Human erythrocyte glucose 6-phosphate dehydrogenase. Evidence for competitive binding of NADP and NADPH.

The tetrameric form of human erythrocyte glucose 6-phosphate dehydrogenase (G6PD) was investigated in respect to interaction of coenzyme at its ron-structural sites. 1:N⁶-ethenoadenine dinucleotide phosphate (ε-NADP), although displaying a lower catalytic efficiency compared to NADP, showed identical binding patterns, i.e. four moles per tetramer with a K_diss of 1.0 μM. Furthermore, spectrofluorometric titrations with NADPH in the absence and in presence of varying NADP concentrations revealed a typically competitive mechanism of binding of NADP (four moles) and NADP (two moles) at the non-structural sites of the tetramer.     

Isolation and partial charaterization of an NADP- and NADPH- binding protein from human erythrocytes.

A simple procedure, exploiting an affinity chromatography step on agarose-linked adenosine 2′,5′-bisphosphate, allows the concurrent purification from human red cell lysates of glucose 6-phosphate dehydrogenase (G6PD) and of another protein (FX). The latter, which has a higher electrophoretic mobility than G6PD, is not identifiable with any of a number of erythrocyte enzymes. It is a holoprotein, composed in its native form of two polypeptide chains of 33,000 M_r each and of one NADP equivalent. Native FX binds NADPH and this binding is competitive with NADP: The corresponding stoichiometry is 0.5 NADPH equivalents per 33,000 M_r (“half-site reactivity”), with a dissociation constant of 1 × 10^?8m. On the basis of competition experiments, the dissociation constant for NADP is estimated to be 1.8 × 10^?7m.     

Binding of pyridine coenzymes to the β-subunit of the voltage sensitive potassium channels

The β-subunit of the voltage-sensitive K⁺ channels shares 15–30% amino acid identity with the sequences of aldo–keto reductases (AKR) genes. However, the AKR properties of the protein remain unknown. To begin to understand its oxidoreductase properties, we examine the pyridine coenzyme binding activity of the protein in vitro. The cDNA of K_vβ2.1 from rat brain was subcloned into a prokaryotic expression vector and overexpressed in Escherichia coli. The purified protein was tetrameric in solution as determined by size exclusion chromatography. The protein displayed high affinity binding to NADPH as determined by fluorometric titration. The K_D values for NADPH of the full-length wild-type protein and the N-terminus deleted protein were 0.1±0.007 and 0.05±0.006 M, respectively — indicating that the cofactor binding domain is restricted to the C-terminus, and is not drastically affected by the absence of the N-terminus amino acids, which form the ball and chain regulating voltage-dependent inactivation of the α-subunit. The protein displayed poor affinity for other coenzymes and the corresponding values of the K_D for NADH and NAD were between 1–3 μM whereas the K_D for FAD was >10 μM. However, relatively high affinity binding was observed with 3-acetyl pyridine NADP, indicating selective recognition of the 2′ phosphate at the binding site. The selectivity of K_vβ2.1 for NADPH over NADP may be significant in regulating the K⁺ channels as a function of the cellular redox state.     

Nucleotide binding affinities of the intact proton-translocating transhydrogenase from Escherichia coli

Transhydrogenase (E.C. 1.6.1.1) couples the redox reaction between NAD(H) and NADP(H) to the transport of protons across a membrane. The enzyme is composed of three components. The dI and dIII components, which house the binding site for NAD(H) and NADP(H), respectively, are peripheral to the membrane, and dII spans the membrane. We have estimated dissociation constants (K_d values) for NADPH (0.87 μM), NADP⁺ (16 μM), NADH (50 μM), and NAD⁺ (100-500 μM) for intact, detergent-dispersed transhydrogenase from Escherichia coli using micro-calorimetry. This is the first complete set of dissociation constants of the physiological nucleotides for any intact transhydrogenase. The K_d values for NAD⁺ and NADH are similar to those previously reported with isolated dI, but the K_d values for NADP⁺ and NADPH are much larger than those previously reported with isolated dIII. There is negative co-operativity between the binding sites of the intact, detergent-dispersed transhydrogenase when both nucleotides are reduced or both are oxidised.     

Identification of nitric oxide and nitrous oxide as products of nitrite reduction by Pseudomonas cytochrome oxidase (nitrate reductase)

The cytosol fraction of rat adrenocortical tissue contains comparatively high levels of two prostaglandin metabolizing enzymes. The first, prostaglandin-9-ketoreductase, utilizes NADPH more effectively than NADH as cofactor, is inhibited by NADP, and exhibits an apparent K_m of 304 μM for PGE₁. 15-hydroxyprostaglandin dehydrogenase, tentatively identified as the type II NADP-dependent isozyme, is inhibited by NADPH but not NADH, and exhibits an apparent K_m of 157 μM when PGE₁ is used as substrate. Changes in specific activities of the two enzymes following ACTH, hypophysectomy, or dexamethasone treatment are inconclusive in defining a chronic regulatory role for adrenocorticotropin.     

The effect of NADP on the subunit structure and activity of spinach chloroplast glyceraldehyde-3-phosphate dehydrogenase

Spinach chloroplast glyceraldehyde phosphate dehydrogenase (d-glyceraldehyde-3-phosphate: NADP oxidoreductase, phosphorylating; EC 1.2.1.13) is an equilibrium mixture of aggregates of a basic protomer (M_r about 145,000) and is active with both NADP and NAD. The enzyme is primarily “tetrameric” (M_r about 600,000), although minor amounts of smaller and larger oligomers are also found. Gel chromatography in buffer containing 30 μm NADP results in depolymerization of the enzyme, mainly to protomers. NAD does not dissociate and counteracts this effect of NADP.The apparent K_m values of the protomers are 7 μm (NADP) and 8 μm (NAD). The aggregates with a M_r > 10⁶ have properties similar to the protomers. The tetramer as first isolated has higher M_m values for NADP (380 μm) and NAD (48 μm), but its apparent affinity for NADP is further decreased by repeated gel filtrations in buffer or by a single one in buffer containing NAD. Such preparations display nonlinear kinetics when NADP is the varied substrate and have a K_m (NADP) of about 1.5–3.3 μm. All these effects are reversible.V values are apparently the same in all enzyme forms and the $\text{V (}\text{NADP}\text{)}\text{V (}\text{NAD}\text{)}$ ratio always approaches 2. Since, however, the enzyme is presumably dissociated by the NADP concentrations required for a “saturating” assay, the significance of V (NADP) seems questionable.     

Reexamination of the interaction between ferredoxin:NADP reductase and NADP

A comparison was made of graphical and subtractive methods for the determination of the dissociation constant of a complex between ferredoxin:NADP reductase and NADP. The subtractive method gave K_d values near 10 μm which are consistent with recently determined values for K_m,NADP in assays of NADP photoreduction by chloroplast membranes. The graphical method gave values which were considerably higher. The difference between the two methods is due to the failure of the graphical method to correct for the amount of each component present in the complex at the low NADP/ flavoprotein ratios necessary for binding studies. A second NADP binding site of much lower affinity (K_d approx 1 mm) was also detected.     

Purification and properties of NADP-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from the green alga Chlamydomonas reinhardtii

NADP-dependent non-phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9), previously described in higher plants, has been now found to be present in eukaryotic green algae, but in neither cyanobacteria nor non-photosynthetic microorganisms. The enzyme from the unicellular green alga Chlamydomonas reinhardtii, strain 6145c, has been purified to apparent electrophoretic homogeneity. The non-phosphorylating enzyme was effectively separated from the NADP-dependent phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) dye-ligand chromatography on Reactive Red-120 agarose. The purified enzyme exhibited an optimum pH in the 8.5–9.0 range and a specific activity of approx. 8 μmol·(mg protein)⁻¹·min⁻¹. The native protein was characterized as a homotetramer with a molecular weight of 190 000, a Stokes radius of 5.2 mn, and an isoelectric point of 6.9. From kinetic studies, K_m-values of 9.8 and 51 μM were calculated for NADP and D-glyceraldehyde 3-phosphate, respectively, an absolute specificity for both substrates being observed. L-Glyceraldehyde 3-phosphate was a potent non-competitive inhibior (K_i, 48 μM). The reaction products NADPH and D-3-phosphoglycerate inhibited enzyme activity in a competitive manner with respect to NADP (K_i, 78 μM) and D-glyceraldehyde 3-phosphate (K_i, 1.2 mM), respectively. Thermal inactivation occurred above 45°C and was effectively prevented by either substrate. The presence of essential vicinal thiol groups is suggested by the inactivation produced by diamide, with D-glyceraldehyde 3-phosphate, but not NADP, behaving as a protective agent. The enzyme's possible physiological role in photosynthetic metabolism is discussed briefly.     

Catalytic mechanism and cofactor preference of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus

Given the rapid rise in antibiotic resistance, including methicillin resistance in Staphylococcus aureus (MRSA), there is an urgent need to characterize novel drug targets. Enzymes of the lysine biosynthesis pathway in bacteria are examples of such targets, including dihydrodipicolinate reductase (DHDPR, E.C. 1.3.1.26), which is the product of an essential bacterial gene. DHDPR catalyzes the NAD(P)H-dependent reduction of dihydrodipicolinate (DHDP) to tetrahydrodipicolinate (THDP) in the lysine biosynthesis pathway. We show that MRSA–DHDPR exhibits a unique nucleotide specificity utilizing NADPH (K_m = 12 μM) as a cofactor more effectively than NADH (K_m = 26 μM). However, the enzyme is inhibited by high concentrations of DHDP when using NADPH as a cofactor, but not with NADH. Isothermal titration calorimetry (ITC) studies reveal that MRSA–DHDPR has ∼20-fold greater binding affinity for NADPH (K_d = 1.5 μM) relative to NADH (K_d = 29 μM). Kinetic investigations in tandem with ITC studies show that the enzyme follows a compulsory-order ternary complex mechanism; with inhibition by DHDP through the formation of a nonproductive ternary complex with NADP⁺. This work describes, for the first time, the catalytic mechanism and cofactor preference of MRSA–DHDPR, and provides insight into rational approaches to inhibiting this valid antimicrobial target.     

Regulation of C4 photosynthesis: Physical and kinetic properties of active (dithiol) and inactive (disulfide) NADP-malate dehydrogenase from Zea mays

NADP-malate dehydrogenase was purified from leaves of Zea mays in the absence of thiol-reducing agents by (NH₄)₂SO₄, polyethylene glycol, and pH fractionation followed by dye-ligand affinity chromatography and gel filtration. The purified enzyme is completely inactive (no activity detected between pH 6 and 9) but can be reactivated by thiol-reducing agents including dithiothreitol and thioredoxin. The active enzyme shows distinctly alkaline pH optima when assayed in either direction; K_m values at pH 8.5 are oxaloacetate, 18 μm; malate, 24 mm; NADPH, 50 μm; and NADP, 45 μm. The reduction of oxaloacetate is inhibited by NADP (competitive with respect to NADPH, K_i = 50 μm). The molecular weight of the native inactive or active enzyme is 150,000 with subunits of M_r 38,000. Active enzyme is much more sensitive (>50-fold) to heat denaturation than is the inactive enzyme and is irreversibly inactivated by N-ethylmaleimide whereas the inactive enzyme is insensitive to this reagent. The active and inactive forms of NADP-malate dehydrogenase are assumed to correspond to dithiol and disulfide forms of the enzyme, respectively. The relative coenzyme-binding affinities of inactive NADP-malate dehydrogenase differ by a factor of 10² from the binding affinities for active NADP-malate dehydrogenase and 10⁴ for non-thiol-regulated NAD-specific malate dehydrogenase. It is proposed that the 100-fold change in differential binding of NADP and NADPH upon conversion of NADP-malate dehydrogenase to the disulfide form may sufficiently alter the equilibrium of the central enzyme-substrate complexes, and hence the catalytic efficiency of the enzyme, to explain the associated loss of activity.     

Red-cell glucose-6-phosphate dehydrogenase of “brown-howler” monkeys (Alouatta fusca)

Physico-chemical properties of erythrocyte glucose-6-phosphate dehydrogenase including erythrocyte G6PD activity, Michaelis constants, K_mG6P and NADP, pH optimum, thermostability and molecular weight were investigated in “brown-howler” monkeys and then compared with the values of human G6PD B(+). The values of Michaelis constants (K_mG6P and NADP) pH optimum were the same as the values of human G6PD B(+). The human G6PD has a dimeric form in the assay conditions employed in the present study, monkey enzyme showing great similariy with human one. Otherwise, the thermostability differed from the human G6PD. The simian enzymatic activity was about four times higher than the human G6PD. A comparison of physico-chemical properties of glucose-6-phosphate dehydrogenase among primates is also presented.     

Purification and Kinetic Characterization of Glucose-6-phosphate Dehydrogenase from Dictyostelium discoideum

Reimers, J. M., Huang, Q., Albe, K. R., and Wright, B. E. 1993. Purification and kinetic characterization of glucose-6-phosphate dehydrogenase from Dictyostelium discoideum. Experimental Mycology 17, 1-6. Glucose-6-phosphate dehydrogenase from Dictyostelium discoideum was purified 650-fold and kinetically characterized. The enzyme catalyzed the conversion of G6P + NADP to 6PG + NADPH stoichiometrically and irreversibly in vitro . The purified enzyme is specific for NADP. Michaelis constants for G6P and NADP were 0.040 and 0.011 mM, respectively. NADPH was found to be a competitive inhibitor with respect to NADP with a K_i of 0.006 mM and a noncompetitive inhibitor with respect to G6P. The data from initial velocity and product inhibition studies were consistent with a sequential mechanism.     

Zinc ion binding to human brain calcium binding proteins. Calmodulin and S100b protein

Comparative studies have been performed on the binding properties of zinc ions to human brain calmodulin and S100b protein. Calmodulin is characterized by two sets of Zn²⁺ binding sites, with K_D ranging from 8.10^?5M to 3.10^?4M. The S100b protein also exhibited two sets of zinc binding sites, with a much higher affinity. K_D = 10^?7 ? 10^?6M. We suggest that S100b protein should no longer be considered only as a “calcium binding protein” but also as a “zinc binding protein”, and that Zn²⁺ ions are involved in the functions of the S100 proteins.     

Potent interaction of histamine and polyamines at microsomal cytochrome P450, nuclei,and chromatin from rat hepatocytes

Histamine and polyamines have been implicated in the mediation of cell proliferation. Our previous work linked the growth-modulatory effects of histamine with its binding to intracellular sites in microsomes and nuclei of various tissues. In this study, we identify cytochrome P450 enzymes as a major component of microsomal intracellular sites in hepatocytes and demonstrate that polyamines compete with high affinity for histamine binding to them. Spectral measurement of histamine binding to P450 in liver microsomes resolved high and intermediate affinity binding sites (K_s1 = 2.4 ± 1.6 μM; K_s2 = 90 ± 17 μM) that corresponded to microsomal binding sites (K_d1 = 1.0 ± 0.9 μM; K_d2 = 57 ± 13 μM) resolved by ³H-histamine binding; additional low affinity (K_d3 ∼ 3 mM), and probably physiologically irrelevant, sites were resolved only by ³H-histamine radioligand studies. As determined spectrally, treatment of microsomes with NADPH/carbon monoxide decreased histamine binding to P450 by about 90% and, as determined by ³H-histamine binding, abolished the high affinity sites and reduced by 85% the number of intermediate sites. Spermine competed potently for ³H-histamine binding: in microsomes, K_i = 9.8 ± 5.8 μM; in nuclei, K_i = 13.7 ± 3.1 μM; in chromatin, K_i = 46 ± 33 nM. Polyamines inhibited the P450/histamine absorbance complex with the rank order of potency: spermine > spermidine ≫ putrescine. In contrast, histamine did not compete for ³H- spermidine binding in nuclei or microsomes, suggesting that polyamines modulate histamine binding allosterically. We propose that certain P450 isozymes that modulate gene function by controlling the level of oxygenated lipids, represent at least one common intracellular target of growth-regulatory endogenous bioamines and, as shown previously, of exogenous growth-modulatory drugs including antiestrogens, antiandrogens, and certain antidepressants and antihistamines. J. Cell. Biochem. 69:233–243, 1998. © 1998 Wiley-Liss, Inc.     

Human sperm glutathione reductase activity in situ reveals limitation in the glutathione antioxidant defense system due to supply of NADPH

In order to characterize further the antilipoperoxidative enzyme system of human sperm, that part of the system designed to provide reducing equivalents for the reduction of highly reactive and potentially damaging lipid hydroperoxides to relatively inert hydroxylipids was examined. The substrate that provides the reducing equivalents directly to glutathione peroxidase (GPX) is reduced glutathione (GSH), which is in turn oxidized to glutathione disulfide (GSSG). The reducing equivalents needed for regeneration of GSH through the action of glutathione reductase (GRD) are provided by NADPH, produced by the action of glucose-6-phosphate dehydrogenase (G6P-DH) on substrates glucose-6-phosphate and NADP⁺. The kinetic properties of the enzymes GRD and G6P-DH were determined by standard enzyme activity assay at 24 and 37°C. At 37°C, the V_max for GRD was found to be 36 nmol/min · 10⁸ cells, with K_m values for GSSG and NAPH of 150 μM and 16 μM, respectively; the V_max for G6P-DH was 3.3 nmol/min · 10⁸ cells with K_m for NADP⁺ of 8 μM. This suggested that G6P-DH activity was limiting in this reductive pathway. The activity of GRD in situ in intact cells was estimated using the thiol-reactive fluorogenic probe ThioGlo-1, which is cell permeant and reacts rapidly with GSH to give a highly fluorescent adduct. Mixing a suspension of human sperm with the fluorogenic reagent at 37°C gave an initial rapid increase in fluorescence, followed by a slower one. The rapid phase is due to reaction with intracellular GSH already present; the slow phase is due to reaction with GSH generated by the GRD-catalyzed reduction of GSSG. Both rates showed first-order kinetics. Calculation of the maximal rate as NADPH oxidation, attributable to in situ GRD activity, gave the value of 1.0 nmol/min · 10⁸ cells, less than the maximum for NADPH production by the dehydrogenase. These results support the suggestion that NADPH production limits the capacity of the pathway leading to hydroperoxide reduction in human sperm. We propose that the antilipoperoxidative defense system of human sperm has just sufficient capacity to allow these cells to fulfill their function but is limited to allow their timely disposal from the female reproductive tract. Mol. Reprod. Dev. 49:400–407, 1998. © 1998 Wiley-Liss, Inc.     

Isolation and characterization of light- and dithiothreitol-modulatable glucose-6-phosphate dehydrogenase from pea chloroplasts

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP oxidoreductase, EC 1.1.1.49) has been purified to electrophoretic homogeneity from pea chloroplasts. The enzyme, which has a Stokes radius of 52 Å, is a tetramer made up of four 56000 Da monomers. The pH optimum is around 8.2. The enzyme is absolutely specific for NADP. The apparent K_m(NADP) is 2.4 ± 0.1 μM. NADPH inhibition of the enzyme is competitive with respect to NADP (mean K_i, 18 ± 5 μM) and is mixed (K_p >K_m, V_max >V_p) with respect to glucose 6-phosphate (mean crossover point, 0.5 ± 0.1 mM). The apparent K_m(glucose 6-phosphate) is 0.37 ± 0.01 mM. The purified enzyme is inactivated in the light in the presence of dilute stroma and washed thylakoids, and by dithiothreitol. Enzyme which has been partially inactivated by treatment with dithiothreitol can be further inactivated in the light in the presence of dilute stroma and washed thylakoids and reactivated in the dark, but only to the extent of the reverse of light inactivation. Dithiothreitol-inactivated enzyme is not reactivated further by addition of crude stroma or oxidized thioredoxin. Dithiothreitol-dependent inactivation of the enzyme follows pseudo-first-order kinetics and shows rate saturation. The enzyme which has been partially inactivated by treatment with dithiothreitol does not differ from the untreated control with respect to thermal and tryptic inactivation. However, enzyme which has been partially light inactivated shows different thermal and tryptic inactivation patterns as compared to the dark control. These observations suggest that the changes in the enzyme brought about by light modulation are not necessarily identical with those brought about by dithiothreitol inactivation.     

Fructose-1, 6-bisphosphate aldolase from rabbit muscle: Different catalytic behavior of the dihydroxyacetone phosphate binding sites at low temperature

The equivalence of the four dihydroxyacetone phosphate binding sites of aldolase was abolished by lowering the temperature. At pH 6.2 and ?13 2C, four binding sites were detected by gel filtration; two sites with a K_diss ?0.1 μm, and a second set of sites with a K_diss = 4 μm. The alteration of the binding was accompanied by the alteration of the catalytic activity. The low-affinity sites were incapable of catalyzing the cleavage of the (3S) CH bond of dihydroxyacetone phosphate, and form only the ketimine phosphate intermediate. The high-affinity sites were still able to cleave the (3S) CH bond of dihydroxyacetone phosphate; however, the eneamine phosphate intermediate formed was almost fully converted into the eneamine-aldehyde … phosphate intermediate, which was the prevailing species at the equilibrium. The mechanism of the half-of-the sites reactivity of aldolase at low temperature has been explained and the nonequivalence of sites in promoting catalysis has been utilized to dissect and characterize the individual partial reactions of the enzyme. In the course of these studies it has been shown that the rate of hydration-dehydration of dihydroxyacetone phosphate at ?24 °C was too slow to measure.     

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Interaction of the enzyme with coenzymes and coenzyme analogs.

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides utilizes either NAD⁺ or NADP⁺ as coenzyme. Kinetic studies showed that NAD⁺ and NADP⁺ interact with different enzyme forms (Olive, C., Geroch, M. E., and Levy, H. R. (1971) J. Biol. Chem.246, 2047–2057). In the present study the techniques of fluorescence quenching and fluorescence enhancement were used to investigate the interaction between Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and coenzymes. In addition, kinetic studies were performed to examine interaction between the enzyme and various coenzyme analogs. The maximum quenching of protein fluorescence is 5% for NADP⁺ and 50% for NAD⁺. The dissociation constant for NADP⁺, determined from fluorescence quenching measurements, is 3 μm, which is similar to the previously determined K_m of 5.7 μm and K_i of 5 μm. The dissociation constant for NAD⁺ is 2.5 mm, which is 24 times larger than the previously determined K_m of 0.106 mm. Glucose 1-phosphate, a substrate-competitive inhibitor, lowers the dissociation constant and maximum fluorescence quenching for NAD⁺ but not for NADP⁺. This suggests that glucose 6-phosphate may act similarly and thus play a role in enabling the enzyme to utilize NAD⁺ under physiological conditions. When NADPH binds to the enzyme its fluorescence is enhanced 2.3-fold. The enzyme was titrated with NADPH in the absence and presence of NAD⁺; binding of these two coenzymes is competitive. The dissociation constant for NADPH from these measurements is 24 μm; the previously determined K_i is 37.6 μm. The dissociation constant for NAD′ is 2.8 mm, in satisfactory agreement with the value obtained from protein fluorescence quenching measurements. Various compounds which resemble either the adenosine or the nicotinamide portion of the coenzyme structure are coenzyme-competitive inhibitors; 2′,5′-ADP, the most inhibitory analog tested, gives NADP⁺-competitive and NAD⁺-noncompetitive inhibition, consistent with the kinetic mechanism previously proposed. By using pairs of coenzyme-competitive inhibitors it was shown in kinetic studies that the two portions of the NAD⁺ structure cannot be accommodated on the enzyme simultaneously unies they are covalently linked. Fluorescence studies showed that there are both “buried” and “exposed” tryptophan residues in the enzyme structure.     

Magnesium reduces affinities of antagonists at rat cortex α2-adrenergic receptors labeled with 3H-clonidine: Evidence for heterogeneity of α2-receptor conformations with respect to antagonists

³H-clonidine labeled two binding sites in rat cortex membranes with apparent K_D values of about 1.0 and 5.9 nM. These sites appeared analogous to “super-high” (SH) and “high” (H) affinity states of the α₂-receptor described in human platelets. 10 mM magnesium increased the number of SH receptors by 30% whereas 100 μM GTP reduced SH₃receptor number by 45% with no significant change in the K_D of ³H-clonidine at α₂(SH) sites. In drug competition studies using 1.0 nM ³H-clonidine, 100 μM GTP reduced the affinity of clonidine and increased the affinity of yohimbine, whereas 10 mM magnesium increased the affinity of clonidine and reduced the affinity of yohimbine. The effect of magnesium on the affinity of several antagonists at cortex ³H-clonidine sites ranged from none (phentolamine) to a 6-fold reduction (piperoxan). These data indicate that different states of the α₂-receptor exhibit different affinities for some antagonists.     

Purification and kinetic mechanism of 5,10-metliyienetetrahydrofolate reductase from sheep liver

5,10-Methylenetetrahydrofolate reductase (EC 1.1.1.68) was purified from the cytosolic fraction of sheep liver by (NH₄)₂ SO₄ fractionation, acid precipitation, DEAE-Sephacel chromatography and Blue Sepharose affinity chromatography. The homogeneity of the enzyme was established by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, ultracentrifugation and Ouchterlony immunodiffusion test. The enzyme was a dimer of molecular weight 1,66,000 ± 5,000 with a subunit molecular weight of 87,000 ±5,000. The enzyme showed hyperbolic saturation pattern with 5-methyltetrahydrofolate.K _0.5 values for 5-methyltetrahydrofolate menadione and NADPH were determined to be 132 ΜM, 2.45 ΜM and 16 ΜM. The parallel set of lines in the Lineweaver-Burk plot, when either NADPH or menadione was varied at different fixed concentrations of the other substrate; non-competitive inhibition, when NADPH was varied at different fixed concentrations of NADP; competitive inhibition, when menadione was varied at different fixed concentrations of NADP and the absence of inhibition by NADP at saturating concentration of menadione, clearly established that the kinetic mechanism of the reaction catalyzed by this enzyme was ping-pong.