CEREBROSIDE GALACTOSIDASE: A METHOD FOR DETERMINATION AND A COMPARISON WITH OTHER LYSOSOMAL ENZYMES IN DEVELOPING RAT BRAIN 1

(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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66425/1/j.1471-4159.1969.tb06849.x.pd

Effect of methionine sulfoximine on methylation of guanine residues in astroglial transfer ribonucleic acids

Culture-grown astrocytes derived from 3-day-old rat brain were incubated in the presence of [ 3 H]guanosine and of the convulsant agent l -methionine- dl -sulfoximine (MSO). The resulting [ 3 H]tRNA was purified from control and MSO-exposed cells at several time points during the incubation and was hydrolyzed to [ 3 H]guanine and four [ 3 H]methyl guanines which were separated by high pressure liquid chromatography. Three of the four [ 3 H]methyl guanines were more highly labeled in the [ 3 H]tRNA of the MSO-exposed cells, relative to that of the control cells throughout the entire incubation period. The findings extend to cultured astrocytes, the stimulatory effect of MSO on the methylation of neural tRNA guanines, previouly observed both in vitro using [ 14 C] S -adenosyl- l -methionine and in vivo using [ methyl 3 -H] l -methionine.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45427/1/11064_2004_Article_BF00964832.pd

Biosynthesis of Polyamines in Mouse Brain: Effects of Methionine Sulfoximine and Adenosylhomocysteine

This study examines the consequences on cerebral polyamine biosynthesis of increases and decreases in cerebral methylation. Increases were elicited by administering the convulsant agent methionine sulfoximine (MSO) and decreases by elevating in vivo the cerebral levels of the methylation inhibitor S-adenosylhomocysteine. Following the intraventricular (i.vt.) administration of one of the two possible polyamine precursors, [1,4-14C]putrescine, the specific radioactivity (sra) of the newly formed [14C]spermidine remained unchanged. Conversely, after i.vt. L-[3,4-14C]methionine, the other polyamine precursor, significantly higher sra values for [14C]spermidine and [14C]spermine were recorded in the brains of the MSO-treated animals. [14C]S-adenosylmethionine in the brain of the MSO-treated animals was also more highly labeled following [1-14C]-methionine, indicating its accelerated formation relative to controls. We also investigated the effect of the administration of adenosine + homocysteine, a treatment that results in elevated brain adenosylhomocysteine levels, on polyamine biosynthesis from [3,4-14C]-methionine. The results of these experiments show both significantly lower sra values for [14C]spermidine and [14C]spermine and significantly higher than control endogenous methionine levels, a clear sign of the existence of a retardation in the conversion of methionine to polyamines under these conditions. In conclusion, the present study demonstrates that while interference with cerebral methylation results in significant alterations of the rate of formation of the methionine moiety of spermidine and spermine, it has no effect on the entry of the putrescine moiety into the two polyamine molecules

UNEQUAL PATTERNS OF DEVELOPMENT OF SUCCINATE-DEHYDROGENASE and ACETYLCHOLINESTERASE IN PURKINJE CELL BODIES and GRANULE CELLS ISOLATED IN BULK FROM THE CEREBELLAR CORTEX OF THE IMMATURE RAT 1

–A preparative procedure for the isolation in bulk of two cellular populations of the cerebellar cortex of the immature rat, the granule cells and the Purkinje cell bodies, is described. The procedure is used to delineate the developmental pattern of succinate-INT-reduclase (EC 1.3.99.1) and acetylcholinesterase (EC 3.1.1.7) in the crucial period of cerebellar maturation, i.e. between 12 and 19 days postnatally. Although the overall yield of neuronal RNA diminished with age, the proportion of RNA in the Purkinje cell body fraction increased while that in the granule cells decreased and microscopic examination of the fractions confirmed this result. The yields of succinate-INT-reductase and of acetylcholinesterase in the fractions paralleled the yields of RNA. A significant finding was the trend toward diminishing specific activities (units/Μg of RNA) with age of both enzymes in the Purkinje cell bodies as against the opposite, upward trend of their specific activities in the granule cells. An additional finding of interest was the different ratio of true acetylcholinesterase/total cholinesterase activity in the two cell types, with the granule cells consistently exhibiting higher true acetylcholinesterase values than the Purkinje cell bodies. The present report thus supplements the histoenzymological data on the developing rat cerebellum in that it reveals specific differences in the enzymatic development of two different cerebellar types, a finding which was greatly facilitated by the availability of the procedure for their bulk isolation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66200/1/j.1471-4159.1974.tb12210.x.pd