A rapid, semi-automated method for detection of Galbeta1-4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) activity using the lectin Sambucus nigra agglutinin.
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.
Halliday
J A
JA
Institute for Molecular Bioscience, University of Queensland, Australia, 4072.
Franks
A H
AH
Ramsdale
T E
TE
Martin
R
R
Palant
E
E
D001331:Automation; D015374:Biosensing Techniques; D002138:Calibration; D006801:Humans; D007700:Kinetics; D037102:Lectins / Q000378:metabolism*; D037121:Plant Lectins; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D054788:Ribosome Inactivating Proteins; D012799:Sialyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D037102:Lectins; D037121:Plant Lectins; D011994:Recombinant Proteins; C050600:Sambucus nigra lectins; D012799:Sialyltransferases; D054788:Ribosome Inactivating Proteins; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase