Purification of phosphatidylethanolamine N-methyltransferase from rat liver

Phosphatidylethanolamine (PE) N-methyltransferase catalyzes the synthesis of phosphatidylcholine by the stepwise transfer of methyl groups from S-adenosylmethionine to the amino head group of PE. PE N-methyltransferase was solubilized from a microsomal membrane fraction of rat liver using the nonionic detergent Triton X-100 and purified to apparent homogeneity. Specific activities of PE N-methyltransferase with PE, phosphatidyl-N-monomethylethanolamine (PMME), and phosphatidyl-N,N-dimethylethanolamine (PDME) as substrates were 0.63, 8.59, and 3.75 mumol/min/mg protein, respectively. The purified enzyme was composed of a single subunit with a molecular mass of 18.3 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Methylation activities dependent on the presence of PE, PMME, and PDME and the 18.3-kDa protein co-eluted when purified PE N-methyltransferase was subjected to gel filtration on Sephacryl S-300 in the presence of 0.1% Triton X-100. All three methylation activities eluted with a Stokes radius 2.1 A greater than that determined for pure Triton micelles (molecular mass difference of 27.4 kDa). Two-dimensional analysis of PE N-methyltransferase employing nonequilibrium pH gradient gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of a single isoform. Analysis of enzyme activity using PE, PMME, and PDME at various Triton X-100 concentrations indicated the enzyme follows the "surface dilution" model proposed for other enzymes that act at the surface of mixed micelle substrates. Initial velocity data for all three lipid substrates (at fixed concentrations of Triton X-100) were highly cooperative in nature. Hill numbers for PMME and PDME ranged from 3 at 0.5 mM Triton to 6 at 2.0 mM Triton. All three methylation activities had a pH optimum of 10. These results provide evidence that a single membrane-bound enzyme catalyzes all three methylation steps for the conversion of PE to phosphatidylcholine.     

Purification and properties of pancreatic glycine N-methyltransferase.

Glycine N-methyltransferase (GNMT) regulates the ratio of S-adenosylmethionine to S-adenosylhomocysteine. It is very abundant in liver cytosol and earlier studies have shown it to be present in high concentrations in the pancreas. We have previously reported that liver GNMT is allosterically inhibited by 5-methyltetrahydrofolate pentaglutamate (5-CH3-H4PteGlu5), and proposed that this represents a metabolic control mechanism which links the de novo synthesis of methyl groups to the methylating ability of the liver. We now report that pancreatic GNMT also contains bound folate in vivo. Purified pancreatic GNMT is inhibited by reduced folate polyglutamates in vitro. The KI for the synthetic (R,S)5-CH3-H4PteGlu5 is 2.4 x 10(-7) M. The natural (S) form of 5-CH3-H4PteGlu5 is tightly bound and has a Kd of 1.3 x 10(-7) M. One mole is bound per enzyme tetramer. These studies suggest that GNMT is important in the regulation of methyl group metabolism in the pancreas as well as in the liver.     

Identification of phosphorylation sites in glycine N-methyltransferase from rat liver

Previous studies have shown that rat glycine N-methyltransferase (GNMT) is phosphorylated in vivo, and could be phosphorylated in vitro on serine residues with a significant increase of enzyme activity, but no phosphorylation sites were identified. In this work the identification of the specific phosphorylation sites of rat GNMT is reported. Three different preparations of rat GNMT were analyzed: (1) purified from liver by standard methods of protein purification, (2) prepared from isolated hepatocytes and from liver tissue by immunoprecipitation, and (3) recombinant protein expressed in Escherichia coli. We measured the molecular weights of protein isoforms using electrospray mass spectrometry and used liquid chromatography-tandem mass spectrometry (LC-MS/MS) of peptides resulting from tryptic and chymotryptic digests. We also performed chemical analysis of phosphoamino acids and protein sequencing. In all samples, the phosphorylated serine residues 71, 182, and 241 were found. In GNMT prepared from liver tissue and hepatocytes an S9 additional residue was found to be phosphorylated. In hepatocytes and in recombinant GNMT S139 was detected. Serine 9 was also identified as a target for cAMP-dependent protein kinase in vitro. The positions of these phosphorylated residues in the tertiary structure of GNMT indicate their possible effect on enzyme conformation and activity.     

Characterization of glycine N-methyltransferase from rabbit liver.

The enzymatic properties of glycine N-methyltransferase from rabbit liver and the effects of endogenous adenosine nucleosides, nucleotides and methyltransferase inhibitors were investigated using a photometrical assay to detect sarcosine with o-dianisidine as a dye. After isolation and purification the denatured enzyme showed a two-banded pattern by SDS-PAGE. The enzyme was highly specific for its substrates with a pH-optimum at pH 8.6. Glycine N-methyltransferase exhibits Michaelis-Menten kinetics for its substrates, S-adenosylmethionine and glycine, respectively. The apparent Km and Vmax values were determined for both the substrates, the other substrate being present at saturating concentrations. The enzyme was strongly inhibited in the presence of S-adenosylhomocysteine, 3-deazaadenosine, and 5'-S-isobutylthio-5'-deoxyadenosine. All other inhibitors investigated, adenosine, 2'-deoxyadenosine, aciclovir, and 5'-N-ethylcarboxamidoadenosine were poor inhibitors of the methylation reaction. Adenine nucleotides and vidarabin were without effect on the enzymatic activity. Based on the kinetic data glycine N-methyltransferase from rabbit liver exhibits appreciable activity at physiological S-adenosylmethionine and S-adenosylhomocysteine levels.     

Purification of messenger RNA coding for glycine methyltransferase from rat liver

Rat liver messenger RNA coding for glycine methyltransferase was associated preferentially with free polysomes. The mRNA was purified about 1000-fold over the total poly(A)-containing RNA by specific immunoadsorption of polysomes to protein A-Sepharose followed by oligo(dT)-cellulose column chromatography. Sodium dodecyl sulfate-gel electrophoresis of the in vitro translation products in a rabbit reticulocyte lysate system revealed only one major band which migrated to the position of the purified glycine methyltransferase subunit. The result shows that the mRNA isolated is nearly homogeneous and suggests that no precursor form of the enzyme existed. The mRNA sedimented at the position slightly smaller than 18 S rRNA in a sucrose density-gradient centrifugation and was shown to contain about 1,300 nucleotides by the Northern blot hybridization analysis with a cDNA probe.     

Purification and properties of asparagine synthetase from rat liver

Asparagine synthetase (L-aspartate: ammonia ligase (AMP-forming, EC 6.3.1.1) activity in rat liver increased when the animals were put on a low casein diet. The enzyme was purified about 280-fold from the supernatant of rat liver homogenate by a procedure comprising ammonium sulfate fractionation, DEAE-Sepharose column chromatography, and Sephadex G-100 gel filtration. The optimal pH of the enzyme was in the range 7.4–7.6 with glutamine as an amide donor. The molecular weight was estimated to be approximately 110 000 by gel filtration. Chloride ion was required for the enzyme activity. The apparent K_m values for L-aspartate, L-glutamine, ammonium chloride, ATP, and Cl⁻ were calculated to be 0.76, 4.3, 10, 0.14, and 1.7 mM, respectively. The activity was inhibited by l-asparagine, nucleoside triphosphates except ATP, and sulflhdryl reagents.It has been observed that the properties of asparagine synthetase from rat liver are not different from those of tumors such as Novikoff hepatoma and RADA 1.     

Purification and properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase was purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polyacrylamide gel electrofocusing, about 710,000-fold with a 35% yield from the liver cytosol of thioacetamide-treated rats. The final specific activity was approximately 24,400 nmol/min/mg of protein. The apparent molecular weight of the enzyme determined by gel filtration analyses on Sephacryl S-200 was 55,000 in the presence of 0.25 M NaCl and 145,000 in its absence. The minimum molecular weight of the enzyme was determined to be 54,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was estimated as 5.7 in the presence of 8 M urea. Some catalytic properties of the enzyme were also studied.     

5-methyltetrahydrofolate is bound in intersubunit areas of rat liver folate-binding protein glycine N-methyltransferase

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. It is abundant in the liver, where it uses excess S-adenosylmethionine (AdoMet) to methylate glycine to N-methylglycine (sarcosine) and produces S-adenosylhomocysteine (AdoHcy), thereby controlling the methylating potential of the cell. GNMT also links utilization of preformed methyl groups, in the form of methionine, to their de novo synthesis, because it is inhibited by a specific form of folate, 5-methyltetrahydrofolate. Although the structure of the enzyme has been elucidated by x-ray crystallography of the apoenzyme and in the presence of the substrate, the location of the folate inhibitor in the tetrameric structure has not been identified. We report here for the first time the crystal structure of rat GNMT complexed with 5-methyltetrahydrofolate. In the GNMT-folate complex, two folate binding sites were located in the intersubunit areas of the tetramer. Each folate binding site is formed primarily by two 1-7 N-terminal regions of one pair of subunits and two 205-218 regions of the other pair of subunits. Both the pteridine and p-aminobenzoyl rings are located in the hydrophobic cavities formed by Tyr5, Leu207, and Met215 residues of all subunits. Binding experiments in solution also confirm that one GNMT tetramer binds two folate molecules. For the enzymatic reaction to take place, the N-terminal fragments of GNMT must have a significant degree of conformational freedom to provide access to the active sites. The presence of the folate in this position provides a mechanism for its inhibition.     

Purification and properties of microsomal UDP-glucuronosyltransferase from rat liver.

p-Nitrophenol conjugating activity associated with liver microsomal UDP-glucuronosyltransferase (EC 2.4.1.17) was purified 150- to 200-fold from cell-free homogenates. The purification scheme included solubilization with the nonionic detergent Lubrol WX, anion exchange chromatography at pH 6.0 and 7.5, and affinity chromatography with UDP-hexanolamine Sepharose 4B. The enzyme purified as a phospholipid-protein complex and was shown to consist of a single polypeptide chain of molecular weight 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated approximately 531 mol of amino acids/59,000 g of enzyme and a molar ratio of nonpolar to polar residues of 1.08. During fractionation, the enzyme displayed instability with such steps as gel filtration, dialysis, or ultrafiltration of dilute samples; however, upon adsorption to ion exchange resins or storage in concentrated form, the enzyme was reasonably stable. The active lipoprotein complex showed both size and charge heterogeneity as judged by gel filtration and electrofocusing. Three forms of the enzyme resolved by isoelectric focusing had isoelectric points which averaged pH 6.68, 6.56, and 6.31. Polypeptide compositions of these electrophoretically distinct phospholipid protein complexes were indistinguishable on the basis of sodium dodecyl sulfate-polyacryl-amide gel electrophoresis, suggesting that the charge heterogeneity may be the result of differences in the phospholipid content of the lipoprotein complex.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

Purification and properties of ATPase inhibitor from rat liver mitochondria

(1) The ATPase inhibitor protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9.

(2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F₁, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria.

(3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg²⁺. ATP at slightly acidic pH.

(4) The inhibitor has a minimal effect on P_i-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimulates P_i-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.     

Purification and properties of gamma-glutamyl transferase from normal rat liver

gamma-Glutamyl transferase ((5-glutamyl)-peptide: amino-acid 5-glutamyltransferase, ED 2.3.2.2) has been partially purified from both whole rat liver (600-fold) and from isolated biliary tract (1200-fold). The most highly purified fraction gave two protein bands on polyacrylamide gel electrophoresis, the major band alone having enzyme activity. The enzyme purified from biliary tract appears identical to that from whole liver preparation according to molecular weight, kinetic parameters and the effects of various inhibitors. Three liver cell-types; parenchymal, Kupffer and biliary tract were isolated by perfusion of the rat liver in situ with collagenase, followed by selective cell isolation. Approx. 80-90% of the total recovered enzyme activity was found in the biliary tract. Nearly 50% of the apparent enzyme activity in the parenchymal cell was attributable to a nonspecific hydrolase.     

Purification and properties of asparagine synthetase from rat liver.

Asparagine synthetase (L-aspartate:ammonia ligase (AMP-forming, EC 6.3.1.1) activity in rat liver increased when the animals were put on a low casein diet. The enzyme was purified about 280-fold from the supernatant of rat liver homogenate by a procedure comprising ammonium sulfate fractionation. DEAE-Sepharose column chromatography, and Sephadex G-100 gel filtration. The optimal pH of the enzyme was in the range 7.4-7.6 with glutamine as an amide donor. The molecular weight was estimated to be approximately 110,000 by gel filtration. Chloride ion was required for the enzyme activity. The apparent Km values for L-aspartate, L-glutamine, ammonium chloride, ATP, and Cl- were calculated to be 0.76, 4.3, 10, 0.14, and 1.7 mM, respectively. The activity was inhibited by L-asparagine, nucleoside triphosphates except ATP, and sulfhydryl reagents. It has been observed that the properties of asparagine synthetase from rat liver are not so different from those of tumors such as Novikoff hepatoma and RADA 1.