Energy-dependent incorporation of sphingolipid precursors and fatty acids in Bacteriodes melaninogenicus.

Washed cells of Bacteroides melaninogenicus are unable to incorporate the sphingolipid precursor 3-ketodihydrosphingosine (3KDS) or dihydrosphingosine into the complete sphingolipids ceramide phosphorylethanolamine (CPE) and ceramide phosphorylglycerol (CPG), whereas growing cultures are able to do so. This result suggested that an energy source was required by washed cells to initiate the incorporation of 3KDS. Investigation of a number of energy sources for B. melaninogenicus showed that glutamine was active in driving the incorporation of 3KDS. This system shows saturation kinetics. Besides glutamine, only asparagine and reduced nicotinamide adenine dinucleotide (NADH) are effective; glutamate and other compounds are inactive. The glutamine-driven system is sensitive to 2,4-dinitrophenol, azide, N,N'- dicyclohexylcarbodiimide, and carbonyl cyanide m-chlorophenylhydrazone. Asparagine plus NADH shows a synergistic effect in stimulating the incorporation of 3KDS into CPE and CPG in washed cells. However, glutamine plus NADH and glutamine plus asparagine show no such synergy. The cytochrome-free mutant of B. melaninogenicus, strain S, incorporates 3KDS in a manner similar to the parent strain when glutamine is used to drive the reaction; NADH or asparagine, however, are ineffective when used with strain S. Vitamin K-depleted cells of B. melaninogenicus are similar to vitamin K-grown cells, when glutamine or NADH is used to drive the 3KDS incorporation. Glutamine and NADH are also effective in stimulating the incorporation of palmitate and acetate by washed cells of B, melaninogenicus. Increased incorporation of these fatty acids into CPE, CPG, 3KDS, and other phospholipids is significantly increased by the presence of glutamine or NADH. Thus, energization of the membrane of B. melaninogenicus by glutamine or the electron transport system by NADH or asparagine is required for sphingolipid and other phospholipid synthesis. The relationship of this energization to possible transport of sphingolipid precursors is discussed.     

Lipids of Bacteroides melaninogenicus

The lipids of Bacteroides melaninogenicus were readily extractable with chloroform-methanol. Three per cent of the fatty acids were not extractable. The neutral lipids contained 4% of the extractable fatty acids, the stench characteristic of these organisms, and 0.5 mumole of vitamin K(2) isoprenologues K(2)-35, K(2)-40, and K(2)-45 per g (dry weight). This is one-fifth to one-tenth of the vitamin K(2) level found in other bacteria. Ninety-six per cent of the extractable fatty acids were associated with the phospholipids (60 mumoles of lipid phosphate/g, dry weight), which consisted of the diacyl lipids phosphatidic acid, phosphatidyl serine, and phosphatidyl ethanolamine (with phosphatidyl glycerol and cardiolipin in one strain). The unusual phosphosphingolipids ceramide phosphorylethanolamine, ceramide phosphorylglycerol, and ceramide phosphorylglycerol phosphate accounted for 50 to 70% of the lipid phosphate. In protoheme-requiring strains, the protoheme concentration in the growth medium regulated the growth rate and the amount of enzymatically reducible cytochrome c. There were no gross changes in the lipid composition in cells containing different levels of enzymatically reducible cytochrome c.     

Role of nucleosides, 5-phosphoribosyl-1-pyrophosphate, and ribose 1-phosphate in the biosynthesis of phosphosphingolipids in Bacteroides melaninogenicus.

Using a deenergized spheroplast system from Bacterioides melaninogenicus, the sphingolipid precursor 3-ketodihydrosphingosine is not incorporated into the complete sphingolipids, ceramide phosphorylethanolamine, or ceramide phosphorylglycerol unless supplied with glutamine, ATP, ADP, or AMP. Adenosine, inosine, and certain other nucleosides were as effective as ATP. Purine bases and ribose, however, were inactive in this system. 5-Phosphoribosyl-1-pyrophosphate and ribose 1-phosphate also stimulated conversion of 3-ketodihydrosphingosine into ceramide phosphorylethanolamine and ceramide phosphorylglycerol, whereas ribose 5-phosphate showed only slight activity. Hypoxanthine was the main product formed from inosine and adenosine but there was no evidence for nucleotide formation. Adenosine stimulated³²P_i incorporation into cell phospholipids indicating that ribose 1-phosphate, formed via purine nucleoside phosphorylase, could be the compound stimulating sphingolipid synthesis in this system.     

Identification of ceramide phosphorylethanolamine and ceramide phosphorylglycerol in the lipids of an anaerobic bacterium

Nearly half the phospholipids isolated from the anerobic bacterium Bacteroides melaninogenicus are phosphosphingolipids. The two major phosphosphingolipids have been characterized as ceramide phosphorylethanolamine and ceramide phosphorylglycerol. The long-chain bases of these phosphosphingolipids appear to have branched and normal saturated carbon chains of 17, 18, and 19 atoms; the phosphate is at the 1-position of the long-chain base. The composition of the amide-linked fatty acids of the phosphosphingolipids differs from that of the ester-linked fatty acids of the diacylphosphoglycerides in having a higher percentage of 14:0, 17:0, and 18:0 acids as well as containing nearly all the monoenoic fatty acids found in the bacterial lipids. The finding of phosphosphingolipids in bacteria is exceedingly rare and to our knowledge ceramide phosphorylglycerol has not been previously found in nature.     

Phospholipid biosynthesis in the anaerobic protozoon Entodinium caudatum.

1. The anaerobic rumen protozoon Entodinium caudatum was incubated either intact or with various radioactive precursors of phospholipids after ultrasonication. 2. Pulse-chase experiments showed a rapid turnover of phosphatidylinositol and much slower turnovers of phosphatidylethanolamine and phosphatidylcholine. 3. E. caudatum imbibed choline very rapidly; this was immediately and exclusively converted into phosphatidylcholine which was shown by radioautography after 10 min to be distributed throughout the cell membranes. 4. Phosphatidylcholine was synthesized through a phosphorylcholine-CDP-choline pathway, the methylation or base-exchange pathways not being present. 5. Under suitable conditions [Me-14C]choline can be substantially (50-60%) converted into CDP-choline by sonicated E. caudatum and this provides an excellent method of preparing this biosynthetic intermediary. 6. [2-14C]Ethanolamine was taken up much less readily than choline. The former was incorporated into phosphatidylethanolamine by the CDP-ethanolamine pathway. 7. Doubly labelled [32P]phosphatidyl[2-3H]ethanolamine was converted into ceramide phosphorylethanolamine and N-(1-carboxyethyl)phosphatidyl-ethanolamine, without change in the isotopic ratio. Ceramide phosphoryl [2-14C]-ethanolamine was converted into phsophatidylethanolamine. 8. Palmitic acid, oleic acid and linoleic acid were taken by E. caudatum cells and incorporated into phospholipids. By contrast, although stearic acid was taken up it was hardly incorporated into phospholipids.     

Formation of ceramide phosphorylethanolamine from phosphatidylethanolamine in the rumen protozoon Entodinium caudatum (Short Communication)

From `pulse'-labelling experiments of Entodinium caudatum with [¹⁴C]ethanolamine and by incubating the organism with [³²P]phosphatidylethanolamine it is concluded that phosphatidylethanolamine can act as a direct precursor of the phosphorylethanolamine moiety of ceramide phosphorylethanolamine. The phosphorylethanolamine is probably never liberated in the free form but is transferred directly to a ceramide or ceramide-containing acceptor. The results are also in agreement with previous conclusions that phosphatidylethanolamine is the direct lipid precursor of N-(1-carboxyethyl)phosphatidylethanolamine.     

Succinate as a Growth Factor for Bacteroides melaninogenicus

Rumen strains of the obligate anaerobe Bacteroides melaninogenicus normally require medium supplemented with both heme and vitamin K. Sodium succinate was found to be an additional growth factor in that this compound can replace the requirement for heme in the presence of vitamin K, allowing good growth of the organism, and succinate can also partially replace the requirement for vitamin K in the presence of heme. The addition of succinate to a medium supplemented with both vitamin K and heme increases the growth rate of the culture. This ability to stimulate growth was specific for succinate, and cells grown without heme but with vitamin K and succinate were insensitive to cyanide. These experiments demonstrate a central role for succinate in the metabolism of B. melaninogenicus.     

Studies on the preferential incorporation of [3H]glycerol over [32P]phosphate into major phospholipids of human platelets

It is well known that platelets readily incorporate radioactive glycerol, but not radioactive phosphate into phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in vitro, thus not in accordance with de novo synthesis according to the Kennedy pathway. In attempts to understand the reason for the discrepancy, gel-filtered platelets were incubated simultaneously with [32P]Pi and [3H]glycerol, and the specific and relative radioactivities of products and intermediates were determined. Both precursors were incorporated into phosphatidylinositol (PI) with a 32P/3H ratio similar to that in glycerol 3-phosphate (in accordance with the Kennedy pathway). However, PC and PE obtained a much lower ratio. The specific 32P radioactivity in phosphorylcholine was similar to that of the gamma-phosphoryl of ATP and 650-times higher than that of PC. The specific 32P radioactivity of phosphorylethanolamine was 20-times less than that of phosphorylcholine. Both mass and 32P labelling of CDP-choline were below the detection limits. It is concluded that the incorporation of [32P]Pi into PC via phosphorylcholine is insignificant while the preferential incorporation of [3H]glycerol could be explained by exchange of diacyl[3H]glycerol in the reversible choline phosphotransferase (CDP-choline: 1,2-diacylglycerol cholinephosphotransferase) reaction. The same mechanism would explain the preferential incorporation of 3H over 32P into PE, although dilution of 32P at the phosphorylethanolamine stage would account for part of the feeble 32P incorporation. Although other mechanisms are also possible, our results clearly show that the appearance of [3H]glycerol in PC and PE is not a reliable method of monitoring de novo synthesis of these phospholipids.     

Quantitative evaluation of two pathways for ethanolamine phosphoglyceride biosynthesis in rat brainin vivo

[³H]Ethanolamine and [³²P]orthophosphate were injected intraventricularly into adult female rats. At varying time intervals after the injection (1–10 min), the animals were killed by means of a microwave apparatus, and phosphorylethanolamine and ethanolamine phosphoglycerides were extracted from the brains and counted after separation. The kinetic constants for phosphorylethanolamine incorporation into ethanolamine lipids were calculated both from³H data and from³²P data. From our results, it seems that base exchange reactions for ethanolamine incorporation into ethanolamine lipids are a pathway active in brainin vivo.     

The catabolism of phosphatidylethanolamine by the rumen protozoon Entodinium caudatum and its conversion into the N-(1-carboxyethyl) derivative

1. The N-(2-hydroxyethyl)alanine esterified to phosphatidic acid in anaerobic ciliate rumen protozoa has the l configuration. 2. Labelling experiments with Entodinium caudatum cultures using [(32)P]P(i) [2-(14)C]ethanolamine and (32)P- and (14)C-labelled phosphatidylethanolamine show that phosphatidylethanolamine is the direct lipid precursor of the N-(2-hydroxyethyl)alanine-containing phospholipid. 3. Labelling experiments with [(14)C]starch, [(14)C]lactate and [(14)C]pyruvate with E. caudatum cultures indicate that a three-carbon glycolytic intermediate is probably the precursor of the N-(1-carboxyethyl) grouping which substitutes on the amino group of phosphatidylethanolamine. 4. [(32)P]phosphatidylethanolamine is catabolized by E. caudatum forming initially glycerylphosphorylethanolamine and subsequently glycerophosphate and P(i). A little phosphorylethanolamine formed may possibly arise from bacterial enzymes ingested by the protozoa.     

Myotube phospholipid synthesis and sarcolemmal ATPase activity in dystrophic (mdx) mouse muscle.

Phospholipid incorporation of 32P by primary myotube cultures and the tissue activity of sarcolemmal Na+/K(+)-transporting ATPase were studied to determine whether the absence of dystrophin from dystrophic (mdx) muscle would affect membrane lipid synthesis and membrane function. The incorporation of 32P by phospholipid as a ratio with total protein was greater in cultured dystrophic cells compared with control cells. The mdx cells also incorporated more 32P than control cells into phosphatidylethanolamine, which is thought to increase prior to myoblast fusion, and less into phosphatidylserine, phosphatidylinositol, and lysophosphatidylcholine. There was no difference in total protein content or [3H]leucine or 32P incorporation into the aqueous fraction of dystrophic and control cells, although dystrophic cells incorporated less [35S]methionine into protein than controls. Isolated sarcolemma from mdx skeletal muscle tissue demonstrated a consistently greater specific activity of ouabain-sensitive Na+/K(+)-transporting ATPase than sarcolemmal preparations from control skeletal muscle. These observations suggest that cytoskeletal changes such as dystrophin deficiency may alter the differentiation of membrane composition and function.     

Lipid metabolism in various regions of squid giant nerve fiber

The purpose of this investigation was to compare the incorporation of radioactivity from various precursors into lipids of different regions of squid giant nerve fiber systems including axoplasm, axon sheath, giant fiber lobes which contain stellate ganglion cell bodies, and the remaining ganglion including giant synapses. To identify the labeled lipids, stellate ganglia including giant fiber lobes and the remaining tissue were first incubated separately with [14C]glucose, [32P]phosphate, [14C]serine, [14C]acetate and [3H]myristate. The radioactivity from glucose, after conversion to glycerol and fatty acids, was incorporated into most lipids, including triacylglycerol, free fatty acids, cardiolipin, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, sphingomyelin and ceramide 2-aminoethylphosphanate [corrected]. The radioactivity from serine was largely incorporated into phosphatidylserine and, to a lesser extent, into other phospholipids, mainly as the base component. The sphingoid bases of ceramide and sphingomyelin were also significantly labeled. Saturated and monounsaturated and, to a lesser extent, polyunsaturated fatty acids of these lipids were synthesized from acetate, glucose and myristate. Among the major lipids, cholesterol was not labeled by any of the radioactive compounds used. Ganglion residues incorporated the most radioactivity in total lipids from either [14C]glucose or [14C]serine, followed by giant fiber lobes and then sheath. Axoplasm incorporated the least. Among various lipids, phosphatidylethanolamine with shorter saturated fatty acids and phosphatidylglycerol contained the most radioactivity from glucose in all regions. Axoplasm was characterized by a higher proportion of glucose radioactivity in ceramide, sphingomyelin and phosphatidylglycerol. Axoplasm and sheath contained a higher proportion of serine radioactivity than did the other two regions in ceramide. Essentially no radioactivity from [14C]galactose was incorporated in any region.     

OBSERVATIONS ON THE BIOSYNTHESIS OF RAT BRAIN CERAMIDE AND CEREBROSIDE IN VIVO

Abstract— The incorporation in vivo of l -[¹⁴C]serine into ceramide and cerebroside of young rat brain has been studied. Acid hydrolysis of labelled ceramide and galactosyl-ceramide followed by selective partitioning of the resulting components indicated that 88 per cent of the radioactivity was present in the long-chain base portion. At early time points (10 min, 20 min) the precursor was incorporated into ceramide and to a lesser degree into glucosyl-ceramide. During time intervals of 5 and 10 h, the specific activity values (d.p.m./μmol) for ceramide and glucosyl-ceramide decreased, while values for galactosyl-ceramide, containing either unsubstituted fatty acids (NFA) or α-hydroxy fatty acids (HFA), increased 50 and 30 per cent, respectively. Analysis of labelled ceramide at all time points studied (10 min-10 h) indicated that l -[¹⁴C]serine was incorporated onto the NFA type. This observation suggests that HFA-ceramide may not be the physiological precursor of HFA-galactosyl-ceramide. In this context, the postulated precursor roles of both ceramide and psychosine in the biosynthesis of brain cerebrosides are discussed.     

Incorporation of labeled small molecules into rubratoxin

A sterile glucose-mineral salts broth was inoculated with conidia of Penicillium rubrum P-13 and P-3290. Radiolabeled compounds were added to some cultures, these being incubated quiescently at 28° C for 14 days. Other stationary cultures were grown for 21 days, received labeled compounds, and were then grown for 5 more days. The remaining cultures were inoculated with 72-h-old mycelial pellets, received labeled materials and were incubated with shaking for 60 h. Rubratoxin was resolved by thin-layer chromatography. Labeled [1¹⁴C]acetate, [1,5¹⁴C]citrate, [2¹⁴C]malonate, [1¹⁴C]glucose, [U¹⁴C]glucose or [1¹⁴C]hexanoate were incorporated into rubratoxins A and B by P. rubrum 3290 and into rubratoxin B by P. rubrum 13. Incorporation of [1¹⁴C]acetate and [2¹⁴C]malonate increased when exogenous unlabeled acetate, malonate, pyruvate, or phosphoenol-pyruvate was added. Acetate incorporation was influenced by cultural conditions, attaining maximum amounts in quiescent cultures which received labeled acetate after 21 days of incubation. Acetate incorporation in shake cultures was enhanced by reduced nicotinamide adenine dinucleotide phosphate (NADPH) and by unlabeled exogenous citrate.Abbreviations GMS glucose-mineral salts - RCM replacement culture medium - TCA tricarboxylic acid - PEP phosphoenolpyruvate - RIC relative isotopic content - PI percent incorporation     

Exchange of 20-kDa myosin light chain-bound phosphate during sustained contraction of arterial smooth muscle

K(+)-contracted porcine carotid arterial muscles containing phosphorylated 20-kDa myosin light chains (LC) were exposed to carrier-free [32P]orthophosphate in K(+)-stimulating solution during sustained contraction. The covalently bound LC phosphate was completely replaced by [32P]phosphate, indicating that myosin light chain phosphatase and kinase have ready access to the bound phosphate during the sustained contraction. On average, 0.38 mol [32P]phosphate was incorporated per mole LC during the sustained K+ contraction. This value was about half of the maximal value for [32P]phosphate incorporation into LC, 0.74 mol/mol, in muscles contracted with K+ for 1 min. Assuming that sustained contraction involves the maximal number of cross-bridges attached to actin, the data suggest that half of the attached cross-bridges contain phosphorylated LC.     

Biosynthetic Precursors of Vitamin K as Growth Promoters for Bacteroides melaninogenicus

The growth of a vitamin K-requiring strain of Bacteroides melaninogenicus was promoted by some postulated and proven biosynthetic precursors of bacterial menaquinones, 1,4-dihydroxy-2-naphthoic acid, shikimic acid, chorismic acid, and 4(2'-carboxyphenyl)-4-oxobutyric acid. Growth of the organism with [2',4-(14)C(2)]-4(2'-carboxy phenyl)-4-oxobutyric acid as the vitamin K replacement gave rise to a mixture of radioactive menaquinone-9 and menaquinone-10; the dilution factor for this incorporation was 1.8.     

Cyclic AMP decreases the phosphorylation state of myelin basic proteins in rat brain cell cultures

Previous work has suggested that myelin basic proteins are phosphorylated prior to their appearance in the myelin sheath (Ulmer, J. B. and Braun, P. E. (1984) Dev. Neurosci. 6, 345-355). In order to corroborate this finding we have examined the phosphorylation of myelin basic proteins in rat brain cell cultures containing 14-17% oligodendrocytes. Incorporation of 32P into the 14-, 17-, 18.5-, and 21.5-kDa myelin basic proteins was observed in cells incubated with 32P at 7, 14, and 21 days in culture. Myelin basic proteins in 14-day cells incorporated 32P linearly until at least 120 min after the addition of isotope. The apparent half-life of myelin basic protein phosphate groups was determined to be approximately 80 min in pulse-chase experiments. However, this value may be an overestimation due to the presence of significant levels of acid-soluble radioactivity in the cells throughout the chase period. The presence of dibutyryl cAMP or 8-bromo-cAMP in the incubation medium substantially inhibited the incorporation of 32P into the myelin basic proteins at all time points studied. The presence of dibutyryl cAMP in the chase medium in pulse-chase experiments resulted in an increase in the turnover rate of [32P] phosphate in the myelin basic proteins. These results indicate that cAMP decreases the phosphorylation state of myelin basic proteins in oligodendrocytes by inhibiting the phosphorylation and/or stimulating the dephosphorylation of myelin basic proteins.     

Succinate metabolism related to lipid synthesis in the housefly Musca domestica L

The metabolism of succinate was examined in the housefly Musca domestica L. The labeled carbons from [2,3-¹⁴C]succinate were readily incorporated into cuticular hydrocarbon and internal lipid, whereas radioactivity from [1,4-¹⁴C]succinate was not incorporated into either fraction. Examination of the incorporation of [2,3-¹⁴C]succinate, [1-¹⁴C]acetate, and [U-¹⁴C]proline into hydrocarbon by radio-gas-liquid chromatography showed that each substrate gave a similar labeling pattern, which suggested that succinate and proline were converted to acetyl-CoA prior to incorporation into hydrocarbons. Carbon-13 nuclear magnetic resonance showed that the labeled carbons from [2,3-¹³C]succinate enriched carbons 1, 2, and 3 of hydrocarbons with carbon-carbon coupling showing that carbons 2 and 3 of succinate were incorporated as an intact unit. Radio-high-performance liquid chromatographic analysis of [2,3-¹⁴C]succinate metabolism by mitochondrial preparations showed that in addition to labeling fumarate, malate, and citrate, considerable radioactivity was also present in the acetate fraction. The data show that succinate was not converted to methylmalonate and did not label hydrocarbon via a methylmalonyl derivative. Malic enzyme was assayed in sonicated mitochondria prepared from the abdomens and thoraces of 1- and 4-day-old insects; higher activity was obtained with NAD⁺ in mitochondria prepared from thoraces, whereas NADP⁺ gave higher activity with abdomen preparations. These data document the metabolism of succinate to acetyl-CoA and not to a methylmalonyl unit prior to incorporation into lipid in the housefly and establish the role of the malic enzyme in this process.     

Effect of secretin on protein phosphorylation in dispersed acini from rat pancreas

Dispersed acini from rat pancreas, incubated in the presence of KH2(32)PO4 to steady state 32P incorporation into cellular proteins, were exposed to secretin. 32P incorporated into selected proteins, separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, reached a plateau by 150 min. Effect of secretin on amylase release, cellular cyclic AMP levels and protein phosphorylation was then examined. Stimulation of amylase release was apparent with 10(-10)M and was maximal with 10(-7)M by 10 min incubation. Almost maximal increase in cellular cyclic AMP levels and 32P incorporation into selected proteins was also observed with 10(-7)M secretin by 10 min in the presence of 10 mM theophyllin. Both secretin (10(-8)M) and dibutyryl cyclic AMP (10(-3)M) induced the phosphorylation of similar proteins analyzed by counting 32P content in each peptide band after SDS gel electrophoresis. Addition of cyclic AMP (10(-6)M) to homogenates of acini also augmented 32P incorporation from [gamma-32P]ATP into similar proteins. These results indicate that secretin enhances protein phosphorylation in pancreatic acinar cells and cyclic AMP may mediate the action of secretin on protein phosphorylation.     

Regulation of phospholipid metabolism in differentiating cells from rat brain cerebral hemipheres in culture. II. Incorporation of [U-14C]ethanolamine into 1-alkenyl,2-acyl-and 1,2 diacyl-ethanolamine phosphoglycerides.

Cultured dissociated cells from rat embryo cerebral hemisphere incorporate [3H]-and [U-14C]ethanolamine into cellular lipids. Nearly all radioactivity in the lipid fractions is incorporated into 1,2-diacylethanolamine phosphoglycerides and 1-alkenyl,2-acylethanolamine phosphoglycerides (plasmalogen). Kinetic data suggest that the rate of labeling of both ethanolamine phospholipids from the phosphorylethanolamine is similar. A relative increase of the plasmalogen labeling is observed when free ethanolamine is continually present in the medium. The rate of incorporation of label from ethanolamine and phosphorylethanolamine into lipids was measured using a double label technique. Based upon these studies, an independent labeling pattern of the ethanolamine moiety of plasmalogens is suggested. A relative delay for the incorporation of label in plasmalogens could be explained by the presence of a variety of cell types which may differ in their capacity for phospholipid biosynthesis. The rate of incorporation of phosphorylethanolamine into the phosphatidylethanolamine was not affected by the presence of high concentrations of either choline or serine.