Original title: Příprava, exprese a charakterizace myší GCPIII
Translated title: Preparation, expression and characterization of mouse GCPIII
Authors: Bäumlová, Adriana ; Šebo, Peter (referee) ; Konvalinka, Jan (advisor)
Document type: Bachelor's theses
Year: 2010
Language: slo
Abstract: [eng] [cze]
English abstract Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a type II transmembrane glycoprotein which has been discovered in nervous system as an enzyme responsible for the hydrolysis of neuropeptide N-acetyl-L-aspartyl-L-glutamate to N-acetyl-L-aspartate and L-glutamate and that has been hypothesized to influence glutamatergic signaling processes. Except for brain, GCPII was mainly found in prostate, kidney, and small intestine. In small intestine, GCPII cleaves terminal glutamates from polyglutamylated folates facilitating thus absorption of dietary folates. In prostate, this enzyme is known as prostate-specific membrane antigen and is used as a cancer marker. Mus musculus is an important model for studing GCPII and its homologs as a therapeutic target. While human GCPII and its paralog GCPIII are relatively well characterized, no biochemical study of their mouse orthologs is available. That is why mouse glutamate carboxypeptidase III (mGCPIII) was cloned, prepared by recombinant expression in insect cells and characterized. We show that pure mouse GCPIII possesses α-glutamate carboxypeptidase activity which is effectively inhibited by specific inhibitor GCPII, 2-PMPA. We also analyzed sensitivity and specifity of monoclonal antibodies against mouse GCPIII. Immunoblots demonstrate that... Slovenský abstrakt Glutamátkarboxypeptidáza II (GCPII, EC 3.4.17.21) je transmembránovým glykoproteínom II typu, ktorý bol objavený v nervovom systéme na základe degradácie N-acetyl-L-aspartyl-L-glutamatu na N-acetyl-L-aspartát a L-glutamát, a preto sa predpokladá jeho účasť na nervovej signalizácií. Okrem mozgu, GCPII bola objavená vo väčšej miere taktiež v prostate, obličkách a tenkom čreve. V tenkom čreve GCPII napomáha vstrebávaniu folátov z potravy tým, že odštepuje koncové glutamáty z polyglutamylovaných folátov. V prostate nie je funkcia tohto proteínu doposiaľ známa, avšak využíva sa ako márker rakoviny. Mus musculus je dôležitým modelom pre štúdium GCPII a jej homológov ako terapeutického ciela. Ľudská GCPII a jej paralóg GCPIII sú pomerne dobre charakterizované, zatiaľ čo žiadna biochemická štúdia o ich myších ortológoch nie je dostupná. Preto sme myšaciu glutamátkarboxypeptidázu III (mGCPIII) naklonovali, pripravili rekombinantnou expresiou v hmyzích bunkách a chrakterizovali. Ukázali sme, že mGCPIII vykazujeα-glutamátkarboxypeptidázovú aktivitu, ktorá je inhibovatelná špecifickým inhibítorom GCPII, 2-PMPA. Taktiež sme testovali citlivosť a špecifitu monoklonálnych protilátok voči myšacej GCPIII. Imunochemická detekcia odhalila, že protilátka GCP-04 rozpoznáva ľudskú GCPII rovnako dobre ako...
Keywords: 2-PMPA; GCPII; GCPIII; glutamate; homologs; 2-PMPA; GCPII; GCPIII; glutamát; homológy

Institution: Charles University Faculties (theses) (web)
Document availability information: Available in the Charles University Digital Repository.
Original record: http://hdl.handle.net/20.500.11956/30157

Permalink: http://www.nusl.cz/ntk/nusl-285646


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Universities and colleges > Public universities > Charles University > Charles University Faculties (theses)
Academic theses (ETDs) > Bachelor's theses