Isolation and characterization of three different forms of arylamidase from rat skeletal muscle

An arylamidase hydrolysing L-leucine-4-nitroanilide was extracted from rat skeletal muscle homogenate and furified by means of anion-exchange chromatography on DEAE-Sephadex A-50 followed by gel filtration on Sephadex G-150 and Sepharose 6B. The enzyme was isolated in the form of three different protein complexes that differ in molecular weight, kinetic data, and sensitivity to metal ions. As studied by SDS-gel electrophoresis and repeated gel chromatography on Sepharose 6B these forms are: 1. a stable monomer (A1) of M_r 122 000; 2. a stable dimer (A2) of M_r 244 000; and 3. a stable polymer (A3) of more than M_r 4·10⁶. The arylamidase was optimally active at pH 7.3 and did not require metal ions. Treatment with 1,10-phenanthroline resulted in complete inactivation, the activity could be restored by the addition of manganous chloride. The sulphhydryl-blocking reagent 4-hydroxymercuribenzoate strongly inactivated the arylamidase, this inhibition could be reversed by the addition of 2-mercaptoethanol. Addition of phenylmethylsulfonyl fluoride had no effect on the enzyme activity. Furthermore, the influence of metal ions as well as the substrate specificity were investigated and compared for all three forms of arylamidase.     

Purification and characterization of a nicotinamide adenine dinucleotide-dependent secondary alcohol dehydrogenase from Candida boidinii

From the yest Candida biodinili grown on glucose a new secondary alcohol dehydrogenase was purified 426-fold by heat treatment, column chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose Cl-6b, and gel filtration on Sephacryl S-300. The purified enzyme was homogeneous as judged by analytical polyacrylamide gel electrophoresis. The molecular weight was found to be 150 000 by sedimentation equilibirum as well as by flitration. The enzyme appears to be composed of four identical subunits (M_r = 38000) as determined by SDS-gel electrophoresis. The enzyme catalyzes the oxidation of isopropanol to acetone in the presence of NAD⁺ as an electron acceptor. The K_m values were found to be 0.099 mM for isopropanoi and 0.14 mM for NDA⁺. Besides isopropanol also other secondary alcohols like butan-2-ol, pentan-2-ol, pentan-3-ol, hexan-2-ol, cyclobutanol, cyclopentanol, and cyclohexanol served as a substrate and were oxidazed to the correponding ketones. Isopropanol seems to be the best substrate for this enzyme which we therefore call isopropanol dehydrogenase. Primary alcohols are not oxidized by the enzyme. The optimum pH for enzymatic activity in the oxidation reaction was found to be 9.0, the optimal temperature is 45°C. The isolectric point of the isopropanol dehydrogenase was found to be pH 4.9. The enzyme is inactivated by mercaptide-forming reagents and chelating agents, 2-mercaptoethanol is an inhibitor. Zinc ions appear necessary for enzyme productuion.     

Purification and characterization of extracellular acid phosphatase of Tetrahymena pyriformis

An extracellular acid phosphatase secreted into the medium during growth of Tetrahymena pryiformis strain W was purified about 900-fold by (NH₄)₂SO₄ precipitation, gel filtration and ion exchange chromatography. The purified acid phosphatase was homogenous as judged by polycrylamide gel electrophoresis and was found to be a glycoprotein. Its carbohydrate content was about 10% of the total protein content. The native enzyme has a molecular weight of 120 000 as determined by gel filtration and 61 000 as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The acid phosphatase thus appears to consist of two subunits of equal size. The amino acid analysis revealed a relatively high content of asparic acid, glutamic acid and leucine. The purified acid phosphatase from Tetrahymena had a rather broad substrate specificity; it hydrolyzed organic phosphates, nucleotide phosphates and hexose phosphates, but had no diesterase activity. The K_m values determined with p-nitrophenyl phosphate, adenosine 5′-phosphate and glucose 6-phosphate were 3.1·10^?4 M, 3.9·10^?4 M and 1.6·10^?3 M, respectively. The optima pH for hydrolysis of three substrates were similar (pH 4.6). Hg²⁺ and Fe³⁺ at 5 mM were inhibitory for the purified acid phosphatase, and fluoride, L-(+)-tartaric acid and molybdate also inhibited its cavity at low concentrations. The enzyme was competitively inhibited by NaF (K_i=5.6·10^?4 M) and by L-(+)-tartaric acid (K_i = 8.5·10^?5 M), while it was inhibited noncompetitively by molybdate K_i = 5.0·10^?6 M). The extracellular acid phosphatase purified from Tetrahymena was indistinguishable from the intracellular enzyme in optimum pH, K_m, thermal stability and inhibition by NaF.     

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.     

Production and Purification of Extracellular D-Xylose Isomerase from an Alkaliphilic, Thermophilic Bacillus sp.

An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50°C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and β-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85°C for 20 min. Divalent metal ions Mg²⁺, Co²⁺, and Mn²⁺ were required for maximum activity of the enzyme. The K_m values for D-xylose and D-glucose at 80°C and pH 7.5 were 6.66 and 142 mM, respectively, while K_cat values were 2.3 × 10² s^-1 and 0.5 × 10² s^-1, respectively.     

Enzymatic biosynthesis of ergotamine and investigation on some aminoacyl-tRNA synthetases in Claviceps

An enzyme system from Claviceps purpurea (Fr.) Tul. catalyzing the incorporation of l-phenylalanine into ergotamine - ergotamine synthetase - was purified 172-fold. This was done by a combination of ammonium sulfate precipitation, gel filtration, ion-exchange chromatography on DEAE-Sepharose CL-6B, and hydroxyapatite chromatography. The activation of ergotamine specific amino acids as well as d-lysergic acid and dihydrolysergic acid via adenylates, as determined by the ATP-³²PP_i exchange, was investigated. Phenylalanyl-tRNA synthetase, catalyzing the same type of activation reaction, could not be separated from ergotamine synthetase by the purification procedure applied. Therefore, at the present stage of enzyme purification, phenylalanine-dependent ATP-³²PP_i exchange cannot be used to measure ergotamine synthetase activity specifically.Phenylalanyl-tRNA synthetase and leucyl-tRNA synthetase were separated into mitochondrial and cytoplasmic isoenzymes by hydroxyapatite chromatography. Their charging activities of procaryotic versus eucaryotic tRNA and their molecular masses were determined.     

Molecular weight of starch synthetase from Oryza sativa leaves

Soluble ADP-glucose: α-1,4-glucan-4-glucosyltransferase with primed activity was extracted from rice leaves and purified by (NH₄)₂SO₄ fractionation, gradient elution on DEAE-cellulose and finally by Sephadex G200 gel filtration or amylopectin-cellulose chromatography. The purified enzyme was essentially homogeneous electrophoretically, but exhibited two peaks corresponding to MW of 22 000 and 67 000 on Sephadex G200 chromatography and five distinct bands on sodium dodecyl sulfate gel electrophoresis with MW of 11·5, 20, 35, 50 and 68 × 10³.     

Protein kinase in Cicer embryonic axes

A dramatic increase in the cAMP-independent protein kinase activity (10-fold) was observed in the first 10 hr of germination of excised embryonic axes of Cicer. The lag phase of enzyme induction was very short since a 6-fold stimulation of protein kinase activity was witnessed after two hr of imbibition of the axes. The increase in protein kinase activity is ascribed to de novo biosynthesis of the enzyme. Conclusive proof for the de novo biosynthesis of protein kinase was obtained by labelling the proteins in vivo with [³⁵S]-sulphate and subsequently recovering the label predominantly in the methionine residues of the purified enzyme. The purification of the enzyme to electrophoretic homogeneity (453-fold) was achieved by ion exchange chromatography followed by affinity chromatography on a Casein-Sepharose column. Polyacrylamide gel electrophoresis of the [³⁵S]-labelled enzyme revealed a single radioactive band by autoradiography, that co-electrophoresed with the Coomassie Blue stained band of protein kinase. The M, of the purified protein kinase is 94 000 as determined by molecular sieving on Sepharose CL-6B. SDS-PAGE data indicated that the enzyme is composed of two subunits of M,s 49 000 and 62 000. Chemical characterization of the reaction product of protein kinase revealed that phosphorylation occurs at serine and threonine residues of the substrate, casein.     

Purification and characterization of a sodium-stimulated β-galactosidase from Bifidobacterium longum CCRC 15708

Summary β-galactosidase from Bifidobacterium longum CCRC 15708 was first extracted by ultrasonication then purified by Q Fast-Flow chromatography and gel chromatography on a Superose 6 HR column. These steps resulted in a purification of 15.7-fold, a yield of 29.3%, and a specific activity of 168.6 U mg⁻¹ protein. The molecular weight was 357 kDa as determined from Native-PAGE. Using o-nitrophenyl-β-d-galactopyranoside (ONPG) as a substrate, the pH and temperature optima of the purified β-galactosidase were 7.0 and 50 °C, respectively. The enzyme was stable at a temperature up to 40 °C and at pH values of 6.5–7.0. K _m and V _max for this purified enzyme were noted to be 0.85 mM and 70.67 U/mg, respectively. Na⁺ and K⁺ stimulated the enzyme up to 10-fold, while Fe³⁺, Fe²⁺, Co²⁺, Cu²⁺, Ca²⁺, Zn²⁺, Mn²⁺ and Mg²⁺ inhibited the activity of β-galactosidase. Furthermore, although glucose, galactose, maltose, or raffinose exerted little or no effect on the β-galactosidase activity, lactose and fructose inhibited the enzyme activity. The effect of lactose on the enzyme activity for ONPG is probably a case of competitive inhibition. A relatively high specific activity of β-galactosidase from B. longum CCRC 15708 could be obtained by Q Fast-Flow chromatography and gel chromatography on a Superose 6 HR column. In some aspects, particularly the activation by monovalent cations, the properties of β-galactosidase of B. longum CCRC 15708 are different from those obtained from other sources. Data collected in the present study are of value and indispensable when β-galactosidase from B. longum CCRC 15708 is employed in practical application.     

Purification and Characterization of Carbonic Anhydrase from Ağrı Balık Lake Trout Gill (Salmo trutta labrax) and Effects of Sulfonamides on Enzyme Activity

Carbonic anhydrase (CA) was purified from A?r? Bal?k Lake trout gill (fCA) by affinity chromatography on a sepharose 4B‐tyrosine‐sulfanilamide column. The fCA enzyme was purified with about a 303.9 purification factor, a specific activity 4130.4 EU (mg‐protein)^–1, and a yield of 79.3 by using sepharose‐4B‐l tyrosine‐sulfanilamide affinity gel chromatography. The molecular weight determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was found to be about 29.9 kDa. The kinetic parameters, K_M and V_max were determined for the 4‐nitrophenyl acetate hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CA enzymes. The K_i constants for mafenide ( 1 ), p‐toluenesulfonamide ( 2 ), 2‐bromo‐benzene sulfonamide ( 3 ), 4‐chlorobenzene sulfonamide ( 4 ), 4‐amino‐6‐chloro‐1–3 benzenedisulfonamide ( 5 ), sulfamethazine ( 6 ), sulfaguanidine ( 7 ), sulfadiazine ( 8 ), and acetozazolamide ( 9 ) were in the range of 7.5–108.75 μM.     

Isolation and properties of carboxypeptidase from leaves of wounded tomato plants

A carboxypeptidase was purified to homogeneity from upper, unwounded leaves of tomato plants in which carboxypeptidase activity had been induced to increase over three-fold by severely wounding the lower leaves. The carboxypeptidase was purified by ammonium sulfate precipitation, affinity chromatography, and finally by gel permeation chromatography. Electrophoresis at pH 4.3 and isoelectric focusing showed only a single band. The isoelectric point was 5.2 and the MW 105 000. Tomato carboxypeptidase possessed both peptidase and esterase activities and it sequentially hydrolysed amino acids from the carboxyl-terminal end of insulin chain B. It was optimally active at pH 6–7 on peptidase substrates, and at pH 8 on esterase substrates. The enzyme was inhibited by diisopropylfluorophosphate and incorporated 1 mol of DFP-[³H]. per mol of enzyme. Both peptidase and esterase activities were strongly inhibited by HgCl₂ but not by p-hydroxymercuribenzoate or iodoacetamide. Carboxypeptidase inhibitor from potatoes did not inhibit the enzyme.     

Purification and characterization of a β-amylase from soya beans

1. β-Amylase obtained by acidic extraction of soya-bean flour was purified by ammonium sulphate precipitation, followed by chromatography on calcium phosphate, diethylaminoethylcellulose, Sephadex G-25 and carboxymethylcellulose. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis on paper and in polyacrylamide gel. 3. The pure enzyme had a nitrogen content of 16·3%, its extinction coefficient, E^1%_1cm., at 280mμ was 17·3 and its specific activity/mg. of enzyme was 880 amylase units. 4. The molecular weight of the pure enzyme was determined as 61700 and its isoelectric point was pH5·85. 5. Preliminary examinations indicated that glutamic acid formed the N-terminus and glycine the C-terminus. 6. The amino acid content of the pure enzyme was established, one molecule consisting of 617 amino acid residues. 7. The pH optimum for pure soya-bean β-amylase is in the range 5–6. Pretreatment of the enzyme at pH3–5 decreases enzyme activity, whereas at pH6–9 it is not affected.     

Purification and composition of Ca2+/Mg2+ ATPase from rat heart plasma membrane

The Ca²⁺/Mg²⁺ ATPase, which is activated by millimolar concentrations of Ca²⁺ or Mg²⁺, was solubilized from rat heart plasma membrane by employing lysophosphatidylcholine, CHAPS, Nal, EDTA and Tris-HCI at pH 7.4. The enzyme was purified by sucrose density gradient, Affi-Gel Blue column and Sepharose 6B column chromatography. The purified enzyme was seen as a single peptide band in the sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of about 90,000. The apparent molecular weight of the holoenzyme as determined under non-dissociating conditions by gel filtration on Sepharose 6B column was about 180,000 indicating two subunits. The enzyme was insensitive to ouabain, verapamil, vanadate, oligomycin, N,N-dicyclohexylcarbodiimide and NaN₃, but was markedly inhibited by 20 µM gramacidin S and 50 µM trifluoperazine. Analysis of the purified Ca²⁺/Mg²⁺ ATPase revealed the presence of 17 amino acids where leucine, glutamic acid and aspartic acid were the major components and histidine, cysteine and methionine were the minor components. The purified enzyme was associated with 19.7 µmol phospholipid/mg protein which was 60 times higher than the phospholipid content in plasma membrane. The cholesterol content in the purified enzyme preparation was 0.75 µmol/mg protein and this represented an 8-fold enrichment over plasma membrane. The glycoprotein nature of the enzyme was evident from the positive periodic acid-Schiff staining of the purified Cau2+/Mg^ATPase in the sodium dodecyl sulfate polyacrylamide gel. The polysaccharide content of the enzyme was enriched 8-fold over plasma membrane; neurominidase treatment decreased the polysaccharide content. Concanavalin A prevented the ATP-dependent inactivation of the purified Ca²⁺/Mg²⁺ ATPase and was found to bind to the purified enzyme with a K_D of 576 nM and Bmax of 4.52 nmol/mg protein. The results indicate that Ca²⁺/Mg²⁺ ATPase is a glycoprotein and contains a large amount of lipids.     

Purification and properties of galactokinase from Tetrahymena thermophila

Galactokinase (EC 2.7.1.6; ATP: d-galactose-1-phosphototransferase) was purified 152-fold with an 11% yield from Tetrahymena thermophila maximally derepressed for enzyme synthesis in late stationary phase. The purification procedure utilized sequential acid precipitation, batch DEAE-Sephacel chromatography, differential ammonium sulfate precipitation and narrow range electrofocusing. The apparent molecular weight of the holoenzyme as determined by gel filtration on Sephadex G-200 is 50 000-55 000. The holoenzyme consists of two subunits of approx. 28 000 daltons each, as determined by SDS-polyacrylamide gel electrophoresis. The native enzyme appears to be a single species with an isoelectric point at pH 5.1 Optimal activity was obtained at pH 7.8 and 41°C, with no added monovalent salt. d-Galactose, 2-deoxygalactose and galactosamine all are suitable carbohydrate substrates for the stereospecific galactokinase; only substitution at the C-2 position of galactose retains enzyme recognition. The enzyme utilizes ATP, 2′-dATP and 3′-dATP as phosphate donors; ADP and adenosine-5′-[γ-thio]triphosphate are inhibitory. The K_m values for galactose and ATP were determined to be 0.60 mM and 0.15 mM, respectively. The enzyme requires a divalent cation for activity, with effectiveness being in the order: Mg²⁺ >Co²⁺ >Mn²⁺ >Fe²⁺. Galactokinases from all eucaryotic sources studied thus far seem to be very similar. Based upon the results reported here, the galactokinases from Tetrahymena and yeast appear to be most similar in their biophysical and biochemical properties.     

An enzyme mediating the conversion of zeatin to dihydrozeatin in phaseolus embryos

A reductase catalyzing the conversion of zeatin to dihydrozeatin was detected in soluble fractions of immature Phaseolus vulgaris embryos. The enzyme was partially purified by ammonium sulfate fractionation and affinity, gel filtration, and anion exchange chromatography. NADPH was the only cofactor required for enzyme activity, and the pH optimum was 7.5 to 8.0. The enzyme did not recognize compounds closely related to zeatin, such as ribosylzeatin, cls-zeatin, O-xylosylzeatin, N⁶-(Δ²-isopentenyl)adenine, or N⁶-(Δ²-isopentenyl)adenosine. No conversion of dihydrozeatin to zeatin by the enzyme was observed. Two forms of the reductase could be separated by either gel filtration or anion exchange high performance liquid chromatography. The high molecular weight isozyme (M_r 55,000 ± 5,000) eluted as the second peak from the anion exchange column, while the low molecular weight isozyme (M_r 25,000± 5000) was less negatively charged. The results suggest that side chain reduction occurs at the free base level. In addition, Phaseolus embryos are useful for the detection of zeatin-specific metabolic enzymes.     

Purification and characterization of a heat-stable alkaline protease from Bacillus stearothermophilus F1

A thermophilic Bacillus stearothermophilus F1 that produced an extremely thermostable alkaline protease was isolated from decomposed oil palm branches. The isolated protease was purified to homogeneity by heat treatment, ultrafiltration and gel filtration chromatography with a 128-fold increase in specific activity and 75% recovery. The protease, which is a serine-type enzyme, has a relative molecular mass of 33 500 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis but only 20 000 by gel-filtration chromatography. The enzyme was optimally active at pH 9.0 and was stable for 24 h at 70° C and in the pH range from 8.0 to 10.0. It was capable of hydrolysing many soluble and insoluble protein substrates but no esterase activity was detected. The enzyme activity was markedly inhibited by Co²⁺ and Hg²⁺, whereas Mg²⁺, Fe²⁺, Cu²⁺, Zn²⁺ and Sr²⁺ had little or no inhibitory effect. However, Mn²⁺ strongly activated the protease activity. The protease exhibited a high degree of thermostability [t _1/2 (85° C) = 4 h, (90° C) = 25 min]. The stability at higher temperatures (85° C and above) was shown to be dependent on the presence of Ca²⁺. Correspondence to: A. B. Salleh     

Purification and properties of carbonic anhydrase from Chlamydomonas reinhardii

Carbonic anhydrase (CA) was purified from the unicellular green alga Chlamydomonas reinhardii, and the purity of the preparation was established by gradient gel electrophoresis. The purified enzyme exhibited a MW of 165 000 and contained 6 atoms of Zn. The subunit MW, as determined by dodecyl sulfate electrophoresis, was 27 000. These results are consistent with a quarternary structure which is hexameric, each monomer containing 1 g atom of Zn. Like spinach CA, and in contrast to other oligomeric plant CAs, a sulfhydryl reducing agent is not needed to stabilize the enzyme. CO₂-hydrase activity was inhibited by both acetazolamide (I₅₀ = 7.8 × 10^?9M) and sulfanilamide (I₅₀ = 1.3 × 10^?5M), as well as by certain inorganic anions. The purified enzyme showed relatively weak esterase activity with p-nitrophenyl acetate but was an extremely effective esterase with 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone as the substrate. Both esterase activities could be completely inhibited by adding acetazolamide. In its gross structural characteristics, the C. reinhardii enzyme resembles the CAs from higher plants. However, in its esterase activity and the inhibition by sulfonamides it is markedly different from plant CAs and bears more resemblance to erythrocyte CAs.     

Purification and Characterization of Two Highly Thermophilic Alkaline Lipases from Thermosyntropha lipolytica

Two thermostable lipases were isolated and characterized from Thermosyntropha lipolytica DSM 11003, an anaerobic, thermophilic, alkali-tolerant bacterium which grows syntrophically with methanogens on lipids such as olive oil, utilizing only the liberated fatty acid moieties but not the glycerol. Lipases LipA and LipB were purified from culture supernatants to gel electrophoretic homogeneity by ammonium sulfate precipitation and hydrophobic interaction column chromatography. The apparent molecular masses of LipA and LipB determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 50 and 57 kDa, respectively. The temperature for maximal activity of LipA and LipB was around 96°C, which is, so far as is known, the highest temperature for maximal activity among lipases, and the pH optima for growth determined at 25°C (pH^25°C optima) were 9.4 and 9.6, respectively. LipA and LipB at 100°C and pH^25°C 8.0 retained 50% activity after 6 and 2 h of incubation, respectively. Both enzymes exhibited high activity with long-chain fatty acid glycerides, yielding maximum activity with trioleate (C_18:1) and, among the p-nitrophenyl esters, with p-nitrophenyl laurate. Hydrolysis of glycerol ester bonds occurred at positions 1 and 3. The activities of both lipases were totally inhibited by 10 mM phenylmethylsulfonyl fluoride and 10 mM EDTA. Metal analysis indicated that both LipA and LipB contain 1 Ca²⁺ and one Mn²⁺ ion per monomeric enzyme unit. The addition of 1 mM MnCl₂ to dialyzed enzyme preparations enhanced the activities at 96°C of both LipA and LipB by threefold and increased the durations of their thermal stability at 60°C and 75°C, respectively, by 4 h.     

Action of sulphite on plant malate dehydrogenase

SO₃^2? acts on NAD- and NADP-dependent malate dehydrogenase in several ways. Firstly, SO₃^2? favours the appearance of low MW species (65 000 and 39 000 daltons) in Sephadex gel chromatography. Secondly, the enzyme form which is obtained by gel chromatography with dithioerythritol plus nucleotide cofactor is changed in the presence of SO₃^2?. This is indicated by the appearance of a linear reaction (instead of curvilinear), and by the abolition of the biphasic sigmoidal kinetics on varying substrate and cofactor concentrations. SO₃^2? causes the loss of negative cooperativity at low substrate or cofactor concentrations. Thus the inhibition of initial velocity at high substrate or cofactor concentrations is even more marked than at lower ones. Thirdly, SO₃^2? strongly reduces the activity in substrate saturating conditions.     

An improved purification and further characterization of the messenger ribonucleic acid for the fatty acid synthetase from rat liver

High purity fatty acid synthetase mRNA has been prepared from rat liver. The translational purity of the mRNA preparation was at least 27% as judged by the percentage of the radioactivity incorporated into acid-insoluble material that was precipitated by anti-fatty acid synthetase antibody. The specific activity of the mRNA was 220-times greater than that reported previously from this laboratory [1]. The large increase in the specific activity was achieved by the repeated use of high resolution linear-log sucrose density gradient centrifugation and the removal of 28 S rRNA by Sepharose 4B chromatography, as well as by the optimization of the K⁺ concentration (160 mM) in the reticulocyte lysate translation system. The mRNA preparation showed a single major band on agarose gel electrophoresis under denaturing conditions, and the translational activity of the fatty acid synthetase mRNA on the gel was found to coincide with this band. The molecular weight of the fatty acid synthetase mRNA is 2.5·10⁶ Da. The mRNA directed the synthesis of fatty acid synthetase with a molecular weight indistinguishable from that of the authentic enzyme subunit (M_r = 240 000). The copurification of the translation product and authentic enzyme revealed that the fatty acid synthetase polypeptides synthesized in the reticulocyte lysate system are assembled in vitro into dimers, the native form of the enzyme.