Cerebroside galactosidase: a method for determination and a comparison with other lysosomal enzymes in developing rat brain.

( 1) A method is described for assaying brain for cerebroside galactosidase activity. The enzyme was liberated by sonication and addition of sodium taurocholate, then by digestion with pancreatic enzymes. It was further purified by precipitation at pH 3. The enzyme was then incubated with an emulsion of galactose-labelled cerebroside in taurocholate and oleate at pH 4.5, and the liberated galactose was determined by scintillation counting. (2) The content of cerebroside galactosidase in rat brain at various ages has been determined. The enzyme was present before cerebroside appears in noticeable amounts (4 days) and the amount rose considerably during the period of active cerebroside deposition and myelination. The amount then remained at a high concentration even in the adult. (3) Comparison with other lysosomal brain enzymes was made in the age study. Nitrophenyl galactoside hydrolase also increased during myelination but levelled off earlier; its activity paralleled the amount of ganglioside. Nitrophenyl glucoside hydrolase started at a lower level and decreased with age. Sulphatase activity rose during myelination, then decreased somewhat after 15 days. Ceramidase followed a pattern similar to that of nitrophenyl galactoside hydrolase; it is suggested that both of these enzymes reflect ganglioside metabolism. (4) The relative amounts of brain enzymes in different states were determined as a function of age in the case of cerebrosidase, nitrophenyl galactoside hydrolase and sulphatase. The proportion found in the high speed supernatant fraction was low but increased after myelination. The proportion that could be 'solubilized' by sonication decreased after myelination but the values differed greatly for the three enzymes. This treatment solubilized one-seventh of the cerebrosidase, half the nitrophenyl galactosidase and three-quarters of the sulphatase. IN THE course of developing a method for purification of rat brain cerebroside galactosidase (cerebrosidase), we observed that there was a positive correlation between the concentration of enzyme and the concentration of cerebroside (BOWEN and RADIN, 1968a). The subcortical region of rat brain contained more of both than did the cortex, and older rats contained more of both than did younger rats. It seemed of interest to obtain further details on the changes with age, especially with regard to the question whether or not cerebrosidase appears in brain before the appearance of cerebroside. This lipid is essentially indetectable before 9 days in rat brain, then increases rapidly (KISHIMOTO, DAVIES and RADIN, 1965). 1 This work supported in part by a Public Health Service Grant, No. NB-03192, from the National a Present address: Unilever Research Laboratory, Isleworth, Middlesex, England. Abbreuiafions used: NPGalH, p-nitrophenyl B-D-galactoside hydrolase; NPGluH, p-nitrophenyl B-D-glucoside hydrolase; Buffer A, 0.05 hf-Tris-HCI, pH 7.4 (at room temperature), -+ 0.01 M-MgCIB -k 8 mhi-mercaptoethanol. Institute for Neurological Diseases and Blindness.