NAD kinase from Bacillus licheniformis: inhibition by NADP and other properties

NAD kinase was purified 180-fold from Bacillus licheniformis to determine the role it plays in NADP turnover in this organism. The enzyme was found to have a pH optimum of 6.8 and an apparent K _m for NAD of 2.7 mM. The ATP saturation curve was not hyperbolic; 5.5 mM ATP was required to reach half maximal activity. Both Mn²⁺ and Ca²⁺ could be substituted for Mg²⁺. Several compounds including nicotinic acid, nicotinamide, nicotinamide mononucleotide, quinolinic acid, NADPH, ADP, AMP and cyclic AMP did not affect NAD kinase activity. In contrast, the enzyme was inhibited by NADP at concentrations typically found in logarithmic cells of B. licheniformis. This inhibition was competitive with NAD and had a K _i of 0.13 mM. It is suggested that in vivo NAD kinase activity is highly dependent on the concentrations of NAD and ATP and the proportion of oxidized and reduced NADP.This paper is dedicated to Sydney C. Rittenberg on the occassion of his retirement, with respect and much affection, in appreciation for his friendship and years of distinguished service as a teacher and scientist     

Kinetic properties of NAD malic enzyme from cauliflower

The kinetic characteristics of NAD malic enzyme purified to homogeneity from cauliflower florets have been examined. Free NAD⁺ is the active form of this coenzyme. Double-reciprocal plots of data obtained by varying NAD⁺ and malate^2? at a saturating concentration of Mg²⁺ or by varying Mg²⁺ and NAD⁺ at a saturating level of malate^2? are of intersecting type. This indicates that NAD malic enzyme obeys a sequential mechanism. Analysis of these sets of data suggests that each of these substrate pairs binds randomly to the enzyme. However, each substrate binds tighter when others are already present on the enzyme. NAD malic enzyme cannot decarboxylate malate^2? in the absence of either Mg²⁺ or NAD⁺. Arrhenius plots of the NAD-linked reaction are concave downward, indicating the existence of two rate-determining steps with activation energies of 26.5 and 14.2 kcal/mol, respectively. In addition to Mg²⁺, the enzyme can also use Mn²⁺ and Co²⁺. Using Co²⁺ in place of Mg²⁺ does not change V_max or K_m,malate^2? but the K_m for metal and NAD⁺ are greatly decreased. At pH 7.0 and above, Mn²⁺ isotherms and malate^2? curves with Mn²⁺ are nonlinear and appear to be composed of two separate saturation curves. NAD malic enzyme is completely and irreversibly inactivated by N-ethylmaleimide. The enzyme is also irreversibly inactivated approximately 50% by KCNO.     

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

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

Properties of NADP-malic enzyme from glumes of developing wheat grains

Properties of partially purified NADP-malic enzyme (EC 1.1.1.40) from glumes of developing wheat grains were examined. The pH optimum for enzyme activity was influenced by malate and shifted from 7.3 to 7.6 when the concentration of malate was increased from 2 to 10 mM. The K_m values, at pH 7.3, for various substrates were: malate, 0.76 mM; NADP, 20 μM and Mn²⁺, 0.06 mM. The requirement of Mn²⁺ cation for enzyme activity could be partially replaced by Mg²⁺ or Co²⁺. Mn²⁺ dependent enzyme activity was inhibited by Pb²⁺, Ni²⁺, Hg²⁺, Zn²⁺, Cd²⁺, Al³⁺ and Fe³⁺. During the reaction, substrate molecules (malate and NADP) reacted with enzyme sequentially. Activity of malic enzyme was inhibited by products of the reaction viz pyruvate, HCO₃^? and NADPH₂. At a limiting fixed concentration of NADP, these products induced a positive cooperative response to increasing concentrations of malate.     

Isopentenyl pyrophosphate isomerase from pig liver

Isopentenyl pyrophosphate isomerase (EC 5.3.3.2) from pig liver has been purified 197-fold. The preparation was estimated to contain less than 10% of contaminating protein. The molecular weight determined by gel filtration was 82,500 ± 3,000 and the isoelectric point from isoelectric focusing was in the range 6.0–6.2. N-terminal analysis showed the presence of both leucine and proline. The pH optimum of the enzyme preparation was 6.3. After dialysis against EDTA, activity was restored by either Mn²⁺ or Mg²⁺, the former being more effective. At the optimum pH and concentration of Mn²⁺, K_m and V were 2.7 μm and 6.7 μmol min^?1 mg^?1, respectively. The enzyme was partially inhibited by a variety of terpene mono- and pyrophosphate esters, by inorganic phosphate ions, and by acetate ions; essentially complete inhibition by sulfhydryl-blocking reagents was observed. ATP partially inhibited, the degree of inhibition showing a sigmoid dependence on ATP concentration. Monothiols and dithiothreitol activated the enzyme, as did mevalonic acid.     

Physical and Kinetic Properties and Regulation of the NAD Malic Enzyme Purified from Leaves of Crassula argentea

The NAD malic enzyme has been purified to near homogeneity from the leaves of Crassula argentea Thunb. The enzyme has two subunits, one of 59,000 daltons, and one of 62,000 daltons. In native gels stained for activity, the enzyme appears to exist in the dimeric, tetrameric, and predominantly the octameric forms.

The enzyme uses either Mg²⁺ or Mn²⁺ as the required divalent cation, and utilizes NADP at a rate less than 20% of that with NAD. With Mn²⁺ the K_m for malate²⁻ is lower than with Mg²⁺, but V_max is lower than with Mg²⁺. In the forward (malate-decarboxylating) direction with NAD, the kinetic parameters are essentially like those observed for the enzyme from C₃ plants. In the reverse reaction, run with Mn²⁺, the activity is 1.5% of that in the forward reaction. The equilibrium constant is 1.1 × 10⁻³ molar.

The kinetic mechanism of the reaction, at least in the forward direction, is sequential, with apparently random binding of all reaction components. Product inhibition patterns confirm this.

The enzyme displays a strong hysteretic lag, which is shortened by high enzyme concentrations, high substrate concentrations, and the presence of the product NADH.

The enzyme is activated by coenzyme A with K_a = 4 micromolar. AMP also shows competitive activation, with K_a = 24 micromolar. The activation by coenzyme A and AMP is additive, implying separate sites for their binding. Phosphoenolpyruvate activates the reaction at low (micromolar) concentrations, but higher concentrations of phosphoenolpyruvate cause deactivation. Fumarate²⁻ is a strong activator, with K_a = 0.3 millimolar. Fructose-1,6-bisphosphate activates the enzyme, but its most pronounced effect is in shortening the lag. Citrate is a competitive inhibitor of malate, with K_i = 4.9 millimolar.

     

Reduced nicotinamide-adenine-dinucleotide-pyrophosphorylase: A novel enzyme in seeds

An enzyme which splits reduced NAD has been partially purified from pea (Pisum sativum, Kelvedon Wonder) seeds. The activity requires orthophosphate and the products are ADP and probably NMN (dihydro NMN?). The enzyme splits the NADH₂ at the pyrophosphate bond and incorporates the phosphate into the AMP residue. NAD, NADP or NADPH₂ could not replace NADH₂. The enzyme is unstable during storage, is activated by Mg²⁺ and by Mn²⁺, and inhibited by Ca²⁺. K⁺, Li⁺ and NH₄⁺ have no effect. The possible role of this enzyme in the synthesis of ATP in seeds at the early stage of germination is discussed.     

Purification and Characterization of Cytosolic NADP Specific Isocitrate Dehydrogenase from Pisum sativum

Cytosolic NADP-specific isocitrate dehydrogenase was isolated from leaves of Pisum sativum. The purified enzyme was obtained by ammonium sulfate fractionation, ion exchange, affinity, and gel filtration chromatography. The purification procedure yields greater than 50% of the total enzyme activity originally present in the crude extract. The enzyme has a native molecular weight of 90 kilodaltons and is resolved into two catalytically active bands by isoelectric focusing. Purified NADP-isocitrate dehydrogenase exhibited K_m values of 23 micromolar for dl-isocitrate and 10 micromolar for NADP, and displayed optimum activity at pH 8.5 with both Mg²⁺ and Mn²⁺.     

Purification and Characterization of NAD Malic Enzyme from Leaves of Eleusine coracana and Panicum dichotomiflorum

NAD malic enzyme (EC 1.1.1.39), which is involved in C₄ photosynthesis, was purified to electrophoretic homogeneity from leaves of Eleusine coracana and to near homogeneity from leaves of Panicum dichotomiflorum. The enzyme from each C₄ species was found to have only one type of subunit by SDS polyacrylamide gel electrophoresis. The M_r of subunits of the enzme from E. coracana and P. dichotommiflorum was 63 and 61 kilodaltons, respectively. The native Mr of the enzyme from each species was determined by gel filtration to be about 500 kilodaltons, indicating that the NAD malic enzyme from C₄ species is an octamer of identical subunits. The purified NAD malic enzyme from each C₄ species showed similar kinetic properties with respect to concentrations of malate and NAD; each had a requirement for Mn²⁺ and activation by fructose- 1,6-bisphosphate (FBP) or CoA. A cooperativity with respect to Mn²⁺ was apparent with both enzymes. The activator (FBP) did not change the Hill value but greatly decreased K_0.5 (the concentration giving half-maximal activity) for Mn²⁺. The enzyme from E. coracana showed a very low level of activity when NADP was used as substrate, but this activity was also stimulated by FBP. Significant differences between the enzymes from E. coracana and P. dichotomiflorum were observed in their responses to the activators and their immunochemical properties. The enzyme from E. coracana was largely dependent on the activators FBP or CoA, regardless of concentration of Mn²⁺. In contrast, the enzyme from P. dichotomiflorum showed significant activity in the absence of the activator, especially at high concentrations of Mn²⁺. Both immunodiffusion and immunoprecipitation, using antiserum raised against the purified NAD malic enzyme from E. coracana, revealed partial antigenic differences between the enzymes from E. coracana and P. dichotomiflorum. The activity of the NAD malic enzyme from Amaranthus edulis, a typical NAD malic enzyme type C₄ dicot, was not inhibited by the antiserum raised against the NAD malic enzyme from E. coracana.     

Characterization of NADP-Isocitrate Dehydrogenase from Nodules of Pigeonpea (Cajanus cajan)

NADP-isocitrate dehydrogenase from nodules of pigeonpea (Cajanus cajan L. cv UPAS-120) was partially purified to about 57 folds and its properties were studied. The enzyme showed an absolute requirement for a divalent cation which was fulfilled either by Mn⁺² or Mg⁺² and to a smaller extent by Co⁺². The enzyme exhibited a sigmoidal response to increasing concentrations of Mn²⁺ (S_0.5=0.3mM). The apparent Km values for isocitrate, NADP and Mg²⁺ were 21, 23 and 280 μM, respectively. It had an optimum pH of 8.0–8.2. The enzyme activity was not affected by various organic acids, amino acids and amides. NADH inhibited the activity non-competitively with respect to NADP. An apparent inhibition by ATP and ADP was due to chelation of divalent cation. NADPH acted competitively against NADP and non-competitively against isocitrate. Glutamate caused uncompetitive inhibition with respect to NADP and competitive against isocitrate. Kinetic studies suggested the reaction mechanism to be probably random sequential. Possible regulation of the enzyme activity in the nodules via cellular redox state and the levels of reaction products is discussed.     

Activation of purified guanylate cyclase by nitric oxide requires heme comparison of heme-deficient,heme-reconstituted and heme-containing forms of soluble enzyme from bovine lung

Bovine lung soluble guanylate cyclase was purified to apparent homogeneity in a form that was deficient in heme. Heme-deficient guanylate cyclase was rapidly and easily reconstituted with heme by reacting enzyme with hematin in the presence of excess dithiothreitol, followed by removal of unbound heme by gel filtration. Bound heme was verified spectrally and NO shifted the absorbance maximum in a manner characteristic of other hemoproteins. Heme-deficient and heme-reconstituted guanylate cyclase were compared with enzyme that had completely retained heme during purification. NO and S-nitroso-N-acetylpenicillamine only marginally activated heme-deficient guanylate cyclase but markedly activated both heme-reconstituted and heme-containg forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 μM hematin in enzyme reaction mixtures containing dithiothreitol. Preformed NO-heme activated all forms of guanylate cyclase in the absence of additional heme. Guanylate cyclase activation was observed in the presence of either MgGTP or MnGTP, although the magnitude of enzyme activation was consistently greater with MgGTP. The apparent K_m for GTP in the presence of excess Mn²⁺ or Mg²⁺ was 10 μM and 85–120 μM, respectively, for unactivated guanylate cyclase. The apparent K_m for GTP in the presence of Mn²⁺ was not altered but the K_m in the presence of Mg²⁺ was lowered to 58 μM with activated enzyme. Maximal velocities were increased by enzyme activators in the presence of either Mg²⁺ or Mn²⁺. The data reported in this study indicate that purified guanylate cyclase binds heme and the latter is required for enzyme activation by NO nitroso compounds.     

Properties of leaf NAD malic enzyme from plants with C4 pathway photosynthesis

C₄ acid decarboxylation in one group of C₄-pathway species is mediated by an NAD malic enzyme. This paper reports on the partial purification and properties of this enzyme from three species of this group, Atriplex spongiosa, Amaranthus edulis, and Panicum miliaceum. Depending upon the conditions, the Atriplex spongiosa enzyme was 5–30% as active with NADP compared with NAD but the enzyme from the other species was specific for NAD. The enzyme from each species had an absolute requirement for Mn²⁺ that could not be replaced by Mg²⁺, and activity was increased several fold by low concentrations of either CoA or acetyl CoA. For the enzyme from Atriplex spongiosa and Amaranthus edulis, there was cooperativity for malate binding and the activators CoA and acetyl CoA functioned to increase the affinity of malate for the enzyme. The Hill coefficients for malate binding were approximately 2 and 4, respectively. However, with the enzyme from Panicum miliaceum, cooperative binding of malate was not apparent and activators operated by increasing V rather than the affinity for malate. Bicarbonate inhibited the enzyme from Atriplex spongiosa and Amaranthus edulis and its effect was inversely related to the concentrations of malate, NAD, and activators. The possible significance of these various allosteric effects on the regulation of the enzyme in vivo is discussed. Reactant concentrations and other conditions required for maximum activity are reported.     

The influence of the divalent cations Mn2+ and Mg2+ on the activation of particulate and digitonin-solubilized adenylate cyclase from rat fat cell membranes

A comparison was made between the activation of membrane-bound adenylate cyclase from rat fat cell membranes and the enzyme solubilized with digitonin. The isoprenaline stimulation of the particulate enzyme was enhanced by GTP, both in the presence of Mg²⁺ and Mn²⁺, but no effect of the metal ion nor of GTP was found on the K_a of isoprenaline. The K_a of sodium fluoride for enzyme stimulation was shifted to 3-fold higher concentrations when Mg²⁺ was replaced by Mn²⁺, whereas V decreased. GTP did not influence the K_a of sodium fluoride but reduced V, irrespective of the metal ion. After digitonin solubilization the enzyme was no longer responsive to isoprenaline or GTP; however, V of the sodium fluoride activation was higher in the presence of Mn²⁺ than in the presence of Mg²⁺, and the K_a was found at 15-fold higher concentrations. Both the solubilized and the particulate adenylate cyclase were inhibited by adenosine; this inhibition was also seen with the fluoride stimulated enzyme. We conclude that solubilization with digitonin did not result in an enzyme preparation which preferentially turns over MnATP²⁺, although the fat cell adenylate cyclase possesses a metal ion regulatory site with a higher affinity for Mn²⁺ than for Mg²⁺. The data suggest that the guanyl nucleotide regulatory site and the sodium fluoride-sensitive site are located on different subunits while there is an interaction between the metal ion regulatory site and the fluoride-sensitive site.     

Regulation of rat-kidney cortex fructose-1,6-bisphosphatase activity. I. Effects of fructose-2,6-bisphosphate and divalent cations

• 1.1. The native rat-kidney cortex Fructose-1,6-BPase is differentially regulated by Mg²⁺ and Mn²⁺.
• 2.2. Mg²⁺ binding to the enzyme is hyperbolic and large concentrations of the cation are non-inhibitory.
• 3.3. Mn²⁺ produces a 10-fold rise in V_max higher than Mg²⁺. [Mn²⁺]_0.5 is much larger than [Mg²⁺]_0.5. At elevated [Mn²⁺] inhibition is observed.
• 4.4. Mg²⁺ and Mn²⁺ produce antagonistic effects on the inhibition of the enzyme by high substrate.
• 5.5. Fru-2,6-P₂ inhibits the enzyme by rising the S_0.5 and favouring a sigmoidal kinetics.
• 6.6. The inhibition by Fru-2,6-P₂ is released by Mg²⁺ and more powerfully by Mn²⁺ increasing the I_0.5.

     

Allosteric regulation of NAD(NADP)-dependent glyceraldehyde-3-phosphate dehydrogenase from Chlorella by α-amino acids,dithiothreitol and ATP

α-Amino acids (glycine, serine, histidine, aspartic acid and cysteine) and dithiothreitol (DTT) have been shown to activate both activities of the NAD(NADP)-dependent glyceraldehyde-3-phosphate dehydrogenase from Chlorella. The activation is allosteric and reaches 200–700%. The Hill coefficient values are close to 2 with all activators. ATP activates NADP-dependent but inhibits NAD-dependent activity, n_app and K values being the same for both enzyme activities. In this case positive cooperativity is also observed (n_app = 2.2). The present findings reveal the possible regulation of GAPD function in Chlorella with each of the coenzymes.     

Physical and kinetic properties of sheep liver pyridoxine kinase

Pyridoxine kinase purified from sheep liver was found to consist of a single polypeptide chain with a molecular weight of 60,000 as determined by gel filtration, sedimentation equilibrium ultracentrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric pH of the enzyme was 5.1, and the pH optimum was between 5.5 and 6.0. The enzyme required divalent cations for activity. At cation concentrations of 80 μm, the enzyme activity with each cation was in the order of Zn²⁺ > Mn²⁺ > Mg²⁺. At cation concentrations of 400 μm, the enzyme activity with each cation was in the order of Mn²⁺ > Zn²⁺ > Mg²⁺. Excess free divalent cation inhibited the enzyme. Pyridoxine kinase also required monovalent cations. The enzyme activation was greatest with K⁺, then Rb⁺ and NH₄⁺, whereas the enzyme had very little activity with Na⁺, Li⁺, or Cs⁺. Na⁺ did not interfere with the activation by K⁺. The activation of the kinase by K⁺, NH₄⁺, and Rb⁺ followed Michaelis-Menten kinetics, and the apparent K_m values for the cations were 8.9, 3.7, and 5.3 mm, respectively. Increasing the potassium concentration lowered the apparent K_m value of the enzyme for pyridoxine and had little or no effect on the K_m for ZnATP^2? or the V of the kinase-catalyzed reaction.     

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.     

Purification and Characterization of a Tripeptidase from Lactococcus lactis subsp. cremoris Wg2

A tripeptidase from a cell extract of Lactococcus lactis subsp. cremoris Wg2 has been purified to homogeneity by DEAE-Sephacel and phenyl-Sepharose chromatography followed by gel filtration over a Sephadex G-100 SF column and a high-performance liquid chromatography TSK G3000 SW column. The enzyme appears to be a dimer with a molecular weight of between 103,000 and 105,000 and is composed of two identical subunits each with a molecular weight of about 52,000. The tripeptidase is capable of hydrolyzing only tripeptides. The enzyme activity is optimal at pH 7.5 and at 55°C. EDTA inhibits the activity, and this can be reactivated with Zn²⁺, Mn²⁺, and partially with Co²⁺. The reducing agents dithiothreitol and β-mercaptoethanol and the divalent cation Cu²⁺ inhibit tripeptidase activity. Kinetic studies indicate that the peptidase hydrolyzes leucyl-leucyl-leucine with a K_m of 0.15 mM and a V_max of 151 μmol/min per mg of protein.     

Fructose-1,6-bisphosphatase in mantle of the sea mussel Mytilus galloprovincialis Lmk—I. Purification and ion requirements

• 1.1. The enzyme fructose-1,6-bisphosphatase was purified from the mantle of the sea mussel Mytilus galloprovincialis Lmk. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The mol. wt and subunit mol. wt of the enzyme were 105,000 and 27,000, respectively.
• 2.2. Divalent cations are essential for the enzyme activity. In the absence of chelating agents, FBPase 1 exhibits hyperbolic kinetics with respect to Mn²⁺, Zn²⁺ and Mg²⁺. The K_m for Mg²⁺ is lower than the physiological concentration of cation in the tissue, whereas its K_m for Mn²⁺ and Zn²⁺ is greater than the respective in vivo concentrations.
• 3.3. The joint action of Mg²⁺ and Zn²⁺ increases the affinity of the enzyme for the substrate Fru-1,6-P₂, though V_max is reduced.
• 4.4. Na⁺ strongly inhibits the enzyme even at very low concentrations. K⁺ has no effect whatsoever.

     

Regulation of ADP-Glucose Pyrophosphorylase from Chlorella vulgaris

ADP-glucose pyrophosphorylase was partially purified from Chlorella vulgaris 11h. 3-Phosphoglycerate activated the enzyme by lowering the Michaelis constant for glucose-1-phosphate (from 0.97 to 0.36 millimolar in the presence of 2 millimolar phosphoglycerate) and ATP (from 0.23 to 0.10 millimolar), as well as increasing the V_max. Saturation curves for 3-phosphoglycerate were hyperbolic and the activator concentration at half V_max value for 3-phosphoglycerate was 0.41 millimolar either in the presence or absence of phosphate. Phosphate inhibited the enzyme in a competitive manner with respect to glucose-1-phosphate, but did not affect the Michaelis constant value for ATP. 3-Phosphoglycerate changed neither the inhibitor concentration at half V_max value of 1.0 millimolar for phosphate nor the hyperbolic inhibition kinetics for phosphate. The enzyme required divalent cations for its activity. The activation curves for Mn²⁺ and Mg²⁺ were highly sigmoidal. The activator concentration at half V_max values for Mn²⁺ and Mg²⁺ were 2.8 and 3.7 millimolar, respectively. With optimal cations, the Michaelis constant values for ATP-Mn and ATP-Mg were 0.1 and 0.4 millimolar, respectively.