Roy M. Bradley

Lactosylceramide galactosidase: Comparison with other sphingolipid hydrolases in developing rat brain

Abstract Four enzyme assays were carried out with brains from rats of age 4 days to about 320 days. The enzymes were acid hydrolases: lactosylceramide galactosidases, galactosylphenol galactosidase, glucosylceramide glucosidase,a nd sphingomyelin phosphocholine hydrolase. The first two activities (based on wet brain weight) rose with age until about 24 days, then declined moderately; this curve parallel somewhat the curve for ganglioside concentration in brain. It is suggested that this parallelism supports the idea that lysosomal enzymes function in normal turnover. The other two enzymes studied showed a steadily declining activity with increasing age. It was found that the brain cytosol contained 12% or less of the galactosidase activiti, the value increasing somewhat in older rats. Most of the particulate galactosidase activities could be dispersed by sonication. The similarities between the two galactosidase activities suggest that most of the hydrolysis of the galactosylphenol is carried out by the lactosylceramide hydrolase. A procedure is given for preparing [ 3 H]lactosylceramide labeled in the galactose portion of the molecule and for the assay of its hydrolase.

GANGLIOSIDE COMPOSITION AND BIOSYNTHESIS IN CULTURED CELLS DERIVED FROM CNS

Abstract— Cultured mouse neuroblastoma cells (clone N18) contained a homologous series of gangliosides, GM3, GM2, GM1 and GD1a; the total lipid bound sialic acid (LBSA) was 3.3 nmol per mg of protein, of which GD1a comprised two‐thirds. In contrast, neonatal hamster astrocytes (clone NN) and human glioblastoma cells (Cox clone) contained mainly GM3, which represented 95% of the 2 nmol of LBSA per mg protein in these cells. When the cells were grown in the presence of [14C]galactose, label was incorporated into all of the gangliosides isolated from the cells. The labeling pattern corresponded to the ganglioside composition of the cell lines; GD1a was more extensively labeled in N18 cells and GM3 was the major labeled ganglioside extracted from glial cells. In addition to in rivo biosynthesis, in vitro synthesis of gangliosides was also determined. The activities of five glycosyltransferases of the ganglioside biosynthetic pathway were measured in homogenates of the three cell lines. The neuroblastoma cells contained all five enzyme activities whereas the two glial cell lines were deficient in UDP‐N‐acetylgalactosamine: GM3N‐acetylgalactosaminyltransferase activity, which catalyzes the synthesis of GM2 from GM3. The results indicated that cells of neuronal origin contain the more complex gangliosides associated with CNS and the requisite biosynthetic enzymes and that cells of glial origin are missing these complex gangliosides and the key glycosyltransferase required for their synthesis.

GM3 (Hematoside) Sphingolipodystrophy

Abstract To characterize further disorders involving storage of glycosphingolipids, we studied an infant who had poor physical and motor development, coarse facies, macroglossia, gingival hypertrop...

Separation of the glycoprotein and ganglioside components of thyrotropin receptor activity in plasma membranes

Abstract The two components of thyroid plasma membranes known to interact with thyrotropin, i.e., a glycoprotein with specific thyrotropin binding activity and the gangliosides of the thyroid membranes, are shown to segregate differently when membranes are solubilized with lithium diiodosalicylate. Individually examined, the interaction of each component with thyrotropin exhibits a different sensitivity to salts. The data suggest that the thyrotropin receptor on the thyroid membrane is a complex which is composed of both glycoprotein and ganglioside components and that its properties are derived from each component.

Response of sphingolipid hydrolases in spleen and liver to increased erythrocytorhexis

Abstract The injection of phenylhydrazine, zymosan, and human erythrocyte stroma caused an increase in the specific activities of various hydrolytic enzymes in rat spleen and liver tissues. The activity of spleen glucocerebrosidase and sphingomyelinase was particularly augmented by the injection of phenylhydrazine whereas the increases obtained with zymosan were not statistically significant. The injection of red-cell stroma brought about a significant increase in the activity of these enzymes; the injection of lipid-extracted stroma had no effect. The injection of partially purified stromal sphingolipids consisting mainly of globoside and sphingomyelin caused an increase in the activity of glucocerebrosidase and sphingomyelinase in liver but not in spleen.

Determination and physiologic disposition of meprobamate.

Summary (1) A specific method for the determination of meprobamate in urine is described. (2) Metabolic studies indicate that about 12 to 20 percent of the unchanged drug is slowly excreted in the urine in 48 hours and that additional products are formed, including several conjugates at least one of which is a glucuronide. (3) Radioactive meprobamate has been prepared and metabolic studies have been performed. The liver is the principal site of metabolic alteration.

EFFECT OF PUROMYCIN ON THE INCORPORATION OF ERYTHRO dl‐(3H)SPHINGOSINE INTO SPHINGOLIPIDS IN VIVO

EXPERIMENTS described previously revealed that chemically synthesized erythro DL[3H]sphingosine when injected intercerebrally into young rats was converted to both ceramide and sphingomyelin (KANFER and GAL, 1966). Under these conditions, radioactivity was not detected in the sphingoglycolipids. In a recent in vivo study it was reported that puromycin drastically inhibited the incorporation of radioactive carbohydrates (i.e. [14C]glucose, [14C]galactose and [%]galactosamine) into gangliosides (KANFER and RICHARDS, 1967). However, there was only a moderate depression of incorporation of radioactivity into the lower phase sphingoglycolipids. As an extension of these earlier observations it appeared of interest to detennine the effect of puromycin on the incorporation of radioactive sphingosine in vivo into sphingolipids. MATERIALS AND M E T H O D S The chemical synthesis of erythro DL-sphingosine specifically labeled with tritium at the A14,15 position from the methyl end of this amino alcohol, has been reported GAL, (1967). Purornycin HC1 was obtained from Nutritional Biochemical Co., Sprague-Dawley rats, 7-8 days old, were used exclusively in these studies. Mode of administration of compounds and experimenfaIprotoco2. The general method was similar to that of FLEXNER, FLEXNER, STELLAR, DE LA HABA and ROBERTS (1962). Puromycin HCl was dissolved in water at a 6nal concentration of 0.5 % and neutralized with NaHCO, prior to use. Each experimental animal was injected with 20 of this solution into the parietal region of the brain while the controls received isotonic saline. Two hours after puromycin administration the radioactive material dissolved in water was injected into the parietal region of each animal in a volume of 20 pl containing 266 pg of erythro ~~-[~H]sphingosine (specific activity = 1.6 X loE dis/min/pmole). At this time, and each hour thereafter for the duration of the study, the experimental animals received 2O-,~l injections of the puromycin solution and the controls saline. The animals were killed 4 hr after administration of the radioactive substrates. The present study contained three groups of at least five animals which received puromycin and three groups of at least four animals which received saline. Each group of animals was worked up separately so that the observations presented were obtained in triplicate. Treatment of tissues. The methods employed for the extraction, chromatography, and analysis of the sphingolipids have been described ( K A W R and RICHARDS, 1967).

The effect of pancreatectomy on fatty acid synthesis in liver of fasted rats.

Abstract 1. 1. Synthesis of long-chain fatty acids from sodium [I- 14 C]acetate was markedly impaired in liver slices and soluble enzyme preparations from rats deprived of food for 40 h. Synthesis of long-chain fatty acids was also reduced in slices from fasted pancreatectomized rats, whereas synthesis in soluble enzyme preparations of livers from these rats was only slightly less than that from normal fed rats. 2. 2. Addition of glucose 6-phosphate accelerated long-chain fatty acid synthesis in enzyme preparations from all three groups of rats. Maximal synthesis in the presence of glucose 6-phosphate was 3–4 times higher in preparations from fasted diabetic than from fasted normal rats. 3. 3. Administration of dexamethasone in vivo increased long-chain fatty acid synthesis in soluble enzyme preparations of liver, but not in liver slices, from fasted normal rats.

GM3 GANGLIOSIDOSIS: A NEW LIPID STORAGE DISEASE

Inherited defects in sphingolipid metabolism are associated with a number of disorders. A one mo. old male (JH) was first seen because of poor physical and motor development, frequent seizures, and an unusual appearance. The first child of young, unrelated Jewish parents, JH weighed 7 1bs. 12 oz. at birth after a normal 8 mo. gestation. He was limp and unresponsive, with coarse facies, macroglossia, gum hypertrophy, squat hands and feet, flexor contractures of the fingers, thickened loose hirsute skin, large inguinal hernia, enlarged liver and spleen and normal fundi. Death at 3 1/2 mo. followed a series of bronchopneumonic episodes. These features suggested GM1, gangliosidosis, which was ruled out by the finding of normal β-galactosidase activity in leukocytes and in a liver biopsy. The activities of acid phosphatase, β-glucosidase, β-N-acetylhexoseaminidase, α-fucosidase, α-mannosidase, and arylsulfatase A were greater than normal. The diagnosis of GM3 gangliosidosis was established by thin-layer chromatographic analysis, which demonstrated the accumulation of GM3 (N-acetylneuraminylgalactosylglucosylceramide) in post mortem samples of brain and liver. The enzymatic basis of the GM3 storage is now under investigation. (Supported in part by the John A. Hartford Fndn. and the Tay-Sachs Assn. of Maryland.)