 and table 1 ). different responses of these two mutants to protoporphyrin ix provides additional information about the mechanism of sgc activation . a previous model , based on spectroscopic data and supported by the properties of αβ phe105 enzyme ( 24 , 25 , 44 ), assumed that the release of the coordinating bond between his105 and heme or ppix insertion to heme - deficient enzyme allows the his105 residue to exert its stimulatory role . this model infers an indispensable role of the histidine 105 residue for the stimulation of sgc . however , the properties of the αβ cys105 mutant presented in the instant disclosure indicate that histidine 105 can be replaced by a structurally unrelated cysteine residue without affecting the ability of sgc to achieve a highly active conformation or be stimulated by protoporphyrin ix . the properties of the αβ cys105 mutant demonstrate that the his105 does not play a role in stimulating the enzyme . we propose that through the formation of a coordinating bond with heme it retains the regulatory domain in a “ restrictive ” state , which assures only low basal activity of the enzyme . a schematic representation of changes in the regulatory domain of wild type and αβ cys105 sgc is shown in fig8 . the parallelogram schematically represents the protoporphyrin portion of the heme group , while the box represents the regulatory domain . changes in the box height represent conformational changes in the regulatory domain . r - putative dtt - sensitive modification of cys105 or other cysteine residue from the regulatory domain . the binding of no to heme allows the transition from the “ restrictive ” conformation of the regulatory domain to a “ permissive ” conformation , which permits stimulation of the enzyme . under this model , the histidine 105 residue has an active role before binding of no , but plays a passive role after no - induced disruption of the heme - coordinating bond . the αβ cys105 mutant is constitutively found in such a “ permissive ” conformation ( fig8 ), which can be inhibited by dtt . the sensitivity of the αβ cys105 mutant to dtt is an important feature of this constitutively active mutant . the his105 → cys substitution introduced an additional thiol in the regulatory domain of sgc . dtt - dependent inhibition of the αβ cys105 enzyme suggests that a thiol modification is essential for constitutively supporting the mutant enzyme in this “ permissive ” conformation . analysis of the mobility of αβ cys105 sgc subunits indicated that no disulfide bonds between α and β subunits or between distant cysteine residues of the same subunit can be detected ( fig6 ). however , this study cannot exclude a close range disulfide bond formation . cys78 and cys214 of the β subunit were identified as residues important for heme binding ( 7 ) and are , most probably , exposed to the heme pocket and in close proximity to cys105 . formation of a disulfide bond between these residues might provide necessary structural changes in the regulatory domain to support a “ permissive ” conformation . alternatively , cys105 could form a mixed thiol with a glutathione or cysteine molecule also resulting in formation of a “ permissive ” conformation . finally , oxidation of cysteine 105 to sulfenic (— soh ) or sulfinic (— so 2 h ) acids may be the modification required for the “ permissive ” conformation . these modifications , schematically represented as “ r ” in fig8 could be reduced by dtt , resulting in the transition of αβcys 105 regulatory domain from a “ permissive ” to a “ dtt - attenuated ” conformation functionally similar to “ restrictive ” conformation of the wild type sgc . the constitutively active αβ cys105 mutant sgc described in this report could be useful to understand the mechanism of sgc activation . although a large body of evidence exists about changes in the heme moiety of the regulatory domain upon no induction , the mechanism that couples these events with the stimulation of enzymatic activity in the catalytic center is still not clear . an interaction between the regulatory domain and the catalytic center can be easily envisioned . however , the functional outcome of such interaction is not evident . the catalytic center can be activated by mechanisms similar to gsa or forskolin activation of adenylyl cyclase ( 45 , 46 ) or gcap activation of photoreceptor gc ( 47 ). gcap is known to regulate the function of the retinal membrane - bound guanylyl cyclase ( 55 , 56 ), and gsa is known to regulate adenylyl cyclase ( 57 ). in these cases , two cyclase homology domains necessary for the functional catalytic center are brought together by stimulatory molecules . alternatively , the putative interaction between the regulatory and catalytic domain can have an inhibitory effect , similar to the inhibitory role of the kinase homology domain ( khd ) of the membrane guanylyl cyclases with a single transmembrane domain ( 48 ). binding of the ligand to membrane gc results in the relief of inhibition of the catalytic center by khd ( 49 ). in view of the foregoing results and discussion , it is proposed that the histidine 105 residue of the β subunit plays a crucial role in maintaining the regulatory domain of sgc in a “ restrictive ” conformation . substitution of this residue with cysteine mimics the transition of the sgc regulatory domain into a “ permissive ” conformation . to our knowledge this is the first disclosure of a sgc enzyme that can be maintained in a stimulated state without the addition of stimulatory ligands such as no or allosteric regulators . ongoing studies of this mutant are expected to further elucidate the mechanism of sgc stimulation . without further elaboration , it is believed that one skilled in the art can , using the description herein , utilize the present invention to its fullest extent . preferred ways in which the compositions and experimental results described herein may be applied are as follows . these applications are intended to be representative or illustrative of other and various embodiments , and should not be construed as constraining the extent of this disclosure in any way whatsoever . small deletion probing of sgc regulatory domain in the context of βcys 105 subunit may permit the identification of functional determinant ( s ) responsible for sgc regulation . previously performed deletions ( 4 , 40 , 50 ) or insertions ( 12 ) in the n - terminal domains of sgc resulted in enzyme with only basal activity due to the loss of heme moiety . thus , deletion probing of regulatory determinants based on the wild type enzyme could result in heme - deficient enzyme and limit the application of this approach . the availability of the mutant α βcys 105 enzyme allows one to perform this analysis without the risk of losing the heme group . analysis of sgc enzymes carrying βcys105 subunit and α subunit containing various deletions in the regulatory domain will permit one to identify the regions in α subunit ( if any ), which will reduce high specific activity of the mutant α βcys 105 enzyme to levels similar to the basal activity of the wild type enzyme . for this purpose , the activity of purified αβ cys105 carrying the mentioned deletions or a cell lysate containing αβ cys105 enzyme with mentioned deletions will be determined and compared with the activity of the αβ cys105 enzyme . analogous studies could be performed for the enzymes carrying , in addition to his105 → cys substitution , various deletions in the regulatory domain in order to identify regulatory determinants in the β subunit . deletions which will only reduce the activity of the αβ cys105 enzyme , without total loss of ability to synthesize cgmp , will point to elements of sgc enzyme determining the activation process . identification of these determinants in α and / or β subunits will allow one to design new drugs specifically targeting these regulatory determinants . such studies could be carried in addition to crystallization of the described αβ cys105 mutant sgc with and without dtt . comparison of such crystal structures , may be a viable approach to determine structural determinants of sgc stimulation . deletion probing of the mutant αβ cys105 enzyme , and crystallographic studies may be carried out using methods and techniques as are known in the art ( 4 , 40 , 50 ). the constitutively active mutant may also be a useful reagent to screen for novel inhibitors of sgc . absence of heme moiety will insure that found inhibitors are not directed towards heme moiety , but towards other structural elements of the enzyme . screening assays are performed substantially as described in assay of sgc activity in materials and methods , modified to include an inhibitor , test compound or substance of interest . alternatively , any other suitable assay method capable of measuring the activity of the αβ cys105 mutant enzyme may be used , applying the knowledge and techniques that are generally known to those of skill in the art . for example , a screening procedure for identifying a heme - independent inhibitor of soluble guanylyl cyclase includes a ) obtaining purified αβ cys105 mutant soluble guanylyl cyclase enzyme or a cell lysate containing αβ cys105 mutant soluble guanylyl cyclase enzyme ; b ) assaying the purified enzyme or cell lysate for formation of cgmp from gtp in the presence of the test compound ; and c ) assaying the purified enzyme or cell lysate for formation of cgmp from gtp in the absence of the test compound . if desired , steps b ) and c ) may be carried out in the presence or absence of various activator ( s ). finally , the results from the foregoing steps are compared to determine whether the test compound inhibits cgmp production by the purified enzyme or cell lysate . the αβ cys105 mutant enzyme is also useful as a reagent for screening test compounds for heme independent activation of sgc . these assays are also carried out substantially as described above , except that the tests with and without the activator or test compound are compared to determine whether the compound enhances cgmp production by the purified enzyme or cell lysate . therapeutic treatment with αβ cys105 sgc , or the β subunit since sgc is known to be important to the body &# 39 ; s regulation of cardiovascular homeostasis , and to play a critical role in neurotransmission , sensory perception , and a variety of pathologies ( e . g ., high blood pressure , atherosclerosis , septic shock ), the αβ cys105 mutant soluble guanylyl cyclase , or its β cys105 subunit , may be employed to initiate , increase and / or sustain the intracellular production of cyclic gmp in a mammalian cell . the procedure may include administering the mutant sgc , or the β cys105 subunit , to the mammalian cells . additionally or alternatively , the procedure may include introducing into the cell an operative gene or coding region of the gene for both α and β cys105 subunits or for the mutant subunit alone . the mutant β subunit , is then constitutively expressed in the cell and cyclic gmp is produced as a result . a method of treating or preventing a mammalian pathophysiologic condition associated with cyclic gmp regulation of a cellular process may include causing the constitutive expression of αβ cys105 mutant sgc in a mammal in need of such treatment or prevention , to initiate , increase and / or sustain intracellular production of cgmp . this may be accomplished by delivering αβ cys105 mutant sgc enzyme , or the β cys105 subunit thereof , to at least one cell of the mammal , preferably a human . alternatively , the method of treating or preventing a mammalian pathophysiologic condition associated with cyclic gmp regulation of a cellular process may include inhibiting cgmp production by administering an inhibitor of soluble guanylyl cyclase that acts independently of the heme moiety of soluble guanylyl cyclase . techniques for in vitro and in vivo delivery of proteins are known to those of skill in the art . gene delivery of the mutant αβ cys105 sgc , or the β subunit only , may be beneficial in disorders where increased cgmp levels are desired . this may be accomplished by delivering operable genes of α or β cys105 sgc subunits , or at least an operable portion of the gene containing the β cys105 subunit , into at least one cell in the mammal . any number of gene delivery methods , which may include , but are not limited to , the administration of naked dna or cationic lipid - 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