Phng Th Ngc Quyn-k22 sinh hc

S dng enzyme transferase trong chun on bnh hc

I. Gii thiu chung v enzyme 1. Enzyme l g? L cc i phn t c bn cht l protein,c kh nng xc tc cho 1 hoc nhiu phn ng sinh ho vi mc c hiu khc nhau. So vi cc cht xc tc v c,enzyme c nhiu c tnh u vit nh: s dng trong iu kin thng vi tc phn ng nhanh, c cht v sn phm c tnh c hiu. v vy, chng ngy cng c ng dng rng ri trong i sng v k thut . Mi enzym c 1 hoc vi trung tm hot ng xc tc cho phn ng ca c cht C ch tc dng ca enzym C ch tc dng ca enzym l lm gim nng lng hot ha ca phn ng cc c cht d dng t c mc nng lng trng thi chuyn tip, t phn ng c th xy ra . Enzym lm gim nng lng hot ha ca phn ng bng cch kt hp vi c cht to phc E-S: E + S ES E + P (E l enzym , S l c cht, P l sn phm ca phn ng). 2. Phn loi Da vo c tnh ca enzyme hi ha sinh quc t(IUB) thng nht phn loi enzyme thnh 6 lp: Oxidoreductase: xc tc phn ng oxi ha kh Transferase: xc tc phn ng chuyn gc hay chuyn nhm t cht ny sang cht khc Hydrolase: xc tc phn ng thy phn cc hp cht hu c Lyase: xc tc phn ng phn ct khng cn nc, hoc loi nc, thm nc vo ni i Isomerase: chuyn v ng phn hoc ni phn Ligase( synthetase): tng hp cc hp cht hu c cao nng. . Enzym vn chuyn nhm (transferase )

L enzyme xc tc phn ng vn chuyn 1 nhm ha hc( ko phi H) gia 2 c cht AX + B A + BX. Gm 8 lp ph rt khc nhau ty theo bn cht ca nhm c chuyn v nh monocarbon, alkyl, glucosyl, cc nhm phosphate, lu hunh

Phng Th Ngc Quyn-k22 sinh hc Cc aminotransferase : chuyn nhm NH2 t acid amin vo acid cetonic . VD: aspartat transaminase , alanin transferase Transcetolase v transaldolase : chuyn n v 2C v 3C vo c cht . VD: transscetolase , transaldolase . Cc acyl-, metyl- , glucosyl transferase , phosphorylase : chuyn cc nhm tng ng vo c cht . VD: acyl CoA cholesterol acyl transferase (ACAT) , glycogen phosphorylase Cc kinase : chuyn gc PO3 t ATP vo c cht .VD: hexokinase Cc thiolase : chuyn nhm CoA SH vo c cht .VD: acyl CoA acetyltransferase( thiolase ).. Cc polymerase : chuyn cc nucleotid t cc nucleotide triphosphat (NTP) vo phn t ADN hoc ARN .VD: cc ADN polymerase , cc ARN polymerase II. Quy trnh tinh sch enzyme

tch v tinh ch enzyme thng c mt lot phng php ha l khc nhau. C th chia thnh 5 nhm phng php sau: Phng php bin tnh chn lc Phng php kt ta phm on Phng php hp th chn lc Phng php sc k Phng php tch h 2 pha nc xy dng phng php tch v lm sch 1 enzyme cn bit la chn v phi hp 1 cch c hiu qu nht cc bin php tch v lm sch khc nhau. C th: 1. _Glutamyltranspeptidase ( GGT) ( EC.2.3.2.2) c tinh ch t cc phn periplasmic ca Escherichia coli K-12. GGT thuc nhm transferase c tm thy trong nhiu sinh vt khc nhau t vi khun n nhiu ng vt c v, GGT xc tc qu trnh chuyn ha cc cht bi tit trong nc tiu v mt, phn ct glutathione ngoi sinh v nito trong E.coli, nm men v ng vt c v. GGT cng c chuyn sang bacillus subtilic bng AND ti t hp sn xut. Nui E.coli( hoc B,subtilis) trong mi trng dinh dng lng ( ng)+ apicilin+ SeMet thm isopropyl-1-Thio-- D -galactopyranoside lc 12 gi. tch ra GGT t mt ct PBE94 thm ammonium sulfate ti 80% bo ha. Ta ny c ha tan trong

Phng Th Ngc Quyn-k22 sinh hc mt lng nh 20 mM Tris HCl (pH 8). on c cha GGT c kt ta bng cch thm ammonium sulfate ti 80% bo ha v bo qun 4 C cho n khi s dng. Kt ta ammonium sulfate ca SeMet GGT c ha tan trong 50 mM Hepes m ti pH 7,0. SeMet GGT c kh mui bng cch lp i lp li v pha long vi h m bng cch s dng b lc n khi cn 6 mg / ml tinh. .Tinh th GGT c sn xut 4 C. Tinh th c ngm trong dung dch bo v lnh v n flash lm mt bng mt dng kh nit 100 K, ni m cc gii php bo v lnh c chun b bi cc thm PEG 4000 v glycerol vi cc h. Hu ht cc tinh th v cc cht nn c chun b theo phng php ngm, cha cc cht bo v lnh.
-Glutamyltranspeptidase ca Escherichia coli K-12 bao gm mt tiu n v ln v mt tiu n v nh, c th c ngn cch vi nhau bng sc k lng hiu nng cao, tiu n v ln v nh tch ra c ti to trong ng nghim s tng hp glutamyltranspeptidase trong tin thn polypeptide sau x l 1 ln duy nht c hiu qu hn xy dng cc cu trc nguyn vn ca -glutamyltranspeptidase

2. GOT: (glutamat oxaloacetat transaminase) c trong gan, c v c trong nt sn h u, c tinh ch bng phng php sc k troa i ion III. ng dng ca tranferase trong chun on bnh hc 1. ng dng trong chun on bnh v gan Gan l mt tng ln nht trong c th. Trong gan c mt h thng enzym rt hon chnh thc hin hot ng tng hp v chuyn ha cc cht protid, gluxit, lipid. Ngi ta gi chung nhng enzym ny l men gan. Cc enzyme l SGOT(AST), SGPT(ALT), GGT , LDH. Trong gan, cc enzym ny c mt trong t bo gan. Mi ngy, c th lun lun c nhng t bo gi cht i v t bo mi sinh ra, Khi nhng t bo gan cht i th enzym trong t bo s c gii phng vo mu. Nh vy, trong mu lun tn ti mt lng men gan nht nh. Khi mt hoc nhiu hn mt trong s cc enzym ny c gi tr cao hn gi tr bnh thng s d ti men gan cao. C ngha l lng enzyme c gii phng vo mu nhiu hn bnh thng phn nh tnh trng cc t bo gan ang b hy hoi. Ch s men gan cng cao hn bnh thng nh gi tnh trng tn thng nhu m gan cng nhiu.

Phng Th Ngc Quyn-k22 sinh hc Bn enzym ch o nh gi tnh trng tn thng ca cc t bo gan l SGOT, SGPT, GGT v LDH. GGT - Gamma Glutamyl Transferase ( gamma GT) (EC2.3.2.2) : L mt enzyme ca mng t bo, tham gia vo qu trnh trao i cc axit amin qua mng t bo, thc hin chc nng chuyn nhm -glutamyl. N c tm thy nhiu m trong nhiu nht l gan v c du hiu chun on bnh trong y hc. GGT chuyn nhm glutamyl cho mt lot cc phn t cht nhn k c nc c bit l L-axitamin cn bng ni mi ng thi GGT cng c tc dng chng oxi hatrong t bo v n lin quan n s trao i cht gluthatione dn ti s hnh thnh cystein. (5-L-glutamyl)-peptide + mt axit amin peptide +5-L-glutamyl- axit amin GGT c chu k bn r khong 10 ngy ty tnh trng sc khe. V cu trc: GGT gm 2 chui polypeptide, mt ln mt b, c trungtaam hot ng nm trong tiu n v b GGT ch yu c s dung chun on bnh tng huyt p. GGT c th c ci l enzym u tin chu tc ng mt khi xy ra cc bnh l gan v ng mt. Bnh thng, men gan ny tn ti trong mu vi gi tr bnh thng l:

Nam : 5-38 U/L N : 5-29 U/L Tr nh: 3 -30 U/L Tr s sinh : Ln gp 5 ln gi tr c thy tr nh.

Khi c th bn ang mc mt s bnh nh: Vim gan , x gan do ru, vim gan nhim trng, vim gan mn tnh, p xe gan, kn sn l gan, tng lipid mu, x gan do mt tin pht, vim ng mt x ha, si mt, ung th biu m ng mtth men gan ny tng ln cao hn bnh thng ngi nghin ru, GGT thng gia tng n c ( khng km theo tng SGOT SGPT, LDH). Lng enzym ny trong mu tng ng vi lng ru hp th. Theo di lng enzym trong cc ln xt nghim mu nh k, thng xuyn gip bc s nh gi thi quen dng ru ca ngi bnh. T y cho php pht hin cc bnh nhn nghin ru theo di bnh nhn nghin ru, gip chn on sm cc vn gan mt bnh nhn nht l x gan do ru v u gan.

Phng Th Ngc Quyn-k22 sinh hc SGOT ( glutamat oxaloacetat transaminase ) hay ASAT ( aspartat transaminase) v SGPT ( glutamat pyruvat transaminase ) hay ALAT ( alanin transaminase ) Chng l cc enzym c trong t bo gan c vai tr c bn xc tc cho nhng phn ng sau: Aspartat + _cetoglutarat Alanin + _cetoglutarat GOT GPT Oxaloacetat + Glutamat Pyruvat + Glutamat

Cc men gan ny c mt lng nht nh trong mu: Khi c tnh trng tn thng t bo gan nh vim gan, u gan, x gan th, cc men ny tng ln trong mu. Xt nghim kim tra lng men gan (SGOT. SGPT,LDH, GGT) trong mu cho php xc nh mt bnh l gan v theo di tin trin ca bnh. T l SGOT/SGPT >1 , kt hp vi tng GGT v Globulin mu km gim nng antithrombin III (mt yu t ng mu) s d on c tnh trng x gan. .Men gan cao ch l mt cn c tin tng hn cho vic chn on bnh chc chn, kt hp vi cc triu chng ca ngi bnh. Tuy nhin SGOT, SGPT, GGT,LDH khng ch tn ti duy nht t bo gan, m n cn c mt trong t bo ca nhiu c quan b phn khc nh tim, thn,ty , c khp, hng cu, bch cu . LDH - Lactac DeHydrogenase : L mt enzyme trong t bo xc tc phn ng chuyn ha nng lng . LDH c mt nhiu trong t bo gan v c gii phng ra huyt tng khi c tnh trng hy hoi t bo gan. C khong 110 -210 U/L lng LDH tn ti trong mu LDH ch yu tng cao trong cc tn thng t bo gan:Vim gan nhim khun, nhim c., vim gan do ru, di cn gan, vim gan do thuc (rifampicin, salycylat, INH) 2. Xc nh nng c cht Phng php ng hc xc nh triglyceride Phng php ny ch xc nh nng c cht di gi tr Km Xc nh glucose

Phng Th Ngc Quyn-k22 sinh hc D glucose + ATP D-glucose -6-phosphate +NADP+ D-glucose-6-phosphate + ADP D-glucono-denta-lactose-6-phosphate + NADPH +

phn ng u, glucose c phosphoryl ha bi hexokinase ,sau glucose-6-phosphate b hydrogen ha bi tc ng ca glucose-6-phosphate dehydrogenase .S to thnh NADPH s c xc nh bng my quang ph. Trilyceride + 3 H2O Glycerol + ATP ADP + phosphoenolpyruvate Pyruvate + NADH + H+ glycerol +3 acid bo glycerol -3-phosphate + ADP ATP + pyruvate L-lactate + NAD+

Cht bo c thy phn bng lipase(EC.3.1.1.3) carboxylesterase (EC.3.1.1.1) to ra glycerol v s c phosphoryl ha bi glycerol kinase(EC.2.7.1.30), ADP to thnh s tip tc c phosphoryl ha n ATP vi phosphoenolpyruvate v pyruvate kinase(EC.2.7.1.40), cui cng pyruvate c hydrogen ha bi Llactate dehydrogenase (EC.1.1.1.27) v NADH gim dn. Xc nh hot tnh ca creatine kinase Creatine phosphate + ADP createne + ATP

ATP to thnh trong phn ng ny nh xc tc ca enzyme creatin kinase. Creatine kinase (CK), cn c gi l creatine phosphokinase (CPK) hoc phospho-creatine kinase, hoc c xem nh l creatine kinase l mt loi enzyme thuc nhm transferase ( EC 2.7.3.2 ) trong nhm Nphosphoryl ca Adenosindiphosphat (ADP) xut hin cc m khc nhau v cc loi t bo. CK xc tc chuyn i ca creatine v bin i adenosine triphosphate (ATP) to ra phosphocreatine (PCR) + adenosine diphosphate (ADP) v ngc li . Phn ng lu tr nng lng trong c bp v m no INCLUDEPICTURE "http://upload.wikimedia.org/wikipedia/commons/thumb/c/c0/Creatine_kinase_rxn.png/500pxCreatine_kinase_rxn.png" \* MERGEFORMATINET INCLUDEPICTURE "http://upload.wikimedia.org/wikipedia/commons/thumb/c/c0/Creatine_kinase_rxn.png/500px-

Phng Th Ngc Quyn-k22 sinh hc Creatine_kinase_rxn.png" \* MERGEFORMATINET

CK c nhiu trong cc m v cc t bo s dng nhiu ATP, c bit l c xng, no , cc t bo tip nhn nh sng ca vng mc , cc t bo lng trong tai trong , tinh trng v c trn , PCR phc v nh mt h cha nng lng nhanh chng v ti sinh ca ATP ti ch, cng nh vn chuyn nng lng trong t bo [3] Nh vy creatine kinase l mt enzyme v cng quan trng. C 4 loi enzyme khc nhau: CK-MM, CK-BB v CK-MB CK-1: CK-2: CK-3: CK-Mt: MB MM tm BB isoenzyme, nng c trong mng tm tm cao ty nht th, thy thy trong trong c c chim ch tim, xng, 15% t t yu hn hn lng trong trong trong CK c ca c no. xng. tim. tim.

isoenzyme, isoenzyme, thy

CK c chu k bn r rt ngn. Hot ng mnh lc 6-12 gi v tr li bnh thng trong vng 24-48 gi. Khi chn thng c bp s duy tr nng CK cao. Hot ng CK l n nh cho 7 ngy ti 4 C v 1 thng -25 C INCLUDEPICTURE "https://ahdc.vet.cornell.edu/clinpath/modules/chem/images/ckastkin.gif" \* MERGEFORMATINET INCLUDEPICTURE

Phng Th Ngc Quyn-k22 sinh hc "https://ahdc.vet.cornell.edu/clinpath/modules/chem/images/ckastkin.gif" \* MERGEFORMATINET

Hnh 1: chc nng c xng (bi Cardinet Guitar Hero III trong "Clinical Biochemistry of Domestic Animals", 5th edition, Kaneko et al, eds, pg 426, 1997; Copyright Elsevier.)

Xt nghim lm sng, creatine kinase trong xt nghim mu l mt du hiu nhn bit ca nhi mu c tim Khi bnh nhn au ngc hoc nu nghi ng. suy thn cp s c kim tra lng CK. Thng thng tng s lng CK l 24-170 IU / l ph n, 24-195 IU / l nam gii, nu CK tng cao l du hiu ca tn thng c bp . C th l du hiu ca chn thng , tiu c vn , nhi mu c tim , vim c v vim c tim . Vi bnh nhn suy gip, CK tang tng ng vi gim huyt thanh T3. V vy, vic c lng huyt thanh CK c xem l c gi tr trong vic kim tra cho bnh nhn suy gipGim CK c th l mt du hiu ca bnh gan do ru v vim khp dng thp .

Phng Th Ngc Quyn-k22 sinh hc

Ti liu tham kho

1. ng Th Thu, Cng ngh enzyme 2. Phm Th Chn Chu, enzyme v ng dng trong cng ngh sinh hc tp 3 3. Wallimann T, Hemmer W (1994). "Creatine kinase in non-muscle tissues and cells". Molecular and Cellular Biochemistry . 133134: 193220. 4. Hekimsoy, Zeliha; Oktem, Iris Kavalali (2005). "Serum creatine kinase levels in overt and subclinical hypothyroidism". Endocrine Research 31

5. Tietz Textbook of Clinical Chemistry. Edited by CA Burtis, ER Ashwood. WB Saunders Company, Philadelphia, 1994 6. Journal of Bacteriology, Dec, 1986, p1325-1331 0021-9193/86/12132507502.000 _glutamyltranspeptidase from E.coli K12: Purification and Propecties
7. http://www.ncbi.nlm.nih.gov/pubmed/16618936