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Unit Code 80341:
Galactose-1-Phosphate Uridyltransferase Biochemical Phenotyping, Erythrocytes

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Method Description

Isoelectric focusing is used to resolve the

isoenzymes of GALT. The band patterns, when used in

conjunction with a quantitative GALT result, can be used to predict

the GALT phenotype of an individual.

 

In isoelectric focusing, a pH gradient is established across an

agarose gel by adding a select mixture of amphoteric molecules

to the gel and applying an electric field to the gel. Each protein

(isoenzyme) has its own unique isoelectric point, a pH at which the

net charge of the protein is equal to zero. Therefore, if a protein is

applied to the gel, it will migrate through the pH gradient in the gel

until it reaches its isoelectric point. There the protein will stop and

"focus" into distinct bands.

 

In this procedure, a red blood cell hemolysate is focused on a

5% agarose gel containing ampholytes of a 5-7 pH range.

The isoenzyme bands are then visualized by applying a substrate

mixture that results in a series of reactions (shown below).

The final product, NADPH, is stained a blue-violet color when it

reacts with phenazine methosulfate (PMS) and 3-(4-5

dimethylthiazol-2-yl)I-2,5-diphenyltetrazolium bromide (MTT).

(Shin YS, Niedermeier HP, Endres W, et al: Agarose gel

isoelectrofocusing of UDP-galactose pyrophosphorylase and

galactose-1-phosphate uridyltransferase: developmental aspect

of UDP-galactose pyrophosphorylase. Clin Chim Acta 1987;166:

27-35, modified to acrylamide as described by Leclerc P, Forest

JC: Electrophoretic determination of isoamylases in serum with

commercially available reagents. Clin Chem 1982;28:37-40)

    

 

                                             GALT

   Gal-1-P UDP-Glu -------------------> UDP-Gal Glu-1-P

 

                                     Phosphoglucomutase

   Glu-1-P Glu-1-6 diP --------------------> Glu-1-6 diP Glu-6-P

 

                              Glu-6-P-Dehydrogenase

   Glu-6-P NADP ----------------------->  Ribose-5-P CO(2) NADPH

 

   NADPH MTT PMS  --------------->  Blue Violet Stain

 

Performing Laboratory Location

Rochester

Key