A FLUOROMETRIC ENZYME ASSAY FOR THE DIAGNOSIS OF SANFILIPPO DISEASE-C(MPS-III-C)

Both the alpha- and beta-anomers of 4-methylumbelliferyl-D-glucosamini de were synthesized and shown to be substrates for the lysosomal acety l-CoA: glucosaminide N-acetyltransferase. Using the beta-anomer, fibro blasts and leukocytes from 11 different Sanfilippo C patients showed < 1 % of mean normal N-acetyltransferase activity. Heterozygotes showed intermediate activities. The enzymatic liberation of the fluorochrome from 4-methylumbelliferyl-beta-D-glucosaminide requires the sequentia l action of the N-acetyltransferase and beta-hexosaminidase. Normal be ta-hexosaminidase activity caused complete hydrolysis of the reaction intermediate 4-methylumbelliferyl-beta-D-N-acetylglucosaminide formed by the N-acetyltransferase. In cell extracts with a beta-hexosaminidas e deficiency, however, a second incubation in the presence of excess b eta-hexosaminidase is needed to avoid underestimation of the N-acetylt ransferase activity.