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.     

Isolation and characterization of a Chinese hamster ovary cell mutant with altered regulation of phosphatidylserine biosynthesis

We have screened approximately 10,000 colonies of Chinese hamster ovary (CHO) cells immobilized on polyester cloth for mutants defective in [14C]ethanolamine incorporation into trichloroacetic acid-precipitable phospholipids. In mutant 29, discovered in this way, the activities of enzymes involved in the CDP-ethanolamine pathway were normal; however, the intracellular pool of phosphorylethanolamine was elevated, being more than 10-fold that in the parental CHO-K1 cells. These results suggested that the reduced incorporation of [14C]ethanolamine into phosphatidylethanolamine in mutant 29 was due to dilution of phosphoryl-[14C]ethanolamine with the increased amount of cellular phosphorylethanolamine. Interestingly, the rate of incorporation of serine into phosphatidylserine and the content of phosphatidylserine in mutant 29 cells were increased 3-fold and 1.5-fold, respectively, compared with the parent cells. The overproduction of phosphorylethanolamine in mutant 29 cells was ascribed to the elevated level of phosphatidylserine biosynthesis, because ethanolamine is produced as a reaction product on the conversion of phosphatidylethanolamine to phosphatidylserine, which is catalyzed by phospholipid-serine base-exchange enzymes. Using both intact cells and the particulate fraction of a cell extract, phosphatidylserine biosynthesis in CHO-K1 cells was shown to be inhibited by phosphatidylserine itself, whereas that in mutant 29 cells was greatly resistant to the inhibition, compared with the parental cells. As a conclusion, it may be assumed that mutant 29 cells have a lesion in the regulation of phosphatidylserine biosynthesis by serine-exchange enzyme activity, which results in the overproduction of phosphatidylserine and phosphorylethanolamine as well.     

THE METABOLISM OF LABELLED ETHANOLAMINE IN NEURONAL AND GLIAL CELLS OF THE RABBIT IN VIVO1

Abstract— Adult rabbits were injected intraventricularly with [¹⁴C]ethanolamine and the incorporation of the base into the phosphatidylethanolamine and ethanolamine plasmalogen (and their water-soluble precursors) of isolated neuronal and glial cells was investigated. All the radioactivity was incorporated into the base moiety of the ethanolamine lipids for the time intervals examined in both types of cells. In neurons, maximum labelling of the two ethanolamine lipids occurred at 7 h after administration, whereas the highest specific radioactivity for glial phosphatidylethanolamine and ethanolamine plasmalogen was reached at 20 and 36 h, respectively. The two lipids had a faster turnover in neurons than in glia, and in both populations incorporated the base at a faster rate than did whole brain tissue. The maximum incorporation rates for phosphorylethanolamine and CDP-ethanolamine were reached in both types of cell at about 6 h after administration but the content of radioactivity per unit protein for phosphorylethanolamine was much higher in glial than in neuronal cells. It is concluded that the site of most active synthesis of ethanolamine phospholipids in vivo is the neuronal cell, with a possible transfer of intact lipid molecule to the glial compartment.     

Preparation of radioactive acetyl-l-carnitine by an enzymatic exchange reaction

A rapid method for the preparation of [1-¹⁴C]acetyl-l-carnitine is described. The method involves exchange of [1-¹⁴C]acetic acid into a pool of unlabeled acetyl-l-carnitine using the enzymes acetyl-CoA synthetase and carnitine acetyltransferase. After isotopic equilibrium is attained, radioactive acetylcarnitine is separated from the other reaction components by chromatography on Dowex 1 (Cl^?) anion exchange resin. One of the procedures used to verify the product [1-¹⁴C]acetyl-l-carnitine can be used to synthesize (3S)-[5-¹⁴C]citric acid.     

Radioenzymatic determination of CMP-N-acetylsialic acid and free N-acetylsialic acid in biological material

Free N-acetylsialic acid (NeuNAc) and CMP-N-acetylsialic acids (CMP-NeuNAc) are extracted from freeze-clamped or liquid nitrogen-frozen biological material by sequential extraction with cold acetone and acetone/water. [¹⁴C]NeuNAc and [¹⁴C]CMP-NeuNAc (20,000 dpm each) are added to the frozen material to correct for small losses occurring during the subsequent steps. NeuNAc and CMP-NeuNAc are separated by anion-exchange chromatography. CMP-NeuNAc is hydrolyzed with formic acid and again chromatographed on an ion-exchange column. The NeuNAc-containing fractions (representing free NeuNAc and CMP-NeuNAc) are converted to [¹⁴C]CMP-NeuNAc in the presence of [¹⁴C]CTP and CMP-NeuNAc synthetase. [¹⁴C]CMP-NeuNAc is separated by paper chromatography and the radioactivity measured by liquid scintillation counting. The amount of NeuNAc is calculated from a calibration curve obtained with NeuNAc standards. The small amounts of [¹⁴C]NeuNAc and [¹⁴C]CMP-NeuNAc added initially do not interfere with the final assay. The method gives reliable values down to 50 pmol/assay, but the sensitivity can be easily increased by a factor of 10. Recoveries, with NeuNAc and CMP-NeuNAc added to biological extracts, were 98.3 and 98.5% for NeuNAc and CMP-NeuNAc, respectively. With this method values of 61.2 ± 12.8 and 24.4 ± 5.2 nmol/g wet wt were found in rat liver for free NeuNAc and CMP-NeuNAc, respectively. Values for free NeuNAc found in human blood plasma were 600 ± 476 and 373 ± 180 pmol/g plasma for healthy persons and patients with breast cancer, respectively. Free CMP-NeuNAc could not be found in plasma.     

Determination of the specific radioactivity of 14C-labeled glutamic acid and glutamine

A new, simple, and accurate method for the sequential determination of the specific radioactivity of [1-¹⁴C]glutamic acid and [1-¹⁴C]glutamine is described. Using this method, radioactivity in H¹⁴CO₃^?, in [¹⁴C]glutamic acid, and in [¹⁴C]-glutamine can be readily determined on a single sample of blood plasma. Radioactivity is released as ¹⁴CO₂ in a stepwise fashion, trapped in the center wells, and counted in a liquid scintillation counter. The applicability of the method is discussed.     

The calcium-stimulated incorporation of ethanolamine and serine into the phospholipids of the housefly Musca domestica

1. The calcium-stimulated incorporation of [2-¹⁴C]ethanolamine and l-[3-¹⁴C]-serine into the phospholipids of homogenates of the fat bodies from larval houseflies (Musca domestica) was studied. 2. Ethanolamine and serine acted as competitive inhibitors with one another. N-Methylethanolamine was not distinguished from ethanolamine by the system. Tris buffer was also a competitor with these compounds, and a number of other amino alcohols were inhibitory, probably competitively. 3. The incorporation of [³²P]phosphorylethanolamine into phospholipids was observed in suspensions of whole fat bodies. This incorporation was stimulated by magnesium. 4. During the incubation of the homogenates, a calcium-stimulated breakdown of phospholipids by a phospholipase A occurred. 5. These results are compared with results published for similar mammalian systems, and their possible physiological significance is discussed.     

The transport of cytidine into rat brain in vivo,and its conversion into cytidine metabolites

Double-labeled cytidine, with a³H/¹⁴C isotope ratio of 20.00, has been intraventricularly injected into the brain of young rats, and its fate followed up to 90 min from administration together with the time-course of labeling. The injected nucleoside enters the brain as an intact molecule and is immediately utilized without prior degradation. Cytidine is actively converted into uridine and CMP, the latter being then transformed by a stepwise mechanism into CDP and CTP, and finally into CDP-choline and CDP-ethanolamine. The results indicate that administered cytidine represents a compound likely to enter metabolic events, which lead to CDP-choline and CDP-ethanolamine synthesis, and presumably to phospholipid production.     

Analysis of glucose 1-phosphate at the picomole level with [C]UTP and uridine 5′-diphosphoglucose pyrophosphorylase

A new sensitive method is described for glucose 1-phosphate analysis. The key reaction is the pyrophosphorolysis of UDP-glucose catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase. The reaction product, [¹⁴C]UDP-glucose, is separated from [¹⁴C]UTP by adsorbing [¹⁴C]UTP selectively onto polyethyleneimine cellulose or by separating both labeled compounds on one-dimensional polyethyleneimine thin-layer chromatograms. The sensitivity of the method for glucose 1-phosphate analysis is 5 pmol. The method has been successfully employed to monitor the level of glucose 1-phosphate in early germination of wheat embryos.     

Ribohomopolymer formation as a source of error in the radioactive precursor incorporation studies of nuclear DNA-dependent RNA synthesis

Isolated rat liver nuclei contain ribohomopolymer polymerases with relative activities in the following order: Poly (A) (100%) > Poly (C) (62%) > Poly (U) (34%) > Poly (G) (13%). Because these enzymes share the same substrates with the nuclear DNA-dependent RNA polymerases in nuclei, labelled precursor is therefore concurrently incorporated into both RNA and ribohomopolymer. Thus, experiments designed to study DNA-dependent RNA synthesis are subjected to error. It is estimated when [¹⁴C]ATP is used as the labelled precursor, the error is as high as 35%; [¹⁴C]CTP, 20%; [¹⁴C]UTP or [¹⁴C]GTP, 10%.     

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.     

Phosphatidylethanolamine is the donor of the phosphorylethanolamine linked to the alpha1,4-linked mannose of yeast GPI structures

Glycosylphosphatidylinositol (GPI) anchors of all species contain the core structure protein-CO-NH-(CH(2))(2)-PO(4)-Manalpha1-2Manalpha1-6Manalpha1-4GlcNalpha1-6inositol-PO(4)-lipid. In recent studies in yeast it was found that gpi10-1 mutants accumulate M2, an abnormal intermediate having the structure Manalpha1-6[NH(2)-(CH(2))(2)-PO(4)-->]Manalpha1-4GlcNalpha1-6(acyl-->)inositol-PO(4)-lipid. It thus was realized that yeast GPI lipids, as their mammalian counterparts, contain an additional phosphorylethanolamine side chain on the alpha1,4-linked mannose. The biosynthetic origin of this phosphorylethanolamine group was investigated using gpi10-1 Deltaept1 Deltacpt1, a strain which is unable to synthesize phosphatidylethanolamine by transferring phosphorylethanolamine from CDP-ethanolamine onto diacylglycerol, but which still can make phosphatidylethanolamine by decarboxylation of phosphatidylserine. Gpi10-1 Deltaept1 Deltacpt1 triple mutants are unable to incorporate [(3)H]ethanolamine into M2 although metabolic labeling with [(3)H]inositol demonstrates that they make as much M2 as gpi10-1. In contrast, when labeled with [(3)H]serine, the triple mutant incorporates more label into M2 than gpi10-1. This result establishes that the phosphorylethanolamine group on the alpha1,4-linked mannose is derived from phosphatidylethanolamine and not from CDP-ethanolamine.     

Use of phosph-cellulose paper disks for the assay of histone acetyltransferase.

A modified method for the assay of histone acetyltransferase is presented. Previously reported methods depended upon the determination of the incorporation of radioactivity from [¹⁴C]acetyl coenzyme A into trichloroacetic acid (TCA)-precipitable material. However, as shown in this paper, [¹⁴C]acetylated histone cannot be precipitated quantitatively by TCA. In the method described in this paper, phospho-cellulose (P-cellulose) paper disks are used as an adsorbent for [¹⁴C]acetylated histone and 0.05 m carbonate buffer, pH 9.2, is used as a washing medium. This P-cellulose disk method allows more quantitative determination of [¹⁴C]acetylated histone than the TCA-precipitation methods.     

UTILIZATION OF ACETATE AND PYRUVATE FOR THE SYNTHESIS OF'TOTAL','BOUND'AND'FREE'ACETYLCHOLINE IN THE ELECTRIC ORGAN OF TORPEDO

—Slices of tissue of the electric organ of Torpedo marmorata were incubated in vitro in a salineurea-sucrose solution containing a labelled precursor of the acetyl moiety of ACh ([1-¹⁴C]glucose, [2-¹⁴C]pyruvate, or [1-¹⁴C]acetate) either alone or in the presence of another unlabelled precursor. The incorporation of ¹⁴C from [1-¹⁴C]acetate into ACh was considerably higher than from the other two substrates. The specific radioactivities (SRA) of the‘total',‘bound’and‘free’ACh were compared in experiments with [2-¹⁴C]pyruvate and [1-¹⁴C]acetate. With both precursors, the SRA of the‘bound’ACh were lower than those of‘total’ACh; consequently, the‘free’ACh pool was more labelled than the‘bound’pool. After short incubations with [2-¹⁴C]pyruvate the SRA of'bound’ACh were closer to the SRA of‘total’ACh than with [1-¹⁴C]acetate. A simple method is described for the labelling of ACh and its separation from other labelled compounds in experiments with the electric organ using [¹⁴C]acetate as the labelled precursor.     

Use of charcoal adsorption for the microassay of acetyl-CoA-hydrolase.

A sensitive and rapid radiochemical micromethod is described for measuring the activity of acetyl-CoA hydrolase (EC 3.1.2.1). [1-¹⁴C]Acetyl-CoA is incubated with tissue homogenates; unhydrolyzed [1-¹⁴C]acetyl-CoA is separated from the radiolabeled product, [1-¹⁴C]acetate, by adsorption to charcoal. The soluble [1-¹⁴C]acetate is measured by liquid scintillation techniques. This procedure makes it possible to measure as little as 0.2 to 0.4 nmol acetate generated per assay.     

A method for the determination of glucose-6-phosphatase activity in rat liver with [U-14C]glucose 6-phosphate as substrate.

A method is described for measuring the activity of glucose-6-phosphatase (EC 3.1.3.9) in rat liver. [U-¹⁴C]Glucose 6-phosphate, as substrate, is converted by the enzyme to [¹⁴C]glucose and inorganic phosphate. The addition of ZnSO₄ and Ba(OH)₂ at the end of the reaction precipitates phosphate and the unreacted [¹⁴C]glucose 6-phosphate, whereas [¹⁴C]glucose is not precipitated. After centrifugation, the amount of [¹⁴C]glucose formed is determined in a liquid scintillation counter.     

Measurement of mouse brain glucose utilization in vivo using [U-14C]glucose

The rate of [2-¹⁴C]glucose uptake has been used as an indication of the status of energy consumption by the rat brain, but the cost of this radiolabel can be prohibitive and the surgical manipulation involved in published methods is extensive. A method for measuring glucose utilization in vivo in mouse brain with [U-¹⁴C]glucose is described in this article. Glucose consumption in whole mouse brain obtained with [U-¹⁴C]glucose or [2-¹⁴C]glucose was 0.650±0.022 and 0.716±0.36 nmol/mg/min, respectively. In all instances the rate obtained with the uniformly labeled isotope was somewhat lower than that found with [2-¹⁴C]glucose. The rate of glucose utilization measured with either isotope was significantly depressed in sodium pentobarbital anesthetized mice. The method described here is advantageous because [U-¹⁴C]glucose is substantially less expensive than [2-¹⁴C]glucose and surgical intervention is avoided.     

A radiochemical method for determination of ethanol oxidation

A radiochemical method for the measurement of ethanol exidation by tissue preparations is described. Ethanol oxidation is determined from the production of [¹⁴C]acetaldehyde, quantified as the semicarbazone derivative, from [1-¹⁴C]ethanol. The assay is quantitative, reproducible and highly correlated with the NADH-enzymic-spectrophotometric procedure.     

Enzymic studies of glycol and glycerol lipids containing O-alkyl bonds in liver and tumor tissues

The utilization of [1-¹⁴C]hexadecyl-[2-³H]ethyleneglycol and [1-¹⁴C]hexadecyl-[2-³H]glycerol as substrates for acyltransferase, phosphotransferase, phosphorylcholine, and phosphorylethanolamine transferase, and O-alkyl cleavage activities in cell-free preparations from normal rat liver and preputial gland tumors of mice was investigated. Our studies demonstrate that alkylethyleneglycols, like alkylglycerols, can serve as substrates for acyltransferases in both the liver and tumor microsomes; the product alkylacylethyleneglycerol can be readily deacylated by pancreatic lipase. A polar lipid was formed from the alkylethyleneglycol by the tumor homogenates in the presence of ATP and Mg²⁺; although the small quantities formed precluded absolute identification, its thin-layer Chromatographic behavior in acidic and basic solvent systems indicated that a free phosphate group was present. As expected, phosphorylbase transferases in these preparations did not utilize either the alkylethyleneglycol or alkylglycerol as substrates. The O-alkyl moiety of hexadecyl-ethyleneglycol was oxidized to hexadecanal by a tetrahydropteridine-dependent cleavage enzyme in rat liver microsomes, whereas in the tumor microsomes this activity was not present. We conclude that alkylethyleneglycols are metabolized in a manner similar to alkylglycerols and perhaps by identical enzymes.     

A sensitive method for the separation and quantitation of (14C)proline and (14C)hydroxyproline from the collagen of rat uterus

A specific and sensitive method is described for the isolation and quantitation of [¹⁴C]proline and [¹⁴C]hydroxyproline from uterine collagen of the immature rat. Selectivity is achieved in this isolation by using a protease-free bacterial collagenase. There is complete release of hydroxyproline from uterine protein if the latter is suspended by sonication prior to treatment with collagenase. There is a consistent recovery of [¹⁴C]proline and [¹⁴C]hydroxyproline when they are added to protein hydrolysates of uterus and then subjected to the procedures required for their isolation and quantitation. It is possible using this method to determine the incorporation of [¹⁴C]proline into collagen of the rat uterus and to quantitate its conversion to [¹⁴C]hydroxyproline. Coupled with the colorimetric methods for proline and hydroxyproline, it is also possible to determine their specific activity.