Likely individuality of the enzymes catalyzing the phosphorylation of choline and ethanolamine.

Dichloroacetate has effects upon hepatic metabolism which are profoundly different from its effects on heart, skeletal muscle, and adipose tissue metabolism. With hepatocytes prepared from meal-fed rats, dichloroacetate was found to activate pyruvate dehydrogenase, to increase the utilization of lactate and pyruvate without effecting an increase in the net utilization of glucose, to increase the rate of fatty acid synthesis, and to decrease slightly [1-¹⁴C]oleate oxidation to ¹⁴CO₂ without decreasing ketone body formation. With hepatocytes isolated from 48-h-starved rats, dichloroacetate was found to activate pyruvate dehydrogenase, to have no influence on net glucose utilization, to inhibit gluconeogenesis slightly with lactate as substrate, and to stimulate gluconeogenesis significantly with alanine as substrate. The stimulation of fatty acid synthesis by dichloroacetate suggests that the activity of pyruvate dehydrogenase can be rate determining for fatty acid synthesis in isolated liver cells. The minor effects of dichloroacetate on gluconeogenesis suggest that the regulation of pyruvate dehydrogenase is only of marginal importance in the control of gluconeogenesis.     

Evidence for the existence of multiple forms of choline (ethanolamine) kinase in rat tissues

In order to characterize the form of choline kinase in rat tissues, both electrophoretic and gel chromatographic patterns of choline kinase activity were compared in the liver, kidney, lung, whole intestine and carbon tetrachloride-induced liver cytosols. Kinetic parameters of the reaction were also compared for the main forms of choline kinase protein from these tissues. The overall results suggested strongly that choline kinase does not exist in one particular active form but exists in multiple forms in rat tissues. In the study present here, the electrophoretic patterns of both choline kinase and ethanolamine kinase activities were compared in rat liver, kidney, lung and intestinal cytosols. The results strongly supported the view that both kinase activities are represented on the same enzyme protein(s) in each of the rat tissues examined.     

Influence of choline and ethanolamine administration on choline and ethanolamine phosphorylating activities of mouse liver and kidney.

The administration of ethanolamine to adult male mice resulted in a significant increase in ethanolamine kinase activity in liver and kidney. Similarly, choline administration resulted in a significant increase in choline kinase activity in liver and kidney. The administration of ethanolamine resulted in enhancement of choline kinase activity concomitantly with ethanolamine kinase activity in liver and kidney. The administration of choline, however, did not result in any significant increase in ethanolamine kinase activity in liver or kidney. Cycloheximide administration along with choline-ethanolamine prevented the increase in kinase activity in liver and kidney. The results obtained have been discussed in relation to the regulatory role of choline kinase and ethanolamine kinase by de novo synthesis in response to enhanced substrate concentration, the secondary nature of choline kinase induction on ethanolamine administration, and possible distinction between choline kinase and ethanolamine kinase.     

A tomato enzyme catalyzing the phosphorylation of 3,4-dihydroxy-2-butanone

A riboflavin biosynthesis ribB mutant of Escherichia coli deficient of 3,4-dihydroxy-2-butanone 4-phosphate synthase was complemented with a cDNA library from Lycopersicon esculentum. The complementing gene was isolated and expressed in E. coli. The resulting protein was shown to specify a 62 kDa protein which phosphorylates dihydroxyacetone, both enantiomers of 3,4-dihydroxy-2-butanone, and several other aldoses and ketoses. Sequence analysis revealed homology to dihydroacetone kinases (dak) genes from plants, animals, fungi and some eubacteria. Genes with similarity to the 5' part of the dak gene from tomato were found in many other eubacteria. The physiological role of the dak gene is still incompletely known.     

Evidence for the existence of a novel enzyme system. myo-Inositol-1-phosphate dehydrogenase in Phaseolus aureus.

A novel enzyme system, myo-inositol-1-phosphate dehydrogenase, has been isolated from germinating mung bean seeds. The dehydrogenation and cleavage of myo-inositol 1-phosphate by this enzyme leads to the synthesis of a pentose phosphate which appears to be ribulose 5-phosphate. The pH optimum of the enzyme is 8.6; NAD+ is required as coenzyme and no other nucleotides can replace NAD+. Mono- or divalent cations are not essential for the enzyme activity. Stoichiometry of the reaction suggests that 2 mol of NAD+ are reduced per mol of ribulose-5-P generated.     

Evidence for the existence of three chick lung tropoelastins

Three tropoelastin polypeptides are identified among the cell-free translation products of chick embryo lung mRNAs and organ cultures extracts. The tropoelastins are distinguished by one and two dimensional gel electrophoretic systems and are all immunoreactive with monospecific chick tropoelastin antiserum. The ratio of the three tropoelastins does not vary significantly between 10 and 16 days of lung embryogenesis. The third tropoelastin (c) is found to co-migrate with tropoelastin b on SDS-polyacrylamide gel electrophoresis but is visible after cyanogen bromide cleavage of reticulocyte lysate proteins. Immunoprecipitates from lung organ culture also contain tropoelastins a, b and c.     

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.     

Affinity purification of ubiquitin-protein ligase on immobilized protein substrates. Evidence for the existence of separate NH2-terminal binding sites on a single enzyme

Previous studies have indicated the existence of separate binding sites of ubiquitin-protein ligase, E3, specific for basic (Type I) or bulky hydrophobic (Type II) NH2-terminal amino acid residues of proteins. Another class (Type III) of protein substrates appeared to interact with E3 at regions other than the NH2 terminus (Reiss, Y., Kaim, D., and Hershko, A. (1988) J. Biol. Chem. 263, 2693-2698). In the present study we have used affinity chromatography on immobilized protein substrates to examine the question of whether the different binding sites belong to one E3 enzyme, or to different E3 species. Another objective was to develop a procedure for the extensive purification of E3. When a crude extract of reticulocytes is applied to Type I or Type II protein substrates linked to Sepharose, E3 becomes strongly bound to the affinity columns and is not eluted with salt at high concentration. However, the enzyme can be specifically eluted by a dipeptide that has an NH2-terminal residue similar to that of matrix-bound protein substrate. A 350-fold purification is obtained in this single step. Preparations of E3 purified on either Type I or Type II protein substrate affinity columns act on both types of protein substrates, indicating that the separate binding sites for basic and hydrophobic NH2-terminal residues belong to one enzyme. Another species of E3 that acts strongly on some Type III protein substrates does not bind to Type I or Type II protein substrate affinity columns.     

Incorporation of choline and ethanolamine into phospholipids in germinating soya bean.

1. Incorporation of [Me-14C]choline and [2-14C]ethanolamine into lipids was studied in germinating soya bean (Glycine max L.) seeds. The precursors are only incorporated into phosphatidylcholine and into phosphatidylethanolamine respectively. 2. Base-labelling via a phospholipase-D type of reaction was eliminated as a significant factor. 3. Cyclo heximide inhibited labelling of phosphatidylcholine from [Me-14C]choline but did not affect labelling of the aqueous choline pool. It had no effect on [2-14C]ethanolamine uptake or incorporation into phosphatidylethanolamine. 4. Hemicholinium-15 at 10mM concentrations decreased uptake and lipid labelling from the both bases. 5. There was no evidence for base competition. 6. The endogenous pool of choline was much larger than that of ethanolamine, which resulted in higher specific radioactivities for phosphatidyl-ethanolamine than for phosphatidylcholine. 7. The results can be interpreted as indicating that the kinase and phosphoryltransferase enzymes of the CDP-base pathways are separate for each phospholipid.     

Quantification of choline and ethanolamine phospholipids in rabbit myocardium

To facilitate evaluation of the influence of myocardial phospholipid metabolites on the development of electrophysiologic abnormalities induced by ischemia, a method for the quantification of choline and ethanolamine phospholipids suitable for accurate and reproducible analysis of small amounts of myocardium was developed. The procedure combines chloroform and methanol extraction of phospholipids after tissue homogenization with subsequent separation by sequential thin-layer and high-performance liquid chromatography. Phosphorus in purified lipid classes was determined with the correction for recovery based on 14C-labeled internal standards.     

Phosphorylation of choline and ethanolamine in Ehrlich ascites-carcinoma cells

1. Ehrlich ascites-cell extracts convert choline and ethanolamine approximately equally well into their respective phosphoryl derivatives. 2. Choline is a potent inhibitor of ethanolamine phosphorylation, but ethanolamine has little effect on choline phosphorylation. 3. 2,3-Dimercaptopropanol, cysteine and Ca(2+) inhibit ethanolamine phosphorylation, but have no detectable effect on choline phosphorylation. 4. Choline-phosphorylating activity in Ehrlich ascites-cell extracts is more stable during storage than ethanolamine-phosphorylating activity. 5. Choline phosphorylation is stimulated in the presence of benzoylcholine, succinylcholine, butyrylcholine and propionylcholine, whereas ethanolamine phosphorylation is inhibited. This relationship is reciprocal: the compounds causing the greatest stimulation of choline phosphorylation bring about the greatest inhibition of ethanolamine phosphorylation.     

The mechanism of action of ethanolamine ammonia-lyase, an adenosylcobalamin-dependent enzyme. Evidence for a carboxyl group at the active site

Ethanolamine ammonia-lyase is an adenosylcobalamin-dependent enzyme that catalyzes the rearrangement of ethanolamine and other vicinal amino alcohols to oxo-compounds and ammonia. Treatment of this enzyme with the sulfhydryl group-blocking reagent methyl methanethiosulfonate produces a species with diminished catalytic activity. When methyl methanethiosulfonate -treated ethanolamine ammonia-lyase was incubated with a carboxyl-blocking reagent consisting of glycine ethyl ester plus a water-soluble carbodiimide, the enzyme lost more than 80% of its residual activity, while at the same time glycine ethyl ester was incorporated into it at a stoichiometry of 6 mol/mol of enzyme. Both the loss of activity and the incorporation of glycine ethyl ester were prevented if ethanolamine was included in the glycine ethyl ester-containing incubation mixture. These results suggest that an active site carboxyl group plays a role in the mechanism of catalysis by ethanolamine ammonia-lyase, and that this carboxyl group is amidated when the enzyme is incubated with glycine ethyl ester plus carbodiimide.     

Biosynthesis of phosphatidylethanolamines and phosphatidylcholines from ethanolamine and choline in rat liver.

1. The kinetics of phosphatidylcholine and phosphatidylethanolamine synthesis in rat liver were followed 5-60 min after the intraportal injection of [14-C]choline and [3-H]-ethanolamine. 2. At all time-intervals the specific radioactivity of CDP-choline was only about half that of phosphorylcholine. This indicated that CDP-choline was formed at a similar rate from phosphorylcholine and phosphatidylcholines, the latter probably through the reverse reaction of cholinephosphotransferase (EC 2.7.8.2.). In view of recent data obtained from experiments in vitro this implies a significant role for the cholinephosphotransferase reaction in the turnover of molecular species of phosphatidylcholine. 3. The specific radioactivity of CDP-ethanolamine was about twice that of phosphorylethanolamine at all time-intervals studied. This supports a previous suggestion that the liver phosphorylethanolamine pool is subject to compartmentation and shows that there is no rapid equilibration between different pools. In contrast with a recent study, no evidence was found for any significant methylation of phosphoryl-or CDP-ethanolamine to the corresponding choline derivative. 4. Quantitative data on the biosynthesis of molecular species of phosphoLIPIDS via CDP derivatives were calculated according to simple kinetic models. They were in the same range as those calculated from earlier data on precusors incorporated via diacylglycerols. 5. The proportion of radioactive phosphatidylethanolamines appearing in the plasma was approximately ten times lower than that for phosphatidylcholines. No selectivity was observed in the transfer into plasma of different molecular species of phosphatidylethanolamine.     

Significance of yeast peroxisomes in the metabolism of choline and ethanolamine

The yeasts Candida utilis and Hansenula polymorpha were able to grow in media containing choline or ethanolamine as the sole nitrogen source. During growth in the presence of these substrates, large peroxisomes developed in the cells, and extracts of choline-grown C. utilis cells contained increased levels of amine oxidase and catalase. Incubation of whole cells with choline in the presence of the amine oxidase inhibitor aminoacetonitrile led to excretion of dimethylamine and methylamine. Cytochemical experiments in which spheroplasts prepared from choline-grown cells were incubated with CeCl₃ and choline, trimethylamine, dimethylamine or methylamine revealed positively stained peroxisomes, whereas in the presence of 1 mM aminoacetonitrile staining was not observed. This indicated that choline was degraded via methylated amines and that peroxisomes played a role in its metabolism. A similar involvement of peroxisomes in choline degradation was observed in H. polymorpha. Cell-free extracts of ethanolamine-grown C. utilis and H. polymorpha also contained increased levels of amine oxidase and catalase. Ethanolamine was oxidized by cell-free extracts of both organisms after growth in the presence of ethanolamine or choline. Incubation of spheroplasts of ethanolamine-or choline-grown C. utilis with CeCl₃ and ethanolamine resulted in positively stained peroxisomes. In this organism peroxisomes were therefore also involved in ethanolamine degradation.K. B. Zwart was supported by the Foundation for Fundamental Biological Research (BION) which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO).