Purification of phospholipid methyltransferase from rat liver microsomal fraction.

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.     

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.     

Activation of partially purified rat liver lipid methyltransferase by phosphorylation

Incubation of partially purified rat liver lipid methyltransferase with MgATP and the catalytic subunit of the cyclic AMP dependent protein kinase results in up to 4-fold activation of the methylation reaction. When (gamma-32p) MgATP is included in the assay mixture, the analysis of the phosphoprotein products by electrophoresis shows the incorporation of 32p into a single protein band of about 50K and pI 4.75. It is concluded that rat liver lipid methyltransferase can be converted from a low activity dephosphorylated form to a high activity phosphorylated form.     

Detection of cannabinoid receptors by photoaffinity labelling.

A novel [125I]-labelled photoaffinity ligand designed to detect cannabinoid binding sites has been used in mouse brain preparations and in cultured S49 mouse lymphoma cells. The ligand, 2-iodo-5'-azido-delta 8-THC, shows a high affinity for sites in both brain (Kd = 5.60 pM) and whole cell (Kd = 9.38 pM) systems. Photolabelling studies with brain samples revealed the existence of four ligand-protein adducts, of estimated molecular weights 85.5, 62.1, 30.0 and 25.5 kDa, that were diminished by prior exposure to 8 microM THC. A similar study with S49 cells gave adducts with apparent molecular weights of 62.1, 34.4, 16.9 and 13.5 kDa. The ligand produces a typical cannabinoid cataleptic response in mice suggesting that possibly one or more of the binding sites may be involved in some of the receptor mediated actions of THC.     

Purification and characterization of rat heart and brain catechol methyltransferase.

In an effort to detect the similarities and differences in the properties of rat heart, brain and liver catechol methyltransferase (S-adenosyl-L-methionine:catechol O-methyltransferase, EC 2.1.1.6), we have determined the cellular distribution of this enzyme activity and extensively purified the soluble and microsomal enzymes present in these tissues. Purification of soluble heart (688-fold) and brain enzymes (240-fold) were achieved using an affinity chromatographic system. The properties of these enzymes were compared with respect to their molecular weights, substrate specificities, inhibitor specificities and immunological properties. The characteristics of the enzyme active sites were investigated using various methyl acceptor substrates and various analogs of S-adenosylmethionine as methyl donors. A series of analogs of S-adenosylhomocysteine was also evaluated as inhibitors of these enzymes. The immunological properties of the purified soluble and microsomal enzymes from heart and brain were investigated using an antibody isolated from rabbits which had been immunized with the soluble rat liver enzyme. In general the properties of catechol methyltransferases isolated from heart and brain were similar to the properties of the enzyme isolated from liver. Some minor differences in substrate and inhibitor specificities were observed which might suggest slight differences in the active sites of these enzymes.     

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 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.     

Purification and characterization of cathepsin J from rat liver.

Cathepsin J has been partially purified [Liao, J. C. R. & Lenney, J. F. (1984) Biochem. Biophys. Res. Commun. 124, 909-916], but its detailed properties are still unknown. In this study, we have purified cathepsin J completely and characterized it. It was purified to homogeneity from the mitochondrial-lysosomal fraction of rat liver by acid treatment, followed by ammonium sulfate precipitation (20-65%), and chromatographies on S-Sepharose, ConA-Sepharose, Affi-gel 501, HPLC DEAE-5PW and HPLC TSK G3000SW. Cathepsin J was found to be a lysosomal high-molecular-mass cysteine protease of about 160 kDa consisted of two different subunits. One subunit (alpha subunit) was a glycoprotein with a molecular mass of 19-24 kDa which was reduced to 19 kDa by treatment with endoglycosidase F. It has the amino acid sequence LPESWDWRNVR at its N-terminus, which was very similar to those at the N-termini of rat cathepsins B, H and L. The other subunit (beta subunit) was a glycoprotein with a molecular mass of 17 kDa, which was reduced to 14 kDa by treatment with endoglycosidase F. It had DTPANETYPDLLG at its N-terminus, which had no similarity with the N-terminal sequences of other cathepsins. Cathepsin J showed strong affinity for synthetic substrates such as N-benzyloxycarbonyl-phenylalanyl-arginine 4-methyl-coumaryl-7-amide and glycyl-arginine beta-naphthylamide. It was activated by thiol reagents and chloride ion and was inhibited by cysteine protease inhibitors. However, its initial inhibition constant Ki(initial) by N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine-3- methylbutylamide (E-64-c) was 1800 nM, which was 100-500 times those of cathepsins B and L. Many properties of cathepsin J were similar to those of cathepsin C (dipeptidylaminopeptidase I) reported as a lysosomal cysteine protease with dipeptidyl-aminopeptidase activity [McDonald, J. K., Reilly, T. J. & Ellis, S. (1964) Biochem. Biophys. Res. Commun. 16, 135-140]. Furthermore, antiserum against rat liver cathepsin C reacted with rat liver cathepsin J. These findings suggested that cathepsin J is identical with cathepsin C.     

Purification of 2,3-oxidosqualene cyclase from rat liver.

A rapid and simple purification of milligram amounts of 2,3-oxidosqualene cyclase, an integral membrane enzyme that catalyzes the cyclization of squalene epoxide to lanosterol, is reported. Several nonionic detergents (Triton X-100, Tween 80, Emulphogene, and lauryl maltoside) were evaluated for solubilization of oxidosqualene cyclase from rat liver microsomes. At a detergent concentration of 5 mg/ml, lauryl maltoside was approximately 10 times more effective than Emulphogene in the solubilization of oxidosqualene cyclase; Triton X-100 and Tween 80 were less effective than Emulphogene as judged by the relative specific activities of the solubilized enzyme. Treatment of microsomes with lauryl maltoside resulted in a selective solubilization of the cyclase with concomitant activation of the enzyme. The solubilized enzyme was purified to homogeneity by fast protein liquid chromatography. The purified enzyme consists of a single subunit that has an apparent molecular weight of 65,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme obeys saturation kinetics and the apparent Km of (2,3)-oxidosqualene is 15 microM; the apparent kcat/Km is 200 M-1.min-1. An improved assay of the enzyme that utilizes high performance liquid chromatography methods is also described.     

Solubilization of peroxidase from porcine thyroids and characterization by photoaffinity labelling

Peroxidase was solubilized without proteolysis from porcine thyroid particulate fraction with the nonionic detergent, 1-O-n-octyl-beta-D-glucopyranoside. The enzyme was able to catalyze the oxidation of guaiacol and the iodination of bovine serum albumin (33 atoms of iodine per molecule protein). Binding studies performed with the partially purified enzyme indicated that the substrates thyroxine (T4) and tyrosine compete for the same binding site on the enzyme. Dissociation constants of 0.9 nM and 0.5 nM were found for T4 and tyrosine, respectively. After photoaffinity labelling with underivatized 125I-labelled T4, gel chromatography on Sephacryl S-1000 revealed a relative molecular weight of about 100 000 for the solubilized enzyme. The peroxidase activity and haem-absorbance peak coeluted from the Sephacryl S-1000 column. SDS-polyacrylamide gel electrophoresis under reducing conditions indicated two major radiolabelled polypeptides, Mr 83 000 and Mr 42 600, as well as a smaller peak at Mr 15 400. The 15 400 molecular weight species is probably not part of the peroxidase complex, since it could partially be removed by Sephadex G-25 prechromatography . Further analyses confirmed that the partially purified enzyme is a haemoprotein absorbing maximally at 412 nm. The Soret band is shifted to 423 nm by reducing agents and the haem-cyanide complex has a maximum absorbance at 416 nm.     

Cellular retinol-binding protein from rat liver. Purification and characterization

Cellular retinol-binding protein has been purified to homogeneity from rat liver. The procedures utilized in the purification included acid precipitation, gel filtration on Sephadex G-75 and G-50, and chromatography on DEAE-cellulose. The binding protein was purified approximately 3,500-fold, based on total soluble liver protein. The protein is a single polypeptide chain with a molecular weight of 14,600 based on information obtained by the techniques of sedimentation equilibrium analysis, gel filtration, and sodium dodecyl sulfate-polyacrylamide electrophoresis. The protein binds retinol with high affinity; the appparent dissociation constant was determined by fluorometric titration to be 1.6 X 10(-8) M. Retinol bound to the protein has an absorption spectrum (lambdamax, 350 nm) considerably altered from the spectrum of retinol in ethanol (lambdamax, 325 nm).     

Purification and partial characterization of cysteine-glutamate transaminase from rat liver.

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and alpha-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of alpha-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.     

Purification and properties of ATPase inhibitor from rat liver mitochondria.

(1) The ATPase inhibitior 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 Pi-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimules Pi-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 characterization of formaldehyde dehydrogenase from rat liver cytosol.

Formaldehyde dehydrogenase was purified to electrophoretic and column chromatographic homogeneity from rat liver cytosolic fraction by a procedure which includes ammonium sulfate precipitation, DEAE-cellulose-, hydroxyapatite-, Mono Q-chromatography, and gel filtration. Its molecular mass was estimated to be 41 kDa by gel filtration and SDS-PAGE, suggesting that it is a monomer. It utilized neither methylglyoxal nor aldehydes except formaldehyde as a substrate. It has been reported that liver class III alcohol dehydrogenase and formaldehyde dehydrogenase are the same enzyme and oxidize formaldehyde and long chain primary alcohols. However, the enzyme examined here did not use n-octanoi as a substrate. The Km values for formaldehyde and NAD+ were 5.09 and 2.34 microM at 25 degrees C, respectively. The amino acid sequences of 10 peptides obtained from the purified enzyme after digestion with either V8 protease or lysyl endopeptidase were determined. From these results, the enzyme was proved to be different from the previously described mammalian formaldehyde dehydrogenase and is the first true formaldehyde dehydrogenase to be isolated from a mammalian source.