ISOLATION AND SOME PROPERTIES OF GLYCATED D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM RABBIT MUSCLE

Glycated D-glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from rabb it muscle and human erythrocytes have been investigated. The specific activity of the non-glycated GAPDH from rabbit muscle is approx. 180 u nits. (One unit is defined as the specific activity required to conver t 1 mu M of substrate/min per mg of enzyme.) The activity of the glyca ted enzyme, consisting of two sugars per tetramer, is lower than that of the non-glycated GAPDH. Non-enzymic transamination of the N-termini of glycated GAPDH (gGADPH) indicates that they are not blocked by gly cation. The rate of modification of thiols (Cys-149) with 5,5'-dithiob is-(2-nitrobenzoic acid) was greater for the glycated than the non-gly cated enzymes. The rate of modification of amino groups of Lys residue s of gGAPDH with o-phthalaldehyde was greater for the non-glycated enz yme. In 0.18 M guanidine-HCl solution, the emission intensity at 410 n m of a fluorescent NAD(+) derivative introduced into the active site d ecreased to 80 %, whereas that of gGAPDH decreased to 50 %. This sugge sts that the glycated sites are near the active site; glycation of the enzyme leads to a change of the microenvironment of Cys-149, alters t he conformation of the active site and decreases the activity.