Identification and Properties of Methyltransferases That Synthesize Phosphatidylcholine in Rat Brain Synaptosomes

Abstract: Rat brain was found to enzymatically methylate phospholipids to form phosphatidylcholine with S -adenosyl- l -methionine serving as the methyl donor. Methyltransferase activity was localized in the microsomes and synaptosomes. In synaptosomes, at least two enzymes were found to be involved in the formation of phosphatidylcholine. The first methyltransferase which catalyzes the methylation of phosphatidylethanolamine to form phosphatidyl- N -monomethylethanolamine was found to have a pH optimum of 7.5, a low K_m for 5-adenosyl- l -methionine and a partial requirement for Mg². Methyltransferase I is tightly bound to membranes. The second methyltransferase (II) catalyzes the successive methylations of phosphatidyl- N -monomethylethanolamine to phosphatidyl- N , N -dimethylethanolamine and then to phosphatidylcholine. In contrast to methyltransferase I, methyltransferase II has a pH optimum of 10.5, a high apparent K_m for S -adenosyl- l -methionine and no requirement for Mg². Methyltransferase II is easily solubilized by sonication. The highest specific activity for both enzymes was found in the synaptosomal plasma membrane.     

Myelin basic protein inhibits histone-specific protein methylase I

Bovine brain myelin basic protein, free of associated proteolytic activity, was found to be a specific inhibitor of histone-specific protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23) purified from bovine brain. 50% of the methyl group incorporation into the histone substrate catalyzed by the methylase I was inhibited by myelin basic protein at a concentration of 0.326 mM. However, neither of the peptide fragments (residues 1-116 and residues 117-170) generated by the chemical cleavage of myelin basic protein at the tryptophan residue retained the inhibitory activity for histone-specific protein methylase I. Proteins such as gamma-globulin, bovine serum albumin, bovine pancreatic ribonuclease and polyarginine did not exhibit significant inhibitory activity toward the enzyme. The Ki value for myelin basic protein was estimated to be 3.42 X 10(-5) M for histone-specific protein methylase I and the nature of the inhibition was uncompetitive toward histone substrate.     

Phosphoprotein phosphatases for myelin basic protein in myelin and cytosol fractions of brain.

Phosphoprotein phosphatase (phosphoprotein phosphohydrolase EC 3.1.3.16) activity for myelin basic protein was found to be present in the myelin fraction of rat brain. The enzyme activity was in a latent form and solubilized by 0.2% Triton X-100 treatment with about 50% increase of activity. The cytosol fraction from bovine brain also had phosphoprotein phosphatase activity for myelin basic protein, which was resolved into at least two peaks of activity on DEAE-cellulose column chromatography. Myelin basic protein was the best substrate for both the solubilized myelin fraction and the cytosol enzymes among the substrate proteins tested. The Km values of the solubilized myelin fraction were 4.2 muM for myelin basic protein, 7.4 muM for arginine-rich histone, 8.0 muM for histone mixture and 14.3 muM for protamine, respectively.     

S-Adenosylmethionine Decarboxylase Activity Is Decreased in the Rat Cortex After Traumatic Brain Injury

Abstract: S -Adenosyl- l -methionine decarboxylase (SAMdc) and l -ornithine decarboxylase (ODC) are major enzymes regulating polyamine synthesis. Following ischemia, putrescine content increases as a result of post-traumatic activation of ODC and inhibition of SAMdc. These alterations are thought to mediate edema and cell death. The purpose of this study was to quantify SAMdc activity and edema in the brain following controlled cortical impact injury. Anesthetized adult male rats underwent a right parietal craniectomy and were subjected to cortical impact injury. Tissues were obtained from three bilateral regions: parietal cortex, motor area (CPm); parietal cortex, somatosensory area (CPs); and the pyriform cortex (CPF). SAMdc activity was determined in the postmitochondrial fraction from homogenates of fresh, unfrozen tissues by measuring the decarboxylation of S -adenosyl- l -[ carboxyl -¹⁴C]methionine. Basal SAMdc activity was determined in unoperated rats, and regional differences were noted: Activity was lower in the CPF than in the CPm and CPs. SAMdc activity decreased to the greatest extent in the ipsilateral CPm (impact site) from 1 to 72 h following traumatic brain injury. Significant edema was found in the ipsilateral CPm 1, 8, 16, 24, and 48 h after injury. Decreased SAMdc activity impairs the conversion of putrescine to polyamines and may contribute to delayed pathological changes in the brain after traumatic injury.     

Osmoprotectant β-alanine betaine synthesis in the Plumbaginaceae: S-adenosyl- l-methionine dependent N-methylation of β-alanine to its betaine is via N-methyl and N,N-dimethyl β-alanines

β -Alanine betaine is an osmoprotective compound accumulated by most members of the plant family Plumbaginaceae. Leaf and root tissues of Limonium latifolium known to accumulate β -alanine betaine readily convert supplied β -alanine to β -alanine betaine. To identify the intermediates and the enzymes involved in β -alanine betaine synthesis, radiotracer experiments using [ 14 C] formate were employed. These studies demonstrate that β -alanine betaine is synthesized from β -alanine via N -methyl and N,N- dimethyl β -alanines. A rapid and sensitive radiometric assay was developed to measure N -methyltransferase (NMT) activities by using [methyl-¹⁴C] or [methyl-³H] S -adenosyl- l -methionine (AdoMet) as the methyl donor. Leaf extracts from β -alanine betaine accumulators – Armeria maritima , L. latifolium and L. ramosissimum – had detectable NMT activities while none were found in L. perezii , a species that does not accumulate β -alanine betaine. The NMT activities were further characterized from the leaves of L. latifolium . The activities had a pH optimum of 8.0, were soluble and inhibited by S -adenosyl- l -homocysteine. Extractable activities were similar from plants grown under control and salinity stress conditions. Radiolabeling with [ 14 C] l -aspartic acid indicated that, unlike in bacteria, decarboxylation of l -aspartic acid is not the source of β -alanine in the Plumbaginaceae.     

m-OCTOPAMINE, p-OCTOPAMINE AND PHENYLETHANOLAMINE IN RAT BRAIN: A SENSITIVE, SPECIFIC ASSAY AND THE EFFECTS OF SOME DRUGS

Abstract— An enzyme radiochemical assay for p -octopamine, m -octopamine (norphenylephrine) and phenylethanolamine based on the N -methylation of these amines by the enzyme phenylethanolamine N -methyl transferase ( S -adenosyl- l -methionine: phenylethanolamine N -methyl transferase (EC 2.1.1.28) has been developed. [³H]Methyl- S -adenosyl- l -methionine was used as methyl donor. The reaction products are converted to their dansyl derivatives and separated by TLC in three different solvent systems prior to liquid scintillation counting. The method exhibits a sensitivity of less than 10 pg for each amine and is suitable for the measurement of endogenous p -octopamine levels in mammalian brain. The highest levels of p -octopamine were found in the hypothalamus (3.4 ng/g) but despite the sensitivity of the assay, neither phenylethanolamine nor m -octopamine could be detected. After MAO inhibition, however, both of these amines were found to be present. p -Octopamine was increased substantially in all brain regions following the administration of an MAO inhibitor, whereas pretreatment with reserpine produced a significant decrease in the hypothalamus.     

METHYLATION OF E. COLI TRANSFER RIBONUCLEIC ACIDS BY A tRNA ADENINE-l-METHYLTRANSFERASE FROM RAT BRAIN CORTEX AND BULK-ISOLATED NEURONS

Abstract— Brain cortices or bulk-isolated neuronal cell bodies prepared from cortices of 8-day old male rats were used as the source of a l-methyl adenine-specific tRNA methyltransferase (tRNA-AMT). Ammonium sulfate fractionation and chromatography on spheroidal hydroxylapatite and Sephadex G-200 yielded an 80-fold purified enzyme, as determined by using E. coli bulk tRNA as substrate. The kinetic parameters of tRNA-AMT for the substrate S -adenosyl- l -methionine (SAM) ( K _m= 6 μM) and the inhibitor, S -adenosyl- l -homocysteine (SAH) ( K _i= 3.4 μ m ) were determined and several SAH analogs tested as inhibitors. S -Adenosyl- l -cysteine (SAC) ( 10 ^-4 m ) and S -adenosyl- d -homocysteine (SADH) (10^-4 m ) produced a 35 and a 21% reduction in enzyme activity, respectively. The effects of Mg²⁺, NH₄⁺ acetate and of the polyamines spermine, putrescine and spermidine on the brain tRNA-AMT mimicked the effects of these agents on hepatic tRNA-AMT (G lick et al , 1975).
Comparing the ability of cerebral tRNA-AMT to methylate E. coli tRNA^glu2, tRNA^val, tRNA^phe and bulk tRNA revealed tRNA^glu2 as the best and tRNA^phe as the least effective substrate.
tRNA-AMT prepared from neuronal cell bodies showed closely similar characteristics to the cortical enzyme. A comparison of the activities of tRNA-AMT in neurons and glial cells revealed higher values in the former.     

UDP-galactose-ceramide galactosyltransferase in rat brain myelin subfractions during development.

The localization and activity of the enzyme UDP-galactose-hydroxy fatty acid-containing ceramide galactosyltransferase is described in rat brain myelin subfractions during development. Other lipid-synthesizing enzymes, such as cerebroside sulphotransferase, UDP-glucose-ceramide glucosyltransferase and CDP-choline-1,2-diacylglycerol cholinephosphotransferase, were also studied for comparison in myelin subfractions and microsomal membranes. The purified myelin was subfractionated by isopycnic sucrose-density-gradient centrifugation. Four myelin subfractions, three floating respectively on 0.55 M- (light-myelin fraction), 0.75 M- (heavy-myelin fraction) and 0.85 M-sucrose (membrane fraction), and a pellet, were isolated and purified. At all ages, 70--75% of the total myelin proteins was found in the heavy-myelin fraction, whereas 2--5% of the protein was recovered in the light-myelin fraction, and about 7--12% in the membrane fraction. Most of the galactosyltransferase was associated with the heavy-myelin and membrane fractions. Other lipid-synthesizing enzymes studied appeared not to associate with purified myelin or myelin subfractions, but were enriched in the microsomal-membrane fraction. During development, the specific activity of the microsomal galactosyltransferase reached a maximum when the animals were about 20 days old and then declined. By contrast the specific activity of the galactosyltransferase in the heavy-myelin and membrane fractions was 3--4 times higher than that of the microsomal membranes in 16-day-old animals. The specific activity of the enzyme in the heavy-myelin fraction sharply declined with age. Chemical and enzymic analyses of the heavy-myelin and membrane myelin subfractions at various ages showed that the membrane fraction contained more proteins in relation to lipids than the heavy-myelin fraction. The membrane fraction was also enriched in phospholipids compared with cholesterol and contrined equivalent amounts of 2':3'-cyclic nucleotide 3'-phosphohydrolase compared with heavy- and light-myelin fractions. The membrane fraction was deficient in myelin basic protein and proteolipid protein and enriched in high-molecular-weight proteins. The specific localization of galactosyltransferase in heavy-myelin and membrane fractions at an early age when myelination is just beginning suggests that it may have some role in the myelination process.     

Endogenous phosphorylation of basic protein in myelin of varying degrees of compaction

Fractions containing myelin of varying degrees of compaction were prepared from human white matter. Protein kinase activity in these fractions was measured by using both endogenous and exogenous myelin basic protein (MBP) as substrates. In both cases, less compact myelin fractions possessed higher levels of protein kinase activity than the compact myelin fraction. In addition, the specific activity of phosphorylated basic protein was greater in the loosely compacted fractions than in compact multilamellar myelin. When basic protein in compact myelin or the myelin fractions was phosphorylated by the endogenous kinase, approximately 70% of the [32P]phosphate was incorporated at a single site, identified as Ser-102. The remaining 30% was found in three other minor sites. Electron microscopy of less compact myelin showed it was composed of fewer lamellae which correlated with a relative decrease in the proportion of cationic charge isomers (microheteromers) when MBP was subjected to gel electrophoresis at alkaline pH. The shift in charge microheterogeneity of basic protein to the less cationic isomers in the less compact myelin fractions correlated with an increase in protein kinase activity and a greater specific activity of phosphorylated basic protein.     

Methylase activities from Haemophilus influenzae that protect Haemophilus parainfluenzae transforming deoxyribonucleic acid from inactivation by Haemophilus influenzae endonuclease R.

Specific methylases that have the properties of deoxyribonucleic acid (DNA) modification enzymes have been isolated from Haemophilus influenzae strain Rd. Two activities ((Methylase IIa and methylase III) were found to protect transforming DNA of H. parainfluenzae from the action of H. influenzae restriction enzymes. To determine the specificty of the protection, a procedure based on biological activity was developed for the separation and purification of the restriction endonucleases from H. influenzae strain Rd. Two endonuclease R activities presumably corresponding to Hind II and Hind III (P. H. Roy and H. O. Smith, 1973; H. O. Smith and K. W. Wilcox, 1970) were characterized by differences in their chromatographic properties, ability to attack T7 DNA, and inactivation of the transforming activity of different markers of H. parainfluenzae DNA. One endonuclease R enzyme (Hind II) attacked T7 DNA and was found to inactivate the dalacin resistance marker (smaller than 0.01% activity remaining) with only a slight effect on the streptomycin resistance marker (83% activity remaining). Methylase IIa treatment protected 40% of the dalacin resistance marker of H. parainfluenzae DNA from inactivation by Hind II. The other restriction activity (Hind III) was inert towards T7 DNA and inactivated the streptomycin resistance marker of H. parainfluenzae DNA (smaller than 0.01% activity remaining) without any effect on the dalacin resistance marker. The methylation of H. parainfluenzae DNA accomplished by methylase III protected 60% of the transforming activity of the streptomycin resistance marker of H. parainfluenzae DNA from the action of Hind III.     

The Regional Concentrations of S-Adenosyl-l-Methionine, S-Adenosyl-l-Homocysteine, and Adenosine in Rat Brain

Abstract: The concentrations of S -adenosyl- l -methionine (SAM), S -adenosyl- l -homocysteine (SAH), and adenosine (Ado) were determined in whole brain and rat brain regions by HPLC. The whole brain contains, respectively, 22 nmol, 1 nmol, and 64 nmol of SAM, SAH, and Ado per g of wet tissue. Their distribution indicated that SAM and SAH levels are highest in brainstem, whereas the Ado level is highest in cortex. With aging the SAM concentrations decrease in whole brain, brainstem, and hypothalamus (–25%) and SAH levels increase by 90% in striatum and by 160% in cerebellum, while Ado levels are increased in all regions by 100–180%.     

Studies of myelin proteins in multiple sclerosis brain tissue

Using radioimmunoassays (RIA) for the myelin specific proteins, myelin proteolipid protein (PLP) and myelin basic protein (MBP) and an enzyme assay for the activity of the myelin marker enzyme 23 cyclic-3 phosphohydrolase (CNPase), we have studied plaque, periplaque and normal appearing white matter (NAWM) regions of multiple sclerosis (MS) brain tissue, as well as normal control brain tissue. We found that all three myelin proteins are decreased in all regions, including NAWM, of MS brain, with a decreasing gradient from NAWM to periplaque to plaque. The NAWM was not significantly different from the periplaque region. Surprisingly, when the ratios of the proteins were calculated, MBP activity, although decreased was found to be relatively preserved.     

THE EFFECT OF dl-ALLYLGLYCINE ON POLYAMINE AND GABA METABOLISM IN MOUSE BRAIN

Abstract— dl -Allylglycine, a potent inhibitor of glutamate decarboxylase in vivo when given intraperitoneally, causes a marked decrease in brain GABA concentration and at the same time a dramatic increase in l -ornithine decarboxylase activity and a simultaneous decrease in S -adenosyl- l -methionine decarboxylase activity followed by putrescine accumulation. It does not, however, alter the degree of GABA formation from putrescine. The timing of the recovery of glutamate decarboxylase activity after the injection of dl -allylglycine is concomitant with that of the GABA concentration, indicating that it is probably glutamate decarboxylase that is solely responsible for making up the GABA deficit caused by dl -allylglycine, and that the changes in polyamine metabolism are associated in some indirect way with the recovery process.     

Localization of the basic protein and lipophilin in the myelin membrane with a non-penetrating reagent.

The localization of proteins in myelin was studied by the use of a non-penetrating reagent. Tritiated 4,4'-diisothiocyano-2,2'-ditritiostilbene disulfonic acid was used to label the isolated myelin membrane. The membrane was labelled, the basic protein and the hydrophobic protein, lipophilin, were isolated. After 10 min of exposure to the reagent, the specific activity of lipophilin was found to be 10 times greater than that of the basic protein. Water shock did not alter the specific activities. However, sonication increased the specific activity of lipophilin but not that of basic protein. When the isolated proteins were labelled with 3H-labelled 4,4'-diisothiocyano-2,2'-ditritiostilbene disulfonic acid, the specific activity of the basic protein was 10 times that of lipophilin. We concluded that the low specific activity of basic protein isolated from the labelled membrane was due to the inaccessible position of this protein in the membrane bilayer.     

Myelin Composition and Activities of CNPase and Na+, K+-ATPase in Hypomyelinated "pt" Mutant Rabbit

A disorder of CNS myelination was found in paralytic tremor ("pt") rabbits. The condition is inherited in a sex-linked recessive mode. Ultrastructurally, an obvious myelin deficiency with aberration of myelin sheath formation is observed. The yield of myelin isolation was reduced to 20-30% of control. Myelin isolated from 4-week-old "pt" rabbits contained reduced amounts of galactosphingolipids and of several myelin protein markers. Moreover, myelin basic protein, analyzed by two-dimensional gel electrophoresis, showed a deficit in its more basic components. All these facts suggest a delay in myelin maturation. Ganglioside content was increased as well as Na+,K+-ATPase specific activity. 2',3'-Cyclic nucleotide phosphodiesterase (CNPase) specific activity was the same in "pt" as in control myelin but differed by having greater sensitivity to detergent activation.     

PRODUCT INHIBITION OF RAT BRAIN HISTAMINE-N-METHYLTRANSFERASE

Abstract— The inhibition of S -adenosylmenthionine: histamine- N -methyltransferase (EC 2.1.1.8; HMT) by its products, 3-methylhistamine (3-MetHm) and S -adenosyl- l -homocysteine (SAH), was examined using a preparation of the enzyme which was partially purified from rat brains. SAH was found in in vitro experiments, to be a competative inhibitor of HMT in relation to S -adenosyl- l -methionine (SAM), with a K _i= 5.6 μM. SAH was shown to be a non-competitive inhibitor with respect to histamine (Hm) ( K _i= 5.0 μM). The K _m's for SAM and Hm were 10.2 and 3.0 μM respectively. On the other hand, 3-MetHm was determined to be a non-competitive inhibitor of HMT with respect to Hm ( K _i= 8.7 μM) and an uncompetitive inhibitor with respect to SAM ( K _i= 9.6 μM). These results suggest that the addition of the substrate to, and the release of products by, HMT occurs sequentially. In the nomenclature Of C leland (1963) the reaction is seemingly of the 'ordered Bi-Bi' type.     

The breakdown of myelin-bound proteins by intra- and extracellular proteases

Changes in protein components of purified myelin were measured following incubation in vitro with purified intra- and extracellular enzymes. Incubation with calf brain cathepsin D did not result in a significant relese of acid-soluble peptides as measured by ninhydrin analysis but was accompanied by a large loss of myelin proteins as determined on SDS-acrylamide gels. After 5 hr at 37°C there was a loss of about 25% for fast and slow basic proteins and the Agrawal proteolipid, but only a 5–10% loss for the Folch-Lees and Wolfgram components. Rat brain cathepsin D prepared by affinity chromatography gave a 30–60% breakdown of basic proteins and proteolipids. In general, breakdown using lyophilized myelin was increased over two-fold as compared to experiments with fresh myelin. Breakdown induced by cathepsin D was completely inhibited by the pentapeptide pepstatin. Incubation of myelin at physiological pH resulted in an endogenous breakdown of about 12% for basic proteins in freshly prepared, and 50% for lyophilized material. Addition of a soluble neutral proteinase that splits hemoglobin did not induce additional breakdown except for a small change in the Folch-Lees component. The extracellular enzymes pepsin and TPCK-treated trypsin resulted in a larger breakdown of all components as compared to brain enzymes. Present results demonstrate that all protein components of myelin are accessible to hydrolases and vulnerable to breakdown to varying extents by brain enzymes. These facts are consistent with the known rates for myelin protein turnover and may have a bearing on changes associated with demyelinating diseases     

Regional Distribution of Methionine Adenosyltransferase in Rat Brain as Measured by a Rapid Radiochemical Method

Abstract: The distribution of methionine adenosyltransferase (MAT) in the CNS of the rat was studied by use of a rapid, sensitive and specific radiochemical method. The S -adenosyl-[methyl-¹⁴C] l -methionine ([¹⁴C]SAM) generated by adenosyl transfer from ATP to [methyl-¹⁴C] l -methionine is quantitated by use of a SAM-consuming transmethylation reaction. Catechol O -methyltransferase (COMT), prepared from rat liver, transfers the methyl-¹⁴C group of SAM to 3,4-dihydroxybenzoic acid. The ¹⁴C-labelled methylation products, vanillic acid and isovanillic acid, are separated from unreacted methionine by solvent extraction and quantitated by liquid scintillation counting. Compared to other methods of MAT determination, which include separation of generated SAM from methionine by ion-exchange chromatography, the assay described exhibited the same high degree of specificity and sensitivity but proved to be less time consuming. MAT activity was found to be uniformly distributed between various brain regions and the pituitary gland of adult male rats. In the pineal gland the enzyme activity is about tenfold higher.     

Localization of the basic protein and lipophilin in the myelin membrane with a non-penetrating reagent

The localization of proteins in myelin was studied by the use of a non-penetrating penetrating reagent. Tritiated 4,4′-diisothiocyano-2,2′-ditritiostilbene disulfonic acid was used to label the isolated myelin membrane. The membrane was labelled, the basic protein and the hydrophobic protein, lipophilin, were isolated. After 10 min of exposure to the reagent, the specific activity of lipophilin was found to be 10 times greater than that of the basic protein. Water shock did not alter the specific activities. However, sonication increased the specific activity of lipophilin but not that of basic protein. When the isolated proteins were labelled with ³H-labelled, 4,4′-diisothiocyano-2,2′-ditritiostilbene disulfonic acid, the specific activity of the basic protein was 10 times that of lipophilin. We concluded that the low specific activity of basic protein isolated from the labelled membrane was due to the inaccessible position of this protein in the membrane bilayer.     

Elevated specific activity of 5'-nucleotidase in a spinal cord myelin fraction from shiverer mice. Comparison with other myelin-associated enzymes and myelin proteins

Myelin partially purified from spinal cords of dysmyelinating mutant (shiverer) mice had almost three-fold the specific activity of 5'-nucleotidase found in the respective myelin fraction from normal mice. The specific activities of two other normally myelin-associated enzymes, 2',3'-cyclic nucleotide-3'-phosphohydrolase and carbonic anhydrase, were only slightly higher in the myelin membranes from shiverers, compared to those from controls. In the mutants, the three enzymes probably occur in oligodendrocyte processes. Hypothetically, the 5'-nucleotidase in the myelin sheath in shiverer and normal mice may be localized in specialized structures.