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.     

Partial purification and properties of diacylglycerol kinase from rat liver cytosol

Diacylglycerol:ATP kinase(EC 2.3.1.-) was highly purified (more than 2000-fold) from rat liver cytosol. The specific activity of the obtained enzyme was about 1.5 μmol phosphatidate formed/mg of protein/min. The purification procedures included ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration on Sephadex G-200, and finally affinity chromatography on ATP-agarose. The activities of diacylglycerol:GTP kinase and monoacylglycerol:ATP kinase were copurified throughout the procedures, forming a single peak together with diacylglycerol: ATP kinase. Furthermore, these kinase activities showed a single peak when the highly purified enzyme was analyzed by a sucrose density gradient centrifugation and polyacrylamide gel electrophoresis. The three kinase activities are, therefore, most likely catalyzed by a single enzyme. The kinase showed an apparent molecular weight of 121,000 on gel filtration and sedimented at 5.1 S in a sucrose gradient centrifugation. The apparent K_m values were 170 μm for ATP, 540 μm for GTP, and 3.0 μm for diacylglycerol. A number of nucleoside triphosphates and diphosphates competitively inhibited the kinase, in particular the activity utilizing GTP. Among the nucleotides tested, ADP was the most potent inhibitor (the apparent K_i:50 μm for diacylglycerol:ATP kinase and 42 μm for diacylglycerol:GTP kinase). The kinase required Mg²⁺ and deoxycholate for its activity, and the optimal pH was 8.0–8.5. No dependence on added phospholipids was observed.     

Human GMP synthetase

• 1.1. The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes. placenta, and liver.
• 2.2. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration.
• 3.3. The K_m values were determined to be 4.9μM for XMP, 270μM for ATP. and 340 μM for glutamine.
• 4.4. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient.
• 5.5. The enzyme was specific for ATP as the energy source.
• 6.6. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound.
• 7.7. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

     

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.     

Isolation and characterization of a tryptic fragment containing the thioesterase segment of fatty acid synthetase from the uropygial gland of goose.

Trypsin treatment of purified fatty acid synthetase from the uropygial gland of goose released a 33,000 molecular weight peptide from the 270,000 molecular weight synthease. A combination of ammonium sulfate precipitation, Sephadex G-100 gel filtration, anion-exchange chromatography with QAE-Sephadex, and cation-exchange chromatography with cellulose phosphate gave rise to the first homogeneous preparation of the 33,000 molecular weight fragment containing fatty acyl-CoA thioesterase activity. Amino acid composition of this peptide was quite similar to that of the intact fatty acid synthetase except for a lower valine content; a partial specific volume of 0.734 was calculated for the thioesterase fragment. The pH optimum for the thioesterase was near 7.5 and the enzyme showed a high degree of preference for CoA esters of fatty acids with 16 or more carbon atoms. Palmitoyl-CoA inhibited the enzyme and therefore the rate of hydrolysis was not proportional to the amount of protein at low concentrations. Inclusion of bovine serum albumin in the reaction mixture prevented this inhibition. Disregarding the substrate inhibition, an apparent K_m of 5 × 10^?5m and a V of 340 nmol/min/mg were calculated. The thioesterase was inhibited by active serine-directed reagents such as phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate as well as by SH-directed reagents as p-chloromercuribenzoate and N-ethylmaleimide. The isolated thioesterase fragment generated antibodies in rabbits and the antithioesterase inhibited the enzymatic activity of fatty acid synthetase. The antithioesterase showed immunoprecipitant lines with fatty acid synthetase from the uropygial gland and the synthetase from the liver of goose. Anti-fatty acid synthetase prepared against the enzyme from the gland cross-reacted with the thioesterase segment. Even though the synthetase from the uropygial gland synthesizes multimethyl-branched fatty acids in vivo, the thioesterase segment of this synthetase appears to be quite similar to that isolated from the rat.     

Ethanolamine kinase from Culex pipiens fatigans

Ethanolamine kinase was partially purified from the larvae of Culex pipiens fatigans and its properties were studied. The enzyme was separated from choline kinase by acetic acid precipitation at pH 5.0 of a 13,000g supernatant of the larval homogenate. Alkaline phosphatase activity was removed from the enzyme preparation by the acid treatment followed by ammonium sulfate fractionation. The enzyme was localized in the cytosolic fraction and had a requirement for Mg²⁺ as a cofactor. The K_m values for ethanolamine and ATP were 4 × 10^?4 and 1.54 × 10^?4m, respectively. The affinity of the enzyme for nucleotide triphosphates was in the order, ATP > ITP > GTP while UTP and CTP were poorly utilized. p-Chloromercuribenzoate and N-ethylmaleimide inhibited the enzyme activity and reduced glutathione protected the enzyme from their inhibition. Choline and serine had no effect on the enzyme activity. The enzyme had a molecular weight of 44, 000 daltons as determined by gel filtration chromatography. Eggs contained the highest specific activity of the enzyme while adult insects had the highest total enzyme activity.     

The purification and properties of a protein kinase and the partial purification of a phosphoprotein phosphatase that inactivate and activate acetyl-CoA carboxylase

the occurrence of a soluble fraction from rat liver that inactivates acetyl-CoA carboxylase was previously reported by this laboratory (1). The purification of this fraction is now reported, and we show that it behaves as a cAMP-independent kinase that inactivates acetyl-CoA carboxylase by phosphorylation. The kinase has a molecular weight of 160,000 and it requires ATP and Mg²⁺ for activity. A partial purification from rat liver cytosol of a Mg²⁺-requiring phosphoprotein phosphatase of high molecular weight (greater than 200,000) which dephosphorylates phosphorylated acetyl-CoA carboxylase with the regeneration of enzyme activity is also reported. The kinase, phosphatase, and acetyl-CoA carboxylase are separable from each other by a combination of ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration.     

Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells.

Purine nucleoside phosphorylase (PNP) was purified from rat hepatoma cells and normal liver tissue utilizing the techniques of ammonium sulfate fractionation, heat treatment, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. Homogeneity was established by disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Purified rat hepatoma and liver PNPs appeared to be identical with respect to subunit and native molecular weight, substrate specificity, heat stability, kinetics and antigenic identity. A native molecular weight of 84,000 was determined by gel filtration. A subunit molecular weight of 29,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point was observed at pH 5.8, and the pH optimum was 7.5. Inosine, guanosine, xanthosine, and 6-mercaptopurine riboside were substrates for the enzymes. The apparent K_m for both inosine and guanosine was about 1.0 × 10^?4m and for phosphate was 4.2 × 10^?4m. Hepatoma and liver PNP showed complete cross-reactivity using antiserum prepared against the liver enzyme.     

Thermostable succinyl-Coenzyme A synthetase from Nitrosomonas europaea ATCC 25978: Purification and properties

The ammonia-oxidizing chemoautotrophic bacterium Nitrosomonas europaea possesses prominant succinate-reducing activity of succinyl-Coenzyme A synthetase (SCS, EC 6.2.1.5). SCS was purified as an electrophoretically homogeneous protein from Nitrosomonas europaea strain ATCC 25978 about 275-fold, with a 3.9% activity yield. The molecular mass of the native enzyme was estimated to be about 130 kDa by gel filtration, whereas SDS-PAGE gave two protein bands with M_r values of 29 (α) and 36 kDa (β). The isoelectric point of the enzyme was 5.3. The apparent K_m values of the enzyme for ATP, succinate and CoA were 0.4 mM, 5 mM and 0.1 mM, respectively. The pH and temperature optima of the SCS were about 5.0 and 55°C, respectively. The SCS was stable in the pH range of 8.0–10.0 and up to 70°C. The enzyme was thermostable; 50% of the enzyme activity was retained at 90–100°C for 10 min. The SCS was activated by Mg²⁺ at 1.0–100 mM, but inhibited by Cu²⁺ (0.1 mM) and SDS (1.0 mM). The enzyme utilized ATP as the preferred substrate.     

Isolation and purification of calcium and magnesium dependent endonuclease from rat liver nuclei

An endonuclease associated with rat liver chromatin was extracted with 0.6 M NaCl and purified by ammonium sulfate fractionation and Sephadex G-100 gel filtration. The enzyme produces single strand scissions on native DNA at neutral pH in the presence of 1 mM CaCl₂ and 5 mM MgCl₂. Alkali-denatured DNA was not nicked by the enzyme. Omission of Ca²⁺ reduced the enzyme activity to about one seventh. Without Ca²⁺, however, Mn²⁺ was more effective than Mg²⁺. The molecular weight of the enzyme is about 27,000.     

Purification and characterization of acetate kinase from veillonella alcalescens ATCC 17748.

Acetate kinase was isolated in highly purified form from Veillonella alcalescens through a combination of ammonium sulfate fractionation, DEAE-Sephadex column chromatography, and gel filtration. It had a specific activity about 60-fold that of crude extracts. Purity of the enzyme preparation was estimated to be 90% as judged by polyacrylamide gel electrophoresis. The molecular weights of the enzyme as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by gel filtration were 43,000 and 66,000, respectively. Succinate was unnecessary for the activity of this enzyme. This result is markedly different from that reported previously (Bowman, C. M. et al., 1976, J. Biol. Chem. 251, 3117–3121). This may, however, be due to the difference in bacterial strains used. The enzyme reaction with propionate was equal to about two-thirds that of its reaction with acetate. Apparent K_m values for ATP, acetate, ADP, and acetylphosphate were about 2, 30,0.3, and 1.2 mm, respectively. Phosphate donors, ATP, and acetylphosphate exhibited cooperativity while phosphate acceptors, ADP, acetate, and propionate did not. The enzyme had a broad pH optimum from 7.2 to 10, and required magnesium ions, whose optimal molar ratio to ATP was 1:1. The activity was inhibited by several SH-inhibitors, but not stimulated by free SH groups.     

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.     

Purification and properties of isocitrate lyase from Candida brassicae E-17

Isocitrate lyase (EC 4.1.3.1) was purified from acetate-grown cells of Candida brassicae E-17, by ammonium sulfate fractionation and DEAE-cellulose and Sephadex G-200 gel filtration column chromatographies. The purified enzyme was electrophoretically homogeneous. The molecular weight of this enzyme was 290,000 by gel filtration, and it was composed of four identical subunits whose molecular weights were 71,000 each. The pH and temperature optima were 6.8 and 37°C, respectively. The enzyme was stable from pH 6.0 to 7.0. The enzyme was activated by Mg²⁺ and the maximum activity was obtained with a concentration of 8 mM Mg²⁺. The enzyme was also activated by Mn²⁺ and Ba²⁺. The activity of this enzyme was stimulated by reducing agents. The K_m values for dl-isocitrate were 1.5 mM in sodium phosphate buffer and 0.62 mM in imidazole-HCl buffer.     

Purification and Characterization of Phosphoribulokinase from Immature Pods of Brassica

Phosphoribulokinase (ATP:D — ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19; PRuK) from immature pods of Brassica was purified to apparent homogeneity with about 31% recovery using ammonium sulphate fractionation, gel filtration through Sepharose CL-6B and ion exchange chromatography on DEAE-Sephadex A-50. The purified enzyme, having molecular mass of about 180 kD, was heterotetramer with subunit molecular mass of 48, 47, 41 and 33 kD. The enzyme had an absolute requirement for a divalent cation Mg²⁺ and a monovalent cation K⁺for optimal activity. At optimum pH of 8.0–8.4, the enzyme showed typical hyperbolic response for both the substrates with Km values of 333 μM and 100 μM, respectively for Ru5P and ATP. The enzyme was inhibited by RU-1, 5-P₂, 6-phosphogluconate and AMP, and activatded by glu-1-P, glu-6-P and Pl. RU-1, 5-P₂ and 6-phosphogluconate inhibited the enzyme competitively with respect to Ru5P and non-competitively with respect to ATP. It appears that the activity of the Brassica pod enzyme besides being controlled at the level of metabolites, is regulated by light and energy status of the cell.     

Purification and properties of glutamine synthetase from the plant cytosol fraction of lupin nodules

Glutamine synthetase from the plant cytosol fraction of lupin nodules was purified 89-fold to apparent homogeneity. The enzyme molecule is composed of eight subunits of M_r 44,700 ± 10%. Kinetic analysis indicates that the reaction mechanism is sequential and there is some evidence that Mg-ATP is the first substrate to bind to the enzyme. Michaelis constants for each substrate using the ammonium-dependent biosynthetic reaction are as follows: ATP, 0.24 mm; l-glutamate, 4.0–4.2 mm; ammonium, 0.16 mm. Using an hydroxamate-forming biosynthetic reaction the K_m ATP is 1.1 mm but the K_m for l-glutamate is not altered. The effect of pH on the K_m for ammonium indicates that NH₃ rather than NH₄⁺ may be the true substrate. At 10 mm Mg²⁺, the pH optimum of the enzyme is between 7.5 and 8, but increasing Mg²⁺ concentrations produce progressively more acidic optima while lower Mg²⁺ concentrations raise the pH optimum. The rate-response curve for Mg²⁺ is sigmoidal becoming bell-shaped in alkaline conditions. The enzyme is inhibited by l-Asp (K_i, 1.4 mm) and less markedly by l-Gln and l-Asn. Inhibition by ADP and AMP is strong, both nucleotides exhibiting K_i values around 0.3 mM. Investigations of the probable physiological conditions within the nodule plant cytosol indicate that in situ glutamine synthetase has an activity greater than that required to support the efflux of amino acid nitrogen from the nodule. A possible role for glutamine synthetase in the control of nodule ammonium assimilation is suggested.     

Degradation of RNA in rat reticulocytes. II. Relationship between RNase and its corresponding inhibitor in rat reticulocyte

A latent RNase was partially purified by ammonium sulfate fractionation and Sephadex G-75 gel filtration from rat reticulocyte lysate. By mixing free RNase and its inhibitor in vitro, the latent RNase was shown to be a complex of each component. The latent RNase was activated by SH reagents, but no dissociation of the complex was observed. It was also activated by 6 m urea and 0.1 m H₂SO₄, and in these cases the enzyme was released as judged by gel filtration. From the results of this and precious paper, a possible mechanism of the regulation of RNase activity in rat reticulocyte is discussed.     

Purification of a cytosolic enzyme from human liver with phospholipid hydroperoxide glutathione peroxidase activity

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein which inhibits peroxidation ofmicrosomes. The human enzyme, which may play an important role in protecting the cell from oxidative damage, has not been purified or characterized. PHGPx was isolated from human liver using ammonium sulphate fractionation, affinity chromatography on bromosulphophthalein-glutathione-agarose, gel filtration on Sephadex G-50, anion exchange chromatography on Mono Q resin and high resolution gel filtration on Superdex 75. The protein was purified about 112,000-fold, and 12 μg, was obtained from 140 g of human liver with a 9% yield. PHGPx was active on hydrogen peroxide, cumene hydroperoxide, linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide. The molecular weight, as estimated from non-denaturing gel filtration, was 16,100. The turnover number (37°C, pH 7.6) on (β-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl)-γ-palmitoyl)-l-α-phosphatidylcholine was 91 mol mo⁻¹ s⁻¹. As reported for pig PHGPx, activity of the enzyme from human liver on cumene hydroperoxide and on linoleic acid hydroperoxide was inhibited by deoxycholate. In the presence of glutathione, the enzyme was a potent inhibitor of ascorbate/Fe induced lipid peroxidation in microsomes derived from human B lymphoblastic AHH-1 TK ± CHol cells but not from human liver microsomes. Human cell line microsomes contained no detectable PHGPx activity. However, microsomes prepared from human liver contained 0.009 U/mg of endogenous PHGPx activity, which is 4–5 times the activity required for maximum inhibition of lipid peroxidation when pure PHGPx was added back to human lymphoblastic cell microsomes. PHGPx from human liver exhibits similar properties to previously described enzymes with PHGPx activity isolated from pig and rat tissues, but does not inhibit peroxidation of human liver microsomes owing to a high level of PHGPx activity already present in these microsomes.     

Alkaline phosphatase from the excretory system of the grasshopper,Poekilocerus bufonius

Alkaline phosphatase from the excretory system of the grasshopper, Poekilocerus bufonius was purified with ammonium sulphate fractionation and chromatography on Bio-Gel A-0.5 m. The specific activity of the enzyme is 152 units/mg of protein. The enzyme is a tetramer and the M_r value of the subunit is 72,000 ± 2500 as shown by gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 9.6 and an apparent K_m value of 0.28 × 10⁻³ M. The activity of the enzyme reached a maximum at 75°C and the enzyme showed stability at 65°C. The enzyme was inhibited by Ca²⁺, Na⁺ and Fe³⁺ and was stimulated by Zn²⁺, Mn²⁺ and Mg²⁺.     

Complete purification and general characterization of FAD synthetase from rat liver

Flavin adenine dinucleotide synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2) was purified about 10,000-fold from the high-speed supernatant of rat liver by a sequence of ammonium sulfate fractionation and column chromatographies on DEAE-Sephadex (A-50), chromatofocusing, FMN-agarose affinity, and Sephadex G-200. The specific activity of the purified enzyme was 133 units (nanomoles of FAD formed per min at 37 degrees C)/mg of protein. This preparation was free from contaminating FAD pyrophosphatase. The apparent molecular weight was estimated to be 97,000 by gel filtration on Sephadex G-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent subunit molecular weight of 53,000. Hence, the enzyme is a dimer of approximately 100,000. The enzyme was found most active at pH 7.1, requires Mg2+, and is essentially irreversible in the direction of FAD formation. Kinetic analysis gave Km values of 9.6 microM for FMN and 53 microM for ATP.     

S-Adenosylmethionine synthetase in bloodstream Trypanosoma brucei

S-adenosylmethionine synthetase was studied from bloodstream forms of Trypanosoma brucei brucei, the agent of African sleeping sickness. Two isoforms of the enzyme were evident from Eadie Hofstee and Hanes-Woolf plots of varying ATP or methionine concentrations. In the range 10–250 μM the K_m for methionine was 20 μM, and this changed to 200 μM for the range 0.5–5.0 mM. In the range 10–250 μM the K_m for ATP was 53 μM, and this changed to 1.75 mM for the range 0.5–5.0 mM. The trypanosome enzyme had a molecular weight of 145 kDa determined by agarose gel filtration. Methionine analogs including selenomethionine, L-2-amino-4-methoxy-cis but-3-enoic acid and ethionine acted as competitive inhibitors of methionine and as weak substrates when tested in the absence of methionine with [¹⁴C]ATP. The enzyme was not inducible in procyclic trypomastigotes in vitro, and the enzyme half-life was > 6 h. T. b. brucei AdoMet synthetase was inhibited by AdoMet (K_i 240 μM). The relative insensitivity of the trypanosome enzyme to control by product inhibition indicates it is markedly different from mammalian isoforms of the enzyme which are highly sensitive to AdoMet. Since trypanosomes treated with the ornithine decarboxylase antagonist DL-α-difluoromethylornithine accumulate AdoMet and dcAdoMet (final concentration ≈ 5 mM), this enzyme may be the critical drug target linking inhibition of polyamine synthesis to disruption of AdoMet metabolism.