An enzymatic determination method for galactosylceramide galactosidase (EC 3.2.1.46) was devised by using an enzymatic amplification reaction, NAD cycling. Gaiactose released by crude enzyme samples (tissue homogenates and cell suspensions) from galactosylceramide quantitatively reduced NAD to NADH by the galactose dehydrogenase reaction; then the NADH was amplified 6000-lO,OOO-fold by NAD cycling and determined fluorometrically. A higher sensitivity of assay was obtained compared with the previous radiometric method. The present method was successfully applied to tissues from patients with Krabbc's disease, whose organs are deficient in galactosidase. The galactosidase reaction rate with a crude sample was not proportional to its concentration. However, the double-reciprocal plot of the reaction rate against the sample concentration became linear and provided a unique value of specific activity to each sample.
Although galactosylceramide galactosidase is known to hydrolyze several natural lipid substrates: galactosylceramide ( 1,2), lactosylceramide (3,4), galactosylsphingosine (5), and monogalactosyldiacylglycerol(6), the radiolabeled galactosylceramide has been used as an original substrate in the radiometric determination method (7-l 1). The radiolabeled substrate is now not commercially available and one has to prepare it (12,13). This enzyme is deficient in patients with Krabbe's globoid cell leukodystrophy ( 14,15), therefore, its assay method is important for diagnosis using crude enzyme samples such as homogenate of human organs, leukocytes, and cultured cells. The available amounts of these samples are generally small, and high sensitivity of the assay method is required, especially when amniotic fluid cells are analyzed for prenatal diagnosis. Thus, considering this requirement and the inconvenience in obtaining radiolabeled substrate, a new nonradiometric method, having a OOOZ2697/82/150044-08%02.00/O