Patent Application: US-64200291-A

Abstract:
a method of purifying calmodulin - dependent nitric oxide synthase provides a homogeneous preparation of the enzyme . the enzyme is used to raise antibodies which are a useful immunohistochemical reagent . the antibodies localize calmodulin - dependent nitric oxide synthase to a number of anatomical sites , including retina , intestine , adrenal gland , and vasculature . however , activated macrophages , which are known to possess a nitric oxide producing activity , do not display an immunoreactive protein of appropriate size on western blots using the antibodies . nucleotide sequences encoding calmodulin - dependent nitric oxide synthase indicate a novel sequence with a flavin binding site consensus sequence .

Description:
nitric oxide synthase ( ec # 1 . 14 . 23 ) from brain has been purified to homogeneity , and its properties determined . it has been used to generate antibodies which have been used to localize the enzyme immunohistochemically . in addition , a cdna encoding the nos enzyme has been isolated and its sequence determined . it is a finding of the present invention that nos requires calmodulin for its enzyme activity . this contrasts with reported requirements of nos isolated from activated macrophages , which require biopterins as a cofactor ( tayeh and marletta ; kwon et al .). macrophage - derived nitric oxide synthase has also been found to differ from that of brain - derived nos immunochemically . antisera raised against brain - derived nos do not detect a similar sized protein in extracts of activated macrophages . the same antisera do , however , detect nos in autonomic nerve fibers in the retina , in cell bodies and nerve fibers in the myenteric plexus of the intestine , in adrenal medulla , and in vascular endothelial cells . thus it appears that there are at least two types of nos proteins in mammalian tissues , one being calmodulin dependent and the other biopterin dependent . according to the present invention , one can purify calmodulin - dependent nos using column chromatography . specifically , it has been found that greater than one - thousand - fold purification can be achieved using an affinity chromatography column . nadph is a necessary cofactor for enzyme activity . if one employs a solid matrix containing an nadph moiety or an nadph analog , such as dextran blue , or 2 &# 39 ;, 5 &# 39 ;- adp agarose or 2 &# 39 ;, 5 &# 39 ;- adp sepharose , then the nos of the present invention binds to the matrix . it can be eluted using a soluble form of nadph or an analog thereof at a concentration of about 1 to about 10 mm . it is desirable that the preparation which is applied to the affinity chromatography column first be partially purified on an ion exchange column , such as , diethylaminoethyl ( deae ) cellulose . other ion exchange columns known in the art can also be used . the nos of the present invention binds to deae - cellulose and can be eluted with a sodium chloride gradient . the greatest peak of activity elutes with between about 70 mm and about 145 mm sodium chloride . combination of these two column chromatography steps on a cleared brain homogenate results in a homogeneous preparation , as determined both by silver staining of an sds / page - separated sample , as well as by western blotting . tissues which can be used as a source of calmodulin - dependent nos include brain , endothelial cells of blood vessels , and adrenal glands . in addition , recombinant host cells containing cdna clones of the nos gene can be used as a source of nos for purification according to the present invention . collection and processing of tissues can be done as is known in the art . typically tissues will be homogenized in buffers containing protease inhibitors . debris can be removed from the homogenate by centrifugation at about 20 , 000 × g for 15 minutes . stability of the enzyme is enhanced by storage in bovine serum albumin ( 1 mg / ml )/ 20 % ( vol / vol ) glycerol at - 70 ° c . stability is also enhanced in the presence of calmodulin . preparations of calmodulin - dependent nos can be obtained which are homogeneous , according to techniques described above . thus preparations having specific activities between about 500 and about 1000 nmol / mg / min are obtained . preparations obtained according to the purification methods of the present invention are substantially free of contaminating proteins . thus they are typically greater than 95 % free of proteins of the same species source as the tissue from which they are extracted . preferably , they are greater than 98 % free of other proteins of the same species source . using recombinant host cells to produce preparations of calmodulin - dependent nos will also readily produce preparations which are substantially free of proteins of the same species source . proteins which may be added back to a preparation to promote stability , such as calmodulin or bovine serum albumin , are not considered in the determination of purity . the presence of contaminating proteins can be determined using silver staining of polyacrylamide gels or western blotting . antibody preparations are made according to techniques which are well known in the art . both polyclonal and monoclonal antibodies are contemplated , production of both of which are well known . according to one method for obtaining a polyclonal antibody preparation , rabbits are immunized with a purified preparation of calmodulin - dependent nos , as described above . the antiserum will preferably be affinity - purified by incubation with purified nos and elution with 4m mgcl in 200 mm tris - hcl buffer ( ph 7 . 4 ). the eluate will desirably be dialysed against phosphate buffered saline with 0 . 1 % triton x100 . antibodies can be used for immunohistochemical localization of nos , or for quantitative assays on biological fluids or samples , such as in an enzyme - linked immunoadsorbent assay or radioimmunoassay . such assays can determine if a tissue is producing an abnormally high or low amount of nos . cdna molecules encoding calmodulin - dependent nos are provided . the coding sequence of the gene is shown in seq id no : 1 . whereas a particular nucleotide sequence is disclosed herein , other nos - coding sequences are also encompassed by the invention , such as those which hybridize to the disclosed sequence , due to a difference in species source , allelic variations , or mutations introduced in the course of genetic manipulations . thus other cdna molecules which code for a closely related nos are also contemplated . in addition , the cdna molecule need not be complete in order to be useful . a portion of the cdna can be used as a hybridization probe in order to quantitate mrna expression , for example . nucleotide probes are typically labeled with a detectable moiety such as a radioactive atom , or an enzyme . whereas the entire gene - coding sequence is about 4 kb , sequences above about 12 to 15 nucleotides can be useful as hybridization probes . the cdna sequence can also be used to hybridize to or amplify non - coding sequences . thus the cdna sequence can be used to isolate introns and regulatory regions important for expression in the body . portions of the disclosed sequence may also be used in polymerase chain reactions as primers . for example , primers can be used to amplify the nos gene to determine if a mutation is present . the polymerase chain reaction is well known in the art . this example describes the method by which nos activity was assayed . no synthase activity was measured by monitoring the conversion of [ 3 h ] arginine to [ 3 h ] citrulline . for routine assays , we added 25 μl of enzyme extract and 25 μl of 100 nm [ 3 h ] arginine to 100 μl of buffer containing 50 mm hepes ( ph 7 . 4 ), 1 mm nadph , 1 mm edta , 1 . 25 mm cacl 2 , 1mm dithiothreitol , and 10 μg of calmodulin per ml . after incubation for 5 min at 22 ° c ., assays were terminated with 2 ml of 20 mm hepes , ph 5 . 5 / 2 mm edta , and were applied to 1 - ml columns of dowex ag502x - 8 ( na + form ), which were eluted with 2 ml of water . [ 3 h ] citrulline was quantified by liquid scintillation spectroscopy of the 4 - ml flow - through . eighteen rat cerebella were homogenized in 100 ml of ice - cold buffer a [ 50 mm tris - hcl , ph 7 . 4 / 1 mm edta / antipain ( 10 mg / liter )/ leupeptin ( 10 mg / liter )/ soybean trypsin inhibitor ( 10 mg / liter )/ pepstatin ( 10 mg / liter )/ chymostatin ( 10 mg / liter )/ phenylmethylsulfonyl fluoride ( 100 mg / liter )], and all subsequent procedures were carried out at 4 ° c . the homogenate was centrifuged at 20 , 000 × g for 15 min , and the supernatant was loaded at 2 ml / min onto a 20 - ml column of diethylminoethyl ( deae ) equilibrated with buffer a . the column was washed with 50 ml of buffer a and eluted with a 100 - ml linear gradient of 0 - 400 mm nacl in buffer a . fractions ( 2 . 5 ml ) were assayed for enzyme activity . fractions containing the first peak of activity from the deae column ( see fig1 ) were pooled and added to 2 ml of 2 &# 39 ;, 5 &# 39 ;- adp agarose equilibrated in buffer b ( 10 mm tris - hcl , ph 7 . 4 / 1 mm edta / 5 mm 2 - mercaptoethanol ). after a 10 - min incubation , the suspension was poured into a fritted column , which was washed with 50 ml of buffer b with 0 . 5m nacl and then with 20 ml of buffer b alone . no synthase was eluted with 8 ml of buffer b containing 10 mm nadph . in our preliminary efforts to purify no synthase , we observed that enzymatic activity adheres to a deae column and can be eluted by 1m nacl . however , with gradient elution of nacl , enzymatic activity was not recovered in eluate fractions , suggesting the separation during purification of the enzyme from an important cofactor . since no formation requires ca 2 + , we speculated that calmodulin might be involved . addition of calmodulin to deae eluate fractions restores enzyme activity . no synthase activity elutes in one sharp , major peak followed by a smaller peak of activity , which is observed reproducibly in multiple experiments . for purification of no synthase , we have focused on the first , major peak of enzyme activity eluting from the deae column , which provides a 5 . 6 - fold purification of enzyme activity with 60 % recovery ( table 1 , fig1 ). further purification utilized affinity chromatography with a 2 &# 39 ;, 5 &# 39 ;- adp - linked agarose column . no synthase activity adheres to this column and is not eluted by 0 . 5m nacl . after the 0 . 5m nacl wash , no synthase activity can be eluted with 10 mm nadph , providing a 1000 - fold purification of enzyme activity in this step . the overall purification of no synthase utilizing two steps , deae chromatography and 2 &# 39 ;, 5 &# 39 ;- adp affinity chromatography , affords a 6000 - fold purification of enzyme activity with 30 % recovery . the purified enzyme eluting from the adp affinity column appears homogeneous , constituting a single band on sds / page ( fig3 ). the molecular mass of this band is ≃ 150 kda . to estimate the molecular mass of the native enzyme , we conducted gel filtration chromatography with a superose - 6 column . no synthase activity of the purified enzyme emerges from the column as a single peak coincident with the peak of protein , with an apparent molecular mass of 200 kda , similar to the elution of β - amylase whose molecular mass is 200 kda . thus , purified no synthase appears to be a monomer . table 1______________________________________purification of no synthase specific activity , protein recovery nmol - mg . sup .- 1 purification ,- fraction μg % min . sup .- 1 fold______________________________________15 , 000 × g 180 , 000 100 0 . 16 1supernatantdeae eluate 20 , 000 60 0 . 9 5 . 62 &# 39 ;, 5 &# 39 ;- adp 9 . 0 30 960 6000agarose eluate______________________________________ enzyme was purified and fractions were assayed as described . data presented are from a typical purification , which was repeated five times with closely similar results . this example shows the effects of varying concentrations of calcium , calmodulin and nadph on no synthase activity . calmodulin is an extremely potent stimulator of no synthase activity ( fig2 a ). in the presence of 1 μm ca 2 + , 50 % of maximal stimulation of enzyme activity is apparent with ≃ 200 nm ca 2 + with maximal enhancement of activity observed at 1 μm ca 2 + and some reduction in activity at concentrations exceeding 100 μm ca 2 + . in the absence of nadph , ca 2 + fails to stimulate no synthase activity . this example demonstrates the effect of calmodulin antagonists on no synthase activity . in crude cerebellar supernatant preparations , calmodulin is not required to demonstrate enzyme activity and added calmodulin ( 1 μm ) has no influence on enzyme activity . however , trifluoperazine , a calmodulin antagonist , inhibits enzyme activity of crude preparations with an ic 50 for trifluoperazine in crude supernatant preparations of vascular endothelial tissue , indicating that regulation of the endothelial and brain enzymes by calmodulin is similar . trifluoperazine exerts multiple effects such as blockade of dopamine receptors . the drugs w - 5 [ n - 6 - aminohexyl )- 1 - naphthalenesulfonamide ] and w - 13 [ n - 4 - aminobutyl )- 5 - chloro - 2 - naphthalenesulfonamide ] are more selective calmodulin antagonists . in crude brain supernatant preparations , w - 5 and w - 13 inhibit no synthase activity with respective ic 50 values of 50 and 25 μm . the purified enzyme has high affinity for arginine with a k m of ≃ 2 μm , similar to what we observed previously in crude supernatant preparations . the v max of the purified enzyme is ≃ 1 μmol per mg of protein per min , similar to the v max values for other nadph - requiring oxidative enzymes ( table 2 ). the k i for mearg inhibition of no synthase activity in the purified enzyme is ≃ 1 . 4 μm , similar to values we observed previously in crude preparations . the ec 50 for calmodulin enhancement of enzyme activity in the pure enzyme , 10 nm , is similar to the value observed in crude preparations . also , the ec 50 for calcium stimulation of the purified enzyme is the same in the pure and crude preparations . the purified enzyme is unstable . when stored at 0 ° c ., 50 % of the enzyme activity is lost in 2 hr , whereas the crude supernatant preparation loses 50 % activity at 0 ° c . in 2 days . stability is enhanced by storing the enzyme in bovine serum albumin ( 1 mg / ml )/ 20 % ( vol / vol ) glycerol at - 70 ° c . when stored in this way , the enzyme loses & lt ; 50 % activity in 7 days . table 2______________________________________properties of no synthase______________________________________arginine , k . sub . m 1 . 5 μmv . sub . max 0 . 96 μmol per min per mg of proteinmearg , k . sub . i 1 . 4 μmca . sup . 2 +, ec . sub . 50 200 nmcalmodulin , ec . sub . 50 10 nmcalmodulin antagonists , ic . sub . 50 trifluoperazine 10 μmw - 5 25 μmw - 13 70 μm______________________________________ purified enzyme was assayed as described . values are means of two to six determinations , which varied by & lt ; 20 %. this example demonstrates the production and use of antibodies which are immunospecific for calmodulin - dependent no synthase . antibodies were raised in two rabbits and affinity purified with purified nos . antiserum was incubated with 50 mg purified antigen ( immobilized in nitrocellulose after transfer from an sds - polyacrylmide gel ), eluted with 4m mgcl in 200 mm tris - hcl ( ph 7 . 4 ) buffer , and dialyzed against phosphate buffered saline with 0 . 1 % tritonx100 . to ensure that the antiserum interacts with catalytically active no synthase ( nos , ec1 . 14 . 23 ), we conducted immunoprecipitation experiments . the antiserum precipitates nos activity in cerebellar homogenates , whether measured by the conversion of arginine to citrulline or by the formation of no , with half - maximal precipitation at 10 μg ml - 1 antiserum igg . in western blot analysis the antiserum interacts with a single band of relative molecular mass 150 , 000 ( m r 150k ), the same as purified nos . the density of the band varies amongst various brain regions and subdivisions of pituitary and adrenal glands in close parallel with the regional distribution of nos catalytic activity . antisera from two rabbits and affinity - purified nos antibodies provide identical distributions by western blot analysis and by immunohistochemical staining . immunoreactivity is absent with pre - immune serum or with serum preabsorbed with purified nos . this example demonstrates the molecular cloning of no synthase coding sequences . the nitric oxide ( no ) synthase enzyme was purified to homogeneity as described in example 1 . the purified enzyme was run on an sds gel and transferred to nitrocellulose . trypsin was added to the nitrocellulose paper containing the enzyme in order to liberate peptide fragments . the peptide fragments were purified by reverse phase hplc . the peptides were sequenced with an automated peptide sequencer . about 15 peptides were sequenced . the above procedures were performed in order to obtain peptides whose sequence could be used as a basis for obtaining oligonucleotide probes for molecular cloning . in molecular cloning one frequently prepares a degenerate oligonucleotide based on the amino acid sequence of peptide fragments . for many proteins a mixture of degenerate oligonucleotide probes contains enough correct nucleotide sequence so that hybridization with the cdna representing the protein to be cloned is possible . when such procedures were carried out for no synthase , no appropriate clones could be identified . accordingly , a new technique was developed in which a non - degenerate oligonucleotide probe was generated by polymerase chain reaction ( pcr ). this was done by taking two of the longest peptides , of 18 and 17 amino acids , and constructing degenerate oligonucleotide primers of 21 nucleotides based on the 7 amino acids at the carboxyl and amino termini of each of the two peptides . these oligonucleotides were employed in a pcr reaction to construct two non - degenerate oligonucleotide primers . these two non - degenerate oligonucleotide primers were employed in a further pcr reaction to obtain a large polynucleotide probe . there was no way of knowing a priori whether one would obtain an appropriate probe , as the two oligonucleotide primers employed might have been located too far apart in the sequence of the no synthase to permit amplification . fortunately , we were able to obtain a 600 base pair amplified product for use as a polynucleotide probe to screen molecular clones . the 600 bp polynucleotide probe was random prime labeled with 32 p - atp and used to screen a commercially obtained rat brain cdna library from stratagene . eight overlapping independent clones were isolated and sequenced by double - stranded dideoxy sequencing . this procedure revealed a 4 kb open reading frame coding for a protein of about 150 kd , which corresponds to the molecular weight of no synthase which we had previously purified . the deduced amino acid sequence has been examined by computer program for homology with other known families of proteins . no major homology has been observed . a flavin binding site consensus sequence has been observed . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 2 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 5108 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna ( iii ) hypothetical : n ( iv ) anti - sense : n ( vi ) original source :( a ) organism : rattus rattus ( f ) tissue type : brain ( ix ) feature :( a ) name / key : cds ( b ) location : 400 .. 4686 ( d ) other information :( xi ) sequence description : seq id no : 1 : 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865agtactggacccctggccaatgtgaggttctcagtgttcggcctcggc3054serthrglyproleualaasnvalargpheservalpheglyleugly870875880 885tctcgggcgtacccccacttctgtgcctttgggcatgcggtggacacc3102serargalatyrprohisphecysalapheglyhisalavalaspthr890895 900ctcctggaggaactgggaggggagaggattctgaagatgagggagggg3150leuleuglugluleuglyglygluargileleulysmetargglugly905910 915gatgagctttgcggacaggaagaagctttcaggacctgggccaagaaa3198aspgluleucysglyglngluglualapheargthrtrpalalyslys920925 930gtcttcaaggcagcctgtgatgtgttctgcgtgggggatgacgtcaac3246valphelysalaalacysaspvalphecysvalglyaspaspvalasn935940945a tcgagaagccgaacaactccctcattagcaatgaccgaagctggaag3294ileglulysproasnasnserleuileserasnaspargsertrplys95095596096 5aggaacaagttccgcctcacgtatgtggcggaagctccagatctgacc3342argasnlyspheargleuthrtyrvalalaglualaproaspleuthr9709759 80caaggtctttccaatgttcacaaaaaacgagtctcggctgctcgactc3390glnglyleuserasnvalhislyslysargvalseralaalaargleu985990995ctcagccgccaaaacctgcaaagccctaagttcagccgatcgaccatc3438leuserargglnasnleuglnserprolyspheserargserthrile100010051010ttc gtgcgtctccacaccaacgggaatcaggagctgcagtaccagcca3486phevalargleuhisthrasnglyasnglngluleuglntyrglnpro101510201025ggggaccacct gggtgtcttccccggcaaccacgaggacctcgtgaat3534glyasphisleuglyvalpheproglyasnhisgluaspleuvalasn1030103510401045gcactc attgaacggctggaggatgcaccgcctgccaaccacgtggtg3582alaleuilegluargleugluaspalaproproalaasnhisvalval105010551060aaggt ggagatgctggaggagaggaacactgctctgggtgtcatcagt3630lysvalglumetleuglugluargasnthralaleuglyvalileser106510701075aattgg aaggatgaatctcgcctcccaccctgcaccatcttccaggcc3678asntrplysaspgluserargleuproprocysthrilepheglnala108010851090ttcaagtacta cctggacatcaccacgccgcccacgcccctgcagctg3726phelystyrtyrleuaspilethrthrproprothrproleuglnleu109511001105cagcagttcgcctctctg gccactaatgagaaagagaagcagcggttg3774glnglnphealaserleualathrasnglulysglulysglnargleu1110111511201125ctggtcctcagcaa ggggctccaggaatatgaggagtggaagtggggc3822leuvalleuserlysglyleuglnglutyrgluglutrplystrpgly113011351140aagaaccccaca atggtggaggtgctggaggagttcccgtccatccag3870lysasnprothrmetvalgluvalleugluglupheproserilegln114511501155atgccggctacact tctcctcactcagctgtcgctgctgcagcctcgc3918metproalathrleuleuleuthrglnleuserleuleuglnproarg116011651170tactactccatcagctcc tctccagacatgtaccccgacgaggtgcac3966tyrtyrserileserserserproaspmettyrproaspgluvalhis117511801185ctcactgtggccatcgtctcctacca cacccgagacggagaaggacca4014leuthrvalalailevalsertyrhisthrargaspglygluglypro1190119512001205gtccaccacggggtgtgctcc tcctggctcaacagaatacaggctgac4062valhishisglyvalcyssersertrpleuasnargileglnalaasp121012151220gatgtagtcccctgcttcgt gagaggtgcccctagcttccacctgcct4110aspvalvalprocysphevalargglyalaproserphehisleupro122512301235cgaaacccccaggtgccttgc atcctggttggcccaggcactggcatc4158argasnproglnvalprocysileleuvalglyproglythrglyile124012451250gcacccttccgaagcttctggcaaca gcgacaatttgacatccaacac4206alapropheargserphetrpglnglnargglnpheaspileglnhis125512601265aaaggaatgaatccgtgccccatggttctggtc ttcgggtgtcgacaa4254lysglymetasnprocysprometvalleuvalpheglycysarggln1270127512801285tccaagatagatcatatctacagagagga gaccctgcaggctaagaac4302serlysileasphisiletyrargglugluthrleuglnalalysasn129012951300aagggcgtcttcagagagctgtacact gcctattcccgggaaccggac4350lysglyvalphearggluleutyrthralatyrserarggluproasp130513101315aggccaaagaaatatgtacaggacgtgct gcaggaacagctggctgag4398argprolyslystyrvalglnaspvalleuglngluglnleualaglu132013251330tctgtgtaccgcgccctgaaggagcaaggaggc cacatttatgtctgt4446servaltyrargalaleulysgluglnglyglyhisiletyrvalcys133513401345ggggacgttaccatggccgccgatgtcctcaaagccatcca gcgcata4494glyaspvalthrmetalaalaaspvalleulysalaileglnargile1350135513601365atgacccagcaggggaaactctcagaggaggacgct ggtgtattcatc4542metthrglnglnglylysleuserglugluaspalaglyvalpheile137013751380agcaggctgagggatgacaaccggtaccacgagga catctttggagtc4590serargleuargaspaspasnargtyrhisgluaspilepheglyval138513901395accctcagaacgtatgaagtgaccaaccgccttaga tctgagtccatc4638thrleuargthrtyrgluvalthrasnargleuargsergluserile140014051410gccttcatcgaagagagcaaaaaagacgcagatgaggtttt cagctcc4686alapheileglugluserlyslysaspalaaspgluvalpheserser141514201425taactggatcctcctgcccccgtgcgtgcgatgtggcggctgccccaagtgcccaagta a4746gggcggccgcaggttgactaaattcggacacacacggctgaaccgagtggccctgctctg4806cctcttgtcctgttgctgtgtcctggtccttcttcctgctctgggctctctcaaccccac4866ccctgggttttctccttgactcttgggctacgatgc atcacccttgtaccctgcagtggc4926tctcacaaaaccgcatcctccccacccccacccgattgctgccaagggcaggttgcggtg4986catggctgttgctcctgttgttggggtctgaaggtggctggcgctgggcctcaggtcacc5046ctgaaccagtccc ttggccacttaagcccccttccaccctctttttatgatggtgtgttt5106gt5108 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 1429 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 2 : metglugluasnthrpheglyvalglnglnileglnproasnvalile151015servalargleuphelysarg lysvalglyglyleuglypheleuval202530lysgluargvalserlysproprovalileileseraspleuilearg3540 45glyglyalaalagluglnserglyleuileglnalaglyaspileile505560leualavalasnaspargproleuvalaspleusertyraspserala65 707580leugluvalleuargglyilealasergluthrhisvalvalleuile859095leuargglyp rogluglyphethrthrhisleugluthrthrphethr100105110glyaspglythrprolysthrileargvalthrglnproleuglypro115 120125prothrlysalavalaspleuserhisglnproseralaserlysasp130135140glnserleualavalaspargvalthrglyleuglyasngly progln145150155160hisalaglnglyhisglyglnglyalaglyservalserglnalaasn165170175glyvalalaileaspprothrmetlysserthrlysalaasnleugln180185190aspileglygluhisaspgluleuleulysgluilegluprovalleu19 5200205serileleuasnserglyserlysalathrasnargglyglyproala210215220lysalaglumetlysaspthrglyileglnv alaspargaspleuasp225230235240glylysserhislysalaproproleuglyglyaspasnaspargval245250 255pheasnaspleutrpglylysaspasnvalprovalileleuasnasn260265270protyrserglulysgluglnserprothrserglylysglnser pro275280285thrlysasnglyserproserargcysproargpheleulysvallys290295300asntrpgluthraspvalva lleuthraspthrleuhisleulysser305310315320thrleugluthrglycysthrgluhisilecysmetglyserilemet325 330335leuproserglnhisthrarglysprogluaspvalargthrlysasp340345350glnleupheproleualalysglupheleuaspg lntyrtyrserser355360365ilelysargpheglyserlysalahismetaspargleuglugluval370375380asnlysglu ilegluserthrserthrtyrglnleulysaspthrglu385390395400leuiletyrglyalalyshisalatrpargasnalaserargcysval 405410415glyargileglntrpserlysleuglnvalpheaspalaargaspcys420425430thrthralahisglymetpheas ntyrilecysasnhisvallystyr435440445alathrasnlysglyasnleuargseralailethrilepheprogln45045546 0argthraspglylyshisasppheargvaltrpasnserglnleuile465470475480argtyralaglytyrlysglnproaspglyserthrleuglyasppro 485490495alaasnvalglnphethrgluilecysileglnglnglytrplysala500505510proargglyarg pheaspvalleuproleuleuleuglnalaasngly515520525asnaspprogluleupheglnileproprogluleuvalleugluval530535 540proilearghisprolyspheasptrpphelysaspleuglyleulys545550555560trptyrglyleuproalavalserasnmetleuleugl uileglygly565570575leuglupheseralacyspropheserglytrptyrmetglythrglu580585590 ileglyvalargasptyrcysaspasnserargtyrasnileleuglu595600605gluvalalalyslysmetaspleuaspmetarglysthrserserleu610 615620trplysaspglnalaleuvalgluileasnilealavalleutyrser625630635640pheglnserasplysvalthrileval asphishisseralathrglu645650655serpheilelyshismetgluasnglutyrargcysargglyglycys660665 670proalaasptrpvaltrpilevalproprometserglyserilethr675680685provalphehisglnglumetleuasntyrargleuthrproserphe 690695700glutyrglnproaspprotrpasnthrhisvaltrplysglythrasn705710715720glythrprothrlys argargalaileglyphelyslysleualaglu725730735alavallyspheseralalysleumetglyglnalametalalysarg740 745750vallysalathrileleutyralathrgluthrglylysserglnala755760765tyralalysthrleucysgluilephelyshisalaphe aspalalys770775780alametsermetgluglutyraspilevalhisleugluhisgluala785790795800leuv alleuvalvalthrserthrpheglyasnglyaspproproglu805810815asnglyglulyspheglycysalaleumetglumetarghisproasn8 20825830servalglnglugluarglyssertyrlysvalargpheasnserval835840845sersertyrseraspserarglysser serglyaspglyproaspleu850855860argaspasnphegluserthrglyproleualaasnvalargpheser865870875 880valpheglyleuglyserargalatyrprohisphecysalaphegly885890895hisalavalaspthrleuleuglugluleuglyglygluargileleu900905910lysmetarggluglyaspgluleucysglyglngluglualaphearg915920925thrtrpalalyslysv alphelysalaalacysaspvalphecysval930935940glyaspaspvalasnileglulysproasnasnserleuileserasn9459509 55960aspargsertrplysargasnlyspheargleuthrtyrvalalaglu965970975alaproaspleuthrglnglyleuserasnvalhis lyslysargval980985990seralaalaargleuleuserargglnasnleuglnserprolysphe99510001005sera rgserthrilephevalargleuhisthrasnglyasnglnglu101010151020leuglntyrglnproglyasphisleuglyvalpheproglyasnhis10251030 10351040gluaspleuvalasnalaleuilegluargleugluaspalapropro104510501055alaasnhisvalvallysvalg lumetleuglugluargasnthrala106010651070leuglyvalileserasntrplysaspgluserargleuproprocys10751080 1085thrilepheglnalaphelystyrtyrleuaspilethrthrpropro109010951100thrproleuglnleuglnglnphealaserleualathrasnglulys1105 111011151120glulysglnargleuleuvalleuserlysglyleuglnglutyrglu112511301135glutrpl ystrpglylysasnprothrmetvalgluvalleugluglu114011451150pheproserileglnmetproalathrleuleuleuthrglnleuser1155 11601165leuleuglnproargtyrtyrserileserserserproaspmettyr117011751180proaspgluvalhisleuthrvalalailevalsert yrhisthrarg1185119011951200aspglygluglyprovalhishisglyvalcyssersertrpleuasn12051210 1215argileglnalaaspaspvalvalprocysphevalargglyalapro122012251230serphehisleuproargasnproglnvalprocysileleuvalgly 123512401245proglythrglyilealapropheargserphetrpglnglnarggln125012551260pheaspileglnhislysglym etasnprocysprometvalleuval1265127012751280pheglycysargglnserlysileasphisiletyrargglugluthr1285 12901295leuglnalalysasnlysglyvalphearggluleutyrthralatyr130013051310serarggluproaspargprolyslystyrvalg lnaspvalleugln131513201325gluglnleualagluservaltyrargalaleulysgluglnglygly133013351340hisilet yrvalcysglyaspvalthrmetalaalaaspvalleulys1345135013551360alaileglnargilemetthrglnglnglylysleuserglugluasp 136513701375alaglyvalpheileserargleuargaspaspasnargtyrhisglu138013851390aspilepheglyvalthrl euargthrtyrgluvalthrasnargleu139514001405argsergluserilealapheileglugluserlyslysaspalaasp14101415 1420gluvalpheserser1425