WWW.LIT.I-DOCX.RU
    -
 

Š ۠ ɠ Ƞ KRP Ƞ Ƞ ՠ ...

Š

ՠɠȠ

KRP  

ɠ

ՠ

03.00.04-

- 2004

Š ۠

ɠ Ƞ KRP

Ƞ

Ƞ ՠ

03.00.04-

-2004 Ǡ : , .. : , .. : , .. , .. : ... 21 2004 11 Ġ208.073.01 ʠǠԠ:

121552,, 3-.,. 15 .

Ѡ -Ǡ 2004 , ..

ߠ

.   , . , , , ., . ., .

. , (). () ̠.̠: , -KRP,. , KRP ,KRP .





  ̠ .   ̠--KRP . , (PDBu) KRP , KRP / .   ,, .

̠ KRP .   , KRP - , - ̠- .

. - ̠KRP .

  :

1. - , .

2. , ̠ KRP - in vitro , PDBu .

3. ̠ KRP .

4. - ̠ ̠ invitro .

5. ̠ .

. , 38 - , - . ̠-, ̠.-̠KRP- .

. . ̠̠KRP , .

. " : "(,,2001),30-,31-32-(,,2001;,,2002;,,2003) .

.25 .

. , , , , . 2 27 .

245 .

۠Ƞ۠

.(Lowry etal, 1951)(Bradford, 1976) .

. 6-12%à(Laemmli, 1970). (20:1,v/w),3,(lOOOOg,5) . -R-250 0,43 15-25 .50-250, . --250 Scion Image (Hewlett-PackardHP-Hex) .

(Towbinetal., 1970).(100/350)PVDF(Amersham,):KRP 110̠Caps,͠11,0,10%,5̠2-,̠-225̠,180̠,͠8,3,20%,0,1%. 1 5% -Ҡ(20̠-HCl,͠7,6, 165̠NaCl,0,05%-20).,1-Ҡ ,. ̠(36,Sigma,), 38--(NewEnglandBiolabs,),,,(Amersham,Pierce,). (Amersham, ) 3,3'- (Sigma, ). 0,430 .

. ̠F-(10 (2 15Ѡ (10̠Mops,͠7,0, 1̠MgCl2,0,1̠, 50̠NaCl,1̠().  .̠(0,4-0,8 (77, 0,6-1,2 KRP,0,7 (40 ,20 (100000g, 30 ) LP42Ti (Beckman,).F-10̠G-10 10̠Mops,͠7,0,10̠NaCl,1̠,0,1̠ N75 (0,5-16 5̠MgCb, 40 13000g. R-250, ]Р - .

invitro.KRP,̠N75 77̠ GST-p38, MBP-XPAK1 GSK-3., , N75 KRP . 30Ѡ: 10̠Mops, ͠7,0,20̠NaCl,1̠,0,2̠1̠Р5̠MgCl2. 0,5-1,0x10/. -30 (20:1, /) -6 ddMeklcaHis6. KRP (40 ) :KRP 1:100 , 1:10 -1, 1:20 GST-p38-.̠ - 10:1 (/ )90.1 ̠Р90. 1 ̠ Р - 20:1 (/) 90 . 77̠ (6 ) . N75 (100 - :N7510:1 .

.̠ ̠(1,6 =13 )   25Ѡ 0,5̠,0,5̠2+,15.̠ ̠ 10̠.̠0,550.̠̠ 300,̠110.. 2,5 .

.

. n itro i v 2Π10 6N1(110,2 ), 10.3. (5-6 )(5% , 15% ),2P-Ser,P-Thr-, DC-Plastikfolien Cellulose (Merck, ). (1000, 4, 60-120 ) Multiphor II (LKB, ). 0,25% .

. , -[ ],R-250.,,,3-4 25%,3-440%25͠200-250 40%25.SpeedVacSCI10(FTSSystems, ),1100̠NH4HCO3͠8,0, 25,ʠ(WorthingtonBiochem.Corp.,), 37Ѡ 2 . 50 12. (9500g,15),,. 5002ΠNH4HCO3.1015%3͠5%.() SilicaGel60(Merck,)Multiphor II(LKB,) 1000 (75, 10). - / / 3͠ / 2Π (150/150/40/160, v/v) .

KodakBiomax .

R5R8.KRP(3) CNBr- Sepharose 4B (Pharmacia Biotech., ) . R5 (--Ser ),,KRP-,, . 2 . R8 (--Ser19). (R5 R8 ) -20Ѡ 1:1000-5000 .

. . coliBL21 (DE3)pLys.---(3)Agoo-0,6-0,8.3 (5000g,20),20 (25%,0,2̠,30̠-HCl,͠8,0,0,2̠,0,5̠PMSF,10̠2, 2 ̠, 2 ̠ ), , Hise- GST- , . (BransonSONIFIER450,BransonUltrasonicsCorp.,). (9000g, 20 ) .KRP40-60% (NH4)2SO4 30̠1,30̠-HCl,͠7,5,5̠2-, 0,1 PMSF Q-Sepharose (Pharmacia Biotech., ) 0,0-0,6̠NaCl.,KRP,, 2̠ (NH4)2SO4, 1 ̠12,30̠Tpnc-SO4,͠7,5,20̠2- - (Pharmacia Biotech., ), 2-0̠ (NH4)2SO4 GST(-S-TpaHC)-,MBP(maltose-bindingprotein)-His6-, , - (Pharmacia Biotech.,),Amilose-resin(NewEnglandBiolabs,)TALON(Clontech,) . -20 .

(SpudichandWatt, 1971) .

(ileum)Smithetal.(1999) .

N75̠. (), pEGFP/KJHJM-210 , : 5' - 5'tgcficatggacttccgagcaaacctgc 3' - 5'-tatctcgaggctgccattctctgcagggg. Ncol Xhol (). - pET21d(+) (Novagen, ). Р Hybaid Omnigene (), Pfii-Ultra - (Promega, ). VectorNTISuite5.5 .

(BL2IpLysDH5x,100)10 ,(42)45.137Ѡ,(4000g,5) . ʠ.coliDH5a.ʠ Qiagen() .

QuikChangeSite-Directed Mutagenasis Kit Stratagene (). Ser19 KRP Ala pET22b(+)/KRP 5'-cagggtcaccaaccgccccgctcaatgc 5'gcattgagcggggcggttggtgaccctg. Thr ̠ N75 Ala 5'-ggcaccccaaaggccccactgcc5'-ggcagtggggcctttggggtgcc .

.   2-33x6. (WPIInstruments,), 37,1,3-1,4, 0,1-0,15 . .

60̠1,20̠,0,1-1 uMPDBu .

,10.0,1 uMPDBu10̠. 1 PD98059(60)/SKF86002(20)(Calbiochem,). 0,1̠PDBu .

۠Ƞ

2+   [ ], . , 2+ 2+-.2+- .(PDBu) , . ̠KRP,-KRP(Krymsky,2001).KRP , ., KRP , ,KRP(Shirinskyetal., 1993) .

-.KRP in vivo, KRP KRP-̠ PDBu (Krymsky, 2001). Ser Ser KRPSer Ser . . , Ser13 KRP Ser828̠(R5)-Ser19 KRPSer834̠(R8). R5KRP,- . KRPKRP,-. R8, KRP,-, Ser13KRP. KRP ̠ ,,Ser Ser19KRPSer828Ser834̠invivo .

Ser13Ser19KRPileum, R5 R8:1)2+-1 ;2)ileum PDBu . KRP (. 1).  , PDBuSer13KRP4-5.4-5-KRPSer , 1.PDBuSer,, KRP.Ser19KRP1,5- PDBu .

̠PDBu(,2001).  2+ - ̠Ser828Ser834 .

̠1,PDBu - 1,5-2- Ser828,Ser13KRP.,Ser KRPinvivo-,,Ѡ .Ser19KRP, Ser828Ser834̠ .

Ser13Ser19KRPinvivo R5R8ileum, KRP, in vitro -. KRP R5, R8 . KRPileum(.2),5001 . ,1/,KRP30, ,KRP (50-80)(Shirinskyetal., 1993). KRP Ser (26,31,77%)Ser (30,83,14%) .

KRP, ̠ Ser Ser, Ser Ser KRP.   ~0,1/ .

,Ser 4 PDBu KRP, .,Ser190,30,45-0,5 / .,Ser19KRP .Ser19invivo ,KRP .

,Ser13 Ser19KRP. KRP . , (Ser13)4,KRP2-2,5. PDBu (,2001). Ser , PDBu.,Ser13,. ѠKRP (VorotnikovetaL, 1996),PDBu- , Ser13 KRP , . , , , G-, Ser13 KRP GTPyS(MacDonaldetal.,2000) .

2+ - - - .PDBu (Horowitzetal., 1996),2+-(Krymsky,2001);Ѡ-(Liebmann,2001). - Ѡ PDBu. , , 0,1̠PDBu42/441'238-(.3,). .

-^-+PDBu-, 2+- .

, 42/44 (60 ̠ PD98059) 38 (20 ̠ SKF86002) ,(.3).-, ,, PDBu 38 p42/44CTkU- .

,- . -,- . Ѡ CPI-17, , ֠(LietaL,1998) .

,-PDBu.- , PDBu (, 2001). - ̠ - ̠invitro .

̠-.  N--̠ -. N75,75N- ,-KRP,̠KRP.Thr43N75Ser19 KRP Ala. in vitro. - (. 4). ̠ - Thr43 N- Ser834,Ser19KRP .

- .̠, - 3. (, ), p44wkl-invitro. ̠ - , , , ̠ KRP-.̠- ̠4 ̠2,8 , 1,5- , Vmax . , ̠ 2+-.   , 1,5- ̠ (KlemkeetaL, 1997;Morrisonetal., 1996) .

-̠ . - Ser 834 Thr43 , . , -̠ Ser 834, - KRP- . -[32]Рp 4 4 a k l -̠.̠ . , ,, ̠ (. 5). ̠, 50%- ,[ ]- . , , , ̠ . , ̠- .

KRP- . KRP-̠KRP(Shirinskyet al., 1993). -̠ - KRP- KRP. , KRP - in vitro (Vorotnikov et aL, 1996).   , -KRP . , , , KRP- ̠ (Gao et al., 2003). 77, KRP-, - N- , , 77̠ - .

, - KRP 77̠ in vitro. , KRP- . , , ̠ . ̠ KRP - () .

KRP̠.̠, -100, ̠ .

60% . , R5 R8 , 90% ̠(. 6) .

KRP , KRP , . ,, Ser828, Ser834, ,,,,KRP- .

-,KRP-̠,., KRP/̠ KRP- , . , (Zhouetal., 1999;VerdeciaetaL,2000).ʠ 14-3-3 , WW- IV , ,--PIN1,, - (Lu et al., 2002). , PIN1- , .

Thr43̠-. 2-7,3058-63N-̠DFRXXL, (SmithetaL, 1999;SmithandStulL,2000)., Thr43̠ ,,DFRXXL-,̠. ̠ 1:20,.N-̠N75 , Thr. ̠,N75 .

, N75 (. 7, ) , 3 , 5,8..7, N75 P-N75 .N750,4/, DFRXXL- (Smith et al., 1999; Smith and StuU, 2000). , N75 lkl ,̠p44 -invitro̠F-F-., ̠ (SellersandPato,1984)., .

Hatchetal. (2001),, ̠,,,, .

DRFXXLAMOTHBOB̠ (Hayakawaetal., 1994;Kudryashovetal., 1999). ,N-,DRFXXL-, . Thr43 N75(. 8).N75^75,F-.4̠N75 .

  , Thr N75 .

̠̠Thr43.  2+/̠̠N- (SellersandPato, 1984;YeetaL, 1997)., /̠ ̠ N-2-41 2+ 43 /̠ (HayakawaetaL, 1999).Thr 2+ 815 N- /̠Ser .Ser ̠2+/̠(ContiandAdelstein,1981)., Thr43 2+/̠ N- . -N75 - N75().-,Thr43 N-̠2+/ .

2+/̠ N75F-,2-N75N75 ( ). , , Thr - /̠ , ̠ , .

, ̠ . [2+],2+/, 2+- - . ̠ , -, DFRXXL- .

̠ PDBu- . ̠ PDBu R5R8 KRP (. ). PDBu 2- Ser, Ser KRP, Ser, Ser19 KRP. ( ) KRP PDBu (. ). Ser 2 PDBu. 2- , , (, 2001), ̠ .

, - KRP-. N-̠(), Thr43, -. , Thr4 Ser 828 2- ̠ PDBu .

, . 2+ 2+ , .. -. , , ,, 2+- , 2+- .

2+ -. , .- G-RhoRho-.2+-.RhoѠ CPI-17, () , 2+ , ֠ - . ̠ -Rho- .

Ѡ , - [Ca2+]j. ̠ ̠ -, 2+- . ̠ . Ser834 KRP- ̠ ̠-KRP77 . ,Ser 834 ֠ ̠ .

,֠,. .

̠ ֠ , (Persechini and Hartshorne, 1981, 1983), -, . ,,KRP-. , ̠ ̠ ֠ .   , . Ser 834 ̠ in vitro . , Thr43 MAP- ̠ in vitro .

,̠,., ̠ , ̠ .

  - .

, , - -. Ϡ7 .,KRP.KRP,6S-, ̠ (Shirinsky et al., 1993;

Silveretal., 1997).   KRP- .-,,,N,KRP(Vorotnikovet al., 1996), - . KRP ,̠(WuetaL, 1998). , Ser13 KRP(WuetaL, 1998;Walkeret al., 2001). , , - , , KRP - - Ser13 (MacDonaldetal.,2000).,KRP- Ser19 Ser13 (Walkeretal., 2001). - Ser13 KRP . , Ser3 KRP ^- , - . -, PDBu Ser13 KRP , , , -. , KRP Ser - ., 9 Ser KRP , , Ser13 .

,,2+-Ѡ-, , - . ,,,KRP .

1., p42/44erkl,2 38 2 .

2. In vitro - Thr43 Ser834 ̠ .

Ser19KRP,Ser834 .

3.,PDBu, Thr43Ser828,Ser834.- Ser19 KRP 30%, Ser1325100% .

4. - ̠ 1,5-2 , ̠. Thr ̠ invitro, Ser ̠ .

5.,̠ + - , Ser13KRP .

lf.. , .. , .. . (2003) , .

, 3 365-420 .

4:

2...,..,..,..,.-..,. .

,...(2004) .,69,,̠04-006 .

3.Khapchaev A.Yu., Shirinsky V.P., andVorotnikov A.V. (2003) Phosphorylation by MAPkinasereducesmyosinlightchainkinasebindingtoactomyosin.J.MuscleRes. CellMotil., 24(4-6):357 .

4.KhapchaevA.Yu.,KrymskyM.A., VilitkevichEX., SidorovaM.V.,BespalovaZh.D.,Wang C.-L.A., Shirinsky V.P., and Vorotnikov A.V. (2002) Phosphorylation affects intracellular interactionsofmyosinlightchainkinaseandthekinaserelatedprotein(telokin).J. Muscle Res.CellMotil.,23( 1):40 .

5.VorotnikovA.V., KrymskyM.A., KhapchaevA.Yu., andShirinskyV.P. (2001) Correlation betweencontractionandphosphorylationofcaldesmonandmyosinlightchainkinaseintonic

andphasicsmoothmusclesstimulatedwithphorbolester.J.MuscleRes.CellMotil,22(7):

597 .

6.Krymsky M.A., Khapchaev A.Yu., Sidorova M.V., Bespalova Zh.D., Shirinsky V.P., and VorotnikovA.V.Correlationbetweenphorbolester-inducedcontractionandphosphorylation ofcaldesmon andmyosin light chainkinase in smooth muscle. Abstracts ofInternational Simposium"BiologicalMotility:NewTrendsinResearch"-Pushchino(Russia),2001 .

7.Vilitkevich EX., Dudnakova T.V., Krymsky MA., Khapchaev A.Yu., Sidorova M.V., Bespalova Zh.DM Shirinsky V.P., and Vorotnikov A.V. Phosphorylation-dependent intracellularinteractionsofmyosinlightchainkinaseandkinaserelatedprotein.Abstractsof International Simposium "Biological Motility: New Trends in Research" - Pushchino (Russia),2001 .




:

Vladimir Ya. Levanidov's Biennial Memorial Meetings 2003 . 2 HAPLOPERLA NAVAS (PLECOPTERA, CHLOROPERLIDAE). ..1, ..2 ...

008674 , , . , ...

1 : - ( ) .00.11 ^ ...

: .. : ; ; ; ...

z R \m ? Ha ! (HE.MEROCALLIS L, BCtJEIlPH 03 00.05 -"" , Q \J 2000 . : 6HOjTon{4ecKiL4 , .....

551.77:561.26(265.2) .. 1,3, .. 2, .. 1 - , . 100- , 159, 690022, - - , . 100-...

. 2016. . 8. . 5962 502.75 (PULSATILLA PRATENSIS MILL.) . . , , kupena07@rambler.ru 20132016 . ...






 
<<     |    
2018 www.lit.i-docx.ru - -

, .
, , , , 1-2 .