Isolation and crystallization of unadenylylated glutamine synthetase from Salmonella typhimurium

The enzyme glutamine synthetase (GS) has been isolated from a mutant strain of Salmonella typhimurium, constructed by Kustu, which lacks the enzymatic activity for adenylylation of glutamine synthetase. Thus the purified GS is uniformly unadenylylated, as confirmed by gel electrophoresis and enzyme assays. It crystallizes readily in many morphologies, at least six of which are distinct polymorphs. The most favorable crystal form for structural studies belongs to space group C2, with unit cell dimensions a = 235.5 A, b = 134.5 A, c = 200.1 A, beta = 102.8 degrees, and with one GS molecule per asymmetric unit. The crystals diffract to about 2.8 A resolution in rotation X-ray photographs and thus appear suitable for structural studies at moderate resolution. These crystals are isomorphous with crystalline GS from Escherichia coli in both adenylylated and unadenylylated states, suggesting that the enzymes from the two bacteria are similar molecules, and that adenylylation does not greatly affect the conformation of the molecule.     

Purification and properties of phosphorylase from baker's yeast

A rapid, reliable method for purification of phosphorylase, yielding 200-400 mg pure phosphorylase from 8 kg of pressed baker's yeast, is described. The enzyme is free of phosphorylase kinase activity but contains traces of phosphorylase phosphatase activity. Phosphorylase constitutes 0.5-0.8% of soluble protein in various strains of yeast assayed immunochemically. The subunit molecular weight (Mr) of yeast phosphorylase is around 100,000. The enzyme is composed of two subunits in various ratios, differing slightly in molecular weight and N-terminal sequence. Both are active. Only the enzyme species containing the larger subunit can form tetramers and higher oligomers. The activated enzyme is dimeric. Correlated with specific activity (1 to 110 U/mg), phosphorylase contained between less than 0.1 to 0.74 covalently bound phosphate per subunit. Inactive forms of phosphorylase could be activated by phosphorylase kinase and [gamma-32P]ATP with concomitant phosphorylation of a single threonine residue in the aminoterminal region of the large subunit. The small subunit was not labeled. The incorporated phosphate could be removed by yeast phosphorylase phosphatase, resulting in loss of activity of phosphorylase, which could be restored by ATP and phosphorylase kinase.     

Purification and crystallization of rat liver fatty acid synthetase

A purification procedure for rat liver fatty acid synthetase has been developed using polyethylene glycol. This procedure results in high yields of the enzyme which is essentially free of endogenous proteolytic nicking and also free of any contaminating proteases. The fatty acid synthetase obtained has a specific activity range of 1.8–2.1 measured at 25 °C and is stable at 4 °C for a few weeks and indefinitely when frozen. Approximately 1 mg of enzyme can be obtained per gram of induced rat liver. The enzyme is pure as determined by sodium dodecyl sulfate-gel electrophoresis, sedimentation velocity, and immunoelectrophoresis. The first crystallization of rat liver fatty acid synthetase is also reported.     

Purification and properties of pyruvate kinase from Mycobacterium smegmatis

Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Mycobacterium smegmatis has been purified to homogeneity through a seven-step procedure with a yield of 16% and specific activity of 220 units/mg protein. The purified enzyme had a molecular weight of 230,700 and was composed of four subunits with identical molecular weights of 57,540. Analysis of amino acid composition revealed a low content of aromatic amino acids. The enzyme exhibited sigmoidal kinetics of varying concentrations of phosphoenolpyruvate, the degree of cooperativity and S_0.5v value for phosphoenolpyruvate being strongly dependent on the pH of the reaction mixture. Among the nucleoside diphosphates acting as substrate for pyruvate kinase, ADP was the best phosphate acceptor, as judged by its lowest K_m value. The enzyme showed an absolute requirement for divalent cations (either Mg²⁺ or Mn²⁺), but monovalent cations were not necessary for activity. Other divalent cations inhibited the Mg²⁺-activated enzyme to varying degrees (Ni²⁺ > Zn²⁺ > Cu²⁺ > Ca²⁺ > Ba²⁺). The differences in the kinetic responses of the enzyme to Mg²⁺ and Mn²⁺ are discussed.     

No evidence of covalent modification of glutamine synthetase in the thermophilic phototropic bacterium Chloroflexus aurantiacus

Abstract Regulation of glutamine synthetase (GS) in the thermophilic green phototrophic bacterium, Chloroflexus aurantiacus , was studied. The enzyme was partially purified from cells grown photosynthetically in media with limiting (1 mM) or non-limiting (10 mM) NH⁺₄-concentrations. GS preparations from both cell types were indistinguishable in respect to pH-optimum of GS-transferase activity, sensitivity to feedback modifiers (AMP, L-alanine, glycine) and lack of Mg-inhibition of transferase activity. In contrast to results obtained with a GS preparation from the facultatively phototrophic bacterium, Rhodopseudomonas sphaeroides , the catalytic properties of Chloroflexus GS did not change during incubation with snake venom phosphodiesterase suggesting the absence of in vivo regulation of Chloroflexus GS by adenylylation/deadenylylation.     

Purification and properties of purine hydroxylase II from Aspergillus nidulans

Purine hydroxylase II from Aspergillus nidulans has been purified to near homogeneity. The enzyme has a pI of 5.7, a molecular weight of 300,000, and two subunits with molecular weight of 153,000 each. The enzyme contains 2 FAD, 2 molybdenum atoms, and 4 (2 Fe-2S) iron-sulfur centers per molecule and exhibits broad specificity for reducing and oxidizing substrates. Among the more notable characteristics are the ability to oxidize hypoxanthine and nicotinic acid but not xanthine and virtually complete inactivity with oxygen. Moreover, while the enzyme is inactivated by borate and methanol, it is very resistant to cyanide and arsenite and it not inactivated by allopurinol. At infinite concentrations of reducing and oxidizing substrates, the Km for hypoxanthine was 119 microM, for nicotinic acid was 136 microM, and for NAD+ was 525 microM.     

Purification, composition, and some properties of rat liver carbamyl phosphate synthetase (ammonia).

Hepatic microsomes prepared from vitamin E deficient and supplemented rats were analyzed for cytochrome P-450 content and drug metabolizing activity. Reduced levels of benzo[α]pyrene hydroxylase and ethylmorphine N-demethylase activities were observed in microsomes derived from rats fed a diet deficient in vitamin E compared to those of control rats. NADPH-mediated destruction of P-450, and pentobarbital and zoxazolamine sleeping times were similar in the two groups. Induction with 3-methylcholanthrene raised the levels of benzo[α]pyrene hydroxylase activity of both supplemented and deficient rats to the same absolute levels. No differences were noted in cytochrome P-450 or P-448 content between control and tocopherol deficient rats, nor did the activity of liver catalase differ between the two dietary groups. Thus, these studies did not demonstrate any impairment of heme protein synthesis in vitamin E deficient rats.     

Association-dissociation of mammalian brain glutamine synthetase: Effects of metal ions and other ligands

Glutamine synthetase from ovine brain has been found to exist in vivo and in vitro as a Mn₄E complex, where E is octameric enzyme [F. C. Wedler, R. B. Denman, and W. G. Roby (1982)Biochemistry24, 6389–6396]. Previously observed anomolous effects of added metal ions and protein concentration on the observed specific activity in vitro can now be explained in terms of association-dissociation of the native octamer. In the absence of glycerol, added to stabilize the enzyme for long-term storage, activity decreases sharply below 4 μg/ml (20 nm octamer) in assay mixtures due to dissociation of octamer to tetramer and thence to inactive monomer. No dimeric species were detectable under any conditions. The octameric species Mn₄EMn₄ could be activated further by Mn(II) to form a species Mn₄EMn₄Mn₈ that has a specific activity of ca. 900 U/mg in the transferase assay. Enzyme with one Mn(II)/subunit, Mn₄EMn₄, associated to octamers more extensively than Mn₄E. At the low concentrations of enzyme at which the tetramer predominates, addition of substrates alone or in pairs caused partial reassociation to octamers, the most effective combinations being ATP and glutamate, ADP and l-glutamine, or ATP and l-methionine sulfoximine. Analysis of the data by the methods of Kurganov or Thomes and co-workers indicate that the tetramer/octamer equilibrium has a K_d value of ca. 2.5 × 10^?6m, comparable to values calculated for other enzyme systems. The specific activities for octamer and monomer in the Mg(II)-dependent transferase assay were calculated to be 200 ± 20 and 0 U/mg, respectively. Direct determination of the specific activity of pure tetramer is hampered by its substrate-promoted reassociation to octamer under assay conditions. Tetramers, produced by 2 m urea and then immobilized on CNBr-activated Sepharose 4B, exhibited a specific activity that was 86% of that of the identically treated octamers. This indicates a specific activity of ca. 172 (±20) for tetramers in solution. Light-scattering experiments showed that, with 1.7–2.0 Mn(II) bound per subunit, the octameric enzyme octamers can associate further to an oligomeric species (Mn₄EMn₄Mn₈)_n, where $\text{n}\text{?}\text{? 5}$. This oligomerization also was promoted strongly by lanthanide ions. Mg(II), however, caused only the association of tetramer to octamer. Analysis of various stereochemical models for the interaction of subunit domains (assuming identical subunits) within tetramers, between tetramers in the octamers, and between octamers indicate that the data are most consistent with isologous, rather than heterologous, interactions to produce octamer. These analyses also predict that formation of oligomers from cubic octamers through weaker, Mn(II)-dependent interactions also are most likely to occur via isologous domains. The available electron micrographic evidence support these hypothetical models. Interactions within tetramers are stronger than those between tetramers, which are stronger than those between octamers.     

Acylation of plant acyl carrier proteins by acyl-acyl carrier protein synthetase from Escherichia coli

The acyl-acyl carrier protein synthetase from Escherichia coli has been examined for its ability to specifically acylate acyl carrier protein (ACP) from higher plants in order to develop an assay for plant ACP, and to prepare labeled acyl-ACP of plant origin. It was found that the E. coli enzyme was able to acylate ACP from spinach, soybean, avocado, corn, and several other plants. The acylation was very specific because, in crude extracts of spinach leaves where ACP represented approximately 0.1% of the total soluble protein, ACP was shown to be the only protein acylated. In contrast to other E. coli enzymes that display 2- to 10-fold lower rates with plant versus bacterial ACP, the kinetic constants (K_m and V_max) for acyl-ACP synthetase were found to be essentially identical for spinach and E. coli ACP when acylated with palmitic acid. Palmitic, myristic, lauric, stearic, and oleic acid could all be esterified to both spinach and E. coli ACP with similar specificity. Procedures are described that allow the assay of ACP in plant extracts at the nanogram level.     

Purification and properties of methylmalonyl coenzyme A mutase from human liver

Methylmalonyl coenzyme A (CoA) mutase has been purified to apparent homogeneity from human liver by a procedure involving column chromatography on DEAE-cellulose, Matrex-Gel Blue A, hydroxylapatite, and Sephadex G-150. The overall purification achieved is 500- to 600-fold, yield 3–5%. Electrophoresis of the native purified protein on nondenaturing polyacrylamide gels shows a single diffuse band coincident with the enzyme activity; dodecyl sulfate/polyacrylamide gels show a single protein band with an apparent molecular weight of 77,500. The native protein has a molecular weight of approximately 150,000 by Sephadex G-150 chromatography, suggesting that it is composed of two identical subunits. The activity of the purified enzyme is stimulated only slightly (10–20%) by the addition of its cofactor, adenosylcobalamin, indicating that the purified enzyme is largely saturated with coenzyme. The spectrum of the enzyme is consistent with the presence of about 1 mole of adenosylcobalamin per mole of subunit. The enzyme displays complex kinetics with respect to dl-methylmalonyl CoA; substrate inhibition by l-methylmalonyl CoA appears to occur. The enzyme activity is stimulated by polyvalent anions (PO₄^3? > SO₄^2? > Cl^?); monovalent cations are without effect, but high concentrations of divalent cations are inhibitory. The enzyme activity is insensitive to N-ethylmaleimide, is rapidly destroyed at temperatures > 50 °C, and shows a broad pH optimum around pH 7.5.     

Metal ion requirement by glutamine synthetase of Escherichia coli in catalysis of gamma-glutamyl transfer.

The requirement for metal ions by glutamine synthetase of Escherichia coli in catalyzing the γ-glutamyl transfer reaction has been investigated. In order of decreasing V at pH 7.0, Cd²⁺, Mn²⁺, Mg²⁺, Ca²⁺, Co²⁺, or Zn²⁺ will support the activity of the unadenylylated enzyme in the presence of ADP. With AMP substituted for ADP to satisfy the nucleotide requirement, only Mn²⁺ or Cd²⁺ will support the activity of the unadenylylated enzyme. Kinetic and equilibrium binding measurements show a 1:1 interaction between the nonconsumable substrate ADP and each enzyme subunit of the dodecamer. (To obtain this result, each enzyme subunit must be active in catalyzing γ-glutamyl transfer.) The stability constant of the unadenylylated subunit for ADP-Mn is 3.5 × 10⁵m^?1, or ～2.86 × 10⁷m^?1 under assay conditions, with arsenate, Mn²⁺, and glutamine being responsible for this large affinity increase. Saturation of two Mn²⁺ ion-binding sites per enzyme subunit is absolutely required for activity expression. While apparently not affecting the affinity of the first Mn²⁺ bound (K′ = 1.89 × 10⁶ M^?1), glutamine increases the stability constant for the second Mn²⁺ bound from 2 × 10⁴ to 5.9 × 10⁵m^?1. Reciprocally, increasing Mn²⁺ concentrations decreases the apparent K_m′ value for glutamine. Glutamine (by producing a net uptake of protons in binding to the enzyme) is responsible for changing the proton release from 3 to about 1 for 2 Mn²⁺ bound per enzyme subunit, with ～0.5 H⁺ displaced in both fast and slow processes. The uv spectral change induced by the binding of the first Mn²⁺ to each enzyme subunit remains unchanged by the presence of glutamine. However, glutamine reduces the half-time of the spectral change or slow proton release from ～30 to ～20 sec at 37 °C. Binding and kinetic results indicate a mechanism involving a random addition of Mn²⁺ to two subunit sites. Saturation of the high-affinity site with Mn²⁺ induces a conformational change to an active configuration, while activity expression depends also on the saturation of a second Mn²⁺ binding site (at or near the catalytic site). Once the first Mn²⁺ binding site of the subunit is saturated, an active enzyme complex can be formed either by the sequential binding of Mn²⁺ and ADP at the second site or by the binding of ADP-Mn complex directly to this site if the concentration of ADP-Mn is greater than 10^?8m in the assay. Some additional observations on the binding of Mg²⁺, Ba²⁺, Ca²⁺, and Zn²⁺ to the enzyme are presented.     

Purification and properties of L-alanine:4,5-dioxovalerate aminotransferase from Chlorella regularis

The enzyme L-alanine:4,5-dioxovalerate aminotransferase (EC 2.6.1.43), which catalyzes the synthesis of 5-aminolevulinic acid, was purified 161-fold from Chlorella regularis. The enzyme also showed L-alanine:glyoxylate aminotransferase activity (EC 2.6.1.44). The activity of glyoxylate aminotransferase was 56-fold greater than that of 4,5-dioxovalerate aminotransferase. The ratio of the two activities remained nearly constant during purification, and when the enzyme was subjected to a variety of treatments. 4,5-Dioxovalerate aminotransferase activity was competitively inhibited by glyoxylate, with a Ki value of 0.5 mM. Double-reciprocal plots of velocity versus 4,5-dioxovalerate with varying L-alanine concentrations indicate a ping-pong reaction mechanism. The apparent Km values for 4,5-dioxovalerate and L-alanine were 0.12 and 3.5 mM, respectively. The enzyme is an acidic protein having an isoelectric point of 4.8. The molecular weight of the enzyme was estimated to be 126,000, with two identical subunits. These results suggest that, in Chlorella, as in bovine liver mitochondria and Euglena, both 4,5-dioxovalerate and glyoxylate aminotransferase activities are associated with the same protein. From the activity ratio of transamination and catalytic properties, it is concluded that this enzyme does not function primarily as a part of the 5-carbon pathway to 5-aminolevulinic acid synthesis.     

Purification and some properties of sn-glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae

An NAD-dependent glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate: NAD⁺ oxidoreductase, EC 1.1.1.8) has been isolated and purified from Saccharomyces cerevisiae by affinity and exclusion chromatography. The enzyme was purified 5100-fold to a specific activity of 158. It has a molecular weight of approximately 31,000, a pH optimum between 6.8 and 7.2, and is sensitive to high-ionic-strength salt solutions. The enzyme is most strongly inhibited by phosphate and chloride ions.     

Method for isolation of aminoacyl-tRNA synthetases from plants: purification and some properties of methionyl,phenylalanyl and arginyl tRNA synthetases from yellow lupin seeds

A method for the simultaneous purification of methionyl-, phenylalanyl- and arginyl-tRNA synthetases from yellow lupin seeds (Lupinus luteus) is described. The method uses ammonium sulphate fractionation, and DEAE-cellulose and DEAE-Sephadex A-50 column chromatography. Molecular weight and kinetic parameters of the pure enzymes are reported.     

Purification and properties of Paracoccus denitrificans azurin

The isolation of an azurin type Cu protein from Paracoccus denitrificans (ATCC 13543) is described and some properties are reported. The purified protein has a molecular weight of 13,790 in a single polypeptide chain and contains one Cu atom per molecule. Its spectrum is typical of Type I, “blue” Cu proteins in showing an intense band at 595 nm; but it also shows a weaker absorption band at 448 nm. Its standard reduction potential has been measured to be +230 mV, which is the lowest potential observed to date for azurins isolated from bacterial sources. The purified protein shows fivefold greater electron transport activity with membrane fragments than with the soluble nitrite reductase of Paracoccus. This argues against the latter as the primary physiological oxidase system for azurin.     

S-adenosylhomocysteine hydrolase from hamster liver: Purification and kinetic properties

3-Deazaadenosine is both an inhibitor of and a substrate for S-adenosylhomocysteine hydrolase. Its administration to rats results in the accumulation of both S-adenosylhomocysteine and 3-deazaadenosylhomocysteine in the liver and other tissues. In hamsters, however, the administration of 3-deazaadenosine results only in the accumulation of 3-deazaadenosylhomocysteine (P. K. Chiang and G. L. Cantoni (1979) Biochem. Pharmacol. 28, 1897). In order to investigate the possible reasons for this difference, S-adenosylhomocysteine hydrolase from hamster liver has been purified to homogeneity and some of its kinetic and physical parameters have been determined. The molecular weight of the native enzyme is 200,000 with a subunit molecular weight of 48,000. The K_m's for adenosine and 3-deazaadenosine are about 1.0 μm, and the V_max's are also similar. The K_m for S-adenosylhomocysteine is 1.0 μm, or more than 10 times smaller than the K_m of the rat liver enzyme. This difference in K_m value may explain the differences in the response of rat and hamster liver to the administration of 3-deazaadenosine. S-Adenosylhomocysteine hydrolase from hamster liver exhibits an interesting kinetic property in that its activity can be affected bimodally by either adenosine or adenosine Anal.ogs. At very low concentrations of these analogs, the activity of S-adenosylhomocysteine hydrolase can be stimulated by 10–30%, and at higher concentrations these same analogs become competitive inhibitors.     

Purification and properties of myo-inositol-1-phosphate dehydrogenase from germinating mung bean seeds

A novel enzyme, myo-inositol-1-phosphate dehydrogenase, which catalyzes the conversion of myo-inositol 1-phosphate to ribulose 5-phosphate has been purified 84-fold from mung bean seedling employing several common techniques. The molecular weight of this purified enzyme has been recorded as 88,500 by Sephadex G-200 column chromatography, and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis one protein band containing three subunits of M_r 32,000 each was discernible. K_m values for NAD⁺ and myo-inositol 1-phosphate have been recorded as 2.8 × 10^?4 and 5.0 × 10^?4m, respectively. Production of NADH in myo-inositol-1-phosphate dehydrogenase reaction has also been evidenced by measurement of NADH fluorescence. Dehydrogenation and decarboxylation of myo-inositol 1-phosphate are mediated by the same enzyme. In fact, the rate of dehydrogenation corroborates with that of decarboxylation. Stoichiometry of this reaction suggests that for the production of 1 mol of ribulose 5-phosphate 2 mol of NAD⁺ are reduced.     

Purification and properties of soybean nodule xanthine dehydrogenase

Xanthine dehydrogenase (EC 1.2.1.37), an essential enzyme for ureide metabolism was purified from the cytosol fraction of soybean nodules. The purified xanthine dehydrogenase was shown to be homogeneous by electrophoresis and a pI of 4.7 was determined by isoelectric focusing. The enzyme had a molecular weight of 285,000 and two subunits of molecular weight 141,000 each. The holoenzyme contained 1.7 (±0.7) mol Mo and 8.1 (±2.0) mol Fe/mol enzyme and the enzyme also contained FMN and is thus a molybdoironflavoprotein. Soybean xanthine dehydrogenase is the second enzyme in plants demonstrated to contain Mo and the first xanthine-oxidizing enzyme reported to contain FMN, rather than FAD as the flavin cofactor.