The base-exchange enzyme activities of sarcolemma and sarcoplasmic reticulum from rat heart

The Ca2+ dependent incorporation of [14C]ethanolamine, L-[14C]serine and [14C]choline into phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine, respectively, were investigated in membrane preparations from rat heart. The ethanolamine and serine base-exchange enzyme-catalyzed reactions were associated with the sarcolemma and sarcoplasmic reticulum. There was a 17.2-fold and 6.8-fold enrichment, respectively, of the serine and the ethanolamine base-exchange enzyme activities in the sarcolemma compared to the starting whole homogenate. The sarcoplasmic reticulum was enriched in the ethanolamine and serine base-exchange enzyme activities. The choline base-exchange enzyme activity of all membranes fractions was negligible compared to the ethanolamine or serine base-exchange enzyme activities. The apparent Km for the ethanolamine and serine base-exchange enzyme in sarcolemma was 14 microM and 25 microM, respectively. The pH optimum for these base-exchange activities was 7.5-8.0. There was a dependence upon Ca2+ for these reactions with a 1 or 4 mM concentration required for maximal activity. The properties of the sarcoplasmic reticulum base-exchange enzymes were similar to the sarcolemmal base-exchange enzymes.     

Incorporation of serine and ethanolamine into the phospholipids in rabbit retina.

The incorporation of serine and ethanolamine into phospholipids in rabbit retinal subcellular fractions and in excised retinas was studied in vitro, and some enzymic properties of the incorporation of phospholipid bases by base exchange were examined in the microsomal fraction. The retina was found to have a higher rate of base exchange for the incorporation of phospholipid bases than other tissues. The retinal microsomal fraction possessed the highest specific activity of base exchange, while the rod outer segment had very little activity. These results suggest that the phospholipids in the rod outer segment may be transferred from the inner segment of the photorecepter cell. The apparent Km values for serine and ethanolamine in the microsomal fraction decreased with decreasing Ca2+ concentration. Although no further increase of incorporation of serine and ethanolamine occurred after 40 min in the microsomal fraction, continuous incorporation of both bases into phospholipids was seen for 3 hr in excised retina. Illumination did not significantly affect the incorporation of serine and ethanolamine in excised retina or in the rod outer segment fraction. Base exchange reaction thus may not play a direct role in the visual process.     

Topographical Distribution of Base Exchange Activities in Rat Brain Subcellular Fractions

Abstract: Enrichment in the base-exchange activities was found in the micro-somal fraction of rat brain, with less activity being associated with nuclei, mitochondria and synaptosomes. The distribution of the choline base exchange in microsomal subfractions differed from that for serine and ethanolamine and these three activities seemed asymmetrically distributed in the microsomes. Choline exchange activity was trypsin-sensitive and presumably was located on the cytoplasmic side of the microsomes, while serine and ethanolamine exchange activities were trypsin-insensitive and were assumed to be located on the luminal side of the microsomes. Treatment of rat brain microsomes with phospholipases A, C and D produced significant losses of membrane-bound base exchange activities. Some activity was restored in phospholipase C-treated microsomes by exogenous phospholipid, but significant restoration was not observed in phospholipase A-treated microsomes by such additions. Exogenous phospholipid stimulated choline and ethanolamine exchange activities, but not serine exchange activity of phospholipase D-treated microsomes. The exchange activities of rat brain microsomes differed in their responses to treatment with phospholipases, choline exchange activity in general being more sensitive than either serine or ethanolamine activities.     

Release of Ethanolamine, but Not of Serine or Choline, in Rat Pontine Nuclei on Stimulation of Afferents from the Cortex, In Vivo

Release of ethanolamine, serine, and choline in rat pontine nuclei on electrical stimulation of afferents from the cortex was investigated using in vivo push-pull cannula techniques. Ethanolamine was determined by using gas chromatographic techniques; serine was measured with a HPLC system; and choline was assayed with a luminescence method. Resting elution rates of ethanolamine, serine, and choline were 50.8 +/- 8.4, 34.8 +/- 12.6, and 1.16 +/- 0.20 pmol/5 min, respectively. Stimulation of the cortico-pontine tract evoked a highly significant 3.4-fold increase in release of ethanolamine, whereas serine and choline release was unaffected. Reactions in membrane phospholipids are most likely involved in the stimulation-dependent release of ethanolamine and special consideration was given to base-exchange reactions. Alternatively, a release from intracellular, possibly synaptic stores cannot be excluded.     

Base-exchange reactions of the phospholipids in cardiac membranes

Canine cardiac microsomes were shown to incorporate the nitrogenous bases, serine, ethanolamine, and choline, into their respective phospholipids by the energy-independent, Ca2+-stimulated base-exchange reactions. The optimal Ca2+ concentration was 2.5 mM. Metal ions other than Ca2+ either inhibited or had no effect on the activities. La3+ and Mn2+ were both potent inhibitors. The pH optimum for the reactions at 2.5 mM Ca2+ was approx. 7.8 and depended upon Ca2+ concentration. Apparent Km values at 2.5 mM Ca2+ were 0.06 mM for L-serine, 0.13 mM for ethanolamine and 0.49 mM for choline. The kinetic and metal ion inhibition studies suggest that the choline-exchange reaction is a separate process from the serine and ethanolamine reactions. The ATP-stimulated Ca2+ binding system of the cardiac membranes was not related to the base-exchange reactions; however, the energy-independent Ca2+ binding to the membranes appears to be related to the exchange reactions.     

The Effects of Various Incubation Temperatures, Particulate Isolation, and Possible Role of Calmodulin on the Activity of the Base Exchange Enzymes of Rat Brain

The involvement of calmodulin in the choline, ethanolamine, and serine exchange activities of rat brain microsomes was investigated. Calmodulin stimulated choline exchange activity to a greater extent than ethanolamine and serine exchange activities. The three base exchange activities were inhibited by antipsychotic drugs believed to prevent calmodulin interaction, but not by calmodulin-binding protein. The solutions employed for tissue homogenization and subsequent isolation of microsomes greatly influenced the base exchange activities. The process of resuspending isolated microsomes and recentrifugation, or "washing," produced major losses of detectable activity. The base exchange enzyme activities were maximal at 45 degrees, and Arrhenius plots revealed a common transition temperature of 31 degrees. The activation energies for the base exchange reactions decreased at temperatures above the observed transition temperature. Kinetic data, Km and Vmax, for the base exchange activities at 27, 37, and 45 degrees are presented.     

Phospholipid base exchange activity in rat liver plasma membranes. Evidence for regulation by G-protein and P2y-purinergic receptor.

Phospholipid base exchange activity using choline as substrate was detected in plasma membranes (PM) and other subcellular fractions of rat liver, with microsomes (MS) showing the highest specific activity. In contrast, phospholipase D activity was only detected in PM. In PM, choline exchanged for phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), whereas ethanolamine exchanged for PE and PS, and serine exchanged for PS. Ca2+ (10 microM or higher) stimulated choline incorporation into PC in MS and PM, whereas Mg2+ (10 microM or higher) stimulated it only in PM. Ethanolamine and serine incorporation into PM phospholipids was also stimulated by Ca2+, and inositol incorporation by Mn2+. Phospholipase D activity was substantial in the presence of EGTA and was slightly stimulated by Ca2+ concentrations less than 500 microM. It was undetectable without Mg2+. Low concentrations of oleate (1 mM or less) stimulated phospholipase D activity. These concentrations inhibited choline base exchange activity, whereas higher concentrations (3-8 mM) were stimulatory. Comparison of the subcellular distribution and Ca2+, Mg2+, and oleate effects on choline base exchange and phospholipase D activities supports the view that they are catalyzed by different enzymes. The incorporation of choline, but not ethanolamine or serine, into the phospholipids of PM, but not MS, was stimulated by micromolar concentrations of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) and other slowly hydrolyzable analogues of GTP. GDP, GMP, and other nucleoside triphosphates and their analogues were ineffective. GTP gamma S stimulation of base exchange activity was dependent upon Mg2+ and was inhibited by high concentrations of guanosine 5'-O-2-(thio)diphosphate. In the presence of low concentrations of GTP gamma S, ATP and its slowly hydrolyzable analogues stimulated base exchange activity. Dose-response curves for these nucleotides revealed a potency order consistent with mediation by purinergic receptors of the P2Y type. Base exchange activity stimulated by ATP plus GTP gamma S or GTP gamma S alone was not altered by treatment with pertussis or cholera toxins. These results suggest that the choline base exchange activity of liver PM is regulated by a pertussis toxin-insensitive G-protein linked to P2Y purinergic receptors.     

The monomethylethanolamine- and dimethylethanolamine-base exchange reactions of rat-brain microsomal fraction

The ability of crude rat-brain microsome preparations to convert DME and MME to their corresponding phospholipid was explored. In common with the other base-exchange reactions, the incorporations of DME and MME were stimulated by about 1-4 mM Ca2+, possessed slightly alkaline pH optima, were energy independent and were unaffected by exogenous phospholipids. The Km values were 0.97 mM and 0.5 mM and the Vmax values were 9.6 nmol/mg protein per h and 6.25 nmol/mg protein per h for DME and MME, respectively. The P3 fraction of the brain and heart had the highest specific activities of particles prepared from several tissues.     

Several lines of evidence demonstrating that Plasmodium falciparum, a parasitic organism, has distinct enzymes for the phosphorylation of choline and ethanolamine

In Plasmodium falciparum-infected erythrocyte homogenates, the specific activity of ethanolamine kinase (7.6 +/- 1.4 nmol phosphoethanolamine/10(7) infected cells per h) was higher than choline kinase specific activity (1.9 +/- 0.2 nmol phosphocholine/10(7) infected cells per h). The Km of choline kinase for choline was 79 +/- 20 microM, and ethanolamine was a weak competitive inhibitor of the reaction (Ki = 92 mM). Ethanolamine kinase had a Km for ethanolamine of 188 +/- 19 microM, and choline was a competitive inhibitor of ethanolamine kinase with a very high Ki of 268 mM. Hemicholinium 3 inhibited choline kinase activity, but had no effect on ethanolamine kinase activity. In contrast, D-2-amino-1-butanol selectively inhibited ethanolamine kinase activity. Furthermore, when the two enzymes were subjected to heat inactivation, 85% of the choline kinase activity was destroyed after 5 min at 50 degrees C, whereas ethanolamine kinase activity was not altered. Our results indicate that the phosphorylation of choline and ethanolamine was catalyzed by two distinct enzymes. The presence of a de novo phosphatidylethanolamine Kennedy pathway in P. falciparum contributes to the bewildering variety of phospholipid biosynthetic pathways in this parasitic organism.     

Purification and properties of an ethanolamine-serine base exchange enzyme of rat brain microsomes

The activity of an ethanolamine and serine base exchange enzyme of rat brain microsomes was copurified to near homogeneity. The purification sequence involved detergent solubilization, Sepharose 4B column chromatography, phenyl-Sepharose 4B column chromatography, glycerol gradient sedimentation, and agarose-polyacrylamide gel electrophoresis under non-denaturing conditions. The ratio of the ethanolamine and serine base exchange activities remained almost constant during purification, and both enzyme activities were enriched 25-fold over the initial microsomal suspension. The final enzyme preparation which contained both enzyme activities showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel, having an apparent molecular mass of about 100 kDa. Serine inhibited the ethanolamine incorporation by this preparation and ethanolamine inhibited the serine incorporation. The competitive nature of this inhibition was apparent from Lineweaver-Burk plots, suggesting that the enzyme catalyzes the incorporation of both ethanolamine and serine into their corresponding phospholipids. The Km and Ki values for ethanolamine were quite similar, being 0.02 and 0.025 mM, respectively. The Km and Ki values for serine were also quite similar being 0.11 and 0.12 mM, respectively. The pH optimum was the same at 7.0 with both substrates. The optimum Ca2+ concentration was 8 mM for serine incorporation.     

Phospholipids in the nuclei and chromatin of the rat thymus at long periods following gamma-irradiation

The phospholipid content of the homogenate, nuclei and chromatin of rat thymus was being studied during three months after fractionated gamma-irradiation (2 Gy X 3 at a week interval). The number of phospholipids in the total fraction of phosphatidyl choline + phosphatidyl serine and phosphatidyl ethanolamine in the nuclei and chromatin of rat thymus was shown to decrease 60 min following the last exposure. In a month the phospholipid content in the nuclei and chromatin increased up to the control level keeping it throughout the entire period of observation.     

In vivo incorporation of l-[14C]serine into phospholipids and proteins of the subcellular fractions of developing rat brain

A study was conducted on the in vivo incorporation of l -[¹⁴C]-serine into the lipids and proteins of the various subcellular fractions of the developing rat brain before and during the stage of active myelination. The total radioactivity in the various fractions at 12 days of age was higher than that at 3 days, while the radioactive specific activity was reversed. The specific activities of the proteins and lipids were higher at 3 days of age with the exception of the subcellular fraction containing myelin. At both ages the lipids of the various cellular fractions had similar specific activities, a finding that suggests a common source for lipid biosynthesis. Incorporation of radioactivity into the various phospholipids was in the following order: phosphatidyl serine > phosphatidyl ethanolamine > phosphatidal serine > sphingomyelin and phosphatidyl choline. Of all the phospholipids, the plasmalogens increased most in total radioactivity during the period when meylination was most active. Serine-containing phospholipids appear to be most tightly bound to proteins. The brain mitochrondrial fraction contained most of the phosphatidyl serine decarboxylase activity with some activity in the nuclei. Biosynthesis of phosphatdyil ethanolamine through decarboxylation of phosphatidyl serine could take place in rat brain. Four unidentified radioactive metabolites were found in the acid-soluble fraction in addition to l -[¹⁴C]serine.     

Composition and fatty acid content of rat ventral prostate phospholipids

The major phospholipids of rat ventral prostate have been separated and examined using thin-layer chromatography, gas chromatography and mass spectrometry. The main phospholipid classes were choline and ethanolamine glycerophospholipids, accounting for 77.9% of total lipid phosphorus. The prostate also contained small amounts of serine glycerophospholipids and sphingomyelin. The relative proportions of fatty acids in the different phospholipid classes were also determined. Arachidonic acid in prostatic phospholipids is contributed primarily by ethanolamine glycerophospholipids. This fraction contained 65-69 mol% plasmalogens, whereas choline and serine glycerophospholipid fractions contained less than 5 mol% plasmalogens. Ethanolamine, choline and serine plasmalogens contained mainly vinyl ethers of palmitic and stearic aldehydes. Ethanolamine plasmalogens also contained the vinyl ether of oleic aldehyde.     

Base exchange reactions of the phospholipids in rat brain particles

A particulate fraction from rat brain catalyzes the incorporation of [(14)C]choline, [(14)C]ethanolamine, and l-[(14)C]serine into phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, respectively. The reaction appears to be energy-independent since Mg(2+), CTP, ATP, and NaF have no stimulatory action. The incorporation is inhibited by EDTA and activated by Ca(2+). The pH optimum for the incorporation of choline is 9.5, for ethanolamine it is 9.0, and for l-serine it is 8.5. Tris, bicine, and imidazole buffers are inhibitory. The incorporations are inhibited by a variety of structurally related alcohols and are stimulated by isoserine (alpha-hydroxy,beta-aminopropionic acid).     

Choline kinase and ethanolamine kinase are separate, soluble enzymes in rat liver.

Choline kinase and ethanolamine kinase are located in the cytosol from rat liver and have been copurified more than 500-fold by affinity chromatography [P. J. Brophy and D. E. Vance (1976) FEBS Lett. 62, 123-125]. Kinetic properties of the two activities were determined. Choline kinase had a Km for choline of 0.033 mM and ethanolamine was a competitive inhibitor (Ki = 6.2 mM). Ethanolamine kinase had a Km for ethanolamine of 7.7 mM and choline was a 'mixed' type of inhibitor with a Ki of 0.037 mM. Both enzymes activities responded in a similar fashion to the adenylate energy charge. Betaine and choline phosphate partially inhibited both kinases with a 93% inhibition of the ethanolamine kinase by 5 mM choline phosphate. CTP and ethanolaminephosphate partially inhibited the ethanolamine kinase, but not the choline kinase. Other metabolites tested had negliglible effects on both kinases. The affinity-column-purified enzyme was analyzed by disc gel electrophoresis which resolved the two activities. Hence, although many of the properties of the two activities are similar, choline kinase and ethanolamine kinase must be separate enzymes. Analysis of rat liver cytosol by disc gel electrophoresis indicated four isoenzymes for choline kinase and ethanolamine kinase.     

Specific pools of phospholipids are used for lipoprotein secretion by cultured rat hepatocytes

Since phospholipids are major components of all serum lipoproteins, the role of phospholipid biosynthesis in lipoprotein secretion from cultured rat hepatocytes has been investigated. In liver, phosphatidylcholine is made both by the CDP-choline pathway and by the methylation of phosphatidylethanolamine, which in turn is derived from both serine (via phosphatidylserine) and ethanolamine (via CDP-ethanolamine). Monolayer cultures of rat hepatocytes were incubated in the presence of [methyl-3H]choline, [1-3H] ethanolamine, or [3-3H]serine. The specific radioactivity of the phospholipids derived from each of these precursors was measured in the cells and in the secreted lipoproteins of the cultured medium. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine derived from [1-3H]ethanolamine were markedly lower (approximately one-half and less than one-tenth, respectively) in the secreted phospholipids than in the cellular phospholipids. Thus, ethanolamine was not an effective precursor of the phospholipids in lipoproteins. On the contrary, the specific radioactivity of phosphatidylcholine made from [methyl-3H]choline was approximately equal in cells and lipoproteins. In addition, over the first 4 h of incubation with [3-3H]serine, the specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were significantly higher in the lipoproteins than in the cells. These data indicate that there is not a random and homogeneous labeling of the phospholipid pools from the radioactive precursors. Instead, specific pools of phospholipids are selected, on the basis of their routes of biosynthesis, for secretion into lipoproteins.     

Incorporation of [1-14C]Palmitic Acid into Neutral Lipids and Phospholipids of Rat Cerebral Cortex In Vitro

Abstract: Incorporation of [1-¹⁴C]palmitic acid into neutral lipids and phospholipids of rat cerebral cortex was examined in vitro in normal Krebs-Ringer bicarbonate buffer containing 3% (wthol) albumin and 0.75 mM palmitic acid. Under standard assay conditions, radioactivity in the triacylglycerol fraction increased rapidly during the first 30 min, and then decreased after 60 min, with corresponding increase in radioactivity in phosphatidyl choline, phosphatidyl ethanolamine, and a fraction of phosphatidyl inositol plus phosphatidyl serine. Diacylglycerol was shown to be an intermediate metabolite. Radioactivity increased in triacylglycerol, and decreased in phosphatidyl choline and phosphatidyl ethanolamine throughout incubation under NZ gas. In the fraction of phosphatidyl inositol plus phosphatidyl serine, radioactivity decreased after 30 min during incubation under N, gas. A possible acylation-deacylation cycle, in which triacylglycerol could be a source of free fatty acids for phospholipids, is discussed.     

Acyltransferase capacity of membranes from cotyledons of germinating peas

The incorporation of 1-[¹⁴C]-palmitate into the lipids of microsomal and mitochondrial membranes from peas (Pisum sativum L., var. Massey Gem) and the relative effects of ATP and coenzyme A(CoA) on the process have been examined. Both mitochondrial and microsomal pellets possessed acyltransferase capacity, which responded similarly to additions of ATP and CoA. Incorporation of 1-[¹⁴C]-palmitate into phospholipid was promoted by ATP alone, but incorporation into triacylglycerols was not. The addition of CoA alone did not promote incorporation. The addition of CoA and ATP further promoted incorporation into phospholipids and also stimulated incorporation into triacylglycerol. It was concluded that some CoA must be membrane-bound and available for phospholipid but not for triacylglycerol synthesis. Phospholipase A, treatment of microsomal and mitochondrial phospholipids, previously labelled with 1-[¹⁴C]-palmitate in the presence of ATP and coenzyme A, showed that incorporation occurred only into the 2-position of phosphatidyl choline and phosphatidyl ethanolamine. There was enough lyso-phosphatidyl choline in the phospholipids of microcomal membranes (obtained from a 100 000 g pellet) to account for the observed incorporations of palmitate. Using microsomal membranes whose fatty acyl groups were pre-labelled by incubation of tissue with 1-[¹⁴C]-acetate, no evidence of acyl exchange was found during subsequent incubations with unlabelled palmitate. Similar observations were made using oleate instead of palmitate. It was concluded that acyl-CoA: 1-acylglycerophosphocholine o-acyltransferase (E.C. 2.3.1.23) was responsible for the observed acyl transfer to phosphatidyl choline. Sucrose gradient analysis of whole homogenates and of the 10 000 g pellet showed that both mitochondrial and rough endoplasmic reticulum possessed acyltransferase capacity, with the bulk of this residing in the mitochondria. The possible significance of this widely distributed membrane activity is briefly discussed.     

Synaptosomal phospholipid pool in rabbit brain and its effect on GABA uptake

Rabbit synaptosomes have been used to study the effect of the base-exchange reaction in membrane phospholipids on -aminobutyric acid (GABA) transport in vitro. The uptake of GABA was measured after a base-exchange reaction with ethanolamine, choline, orl-serine and after subsequent displacement of these exchanged moieties from lipid by bases of similar or different structures which were added to the synaptosomal medium. Serine incorporation stimulated GABA transport, but its displacement from membrane lipid by choline or ethanolamine induced an inhibition of GABA transport. Ethanolamine incorporation inhibited GABA transport, but its displacement by serine or choline resulted in stimulation of GABA uptake. Choline incorporation also inhibited GABA transport, although less than ethanolamine. The pool size of synaptosomal phospholipids, presumably involved in GABA uptake, accounted for 0.2 to 10% of the total content of membrane phospholipid. Thus, alteration of phospholipid compositior by exchange of the lipid hydrophilic head-groups influences the extent GABA uptake into rabbit synaptosomes.     

Purification and characterization of choline/ethanolamine kinase from rat liver

Choline kinase, the first enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis, was purified 26,000-fold from rat liver to a specific activity of 143,000 nmol.min-1.mg-1 protein. The subunit molecular mass was 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the apparent native molecular mass was 160 kDa by size exclusion chromatography, suggesting a tetrameric structure. Two peaks of choline kinase activity were obtained by chromatofocusing. These isoforms eluted at pH 4.7 (CKI) and 4.5 (CKII). CKII appeared to be homogeneous by sodium dodecyl sulfate gel electrophoresis. Peptide mapping of two isoforms indicated a high degree of similarity, although there were peptides not common to both. Ethanolamine kinase activity copurified with both isoforms. The ratio of choline to ethanolamine kinase activity was 3.7 +/- 0.7 throughout the purification procedure. Choline and ethanolamine were mutually competitive inhibitors. The respective Km values, 0.013 and 1.2 mM, were similar to the Ki values of 0.014 and 2.2 mM. An antibody raised against CKII immunoprecipitated both choline and ethanolamine kinase activities from liver cytosol at the same titer. These data suggest that both activities reside on the same protein and occur at the same active site. Similarly, both activities were immunoprecipitated from brain, lung, and kidney cytosols. Western blot analysis showed both purified liver isoforms, as well as brain, lung and kidney enzymes, to have a molecular mass of 47 kDa.