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An enzyme system consisting of washed acetone powder of pig brain, adenosine triphosphate, nicotinamide adenine dinucleotide, magnesium, fumaric acid, and ascorbic acid was found to catalyze the oxidative decarboxylation of 2-ketostearic acid. The products were carbon dioxide and heptadecanoic acid. Washing the enzyme powder with ethylene-diaminetetraacetate and other chelating agents destroyed the activity. Further properties of the enzyme system are described. It is believed that this enzyme system accounts in part for the 1-carbon degradation route for brain fatty acids.

The 2-hydroxy fatty acids tend to yield streaks in thin-layer chromatography on silica gel plates. If potassium oxalate is included with binder-free silica gel, good spots are obtained. Similar difficulties are found in paper chromatography of the fatty acid derivatives of coenzyme A, especially with long-chain acids. The same thin-layer system gives good spots with these compounds.

Amides resembling ceramide (fatty acyl sphingosine) were synthesized and tested for their effects on rat brain cerebrosidase (galactosyl ceramide beta-galactosidase). The best inhibitor was N-decanoyl dl-erythro-3-phenyl-2-aminopro-panediol, which exhibited a K(i) of 0.4 mm. A Lineweaver-Burk plot indicated that the amide acted as a noncompetitive inhibitor, presumably by attachment to a site other than the substrate-active site. Preincubation did not affect the degree of inhibition, and inhibition was independent of incubation duration; these observations suggest that the inhibitor does not combine with the enzyme irreversibly. Structural variations produced decreased inhibitory activity: loss of one of the hydroxyl groups, replacement of the aromatic side chain with an aliphatic or substituted phenyl group, or isomeric inversion of the 3-hydroxyl group. It appears that the best activity is obtained with a substance most closely resembling natural ceramide. The cerebrosidases of rat spleen, kidney, and liver are also inhibited by the same amide.

The galactoside bond in cerebroside was found to be cleaved by an enzyme in rat and pig brain. Emulsified stearoyl-(14)C psychosine was used as the substrate and the extent of cleavage was studied by isolating and counting the stearoyl sphingosine (ceramide) formed. The reaction products, ceramide and galactose, were characterized by column and thin-layer chromatography. Cerebroside containing galactose-(3)H was also used to show liberation of galactose. Cholic acid was found to be required for activation of the enzyme, which has a pH optimum of 4.5. Similar cerebrosidase activity was found in spleen, kidney, and lung of rat; liver and heart showed very slight activity. The partially purified enzyme from pig brain also formed ceramide from ceramide lactoside, ceramide glucoside, and cerebronoyl psychosine. The enzyme was active toward o-nitrophenyl galactoside and could be fractionated by Sephadex chromatography into a fraction active toward the nitrophenyl galactoside only and a fraction active toward both this substrate and ceramide galactoside. Human spleen, normal and Gaucher, exhibited cerebrosidase activity.

In a previously described method for determining the activity of cerebroside galactosidase, the enzyme preparation was incubated with an emulsion of cerebroside which had been labeled in the galactose moiety. The liberated galactose was separated from the emulsion by liquid-liquid partitioning, but the presence of detergent necessitated the use of careful agitation and a backwash in order to reduce the contamination of the aqueous layer with excess emulsified substrate. This problem is eliminated by adding a large amount of lipid to reduce the emulsifying power of the detergent. It may be that other lipid hydrolase assays, based on the same principle, would benefit by this approach. Some additional improvements in the assay system are described.

A new method is described for the determination of brain gangliosides by measuring stearic acid, the chief acid of gangliosides, in an appropriately purified brain extract. The method includes extraction of tissue with chloroform-methanol, extraction of gangliosides from the extract with 0.1 M KCl, evaporation of the aqueous phase, methanolysis, and gas-liquid chromatography of the resultant methyl esters with a double internal standard. The method depends on the simple composition of ganglioside fatty acids (80% stearic acid) and allows determination of less than 0.05 micromole of gangliosides. Interfering lipids are removed from the ganglioside extract by washing with chloroform-methanol-water. The effects of contamination with nonlipid N-acetylneuraminic acid are avoided.

Glucocerebrosidase activity was reconstituted in vitro from a soluble glycoprotein factor (P) and a particle-bound factor (C). The physiological significance of the system is discussed.

A device that lowers a micropipet into position just above a thin-layer plate is described. Use of a self-filling pipet and foot-operated bellows allows one to apply many portions of a sample to the same point at high speed without damaging the adsorbent coating.