J.T.R. Clarke

Localization of neutral, magnesium-stimulated sphingomyelinase in plasma membrane of cultured neuroblastoma cells

Abstract A parallel is shown between the distribution of neutral sphingomyelinase and plasma membrane enzymes (5′-nucleotidase and (Na+ + K+)-activated ATPase) in cultured neuroblastoma cells. In contrast there is no evidence of localization in lysosomes (β-hexosaminidase and acid sphingomyelinase), mitochondria (carnitine palmitoyltransferase), or cytosol. Activity in the microsomal fraction is attributed primarily to plasma membrane contamination.

Catabolism of Exogenous and Endogenous Sphingomyelin and Phosphatidylcholine by Homogenates and Subcellular Fractions of Cultured Neuroblastoma Cells. Effects of Anesthetics

Abstract: Cultured murine neuroblastoma cells contain a neutral, Mg2+‐stimulated sphingomyelinase and an alkaline phosphatidylcholine‐hydrolyzing activity that are enriched in the plasma membrane fraction. The reaction products of sphingomyelin catabolism are phosphocholine and ceramide and those of phosphatidylcholine, glycerophosphocholine and fatty acid. These reactions were studied with endogenous as well as exogenous liposomal substrates. With both exogenous and endogenous substrates, the sphingomyelinase activity was stimulated two‐to threefold by Mg2+ and a further three‐to fourfold by volatile anesthetic agents. Stimulation was concentration‐dependent and corresponded to anesthetic potency: methoxyflurane > halothane > enflurane. Greater than 80% of the plasma membrane sphingomyelin was hydrolyzed within 2 h in the presence of Mg2+ and anesthetic. In contrast, the activity with exogenous and endogenous phosphatidylcholine was unaffected by Mg2+ or Ca2+ and was markedly inhibited (50–80%) by anesthetic agents. The degree of inhibition was concentration‐dependent and corresponded to anesthetic potency. The quantitative importance of choline‐containing lipids in cell membranes, the relatively exclusive localization of the neutral Mg2+ ‐stimulated sphingomyelinase in cells of neural origin, the totally different type of hydrolytic attack on phosphatidylcholine, and the reciprocal effects of anesthetics on the hydrolysis of these two lipids strongly suggest important roles for these activities in cell membranes in general and in the neuron in particular.

Phospholipid transfer protein-mediated incorporation and subcellular distribution of exogenous phosphatidylcholine and spingomyelin in cultured neuroblastoma cells

Murine neuroblastoma cells (strain N1E-115) were incubated with 2-[fatty acyl-14 C]acylphosphatidylcholine/sphingomyelin or phosphatidylcholine/[choline-3H]sphingomyelin liposomes (1:1, mol/mol; 1.2 mumol total lipid/mg cell protein) in the presence of partially purified rat liver phospholipid transfer protein (2.5 mg/ml), cytochalasin B (50 microM) and 2-deoxyglucose (50 mM) for 10 min. Washed cells were chased for periods of up to 45 min at 37 degrees C with medium containing transfer protein and unlabeled liposomes. Total transfer protein-dependent incorporation of [14C]phosphatidylcholine ([14C] PC) and [3H]sphingomyelin was 136.7 +/- 26.5(n = 5) and 23.7 +/- 5.4(n = 6) nmol/mg protein per 10 min incubation, respectively, (mean +/- S.D.). Incorporation of [14C]PC into the mitochondrial membrane fraction was 128-fold greater (nmol/mg protein) than incorporation of [3H]sphingomyelin. In contrast, incorporation of [3H]sphingomyelin into a fraction enriched in plasma membrane and into microsomes was 1.4- and 2.6-fold greater, respectively, than incorporation of [14C]PC. During the chase periods, the specific activities of total cellular phospholipids decreased as intact [14C]PC and [3H]sphingomyelin accumulated in the culture medium. In the case of cells labeled with [14C]PC, the effect was due primarily to a decrease in the amount of labeled phospholipid in the mitochondrial fraction; in the case of cells labeled with [3H]sphingomyelin, the decrease in activity was greatest in microsomal and plasma membrane phospholipids. The rate and extent of non-endocytotic incorporation of exogenous phosphatidylcholine into the cell membrane of cultured neuroblastoma cells, and its subsequent subcellular disposition, is different from that of exogenous sphingomyelin. Whereas PC is evidently incorporated into and turned over most rapidly in fraction enriched in mitochondrial membranes, sphingomyelin appears to be preferentially incorporated into microsomal and plasma membrane.

Inability of rabbit peritoneal polymorphonuclear leukocytes to synthesize arachidonic acid from linoletc acid

In PMN leukocytes isolated from rabbit peritoneal exudate the major phospholipids were choline phosphoglycerides (40%), ethanolamine phosphoglycerides (26%) and sphingomyelin (20%) with lesser amounts (3-6%) of serine and inositol phosphoglycerides. The essential fatty acid, linoleic acid, predominated (greater than 35%) in each phospholipid except in inositol phosphoglycerides where it was slightly less than arachidonate and in sphingomyelin where saturated acids predominated. However, on a total mass basis there was more arachidonate in ethanolamine and choline phosphoglycerides than in inositol phosphoglycerides. The uptake, incorporation and metabolism of [1-14C] fatty acids of varying chain length and degrees of unsaturation were examined. All fatty acids were taken up but incorporation of saturated acids varied inversely with chain length. Arachidic acid and trans-isomers of 18:1 and 18:2 were esterified primarily to triacylglycerol whereas phospholipids contained a large portion of the other acids. Icosatrienoic and arachidonic acids were esterified to ethanolamine, serine and inositol phosphoglycerides to a comparatively greater extent, reflecting the normal distribution of these fatty acids. PMN leukocytes had a low capacity for delta 9 desaturation and chain elongation and no delta 6 or delta 5 desaturation could be detected. Thus, PMN leukocytes lack the ability to form arachidonate from 18:2 precursor molecules available in the cellular neutral lipids and phospholipids and arachidonate per se is an essential fatty acid for these cells.

Studies on the turnover of endogenous choline-containing phospholipids of cultured neuroblastoma cells

Previous studies on neuroblastoma cells in culture showed that the presence of partially purified rat liver phospholipid-transfer protein had a marked differential effect on the uptake and apparent subcellular distribution of radioactively labeled sphingomyelin and phosphatidylcholine (PC) added to the medium as mixed phospholipid (PC/sphingomyelin) liposomes. To determine the effect of phospholipid-transfer protein and exogenous phospholipids on the turnover and subcellular distribution of endogenous phospholipids, neuroblastoma cells were preincubated for 48 h in the presence of [methyl-3H]choline and washed. Aliquots of prelabeled cells were reincubated immediately in medium containing phospholipid-transfer protein mixed phospholipid liposomes, cytochalasin B and 2-deoxyglucose for 45 min at 37 degrees C; additional aliquots were chased first for 2 or 18 h with unlabeled choline before reincubation. The extent of labeled phospholipid degradation and accumulation in the medium, and the subcellular distribution of cell-associated labeled choline-containing phospholipids were determined. During incubation with phospholipid-transfer protein and mixed phospholipid liposomes, 25-35% of the cell-associated radioactive label from prelabeled cells, chased or unchased, was lost to the medium in 45 min. Over 50% of the label appearing in the medium was in water-soluble phospholipid degradation products. The loss of cell-associated label into the medium from unchased cells was stimulated significantly by phospholipid-transfer protein; however, prelabeled cells which had been chased for 18 h with unlabeled choline were unaffected by the presence of transfer protein. Endogenously synthesized radioactively labeled PC and sphingomyelin were distributed throughout all subcellular membranes, but least of all in the crude mitochondrial membrane fraction. Analysis of the subcellular distribution of cell-associated label remaining in chased or unchased cells after 45 min incubation with PC/sphingomyelin liposomes showed proportionate losses from all membrane fractions, except the crude mitochondrial fraction, which showed relative retention of labeled phospholipid. Phospholipid-transfer protein had no effect. The results are in distinct contrast to observations on the turnover, metabolism and subcellular distribution of labeled exogenous phospholipids under the same conditions, indicating that exogenous phospholipids do not intermix freely with any quantitatively major pool of endogenous phospholipid.

Abnormal neutral lipase activity in acid-lipase-deficient cultured human fibroblasts.

Acid and neutral lipase activities of homogenized or sonicated cultured fibroblasts were examined using [2-3H]glycerol triolein, glycerol tri[1-14C]oleate or cholesterol [1-14C]oleate as substrates. In normal fibroblasts, optimal conditions for acid lipase activity were pH 4.5–5.0, 0.15–0.2 mM triacylglycerol, and 0.25% Triton X-100. Fatty acid release was linear to 2 h and between 0.2–2.0 mg fibroblast protein/ml. For the neutral lipase, activity was optimal at pH 6.0–7.0, >1.5 mM triacylglycerol or cholesterol oleate (suspended in 8 mg albumin/ml), and 160 μ g phosphatidylserine/ml. The reaction was linear to 60–120 min, and up to 1.0 mg protein/ml. In contrast to the situation at neutral pH, very little [3H]glycerol was released under acid conditions, suggesting little monoglyceride lipase activity at acid pH. Acid lipase activity in fibroblasts from Wolmans disease (WD) or cholesterol ester storage disease (CESD) patients was <1% of activity in normal fibroblasts. Neutral lipase activity (as % of control values) was <15%, <15%, and undetectable when measured as fatty acid release or diglyceride or monoglyceride appearance, respectively, and was less than 25% of controls when cholesterol oleate was the substrate. Neutral lipase activity in mixtures of control and WD fibroblast homogenates was similar to that predicted from individual activities. The triacylglycerol content of the control and mutant cells was within normal limits, and cholesterol content was only slightly elevated, indicating that endogenous dilution of substrate was not the reason for the low activity in mutant cells. Hydrolysis of labeled endogenous triacylglycerol was reduced by >75–80% in the WD cells, indicating that low levels of activity measured with the exogenous substrate were not due solely to lack of accessibility to the neutral lipase. These results suggest a close relationship between the acid and neutral activities of the normal cell in that mutations which affect one apparently affect the activity of the other.

Effects of glycine and glyoxylate on cerebral glucose oxidation in vitro

In Patients with nonketotic hyperglycinemia (NKHG), the principal route of glycine catabolism in the CNS is interrupted by an hereditary defect in the glycine cleavage reaction [5, 10‐methylenetetrahydrofolate: ammonia hy‐droxymethyltransferase (decarboxylating, oxidizing); EC 2.1.2.10] (ANDO el al, 1968; Perryet al, 1975). The resulting accumulation of glycine in cerebral tissue of affected infants is invariably associated with the development of severe brain damage, though the mechanism of the damage is still unknown.

Uptake of radiolabeled galactosyl-(α1 → 4)-galactosyl-(β1 → 4)-glucosylceramide by human serum lipoproteins in vitro

Abstract Human serum was exposed to various amounts of [6- 3 H]galactosyl-(α1 → 4)-galactosyl-(β1 → 4)-glucosylceramide under standardized conditions in vitro, and the uptake of the lipid by serum lipoproteins was determined. Of the bound glycolipid, 2% was isolated with very low density, 24% with low density-, 47% with high density lipoproteins and 27% with the ultracentrifugal residue. The distribution was different from the distribution of endogenous galactosyl-galactosylglucosylceramide, indicating that the glycolipid is probably an integral part of the lipoprotein complexes in vivo.

Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells

To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22h in the presence ofd-[1-3H]galactose or [3H]GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipidsialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellular membrane fractions studied was recovered from plasma membrane and only 10–15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous [3H]GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid.

Preparation and characterization of antibody to galactosyl(α1 → 4)galactosyl(β1 → 4)glucosylceramide

Abstract Antiserum against galactosyl(α1 → 4)galactosyl(β1 → 4)glucosylceramide (globotriaosylceramide, Gb3) was raised in rabbits by the administration of four weekly intramuscular injections of 1.5 mg of the purified glycolipid along with bovine serum albumin and Freunds complete adjuvant. AntiGb3 activity was quantitated initially by immunoprecipitation employing Gb3 mixed with 100-fold excess of lecithin and cholesterol (1 : 1 or 1 : 2, by wt.) as antigen. Subsequently, complement fixation tests done with antigen preparations containing Gb3/lecithin/cholesterol (1 : 6 : 20, by wt.) showed antiGb3 titres of up to 1 : 8192. Fractionation of the antiserum by BioGel A5m chromatography indicated the antibody was an IgM immunoglobulin. The partially purified antibody stimulated complement-dependent release of glucose from glucose-containing liposomes prepared with sphingomyelin/cholesterol/dicetylphosphate/Gb3 (molar ratio, 100 : 75 : 11 : 1). The antibody crossreacted with the trisaccharide, Gal(α1 → 4)Gal(β1 → 4)Glc, but not with galactosylceramide, lactosylceramide, GM1 ganglioside, globotetraosylceramide, digalactosyldiglyceride or a number of low molecular weight saccharides.