Purification of the glutamine synthetase II isozyme of Drosophila melanogaster and structural and functional comparison of glutamine synthetases I and II

Glutamine synthetase II was purified from Drosophila melanogaster adults. It was completely separable from the isozyme glutamine synthetase I by means of DEAE chromatography. The complete enzyme has an apparent molecular weight of 360,000. After two-dimensional electrophoresis it gave a single molecular species with an apparent molecular weight of 42,000. Structural analysis of the two isozymes showed that they are different both in subunit molecular weight and in isoelectric point. Peptide maps of the purified subunits showed considerable dissimilarity. Glutamine synthetase II is more active than glutamine synthetase I in the transferase assay, while the opposite is true in the biosynthetic assay. The kinetic parameters were determined, showing again noteworthy differences between the two isozymes. We therefore conclude that two forms of glutamine synthetase are present in Drosophila, with different primary structures, different kinetic behavior, and the possibility of different functional properties.     

Purification and properties of glutamine synthetase from Saccharomyces cerevisiae

We have purified glutamine synthetase over 130-fold from Saccharomyces cerevisiae. The enzyme exhibits a Km for glutamate of 6.3 mM and a Km for ATP of 1.3 mM in the biosynthetic reaction, with a pH optimum from 6.1 to 7.0. Ten to twelve 43,000 molecular weight subunits comprise the active enzyme of 470,000 molecular weight. Rabbit antibodies prepared against the purified enzyme were used to show that induction of enzyme activity correlates with de novo synthesis of the enzyme subunit.     

Heterogeneity of Glutamine Synthetase Polypeptides in Phaseolus vulgaris L

Glutamine synthetases from roots, nodules, and leaves of Phaseolus vulgaris L. have been purified to homogeneity and their polypeptide composition determined.

The leaf enzyme is composed of six polypeptides. The cytosolic fraction contains two 43,000 dalton polypeptides and the chloroplastic enzyme is formed by four 45,000 dalton polypeptides. Root glutamine synthetase consists only of the same two polypeptides of 43,000 dalton that are present in the leaf enzyme. The nodule enzyme is formed by two polypeptides of 43,000 dalton, one is common to the leaf and root enzyme but the other is specific for N₂-fixing nodule tissue. The two glutamine synthetase forms of the nodule contain a different proportion of the 43,000 dalton polypeptides.

     

Anthranilate synthetase, an enzyme specified by the tryptophan operon of Escherichia coli: purification and characterization of component I

A procedure employed in the purification of anthranilate synthetase component I of Escherichia coli is described. The purified component appears homogeneous by starch gel electrophoresis and by sedimentation analysis. A molecular weight of 60,000 was estimated by gel filtration of Sephadex G-100. This value is consistent with the molecular weight estimated from the sedimentation and diffusion coefficients. Purified anthranilate synthetase component I cannot use glutamine as substrate and thus has no activity in the reaction of chorismate + l-glutamine --> anthranilate; however, it is active when ammonium sulfate is provided as amino donor. Sucrose density gradient analyses showed that ammonium sulfate does not affect the sedimentation velocity of component I. The ultraviolet absorption and fluorescence spectra of the purified component indicated that it contains tryptophan. Peptide pattern and extract complementation evidence suggested that the protein is a single polypeptide chain. Enzyme activity measurements indicated that wild-type E. coli produces equimolar amounts of at least four of the five polypeptides specified by the operon. Purified anthranilate synthetase component I is inhibited by l-tryptophan.     

Properties of the Bacillus licheniformis A5 glutamine synthetase purified from cells grown in the presence of ammonia or nitrate.

The glutamine synthetase from Bacillus licheniformis A5 was purified by using a combination of polyethylene glycol precipitation and chromatography on Bio-Gel A 1.5m. The resulting preparation was judged to be homogeneous by the criteria of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, equilibrium analytical ultracentrifugation, and electron microscopic analysis. The enzyme is a dodecamer with a molecular weight of approximately 616,000, and its subunit molecular weight is 51,000. Under optimal assay conditions (pH 6.6, 37 degrees C) apparent Km values for glutamate, ammonia, and manganese.adenosine 5'-triphosphate (1:1 ratio) were 3.6, 0.4, and 0.9 mM, respectively. Glutamine synthetase activity was inhibited approximately 50% by the addition of 5 mM glutamine, alanine, glycine, serine, alpha-ketoglutarate, carbamyl phosphate, adenosine 5'-diphosphate, or inosine 5'-triphosphate to the standard glutamine synthetase assay system, whereas 5 mM adenosine 5'-monophosphate or pyrophosphate caused approximately 90% inhibition of enzyme activity. Phosphorylribosyl pyrophosphate at 5 mM enhanced activity approximately 60%. We were unable to detect any physical or kinetic differences in the properties of the enzyme when it was purified from cells grown in the presence of ammonia or nitrate as sole nitrogen source. The data indicate that B. licheniformis A5 contains one species of glutamine synthetase whose catalytic activity is not regulated by a covalent modification system.     

Early steps in the path of nascent ribonucleic acid across the surface of ribonucleic acid polymerase, determined by photoaffinity labeling

The photoaffinity probes beta-(4-azidophenyl) adenosine 5'-diphosphate (N3PhppA) and beta-(4-azidophenyl) adenylyl-(3'--5')-uridine 5'-diphosphate (N3PhppApU) were used to determine the RNA polymerase subunit contacts made by the 5' ends of three nascent RNA chains. Ternary enzyme-poly[d(A-T)].oligonucleotide complexes were prepared in which the nascent oligonucleotide contained a photoaffinity label at the 5' end and a 32P radiolabel only at the 3' end. The length of the RNA was fixed at two, three, or four nucleotides. Photolysis of the ternary complexes was followed by dissociation, polyacrylamide gel electrophoresis, autoradiography, and scintillation counting. With a dinucleotide probe, the enzyme subunits labeled were beta' (71%) and sigma (21%). Photolysis of the ternary complex containing trinucleotide RNA also resulted in labeling of the beta' (64%) and sigma (35%) subunits. With a tetranucleotide, the beta' subunit was very heavily labeled (88%), and a small amount of labeling of the beta (7%) and sigma (4%) subunits was observed. The alpha subunit was not labeled with any of the probes. These results imply that a conformational change, possibly involving dissociation of the sigma subunit, occurs in the enzyme as the ribonucleotide is elongated from a tri- to a tetranucleotide.     

Purification and characterization of DNase VII, a 3' leads to 5'-directed exonuclease from human placenta

An exonuclease, DNase VII, has been purified 6000-fold from human placenta. The enzyme has an apparent molecular weight of 43,000, requires Mg2+ for activity, and has a pH optimum of 7.8. The enzyme hydrolyzes single-stranded and nicked duplex DNA at the same rate proceeding in a 3' leads to 5' direction liberating 5'-mononucleotides. It does not measurably hydrolyze polyribonucleotides.     

Purification and properties of glutamine synthetase from liver of Squalus acanthias

Ammonia assimilation for urea synthesis by liver mitochondria in marine elasmobranchs involves, initially, formation of glutamine which is subsequently utilized for mitochondrial carbamoyl phosphate synthesis [P. M. Anderson and C. A. Casey (1984) J. Biol. Chem. 259, 456-462]. The purpose of this study was to determine if the glutamine synthetase catalyzing this first step in urea synthesis has properties uniquely related to this function. Glutamine synthetase has been highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme has a molecular weight of approximately 400,000 in the presence of Mg2+, MgATP, and L-glutamate, but dissociates reversibly to a species with a molecular weight of approximately 200,000 in the absence of MgATP and L-glutamate. Association with the glutamine- and acetylglutamate-dependent carbamoyl phosphate synthetase, also located in the mitochondria, could not be demonstrated. The subunit molecular weight is approximately 46,000. The pH optimum of the biosynthesis reaction is 7.1-7.4. The purified enzyme is stabilized by MgATP and glutamate and by ethylene glycol, and is activated by 5-10% ethylene glycol. The apparent Km values for MgATP, L-glutamate, and ammonia (NH4+-NH3) are 0.7, 11.0, and 0.015 mM, respectively. Mg2+ in excess of that required to complex ATP as MgATP is required for maximal activity; Mn2+ cannot replace Mg2+. The enzyme is activated by low concentrations of chloride, bromide, or iodide; this effect appears to be related to decreases in the apparent Km for glutamate. The enzyme is inhibited by physiological concentrations of urea, but is not significantly affected by physiological concentrations of trimethylamine-N-oxide. Except for activation by halogen anions and the very low apparent Km for ammonia, this elasmobranch glutamine synthetase has properties similar to those reported for mammalian and avian glutamine synthetases. The very low apparent Km for ammonia may be specifically related to the unique role of this glutamine synthetase in mitochondrial assimilation of ammonia for urea synthesis.     

Regulation of the activity of the Bacillus licheniformis A5 glutamine synthetase

The regulation of glutamine synthetase activity by positive and negative effectors of enzyme activity singularly and in combinations was studied by using a homogeneous enzyme preparation from Bacillus licheniformis A5. Phosphorylribosyl pyrophosphate at concentrations greater than 2mM stimulated glutamine synthetase activity by approximately 70%. The concentration of phosphorylribosyl pyrophosphate required for half-maximal stimulation of enzyme activity was 0.4 mM. Results obtained from studies of fractional inhibition of glutamine synthetase activity were consistent with the presence of one allosteric site for glutamine binding (apparent I0.5, 2.2mM) per active enzyme unit at a glutamate concentration of 50 mM. At a glutamate concentration of 30 mM or less, the data were consistent with the enzyme containing two binding sites for glutamine (one of which was an allosteric site with an apparent I0.5 of 0.4 mM). Bases on an analysis of the response of glutamine synthetase activity to positive and negative effectors in vitro and to the intracellular concentration of these effectors in vivo, the primary modulators of glutamine synthetase activity in B. licheniformis A5 appear to be glutamine and alanine (apparent I0.5, 5.2mM).     

Glutamine synthetase from a cyanobacterium, Phormidium lapideum: purification, characterization, and comparison with other cyanobacterial enzymes

Glutamine synthetase has been purified to homogeneity from cell extracts of a non-N2-fixing filamentous cyanobacterium, Phormidium lapideum. The subunit molecular weight of the enzyme was determined as about 59,000 by sodium dodecyl sulfate gel electrophoresis. Electron micrographs of the Phormidium enzyme revealed a two-layered structure of regular hexagons (12 subunits per molecule), which markedly resembles the three-dimensional polypeptide backbone structure of the Salmonella typhimurium glutamine synthetase established by X-ray crystallography (Almassy, Janson, Hamlin, Xuong, & Eisenberg (1986) Nature 323, 304-309). The N-terminal amino acid sequence of the Phormidium enzyme shows very high similarity with that of the enzyme from an N2-fixing cyanobacterium, Anabaena 7120; 18 residues are common in 23 residues compared. Strong immunocross-reactions between the antibody against the purified Phormidium glutamine synthetase and other cyanobacterial enzymes except the Anacystis enzyme were observed. The apparent Michaelis constants for NH3, L-glutamate, and ATP were determined to be 0.29, 7.4, and 1.7 mM, respectively. Divalent metal ions such as Mg2+ and Mn2+ activated the enzyme in the biosynthetic reaction, whereas various amino acids and glutamate analogs strongly inhibited the enzyme.     

RNA polymerase from the fungus, Aspergillus nidulans. Large-scale purification of DNA-dependent RNA polymerase I (or A).

The DNA-dependent RNA polymerase I (or A) from the lower eukaryote Aspergillus nidulans has been purified on a large scale to apparent homogeneity by homogenizing the fungal hyphae in liquid nitrogen, extraction of the enzyme at high salt concentration, precipitation of RNA polymerase activity with polymin P (a polyethylene imine), elution of the RNA polymerase from the polymin P precipitate, ammonium sulphate precipitation, molecular sieving on Bio-Gel A-1.5m, binding to ion-exchangers and DNA-cellulose affinity chromatography. By this procedure 1.6 mg of RNA polymerase I can be purified over 2000-fold from 500 g wet weight of starting material with a yield of 30--35%. The isolated RNA polymerase I is stable for several months at -20 degrees C. The subunit compostion has been resolved by polyacrylamide gel electrophoresis on two-dimensional gels, using either non-denaturing of 8 M urea (pH 8.7) cylindrical gels in the first dimension and sodium dodecyl sulphate slab gels in the second dimension. The putative subunits have molecular weights of 190,000, 135,000, 63,000, 62,000, 43,000, 29,000, (28,000), 16,000 and probably 13,000 and 12,000. Two distinct forms of RNA polymerase I (Ia and Ib) have been resolved by DEAE-Sephadex A-25 chromatography showing ample differences in enzymatic properties and subunit pattern. Additional information is given on RNA polymerase II (or B) which appears to be highly insensitive to alpha-amanitin at concentrations up to 400 micrograms/ml.     

Regulation of a Ligand-Mediated Association-Dissociation System of Anthranilate Synthesis in Clostridium butyricum

The anthranilate synthetase of Clostridium butyricum is composed of two nonidentical subunits of unequal size. An enzyme complex consisting of both subunits is required for glutamine utilization in the formation of anthranilic acid. Formation of anthranilate will proceed in the presence of partially pure subunit I provided ammonia is available in place of glutamine. Partially pure subunit II neither catalyzes the formation of anthranilate nor possesses anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase activity. The enzyme complex is stabilized by high subunit concentrations and by the presence of glutamine. High KCl concentrations promote dissociation of the enzyme into its component subunits. The synthesis of subunits I and II is coordinately controlled with the synthesis of the enzymes mediating reactions 4 and 5 of the tryptophan pathway. When using gel filtration procedures, the molecular weights of the large (I) and small (II) subunits were estimated to be 127,000 and 15,000, respectively. Partially pure anthranilate synthetase subunits were obtained from two spontaneous mutants resistant to growth inhibition by 5-methyltryptophan. One mutant, strain mtr-8, possessed an anthranilate synthetase that was resistant to feedback inhibition by tryptophan and by three tryptophan analogues: 5-methyl-tryptophan, 4- and 5-fluorotryptophan. Reconstruction experiments carried out by using partially purified enzyme subunits obtained from wild-type, mutant mtr-8 and mutant mtr-4 cells indicate that resistance of the enzyme from mutant mtr-8 to feedback inhibition by tryptophan or its analogues was the result of an alteration in the large (I) subunit. Mutant mtr-8 incorporates [(14)C]tryptophan into cell protein at a rate comparable with wild-type cells. Mutant mtr-4 failed to incorporate significant amounts of [(14)C]tryptophan into cell protein. We conclude that strain mtr-4 is resistant to growth inhibition by 5-methyltryptophan because it fails to transport the analogue into the cell. Although mutant mtr-8 was isolated as a spontaneous mutant having two different properties (altered regulatory properties and an anthranilate synthetase with altered sensitivity to feedback inhibition), we have no direct evidence that this was the result of a single mutational event.     

Nucleotide sequence of yeast gene CP A1 encoding the small subunit of arginine-pathway carbamoyl-phosphate synthetase. Homology of the deduced amino acid sequence to other glutamine amidotransferases

A yeast DNA fragment carrying the gene CP A1 encoding the small subunit of the arginine pathway carbamoyl-phosphate synthetase has been sequenced. Only one continuous coding sequence on this fragment was long enough to account for the presumed molecular mass of CP A1 protein product. It codes for a polypeptide of 411 amino acids having a relative molecular mass, Mr, of 45 358 and showing extensive homology with the product of carA, the homologous Escherichia coli gene. CP A1 and carA products are glutamine amidotransferases which bind glutamine and transfer its amide group to the large subunits where it is used for the synthesis of carbamoyl-phosphate. A comparison of the amino acid sequences of CP A1 polypeptide with the glutamine amidotransferase domains of anthranilate and p-amino-benzoate synthetases from various sources has revealed the presence in each of these sequences of three highly conserved regions of 8, 11 and 6 amino acids respectively. The 11-residue oligopeptide contains a cysteine which is considered as the active-site residue involved in the binding of glutamine. The distances (number of amino acid residues) which separate these homology regions are accurately conserved in these various enzymes. These observations provide support for the hypothesis that these synthetases have arisen by the combination of a common ancestral glutamine amidotransferase subunit with distinct ammonia-dependent synthetases. Little homology was detected with the amide transfer domain of glutamine phosphoribosyldiphosphate amidotransferase which may be the result of a convergent evolutionary process. The flanking regions of gene CP A1 have been sequenced, 803 base pairs being determined on the 5' side and 382 on the 3' side. Several features of the 5'-upstream region of CP A1 potentially related to the control of its expression have been noticed including the presence of two copies of the consensus sequence d(T-G-A-C-T-C) previously identified in several genes subject to the general control of amino acid biosynthesis.     

Adenosine-3':5'-monophosphate-binding proteins from bovine kidney. Isolation by affinity chromatography and limited proteolysis of the regulatory subunit of protein kinase II.

Affinity chromatography on cyclic AMP columns allowed a two-step isolation of the cyclic-AMP-binding proteins from bovine kidney cytosol. An AMP-binding protein (apparent molecular weight approximately 60 000) and large amounts of a low affinity binding protein ('P35'; apparent subunit size approximately 35 000) were obtained in practically pure form besides the high affinity binding proteins of the R type. Among the R proteins the dimer R2 of the regulatory subunit of protein kinase II (apparent subunit size approximately 54 000) represented the bulk material. Small amounts of monomer, of higher aggregates, and of a protein 'P49' (subunit size approximately 49 000) presumably identical with the regulatory subunit of protein kinase I were also detected. The R protein fraction of kidney also contained a high affinity binding protein of smaller size (designated as R'; molecular weight approximately 37 000) which appeared to be derived from protein R2 of protein kinase II by limited proteolysis. At all stages of purification, R protein and its aggregates could be quantitatively transformed into R' protein (or a closely related polypeptide) by several proteases including the relatively unspecific proteinase K. The degradation product exhibited unchanged cyclic-AMP-binding capacities but had largely lost the ability to inhibit the catalytic subunit C of protein kinase, to be phosphorylated by C, and to form a dimer. Preliminary experiments indicate that protein R' may be a natural component of kidney tissue.     

A multifunctional protein possessing glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthetase activities in de novo purine biosynthesis

Three activities on the pathway of purine biosynthesis de novo in chicken liver, namely, glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthetase, have been found to reside on the same polypeptide chain. Three diverse purification schemes, utilizing three different affinity resins, give rise to the same protein since the final material has identical specific activities for all three enzymatic reactions and a molecular weight on sodium dodecyl sulfate gels of about 110 000. A single antibody preparation precipitates all three activities and binds to the multifunctional protein obtained by two methods in Western blots. Partial chymotryptic digestion of the purified protein gives rise to two fragments, one possessing glycinamide ribonucleotide synthetase activity and the other containing glycinamide ribonucleotide transformylase activity.     

Purification and the effect of peptide N-glycosidase F on lysosomal membrane-bound glucocerebrosidase from human cultured fibroblasts.

Glucocerebrosidase was purified from human cultured dermal fibroblasts more than 2200-fold to apparent homogeneity using high performance Alkyl-Superose HR 5/5 hydrophobic interaction and Bio-Sil TSK-250 gel permeation column chromatography. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis and protein staining of the catalytically active and concentrated enzyme fractions from the gel permeation columns revealed the presence of one band of Mr 64,000. The glucocerebrosidase preparation purified to homogeneity was digested with peptide N-glycosidase F that cleaves N-linked oligosaccharide structures from glycoproteins. The molecular weight of glucocerebrosidase after digestion with peptide N-glycosidase F was reduced to Mr 57,000, suggesting that the mature enzyme is a glycoprotein and that N-linked oligosaccharide constitutes a minimum of about 10% of the total molecular weight of the polypeptide. These findings are compatible with the hypothesis that glucocerebrosidase was initially synthesized as a precursor polypeptide which was subsequently glycosylated to become the mature enzyme.     

Phosphorylation of lens membranes with a cyclic AMP-dependent protein kinase purified from the bovine lens

We report the phosphorylation of lens membranes with a cAMP-dependent protein kinase isolated from bovine lenses. The holoenzyme was eluted from DEAE agarose at less than 100 mM NaCl and from gel filtration columns with a relative molecular weight of 180 000. The regulatory subunit was identified with the affinity label 8-azido-[32P]cAMP. Four focusing variants with relative molecular weights of 49 000 were seen on two-dimensional gels. The catalytic subunit was purified approx. 5000-fold and migrated at 42 000 Mr on SDS gels. Based on these observations, the enzyme is classified as a Type I cAMP-dependent protein kinase. Purified lens plasma membranes were incubated with the holoenzyme or its catalytic subunit in the presence of 32P-labeled ATP. Several membrane proteins, including the major lens membrane polypeptide, MP26, were shown to be substrates for the kinase in this reaction. MP26 appears to be the major component of intercellular junctions in the lens. Studies with protease treatments on labeled membranes appeared to localize the phosphorylation sites to the cytoplasmic side of the membrane.     

Cell-free translation of virion RNA from nondefective and transformation-defective Rous sarcoma viruses.

Nondefective and transformation-defective virion subunit RNAs from two strains of Rous sarcoma virus (RSV) were translated in cell-free systems derived from Krebs IIA ascites cells, wheat germ, and L-cells. In each case the predominant viral-specific product was a polypeptide of molecular weight 76,000 that is related to the internal viral group-specific antigens, as judged by immunoprecipitation with monospecific antisera and tryptic peptide fingerprinting. No difference could be detected between the translation products of 35S RNA from nondefective and transformation-defective RSV virions, nor of 35S RNA from different strains of RSV. The 76,000-molecular-weight polypeptide synthesized in response to 35S RNA in vitro was labeled with formyl-methionine from initiator tRNA. Models for viral protein synthesis are discussed in the light of these results, and arguments positioning the group-specific antigen gene at the 5' end of the 35S RNA are presented.     

A major polypeptide component of rat liver mitochondria: carbamyl phosphate synthetase.

One of the major components of rat liver mitochondria detected by gel electrophoresis in sodium dodecyl sulfate is a 165,000 molecular weight polypeptide that makes up 15 to 20% of the total mitochondrial protein. This component appears to be a single molecular species. Evidence is presented here for the identification of this protein with the polypeptide chain of a urea cycle enzyme, carbamoylphosphate synthetase I (EC 2.7.2.5). The 165,000 molecular weight polypeptide was solubilized from mitochondria with Triton X-100 and purified to 90% homogeneity by DEAE-cellulose chromatography. This component co-migrated with carbamyl phosphate synthetase activity when mitochondrial proteins were separated by gel filtration or sucrose gradient centifugation. The identification of the 165,000 molecular weight polypeptide with this activity was also supported by the presence or absence of this protein in a variety of rat tissue mitochondria, in liver and kidney mitochondria from various ureotelic and nonureotelic species, and in fetal rat liver mitochondria.