RESUMEN
Peptide T is currently in phase II clinical trials for the treatment of AIDS-associated dementia. Its putative mode of action is inhibition of binding of the HIV envelope protein (gp120) to its cellular receptor (CD4), thus preventing viral infectivity and gp120-induced neuronal toxicity. However, a number of reports have appeared in the literature which have failed to observe any inhibitory activity of Peptide T on CD4-gp120 binding, thus casting doubt on this hypothesis. This study uses a novel biosensor technique to demonstrate that Peptide T does bind to CD4 and that this binding can be specifically inhibited by an anti-CD4 monoclonal antibody. A detailed analysis of the kinetics of the interaction is presented.
Asunto(s)
Antígenos CD4/metabolismo , Péptido T/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/metabolismo , Antígenos CD4/química , Cinética , Matemática , Datos de Secuencia Molecular , Péptido T/química , Unión ProteicaRESUMEN
Surface plasmon resonance techniques have been used to examine the kinetics of binding for two RNA fragments to an RNA binding domain of HIV-1 REv. RBE3 RNA elicited an apparent dissociation constant (KD) of 121 nM while RREIIB41-79 RNA exhibited an apparent dissociation constant (KD) of 2.5 nM. The dissociation rates for both RNA fragments were comparable. However, the shorter sequence, RBE3, exhibited considerably slower association kinetics. A series of known inhibitors were assayed against these RNA' and the derived K1's were consistent with those reported in the literature, validating the method for routine inhibitor assays.
Asunto(s)
Productos del Gen rev/metabolismo , Genes env , VIH-1/genética , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Bioensayo , Técnicas Biosensibles , Productos del Gen rev/química , Productos del Gen rev/genética , Cinética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , ARN Mensajero/química , ARN Mensajero/genética , ARN Viral/química , ARN Viral/genética , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
Sialyltransferase activity has traditionally been studied by determining the rate at which the enzyme transfers a labeled donor sugar to an acceptor substrate. These types of assays can be difficult to quantitate, and the separation of untransfered donor sugar from the sialylated acceptor is time-consuming. The biosensor-based method described here is both rapid and semi-automated. The NeuAc-alpha2-6Gal-R-specific lectin Sambucus nigra agglutinin (SNA) immobilized to the carboxymethyl dextran surface of a BIAcore sensor chip was used to detect and measure the formation of the NeuAc-alpha2-6Gal-R moieties. The sialyltransferase assays were carried out using modified protocols based on the method described in Rearick, J.I., Sadler, J.E., Paulson, J.C., and Hill, R.L. (1979) Enzymatic characterization of betaD-galactoside alpha2-3 sialyltransferase from porcine submaxillary gland. J. Biol. Chem., 254, 4444-4451. The complete assay mixture was simply diluted before injection into the instrument. All injections were performed automatically using the robotics of the BIAcore instrument. Using this technique it is possible to detect product from 0.4 microU of commercial Galbeta1-4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) (ST6Gal I). One unit of sialyltransferase is defined as the quantity that will transfer 1 micromol of N-acetylneuraminic acid from cytidine monophosphate (CMP)-N-acetylneuraminic acid to asialofetuin per min at pH 6.5 and 37 degrees C. The method described here requires as little as 10 microl total assay volume, thus reducing the consumption of reagents. In addition, the sample is completely recoverable from the sensor chip surface, which allows for downstream analysis of the reaction product if desired. This method eliminates the need for labeled donor and acceptor molecules and does not require the separation of the substrates from the product before analysis. Although some kinetic properties of the enzyme can be estimated using this method, further development and validation is required. The method is most useful in determining qualitative estimates of ST6Gal I activity in tissue extracts and in characterizing the production of enzymes in cultured cell systems. The use of a microtiter plate assay format enables the rapid screening of multiple fractions for sialyltransferase activity.