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.     

Structural aspects of the soluble NAD-dependent hydrogenase isolated from Alcaligenes eutrophus H16 and from Nocardia opaca 1b

The soluble NAD-dependent hydrogenase (hydrogen-NAD oxidoreductase, EC 1.12.1.2), consisting of four non-identical subunits, was isolated from Alcaligenes eutrophus H16 and from Nocardia opaca 1b and analyzed by a HPLC gel permeation technique and electron microscopy. The tetrameric enzyme particles from both origins, as determined from negatively stained electron microscopic samples, were found to be elongated and very similar in shape and size. The A. eutrophus enzyme was measured in more detail. It exhibited dimensions of 12.7 nm by 5.5 nm (axial ratio 2.3:1). Dissociation into smaller particles and unspecific aggregation combined with partial inactivation were observed in the presence of the inhibitor NADH. Kept in buffer without added nickel, the enzyme was partially dissociated. Reassociation of tetramers without restored enzyme activity was achieved by addition of 0.5 mM NiCl₂. A working model for the structural organization of the tetrameric enzyme particle is presented.     

Bimodal agonism in heteromeric cyclic nucleotide-gated channels

Direct binding of cGMP or cAMP to tetrameric cyclic nucleotide-gated (CNG) channels will normally promote the open (conductive) conformation. However, the catfish CNGA2 subtype exhibits bimodal agonism, whereby open probability (P_o) increases with initial cGMP binding events ("pro" action) but decreases with subsequent cGMP binding events ("con" action) that occur at concentrations above 3 mM. We constructed, and heterologously expressed, chimeric CNG channel subunits with sequence substitutions in the binding domain (BD), and tested their activation using patch-clamp of cell-free membranes. A normal subunit with the rat CNGA4 BD (with only pro action) could be converted into a bimodal subunit (both pro and con action) by replacing the N-terminal portion of the BD with catfish CNGA2 sequence. We then fused two bimodal and two normal subunits in tandem tetramers, to form heteromeric CNG channels with bimodal pseudo-subunits either adjacent (cis) or diagonally opposite (trans). The cis tetramer showed con action, with a mean ratio of steady-state conductances g_(30mMcGMP) / g_(3mMcGMP) = 0.87, demonstrating bimodal agonism in a heteromeric CNG channel for the first time. In contrast, trans tetramers showed normal cGMP agonism up to 30 mM cGMP with mean g_(30mMcGMP) / g_(3mMcGMP) = 1.02, although a minority of oocytes (4 of 15) expressed anomalous channel populations with con action. Rearranging subunits in a heteromer thus influences a channel's P_o at high cGMP concentration. The sensitivity of con action to neighbouring subunits implies a cooperative mechanism.     

Characterization of a tetrameric G4 form of acetylcholinesterase from bovine brain: a comparison with the dimeric G2 form of the electric organ

Globular forms (G forms) of acetylcholinesterase (AChE) are formed by monomers, dimers and tetramers of the catalytic subunits (G₁, G₂ and G₄). In this work the hydrophobic G₂ and G₄ AChE forms were purified to homogeneity from Discopyge electric organ and bovine caudate nucleus and studied from different points of view, including: velocity sedimentation, affinity to lectins and SDS-polyacrylamide gel electrophoresis under reducing and non-reducing conditions. The polypeptide composition of Discopyge electric organ G₂ is similar to Torpedo, however the pattern of the brain G₄ AChE is much complex. Under non-reducing conditions the catalytic subunit possesses a molecular weight of 65 kDa, however this value increases to 68 kDa after reduction, suggesting that intrachain-disulfide bonds are important in the folding of the catalytic subunits of the AChE. Also it was found that after mild proteolysis; the (¹²⁵I)-TID-20 kDa fragment decreased its molecular weight to approximately 10 kDa with little loss of AChE activity. Finally, we suggest a model for the organization of the different domains of the hydrophobic anchor fragment of the G₄ form.     

Interrelationships among human aldo-keto reductase: immunochemical, kinetic and structural properties

We have propsed earlier a three gene loci model to explain the expression of the aldo-keto reductases in human tissues. According to this model, aldose reductase is a monomer of α subunits, aldehyde reductase I is a dimer of α, β subunits, and aldehyde reductase II is a monomer of δ subunits. Using immunoaffinity methods, we have isolated the subunits of aldehyde reductase I (α and β) and characterized them by immunocompetition studies. It is observed that the two subunits of aldehyde reductase I are weakly held together in the holoenzyme and can be dissociated under high ionic conditions. Aldose reductase (α subunits) was generated from human placenta and liver aldehyde reductase I by ammonium sulfate (80% saturation). The kinetic, structural and immunological properties of the generated aldose reductase are similar to the aldose reductase obtained from the human erythrocytes and bovine lens. The main characteristic of the generated enzyme is the requirement of Li₂SO₄(0.4 M) for the expression of maximum enzyme activity, and its K_m for glucose is less than 50 mM, whereas the parent enzyme, aldehyde reductase I, is completely inhibited by 0.4 M Li₂SO₄ and its K_m for glucose is more than 200 mM. The β subunits of aldehyde reductase I did not have enzyme activity but cross-reacted with anti-aldehyde reductase I antiserum. The β subunits hybridized with the α subunits of placenta aldehyde I, and aldose reductase purified from human brain and bovine lens. The hybridized enzyme had the characteristics properties of placenta aldehyde reductase I.     

A simple method of purification of a soluble oligomycin-insensitive mitochondrial ATPase.

A simple method for the purification of the soluble oligomycin-insensitive mitochondrial ATPase from heart is described. It consists of adsorption of the of the enzyme to Sepharose hexylammonium followed by elution with KCl and a precipitation step with (NH₄)₂SO₄. In sodium dodec'yl sulfate gels, the enzyme shows the A, B, C, D, and E subunits; however, the D and E subunits appear only when the gels are loaded with a high concentration of protein. The K_m for Mg-ATP is approximately 0.6 mm and is inhibited by ADP.     

On site heterogeneity in sturgeon muscle GPDH: a kinetic approach

The kinetics and stoichiometry of the reaction of sturgeon muscle glyceraldchyde-3-PO₄-dehydrogenase (GPDH) with the disulfide interchange reagent bis(2,2' dithio-bis(5-nitrobenzoate) (DTNB) has been studied in detail. The native enzyme, a tetramer of covalently identical subunits, reacts relatively rapidly with precisely four equivalents of reagent, although there are three cysteine residues per subunit (12 per tetratner). Reaction of these four cystcines leads to total catalytic inactivation; the extent of inactivation is proportional to the fractional reaction. The rate of reaction is dependent on the extent of bound NAD: reactivity being very much greater at unliganded sites. The reaction with apo-enzyme is fastest, bimolecular and monophasic. Over a wide range of NAD concentration, however, the reaction of enzyme with a large molar excess of reagent is precisely biphasic, and each individual kinetic experiment can be analytically described by two pseudo first-order (NAD concentration-dependent) rate constants and two unequal NAD concentration-insensitive amplitudes. The biphasicity in rate is quantitatively explainable on the basis of a C₂ symmetry for the tetrameric subunits with a tighter binding of NAD at two of the four sites, if high reactivity is exclusively dependent on the absence of bound NAD. The inequality in the two amplitudes, however, requires either a more complex or a more dynamic model. Arguments are presented for the appropriateness of a C₂ symmetry model in which intramolecular transconformational isomerization of tight and loose NAD binding sites is possible. The equilibrium constant for the isomerization is estimable from the macroscopic specific rates and amplitudes. This “flip-over” C₂ symmetry model is apropos to all situations of negative cooperativity in ligand binding to tetramers, as is discussed.     

Properties of water-insoluble matrix-bound lactate dehydrogenase.

Lactate dehydrogenase enzyme was immobilized by binding to a cyanogen bromideactivated Sepharose 4B-200 in 0.1 m phosphate buffer, pH 8.5. The immobilized enzyme was found to have lower K_m values for its substrates. K_m values for pyruvate and lactate were 8 × 10 ^?5m and 4 × 10^?3m, respectively, an order of magnitude less than the value for the native (free) enzyme. Chicken heart (H₄) lactate dehydrogenase was found to lose nearly all its substrate inhibition characteristics as a result of immobilization. The covalently bound muscle-type subunits of lactate dehydrogenase showed more favorable interaction with the muscle type than with the heart type subunits. An increase in thermal and acid stability of the dogfish muscle (M₄) lactate dehydrogenase as well as a decrease in the percentage of inhibition of enzyme activity by rabbit antisera and in the complement fixation was observed as a result of immobilization. The changes in the properties of the enzyme as a result of immobilization may be attributable to hindrance produced by the insoluble matrix as well as conformational changes in the enzyme molecules.     

Ribosome crystallization in chicken embryos. I. Isolation, characterization, and in vitro activity of ribosome tetramers

Isolated tetrameric particles (166S) derived from the crystalline lattices known to appear in hypothermic chicken embryos consist of mature 80S ribosomes which contain all species of ribosomal RNA and a complete set of ribosomal proteins. Ribosome tetramers are not a special type of polysomes since in solutions of high ionic strengths (500 mM KCl and 50 nM triethanolamine-HCl buffer) containing 5 mM MgCl₂ they dissociate into 40S and 60S ribosomal subunits, without the need of puromycin, and at a concentration of Mg⁺⁺ higher than 3 mM they are not disassembled by mild RNase treatment. Tetramers spontaneously disassemble into 80S monomers when the Mg⁺⁺ concentration is lowered to 1 mM at relatively low ionic strength. Tetramers failed to couple in vitro puromycin-³H into an acid-insoluble product, indicating the lack of nascent polypeptide chains. Although tetramers have no endogenous messenger RNA activity, they can be programmed in vitro with polyuridylic acid (poly U) to synthesize polyphenylalanine. All ribosomes within a tetramer can accept poly U, without the need of disassembly of the tetramers into monomers or subunits.     

Subunit interactions in pig-kidney fructose-1,6-bisphosphatase: Binding of substrate induces a second class of site with lowered affinity and catalytic activity

Background

Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P₂ and by high concentrations of its substrate Fru-1,6-P₂. The mechanism that produces substrate inhibition continues to be obscure.

Methods

Four types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P₂ and Fru-1,6-P₂ on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244.

Results

The kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P₂ induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P₂, competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a “stapler” that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition.

Conclusions

Binding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits.

General significance

Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia.     

Pyruvate kinase isozymes in adult tissues and eggs of Rana pipiens. Comparative studies on the properties of the different isozymes

The properties of the isozymes of pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) found in unfertilized frog egg have been compared to those found in adult tissues of Rana pipiens. Chromatographic, kinetic, and electrophoretic data indicate that, of the five electrophoretic forms found in egg, the isozyme with the least anodic mobility (isozyme I) is the same molecular species as the only isozyme found in heart, and the egg isozyme with the greatest anodic mobility (isozyme V) is identical to the major isozyme found in liver.The activity of egg isozyme I was markedly inhibited by the antibody to the skeletal muscle enzyme, which has been shown previously to cross-react with the cardiac enzyme, but was unaffected by the antibody to liver isozyme V; the opposite effects were observed with egg isozyme V. The antibody to the skeletal muscle enzyme inhibited egg isozymes II > III > IV whereas the antibody to the liver enzyme gave the reverse inhibitory pattern, e.g., isozyme IV > III > II.In vitro dissociation-reassociation of mixtures of isozyme I and V led to the formation of the other three isozymes. Similar experiments performed individually with either egg isozyme III or IV resulted in the production of predominantly isozymes III, II, and I due to the instability of isozyme V during the hybridization procedure.The above results indicate that isozymes I and V are tetramers of the respective parental subunits and that isozymes II, III, and IV are hybrid molecules with subunit assignments of (I₃V₁), I₂V₂), and (I₁V₃), respectively.     

Comparative Enzymology of the Glyceraldehyde 3-Phosphate Dehydrogenases from Pisum sativum

Glyceraldehyde 3-phosphate dehydrogenases (EC 1.2.1.12 and 1.2.1.13) have been purified from the seed, root, etiolated, and green shoot of peas (Pisum sativum). These enzymes are tetramers of 140,000 daltons, with subunits of 35,000 daltons. The enzymes differ in isoelectric point. The seed enzyme has a pI of 5.1, and the root enzyme has a pI of 4.5. The cytoplasmic enzyme from etiolated shoots is slightly acidic with a pI of 5.7 to 6.1 and is found in two separable forms. The chloroplast enzyme (from green shoots) is most basic with a pI of 8.0.     

Degradation of poly(adenosine diphosphate ribose) by homogenates of various normal tissues and tumors of rats.

The extent of increase in the activity of phenylalanine ammonia-lyase upon illumination for 15 hr of cultured Petroselinum hortense cells was greatly dependent upon the age of the culture. Two distinct peaks in specific activity were observed during the growth cycle, one occurring at the beginning and the other at the end of the period of increase in cell fresh weight. High yields in both cell fresh weight and enzyme activity were obtained with the second peak shortly before the stationary phase of the culture was reached. Cells were harvested at this stage and stored at ?20 °C.The enzyme was purified from the frozen cells to apparent homogeneity by precipitation with (NH₄)₂SO₄, chromatography on DEAE-cellulose, Sephadex G-200 and hydroxyapatite columns, and preparative polyacrylamide gel electrophoresis. An over-all yield of 16% was achieved with a 440-fold increase in the specific activity by purification of the enzyme through the hydroxyapatite step.Upon analytical polyacrylamide gel electrophoresis either in the presence or in the absence of sodium dodecyl sulfate, the purified protein migrated essentially as a single band. Molecular weights of about 330,000 and 83,000, respectively, were estimated for the enzyme and for its protein subunits. Thus, the enzyme molecule seems to be composed of four probably identical protein subunits. Two Michaelis constants for l-phenylalanine (K_m^L, 3.2 × 10^?5 M, and K_m^H, 2.4 × 10^?4 M) and a Hill coefficient of h = 0.6 were obtained. This suggests that the enzyme is subject to regulation of its catalytic properties by negative cooperativity of the protein subunits.     

The quaternary structure of Escherichia coli N-acetylneuraminate lyase is essential for functional expression

As part of a general study into the impact of quaternary structure on enzyme function, a library of 31 point mutations were engineered at the dimer-dimer interface of the homotetrameric (β/α)₈-barrel protein, N-acetylneuraminate lyase (NAL, EC 4.1.3.3). Disruption of the interface generated either soluble tetramers or putative dimers that were absolutely insoluble and inactive. Intriguingly, the soluble tetramers were found to have widely varying k_cat values, hinting at a role for the interface in catalysis. Leucine 171 was identified as essential to interface integrity. We conclude that the dimer-dimer interface of NAL is intolerant to mutation and essential for functional expression.     

Co-expression of α and β subunits of the 3-methylcrotonyl-coenzyme A carboxylase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Pseudomonas aeruginosa is a versatile bacterium that can grow using citronellol or leucine as sole carbon source. For both compounds the degradation pathways converge at the key enzyme 3-methylcrotonyl coenzyme-A carboxylase (MCCase). This enzyme is a complex formed by two subunits (α and β), encoded by the liuD and liuB genes, respectively; both are essential for enzyme function. Previously, both subunits had been separately expressed and then the complex re-constituted, however this methodology is laborious and produces low yield of active enzyme. In this work, the MCCase subunits were co-expressed in the same plasmid and purified in one step by affinity chromatography using the LiuD-His tag protein, interacting with the LiuB-S tag recombinant protein. The purified enzyme lost most of the activity within few hours of storage. The co-expressed subunits formed an (αβ)₄ complex that suffered a modification of its oligomerization state after storage, which probably contributed to the loss on activity observed. The recombinant MCCase enzyme presented optimum pH and temperature values of 9.0 and 30o C, respectively. Functionally, MCCase showed Michaelian kinetics behavior with a K_m for its substrate and V_max of 168 μM and 430 nmoles mg⁻¹min⁻¹, respectively. The results suggest that the co-expression and co-purification of the subunits is a suitable procedure to obtain the active complex of the MCCase from Pseudomonas aeruginosa in a single step.     

Purification and characterization of diamine oxidase from rice embryos

Diamine oxidase of rice seedlings has been purified 1800-fold to homogeneity. The MW of the enzyme as determined by Sephadex G-100 gel filtration was 12.3 × 10⁴ and the enzyme contained two identical subunits each with a MW of 6.12 × 10⁴. The optimal temperature and pH for the enzyme were 30° and 7.5 respectively and the enzyme followed typical Michaelis kinetics with a K_m of 10^?5 M. Each enzyme molecule contained four molecules of FAD.     

Active site ligand stabilization of quaternary structures of glutamine synthetase from Escherichia coli

Auto-inactivated EScherichia coli glutamine synthetase contains 1 eq each of L-methionine-S-sulfoximine phosphate and ADP and 2 eq of Mn2+ tightly bound to the active site of each subunit of the dodecameric enzyme (Maurizi, M. R., and Ginsburg, A. (1982) J. Biol. Chem. 257, 4271-4278). Complete dissociation and unfolding in 6 M guanidine HCl at pH 7.2 and 37 degrees C requires greater than 4 h for the auto-inactivated enzyme complex (less than 1 min for uncomplexed enzyme). Release of ligands and dissociation and unfolding of the protein occur in parallel but follow non-first order kinetics, suggesting stable intermediates and multiple pathways for the dissociation reactions. Treatment of Partially inactivated glutamine synthetase (2-6 autoinactivated subunits/dodecamer) with EDTA and dithiobisnitrobenzoic acid at pH 8 modifies approximately 2 of the 4 sulfhydryl groups of unliganded subunits and causes dissociation of the enzyme to stable oligomeric intermediates with 4, 6, 8, and 10 subunits, containing equal numbers of uncomplexed subunits and autoinactivated subunits. With greater than 70% inactivated enzyme, no dissociation occurs under these conditions. Electron micrographs of oligomers, presented in the appendix (Haschemeyer, R. H., Wall, J. S., Hainfeld, J., and Maurizi, M. R., (1982) J. Biol. Chem. 257, 7252-7253) suggest that dissociation of partially liganded dodecamers occurs by cleavage of intra-ring subunit contacts across both hexagonal rings and that these intra-ring subunit contacts across both hexagonal rings and that these intra-ring subunit interactions are stabilized by active site ligand binding. Isolated tetramers (Mr = 200,000; s20,w = 9.5 S) retain sufficient native structure to express significant enzymatic activity; tetramers reassociate to dodecamers and show a 5-fold increase in activity upon removal of the thionitrobenzoate groups with 2-mercaptoethanol. Thus, the tight binding of ligands to the subunit active site strengthens both intra- and inter-subunit bonding domains in dodecameric glutamine synthetase.     

Effect of Sodium Dodecyl Sulfate on D-Glucose-isomerizing Enzyme from Bacillus coagulans,Strain HN-68

Crystalline D-glucose-isomerizing enzyme from Bacillus coagulans, strain NH–68 has been shown to consist of subunits by the method of electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide gels.

The dissociation behavior of the enzyme has been characterized. The enzyme dissociates into inactive subunits by the preincubation with 0.05% SDS in the presence of 5 × 10^?3M MnCl₂ or CoCl₂, but not in the absence of these metal salts. In 8 м urea, however, the enzyme does not dissociate into subunits and the activity is completely recovered by dilution of the urea. Metal salts, such as MnCl₂ and CoCl₂, also do not affect activity in the presence of urea.