Matthew W. Spence

Evidence that neutral spingomyelinase of cultured murine neuroblastoma cells is oriented externally on the plasma membrane

The activity of the neutral, Mg2+-stimulated sphingomyelinase of cultured neuroblastoma cells (N1E-115) is enriched in the plasma membrane fraction and is reduced following treatment of intact or broken cells with trypsin, alpha-chymotrypsin, papain, and protease. Two protease-sensitive enzymes of the cell interior (lactate dehydrogenase and NADPH-cytochrome c reductase) are not affected by protease treatment of intact cells. These results indicate that the neutral, Mg2+-stimulated sphingomyelinase is oriented externally on the plasma membrane of the cultured neuroblastoma cell.

Acid and neutral sphingomyelinases of rat brain. Activity in developing brain and regional distribution in adult brain

SPHINGOMYELINASE catalyses the hydrolysis of sphingomyelin to ceramide and phosphorylcholine (BARNHOLZ et 01.. 1966; HELLER & SHAPIRO, 1966; KANFER et a/., 1966; SCHNEIDER & KENNEDY, 1967). Two sphingomyelinases have been described in brain. One exhibits an acid pH optimum (pH 5.0), is relatively stable to manipulation and storage, and has several isoenzyme forms (BARNHOLZ et al., 1966; CALLAHAN et al., 1974; RAO & SPENCE, 1976; YAMAGUCHI & SUZUKI. 1977). It is widely distributed in mammalian tissues (KANFER et a/., 1966: RACHMILEW~Z et a/.. 1967; ROELFZEMA er al.. 1973) and appears to be a constituent mainly of lysosomes (WEINREB et a/., 1968; FOWLER, 1969). Recently, a second sphingomyelinase (sph’ase 7.4) of brain that has a neutral pH optimum (pH 7.4) and requires Mgz+ for optimal activity has been described (RAO & SPENCE, 1976; GATT. 1976). Differences in several properties (RAO & SPENCE, 1976) indicate that the acid (sph’ase 5.0) and neutral (sph’ase 7.4) activities represent separate enzymes. Unlike sph’ase 5.0, sph’ase 7.4 is concentrated in the brain, and little activity is observed in spleen (SCHNEIDER & KENNEDY, 1967; RAO & SPENCE. 1976) liver or leukocytes (RAO & SPENCE, 1976). This concentration in brain suggests that the role of the enzyme(s) is related to specific functions of that organ. Information relevant to the physiological role of tissue elements can be obtained from studies relating developmental changes in concentration or activity to those in structure and/or function. This is particularly so in brain where there are relatively massive changes in structure and function during development, and the timing of these events in several species is well known (DAVISON & DOBBING, 1968). To learn more about the role of sph’ase 7.4 in brain, we studied the changes in sph’ase 7.4 activity with development and compared these to the changes in sph’ase 5.0 activity. Additional studies of the regional distribution of the two activities in brain have also been carried out.

Interaction of (n−3) and (n−6) fatty acids in desaturation and chain elongation of essential fatty acids in cultured glioma cells

Recent research in various biological systems has revived interest in interactions between the (n−6) and (n−3) essential fatty acids. We have utilized cultured glioma cells to show that linolenic acid, 18∶3(n−3), is rapidly desaturated and chain elongated; 20∶5(n−3) is the major product and accumulates almost exclusively in phospholipids. We examined effects of various (n−6), (n−3), (n−9) and (n−7) fatty acids at 40 μM concentration on desaturation and chain elongation processes using [1-14C]18∶3(n−3) as substrate. In general, monoenoic fatty acids were without effect. The (n−6) fatty acids (18∶2, 18∶3, 20∶3, 20∶4 and 22∶4) had little effect on total product formed. There was a shift of labeled product to triacylglycerol, and in phospholipids, slightly enhanced conversion of 20∶5 to 22∶5 was evident. In contrast, 22∶6(n−3) was inhibitory, whereas 20∶3(n−3) and 20∶5(n−3) had much less effect. At concentrations <75 μM, all acids were inhibitory. Most products were esterified to phosphatidylcholine, but phosphatidylethanolamine also contained a major portion of 20∶5 and 22∶5. We provide a condensed overview of how the (n−6) and (n−3) fatty acids interact to modify relative rates of desaturation and chain elongation, depending on the essential fatty acid precursor. Thus, the balance between these dietary acids can markedly influence enzymes providing crucial membrane components and substrates for biologically active oxygenated derivatives.

Dissociation of phosphorylation and translocation of a myristoylated protein kinase C substrate (MARCKS protein) in C6 glioma and N1E-115 neuroblastoma cells.

Abstract: An 80‐kDa protein labeled with [3H]myristic acid in C6 glioma and N1E‐115 neuroblastoma cells has been identified as the myristoylated alanine‐rich C kinase substrate (MARCKS protein) on the basis of its calmodulin‐binding, acidic nature, heat stability, and immunochemical properties. When C6 cells preincubated with [3H]myristate were treated with 200 nM 4β‐12‐O‐tetradecanoylphorbol 13‐acetate (β‐TPA), labeled MARCKS was rapidly increased in the soluble digitonin fraction (maximal, fivefold at 10 min) with a concomitant decrease in the Triton X‐100–soluble membrane fraction. However, phosphorylation of this protein was increased in the presence of β‐TPA to a similar extent in both fractions (maximal, fourfold at 30 min). In contrast, β‐TPA–stimulated phosphorylation of MARCKS in N1E‐115 cells was confined to the membrane fraction only and no change in the distribution of the myristoylated protein was noted relative to α‐TPA controls. These results indicate that although phosphorylation of MARCKS by protein kinase C occurs in both cell lines, it is not directly associated with translocation from membrane to cytosol, which occurs in C6 cells only. The cell‐specific translocation of MARCKS appears to correlate with previously demonstrated differential effects of phorbol esters on stimulation of phosphatidylcholine turnover in these two cell lines.

Turnover of phospholipid fatty acyl chains in cultured neuroblastoma cells: involvement of deacylation-reacylation and de novo synthesis in plasma membranes

Cultured neuroblastoma cells (NIE-115) rapidly incorporated the essential fatty acid, linoleic acid (18:2 (n = 6), into membrane phospholipids. Fatty acid label appeared rapidly (2-10 min) in plasma membrane phospholipids without evidence of an initial lag. Specific activity (nmol fatty acid/mumol phospholipid) was 1.5-2-fold higher in microsomes than in plasma membrane. In these membrane fractions phosphatidylcholine had at least 2-fold higher specific activity than other phospholipids. With 32P as radioactive precursor, the specific activity of phosphatidylinositol was 2-fold higher compared to other phospholipids in both plasma membrane and microsomes. Thus a differential turnover of fatty acyl and head group moieties of both phospholipids was suggested. This was confirmed in dual-label (3H fatty acid and 32P), pulse-chase studies that showed a relatively rapid loss of fatty acyl chains compared to the head group of phosphatidylcholine; the opposite occurred with phosphatidylinositol. A high loss of fatty acyl chain relative to phosphorus indicated involvement of deacylation-reacylation in fatty acyl chain turnover. The patterns of label loss in pulse-chase experiments at 37 and 10 degrees C indicated some independent synthesis and modification of plasma membrane phospholipids at the plasma membrane. Lysophosphatidylcholine acyltransferase and choline phosphotransferase activities were demonstrated in isolated plasma membrane in vitro. Thus, studies with intact cells and with isolated membrane fractions suggested that neuroblastoma plasma membranes possess enzyme activities capable of altering phospholipid fatty acyl chain composition by deacylation-reacylation and de novo synthesis at the plasma membrane itself.

Phosphatidylcholine metabolism in cultured cells: catabolism via glycerophosphocholine

The catabolism of phosphatidylcholine (PtdCho) has been studied in cultured murine neuroblastoma (N1E-115), C6 glioma, rat brain primary glia, and human fibroblast cells. Cells were pulse labelled for 96 h with [methyl-3H]choline followed by a chase for up to 24 h in medium containing 4 mM choline. Measurement of the radioactivity and mass of choline-containing compounds in these cells indicated that the major degradative pathway is PtdCho----lysophosphatidylcholine (lysoPtdCho)----glycerophosphocholine (GroPCho)----choline. At all times during the chase, PtdCho, sphingomyelin and lysoPtdCho comprised 72-92% of the cell-associated radioactivity; the remaining 10-30% was water-soluble and was chiefly GroPCho (30-80%) in all cell lines. In fibroblasts, however, phosphocholine (PCho) was also a major labelled water-soluble component (33-54%). The specific activity of GroPCho closely parallelled that of PtdCho in fibroblasts, but decreased faster than PtdCho in C6 and N1E-115 cells. We postulate that this may be due to distinct pools of PtdCho in the cell with differing rates of turnover. The changes in specific activity of PCho suggest that the major portion is formed by synthesis rather than as a degradative product. However, the inability to reduce the specific activity of this fraction to that of the intracellular choline suggests that a portion may be derived from either PtdCho or GroPCho.

Neutral and acid sphingomyelinases: somatotopographical distribution in human brain and distribution in rat organs. A possible relationship with the dopamine system.

Acid and neutral sphingomyelinase activities have been measured in 22 regions of human brain, and in several rat organs. In general, acid sphingomyelinase activity was similar in most brain regions examined. By contrast neutral sphingomyelinase activity decreased 30-fold between the globus pallidus and white matter. In grey matter structures activity decreased in the order globus pallidus greater than substantia nigra greater than or equal to putamen greater than head of caudate greater than thalamus greater than cortical structures. Under the conditions of assay and in the presence of several possible donors or acceptors, there was no evidence of transfer of phosphoryl-choline to other lipid acceptors. Acid sphingomyelinase was ubiquitously distributed in all rat tissues examined, highest in liver and lowest in adipose tissue. Neutral sphingomyelinase activity was highest in brain; activity from 25 to 10% of that in brain was observed in testis, adrenal gland and aorta. Activity in the other organs examined was less than 10% of that in brain. We suggest that the neutral enzyme serves a special function in brain, perhaps related to the dopaminergic systems.

Biosynthesis of fatty acids in vitro by homogenate of developing rat brain: desaturation and chain-elongation

Abstract 1. 1. Chain-elongation of [1-14C]16:1(n−7) to 18:1(n−7) in rat brain homogenates was dependent on exogenous NADPH and malonyl-CoA; slight additive stimulation occurred when NADH and acetyl CoA were added. Activity was greatest in developing brain and lower in the adult. 2. 2. Chain-elongation of [1-14C]16:0, 18:0, and 18:1 showed a similar trend. 16:0 exceeded 18:0 elongation 5–20-fold at all ages examined; 18:1 elongation was even less. 3. 3. The formation of 18:1 from [1-14C]16:0 by desaturation and chain-elongation was higher in developing than in adult brain, and was stimulated 2-fold (adult) to 20–30-fold (fetal to 20 days) by NADPH and malonyl CoA. 18:1(n−7) accounted for 12–19% of total 18:1 formed at all ages. Thus, in rat brain the major pathway of 18:1 formation appears to be 16:0 → 18:0 → 18:1(n−9), and the minor pathway 16:0 → 16:1 → 18:l(n−7). 4. 4. Δ6 desaturation of exogenous [1-14C]16:0 and 18:0, and of 18:0 formed endogenously from exogenous 16:0, occurred between fetal stage and 20 days of age only. At birth, this activity accounted for 62% of the 16:1 formed from 16:0, and 28% of the 18:1 formed from 18:0. 5. 5. The previous demonstration of Δ9 desaturating activity, and the present findings of (a) chain-elongation of 16- and 18-carbon saturates and monoenes and (b) Δ6 desaturation of 16:0 and 18:0, show that developing rat brain has the necessary enzymes for forming all the major 16:1 and 18:1 monoenoic fatty-acid isomers.

Lysophosphatidylcholine as an intermediate in phosphatidylcholine metabolism and glycerophosphocholine synthesis in cultured cells: an evaluation of the roles of 1-acyl- and 2-acyl-lysophosphatidylcholine

Previous studies in our laboratory have shown that the principal pathway of phosphatidylcholine (PtdCho) degradation in cultured mouse N1E-115 neuroblastoma, C6 rat glioma, primary rat brain glia and human fibroblasts is PtdCho----lysophosphatidylcholine (lysoPtdCho)----glycerophosphocholine (GroPCho)----glycerophosphate plus choline (Morash, S.C. et al. (1988) Biochim. Biophys. Acta 961, 194-202). GroPCho is the first quantitatively major degradation product in this pathway, and could be formed by phospholipases A1 or A2, followed by lysophospholipase, or by a co-ordinated attack releasing both fatty acids by phospholipase B. The quality and quantities of lysoPtdCho present in cells reflect the nature of the initial hydrolysis step (A1 or A2), specificities of the lysophospholipases, and activities of acyltransferases that form PtdCho from lysoPtdCho. The present study was undertaken to elucidate the relative importance of these pathways by examining the fate of exogenous 1-acyl and 2-acyl-lysoPtdCho incubated with N1E-115 and C6 cells in culture. By fatty acid composition, endogenous lysoPtdCho was found to be mainly 1-acyl in both cell types based on a predominance of saturated acyl species; this suggested either preferential further deacylation or reacylation of 2-acyl-lysoPtdCho, or that 2-acyl-lysoPtdCho was not formed. Exogenous 1- and 2-acyl-lysoPtdCho specifically radiolabelled with choline and/or fatty acid were incubated either singly or as equimolar mixtures with cells. Cell association was rapid and not reversible by washing and both species were taken up at similar rates. The 2-acyl species was acylated to PtdCho faster than the 1-acyl species in both cell lines. Acylation of both lyso species was higher in C6 compared to N1E-115 cells. Hydrolysis of lysoPtdCho to GroPCho was higher in N1E-115 cells and with 1-acyl-lysoPtdCho. Transacylation between two molecules of lysoPtdCho was a minor pathway. These results document the variety and relative importance of reactions of lysoPtdCho metabolism; under similar conditions, 1- and 2-acyl-lysoPtdCho are handled differently. Both species turn over actively, but only the 1-acyl species accumulates while 2-acyl-lysoPtdCho is likely to be reacylated to form PtdCho.

Failure to Correct the Metabolic Defect by Renal Allotransplantation in Fabry's Disease

Plasma neutral glycolipid levels and plasma and leukocyte alpha-galactosidase activities were measured serially before and after renal allotransplantation in two men, aged 47 and 45 years, with renal failure due to Fabrys disease. The patients were followed posttransplantation for 92 and 64 weeks, respectively. No significant elevation of plasma or leukocyte alpha-galactosidase activities above levels in untreated men with Fabrys disease or decrease in the levels of trihexosyl ceramide was observed in either patient. The results do not support the use of renal allotransplantation for enzyme replacement in Fabrys disease.