Interaction of acetyl phosphate and carbamyl phosphate with plant phosphoenolpyruvate carboxylase.

Acetyl phosphate produced an increase in the maximum velocity (Vmax. for the carboxylation of phosphoenolpyruvate catalysed by phosphoenolpyruvate carboxylase. The limiting Vmax. was 22.2 mumol X min-1 X mg-1 (185% of the value without acetyl phosphate). This compound also decreased the Km for phosphoenolpyruvate to 0.18 mM. The apparent activation constants for acetyl phosphate were 1.6 mM and 0.62 mM in the presence of 0.5 and 4 mM-phosphoenolpyruvate respectively. Carbamyl phosphate produced an increase in Vmax. and Km for phosphoenolpyruvate. The variation of Vmax./Km with carbamyl phosphate concentration could be described by a model in which this compound interacts with the carboxylase at two different types of sites: an allosteric activator site(s) and the substrate-binding site(s). Carbamyl phosphate was hydrolysed by the action of phosphoenolpyruvate carboxylase. The hydrolysis produced Pi and NH4+ in a 1:1 relationship. Values of Vmax. and Km were 0.11 +/- 0.01 mumol of Pi X min-1 X mg-1 and 1.4 +/- 0.1 mM, respectively, in the presence of 10 mM-NaHCO3. If HCO3- was not added, these values were 0.075 +/- 0.014 mumol of Pi X min-1 X mg-1 and 0.76 +/- 0.06 mM. Vmax./Km showed no variation between pH 6.5 and 8.5. The reaction required Mg2+; the activation constants were 0.77 and 0.31 mM at pH 6.5 and 8.5 respectively. Presumably, carbamyl phosphate is hydrolysed by phosphoenolpyruvate carboxylase by a reaction the mechanism of which is related to that of the carboxylation of phosphoenolpyruvate.     

Structure and kinetics of phosphonopyruvate hydrolase from Variovorax sp. Pal2: new insight into the divergence of catalysis within the PEP mutase/isocitrate lyase superfamily

Phosphonopyruvate (P-pyr) hydrolase (PPH), a member of the phosphoenolpyruvate (PEP) mutase/isocitrate lyase (PEPM/ICL) superfamily, hydrolyzes P-pyr and shares the highest sequence identity and functional similarity with PEPM. Recombinant PPH from Variovorax sp. Pal2 was expressed in Escherichia coli and purified to homogeneity. Analytical gel filtration indicated that the protein exists in solution predominantly as a tetramer. The PPH pH rate profile indicates maximal activity over a broad pH range. The steady-state kinetic constants determined for a rapid equilibrium ordered kinetic mechanism with Mg2+ binding first (Kd = 140 +/- 40 microM), are kcat = 105 +/- 2 s(-1) and P-pyr Km = 5 +/- 1 microM. PEP (slow substrate kcat = 2 x 10(-4) s(-1)), oxalate, and sulfopyruvate are competitive inhibitors with Ki values of 2.0 +/- 0.1 mM, 17 +/- 1 microM, and 210 +/- 10 microM, respectively. Three PPH crystal structures have been determined, that of a ligand-free enzyme, the enzyme bound to Mg2+ and oxalate (inhibitor), and the enzyme bound to Mg2+ and P-pyr (substrate). The complex with the inhibitor was obtained by cocrystallization, whereas that with the substrate was obtained by briefly soaking crystals of the ligand-free enzyme with P-pyr prior to flash cooling. The PPH structure resembles that of the other members of the PEPM/ICL superfamily and is most similar to the functionally related enzyme, PEPM. Each monomer of the dimer of dimers exhibits an (alpha/beta)8 barrel fold with the eighth helix swapped between two molecules of the dimer. Both P-pyr and oxalate are anchored to the active site by Mg2+. The loop capping the active site is disordered in all three structures, in contrast to PEPM, where the equivalent loop adopts an open or disordered conformation in the unbound state but sequesters the inhibitor from solvent in the bound state. Crystal packing may have favored the open conformation of PPH even when the enzyme was cocrystallized with the oxalate inhibitor. Structure alignment of PPH with other superfamily members revealed two pairs of invariant or conservatively replaced residues that anchor the flexible gating loop. The proposed PPH catalytic mechanism is analogous to that of PEPM but includes activation of a water nucleophile with the loop Thr118 residue.     

Purification and characterization of cytosolic pyruvate kinase from Brassica napus (rapeseed) suspension cell cultures: implications for the integration of glycolysis with nitrogen assimilation.

Cytosolic pyruvate kinase (PKc) from Brassica napus suspension cells was purified 201-fold to electrophoretic homogeneity and a final specific activity of 51 micromol phosphoenolpyruvate utilized per min per mg protein. SDS/PAGE and gel filtration analyses of the final preparation indicated that this PKc is a 220-kDa homotetramer composed of 56-kDa subunits. The enzyme was relatively heat-stable and displayed a broad pH optimum of pH 6.8. PKc activity was absolutely dependent upon the simultaneous presence of a bivalent and univalent cation, with Mg2+ and K+ fulfilling this requirement. Hyperbolic saturation kinetics were observed for phosphoenolpyruvate, ADP, Mg2+ and K+ (apparent Km values = 0.12, 0.075, 0.21 and 0.48 mM, respectively). Although the enzyme utilized UDP, CDP and IDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate, oxalate, and the flavonoids rutin and quercetin were the most effective inhibitors (I50 values = 4, 0.3, 0.07, and 0.10 mM, respectively). L-Aspartate functioned as an activator (Ka = 0.31 mM) by causing a 40% increase in Vmax while completely reversing the inhibition of PKc by L-glutamate. Reciprocal control by L-aspartate and L-glutamate is specific for these amino acids and provides a rationale for the in vivo activation of PKc that occurs during periods of enhanced NH +4-assimilation. Allosteric features of B. napus PKc are compared with those of B. napus phosphoenolpyruvate carboxylase. A model is presented that highlights the pivotal role of L-aspartate and L-glutamate in the coordinate regulation of these key phosphoenolpyruvate utilizing cytosolic enzymes.     

Photosynthetic phosphoenolpyruvate carboxylases: characteristics of alloenzymes from leaves of c(3) and c(1) plants

A detailed comparison of green leaf phosphoenolpyruvate carboxylases from the C(4)-species Atriplex spongiosa and the C(3)-species Atriplex hastata revealed significant physical and kinetic differences. The two alloenzymes can be separated by anion exchange chromatography but have comparable molecular weights (350,000). Maximal velocity estimates were 38.0 and 1.48 micromoles per minute per milligram of chlorophyll for the carboxylases of A. spongiosa and A. hastata, respectively. Km phosphoenolpyruvate estimates were 0.49 and 0.08 mm for the C(4)A. spongiosa and C(3)A. hastata and the Km Mg estimates were 0.33 mm for the C(4) species and 0.017 mm for the C(3) species. The activity of the phosphoenolpyruvate carboxylase of A. spongiosa is more sensitive to chloride and phosphate than the phosphoenolpyruvate carboxylase of A. hastata, but both are equally sensitive to Mg chelating substances such as ATP, ADP, and citrate if assayed at their respective Km Mg values. A survey of the phosphoenolpyruvate carboxylases from 18 C(4) and C(3) species resulted in mean maximal velocity estimates of 29.0 +/- 13.2 and 1.50 +/- 0.57 micromoles per minute per milligram of chlorophyll for the C(4) species and C(3) species, respectively. Km phosphoenolpyruvate estimates were 0.59 +/- 0.35 mm and 0.14 +/- 0.07 mm for the C(4) and C(3), and Km Mg estimates were 0.50 +/- 0.30 and 0.097 +/- 0.057 mm for C(4) and C(3). All differences between means were significant at the 0.01 confidence level, supporting our hypothesis that the phosphoenolpyruvate carboxylase alloenzymes of C(4) and C(3) plants are functionally different and are associated with different photosynthetic roles. Both function in the photosynthetic production of C(4) acids, the phosphoenolpyruvate carboxylase of C(4) species largely producing malate or aspartate (or both) as a photosynthetic intermediate and the phosphoenolpyruvate carboxylase of C(3) species producing malate or aspartate (or both) as a photosynthetic product.     

Mechanistic studies of phosphoenolpyruvate carboxylase from Zea mays utilizing formate as an alternate substrate for bicarbonate.

Formate is an alternate substrate for bicarbonate in the reaction with PEP catalyzed by phosphoenolpyruvate carboxylase from Zea mays, producing formyl phosphate and pyruvate. The Km for formate is 25 +/- 2 mM, and the maximum velocity is 1% of that for bicarbonate at pH 8.0. Use of [18O]formate produces inorganic phosphate containing 1 equiv of 18O, but no label is incorporated into residual phosphoenolpyruvate. PEP carboxylase catalyzes the hydrolysis of phosphoglycolate or L-phospholactate 2000 times more slowly and D-phospholactate 4000 times more slowly than the reaction between bicarbonate and PEP.     

Bacterial acetone carboxylase is a manganese-dependent metalloenzyme

Bacterial acetone carboxylase catalyzes the ATP-dependent carboxylation of acetone to acetoacetate with the concomitant production of AMP and two inorganic phosphates. The importance of manganese in Rhodobacter capsulatus acetone carboxylase has been established through a combination of physiological, biochemical, and spectroscopic studies. Depletion of manganese from the R. capsulatus growth medium resulted in inhibition of acetone-dependent but not malate-dependent cell growth. Under normal growth conditions (0.5 microm Mn2+ in medium), growth with acetone as the carbon source resulted in a 4-fold increase in intracellular protein-bound manganese over malate-grown cells and the appearance of a Mn2+ EPR signal centered at g = 2 that was absent in malate-grown cells. Acetone carboxylase purified from cells grown with 50 microm Mn2+ had a 1.6-fold higher specific activity and 1.9-fold higher manganese content than cells grown with 0.5 microm Mn2+, consistently yielding a stoichiometry of 1.9 manganese/alpha2beta2gamma2 multimer, or 0.95 manganese/alphabetagamma protomer. Manganese in acetone carboxylase was tightly bound and not removed upon dialysis against various metal ion chelators. The addition of acetone to malate-grown cells grown in medium depleted of manganese resulted in the high level synthesis of acetone carboxylase (15-20% soluble protein), which, upon purification, exhibited 7% of the activity and 6% of the manganese content of the enzyme purified from acetone-grown cells. EPR analysis of purified acetone carboxylase indicates the presence of a mononuclear Mn2+ center, with possible spin coupling of two mononuclear sites. The addition of Mg.ATP or Mg.AMP resulted in EPR spectral changes, whereas the addition of acetone, CO2, inorganic phosphate, and acetoacetate did not perturb the EPR. These studies demonstrate that manganese is essential for acetone carboxylation and suggest a role for manganese in nucleotide binding and activation.     

Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP-utilizing enzymes.

The synthesis of 10 new phosphoenolpyruvate (PEP) analogues with modifications in the phosphate and the carboxylate function is described. Included are two potential irreversible inhibitors of PEP-utilizing enzymes. One incorporates a reactive chloromethylphosphonate function replacing the phosphate group of PEP. The second contains a chloromethyl group substituting for the carboxylate function of PEP. An improved procedure for the preparation of the known (Z)- and (E)-3-chloro-PEP is also given. The isomers were obtained as a 4 : 1 mixture, resolved by anion-exchange chromatography after the last reaction step. The stereochemistry of the two isomers was unequivocally assigned from the (3)J(H-C) coupling constants between the carboxylate carbons and the vinyl protons. All of these and other known PEP-analogues were tested as reversible and irreversible inhibitors of Mg2+- and Mn2+- activated PEP-utilizing enzymes: enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), pyruvate kinase, PEP carboxylase and enolase. Without exception, the most potent inhibitors were those with substitution of a vinyl proton. Modification of the phosphate and the carboxylate groups resulted in less effective compounds. Enzyme I was the least tolerant to such modifications. Among the carboxylate-modified analogues, only those replaced by a negatively charged group inhibited pyruvate kinase and enolase. Remarkably, the activity of PEP carboxylase was stimulated by derivatives with neutral groups at this position in the presence of Mg2+, but not with Mn2+. For the irreversible inhibition of these enzymes, (Z)-3-Cl-PEP was found to be a very fast-acting and efficient suicide inhibitor of enzyme I (t(1/2) = 0.7 min).     

Studies on 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe)from Escherichia coli K12. 2. Kinetic properties.

1. Co2+ is not a cofactor for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe). 2. The following analogues of phosphoenolpyruvate were tested as inhibitors of 3-deoxy-D-arabinoheptolosonate-7-phosphate synthetase(phe): pyruvate, lactate, glycerate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-methylphosphoenolpyruvate, 3-ethylphosphoenolpyruvate and 3,3-demethylphosphoenolpyruvate. The rusults obtained indicate that the binding of phosphoenolpyruvate to the enzyme requires a phosphoryl group on the C-2 position of the substrate and one free hydrogen atom at the C-3 position. 3. The dead-end inhibition pattern observed with the substrate analogue 2-phosphoglycerate when either phosphoenolpyruvate or erythrose 4-phosphate was the variable substrate is inconsistent with a ping-pong mechanism and indicates that the reaction mechanism for this enzyme must be sequential. The following kinetic constants were determined:Km for phosphoenolpyruvate, 0.08 +/- 0.04 mM; Km for erythrose 4-phosphate, 0.9 +/- 0.3 mM; K is for competitive inhibition by 2-phosphoglycerate with respect to phosphoenolpyruvate, 1.0 +/- 0.1 mM. 4. The enzyme was observed to have a bell-shaped pH PROFILE WITH A PH OPTIMUM OF 7.0. The effects of pH ON V and V/(Km for phosphoenolpyruvate) indicated that an ionizing group of pKa 8.0-8.1 is involved in the catalytic activity of the enzyme. The pKa of this group is unaffected by the binding of phosphoenolpyruvate.     

(E)-3-Cyanophosphoenolpyruvate, a new inhibitor of phosphoenolpyruvate-dependent enzymes

(E)-3-Cyanophosphoenolpyruvate has been synthesized by reacting dimethyl chlorophosphate with the potassium enolate of ethyl cyanopyruvate. The resulting trialkyl ester was deesterified with bromotrimethylsilane followed by potassium hydroxide. Subsequent treatment with Dowex-50-H+ resin and cyclohexylamine afforded the tricyclohexylammonium salt; only the E geometric isomer was obtained. This compound can be photoisomerized to a 70:30 E:Z mixture. (E)-3-Cyanophosphoenolpyruvate is an excellent competitive inhibitor of phosphoenolpyruvate carboxylase [KI(Mn2+) = 16 microM, KI(Mg2+) = 1360 microM], pyruvate kinase [KI(Mn2+) = 0.085 microM, KI(Mg2+) = 0.76 microM], and enolase [KI(Mn2+) = 360 microM, KI(Mg2+) = 280 microM]. The compound is a substrate for pyruvate kinase (Vmax approximately 1% of phosphoenolpyruvate rate), but not for the other two enzymes. No irreversible inactivation is observed with phosphoenolpyruvate carboxylase of pyruvate kinase.     

The inhibition of ATP-dependent shape change of human erythrocyte ghosts correlates with an inhibition of Mg(2+)-ATPase activity by fluoride and aluminofluoride complexes.

The vanadate-sensitive Mg(2+)-dependent ATPase activity of the human erythrocyte ghost is believed to be involved in the shape change events that convert echinocytic ghosts to smoothed forms (biconcave discs and stomatocytes). At physiological salt concentration, pH 7.4, 2 mM ATP, 5 mM Mg2+ and 1 mM EGTA, the Mg(2+)-ATPase activity of ghosts was inhibited strongly by millimolar concentrations of sodium fluoride: I50 = 1.31 +/- 0.23 mM (mean +/- S.D.; n = 12). The addition of aluminium chloride to 15 microM reduced the concentration of NaF required for 50% inhibition to 0.76 +/- 0.21 mM (n = 10). Aluminium alone had only a small inhibitory effect on the ATPase activity (13 +/- 9%; n = 10). Desferrioxamine, a strong chelator of tervalent aluminium ion, failed to reverse the inhibition by fluoride and reversed the inhibition in the presence of aluminium and fluoride back to those values obtained with fluoride alone. Of several metal salts tested only beryllium sulfate was able to replace aluminium as an effective inhibitor in the presence of fluoride. Inhibition of the Mg(2+)-ATPase activity by fluoride and the aluminofluoride complexes correlated with an inhibition of the rate of MgATP-dependent change in red cell ghost shape from echinocytes to smoothed forms. All gross morphological changes of the smoothing process were affected, including the production of discocytes, stomatocytes and endocyctic vesicles.     

Activation of acetyl-CoA carboxylase. Purification and properties of a Mn2+-dependent phosphatase

Acetyl-CoA carboxylase was isolated from rat liver by polyethylene glycol precipitation and avidin affinity chromatography. Sodium dodecyl sulfate electrophoresis of the enzyme gives one protein band (Mr 250,000). Phosphate analysis of the carboxylase showed the presence of 8.3 mol of phosphate/mol of subunit (Mr 250,000). The purified carboxylase has low activity in the absence of citrate (specific activity = 0.3 units/mg). However, addition of 10 mM citrate activates the carboxylase 10-fold, with half-maximal activation observed at 2 mM citrate, well above the physiological citrate level. Using this carboxylase as a substrate, we have isolated from rat liver a protein that activates the enzyme about 10-fold. This protein has been purified to near homogeneity (Mr 90,000). Incubation of this protein with 32P-labeled acetyl-CoA carboxylase results in a time-dependent activation of carboxylase with concomitant release of 32Pi, indicating that this protein is a phosphoprotein phosphatase. Both activation and dephosphorylation are dependent on Mn2+, but not citrate. This phosphatase does not hydrolyze p-nitrophenyl phosphate but does show high affinity for acetyl-CoA carboxylase (Km = 0.2 microM) as compared to its action on phosphorylase a (Km = 5.5 microM) and phosphohistone (Km = 20 microM). Activated acetyl-CoA carboxylase was isolated after dephosphorylation by the phosphatase. Such preparations contain about 5 mol of phosphate/mol of subunit and have specific activities of 2.6-3.0 units/mg in the absence of citrate. These activities are comparable to those of the phosphorylated carboxylase in the presence of 10 mM citrate. Thus, dephosphorylation by the Mn2+-dependent phosphatase renders the carboxylase citrate-independent, as compared to the phosphorylated form, which is citrate-dependent. To our knowledge this is the first report of a preparation of animal acetyl-CoA carboxylase that has substantial catalytic activity independent of citrate.     

Divergent effects of the malignant hyperthermia-susceptible Arg(615)-->Cys mutation on the Ca(2+) and Mg(2+) dependence of the RyR1

The sarcoplasmic reticulum (SR) Ca(2+) release channel (RyR1) from malignant hyperthermia-susceptible (MHS) porcine skeletal muscle has a decreased sensitivity to inhibition by Mg(2+). This diminished Mg(2+) inhibition has been attributed to a lower Mg(2+) affinity of the inhibition (I) site. To determine whether alterations in the Ca(2+) and Mg(2+) affinity of the activation (A) site contribute to the altered Mg(2+) inhibition, we estimated the Ca(2+) and Mg(2+) affinities of the A- and I-sites of normal and MHS RyR1. Compared with normal SR, MHS SR required less Ca(2+) to half-maximally activate [(3)H]ryanodine binding (K(A,Ca): MHS = 0.17 +/- 0.01 microM; normal = 0.29 +/- 0.02 microM) and more Ca(2+) to half-maximally inhibit ryanodine binding (K(I,Ca): MHS = 519.3 +/- 48.7 microM; normal = 293.3 +/- 24.2 microM). The apparent Mg(2+) affinity constants of the MHS RyR1 A- and I-sites were approximately twice those of the A- and I-sites of the normal RyR1 (K(A,Mg): MHS = 44.36 +/- 4.54 microM; normal = 21.59 +/- 1.66 microM; K(I,Mg): MHS = 660.8 +/- 53.0 microM; normal = 299.2 +/- 24.5 microM). Thus, the reduced Mg(2+) inhibition of the MHS RyR1 compared with the normal RyR1 is due to both an enhanced selectivity of the MHS RyR1 A-site for Ca(2+) over Mg(2+) and a reduced Mg(2+) affinity of the I-site.     

Differentiation of the slow-binding mechanism for magnesium ion activation and zinc ion inhibition of human placental alkaline phosphatase

The binding mechanism of Mg(2+) at the M3 site of human placental alkaline phosphatase was found to be a slow-binding process with a low binding affinity (K(Mg(app.)) = 3.32 mM). Quenching of the intrinsic fluorescence of the Mg(2+)-free and Mg(2+)-containing enzymes by acrylamide showed almost identical dynamic quenching constant (K(sv) = 4.44 +/- 0.09 M(-1)), indicating that there is no gross conformational difference between the M3-free and the M3-Mg(2+) enzymes. However, Zn(2+) was found to have a high affinity with the M3 site (K(Zn(app.)) = 0.11 mM) and was observed as a time-dependent inhibitor of the enzyme. The dependence of the observed transition rate from higher activity to lower activity (k(obs)) at different zinc concentrations resulted in a hyperbolic curve suggesting that zinc ion induces a slow conformational change of the enzyme, which locks the enzyme in a conformation (M3'-Zn) having an extremely high affinity for the Zn(2+) (K*(Zn(app.)) = 0.33 microM). The conformation of the M3'-Zn enzyme, however, is unfavorable for the catalysis by the enzyme. Both Mg(2+) activation and Zn(2+) inhibition of the enzyme are reversible processes. Structural information indicates that the M3 site, which is octahedrally coordinated to Mg(2+), has been converted to a distorted tetrahedral coordination when zinc ion substitutes for magnesium ion at the M3 site. This conformation of the enzyme has a small dynamic quenching constant for acrylamide (K(sv) = 3.86 +/- 0.04 M(-1)), suggesting a conformational change. Both Mg(2+) and phosphate prevent the enzyme from reaching this inactive structure. GTP plays an important role in reactivating the Zn-inhibited enzyme activity. We propose that, under physiological conditions, magnesium ion may play an important modulatory role in the cell for protecting the enzyme by retaining a favorable geometry of the active site needed for catalysis.     

Dissociation and characterization of enzymes from a multienzyme complex involved in CO2 fixation.

A multienzyme complex from Euglena, molecular weight about 360,000, containing phosphoenolpyruvate carboxylase, malate dehydrogenase, and acetyl-coenzyme A carboxylase has been dissociated into active constituent enzymes. The respective molecular weights are 183,000, 67,000, and 127,000. The malate dehydrogenase contained in the complex is electrophoretically distinct from other malate dehydrogenase isozymes found in Euglena. The K-m for HCO3minus of the free and complexed acetyl-CoA carboxylase is 4.2-5.4 mM, and the substrate dependency for acetyl-CoA describes a sigmoidal relationship. The HCO3minus K-m for the free phosphoenolpyruvate carboxylase is 7.3-5.4 mM while that for the same enzyme contained in the complex is 0.7-1.3 mM. Both the free and complexed forms ofphosphoenolpyruvate carboxylase have a K-m for phosphoenolpyruvate of 0.9-1.7 mM. The latter enzyme in both the complex and free forms is stimulated by NADH, acetyl-CoA, and ATP. In the free phosphoenolpyruvate carboxylase, the stimulation passes through a maximum depending on effector concentration. The effect of NADH is to increase V-max while K-m values remain unmodified.     

Multiple affinity states of opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells. Opiate agonist association rate is a function of receptor occupancy

The existence of multiple affinity states for the opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells has been demonstrated by competition binding studies with tritiated diprenorphine and [D-Ala2, D-Leu5]enkephalin (DADLE). In the presence of 10 mM Mg2+, all receptors exist in a high affinity state with Kd = 1.88 +/- 0.16 nM. Addition of 10 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) decreased the affinity of DADLE to Kd = 8.08 +/- 0.93 nM. However, in the presence of 100 mM Na+, which is required for opiate inhibition of adenylate cyclase activity, analysis of competition binding data revealed three sites: the first, consisting of 17.5% of total receptor population has a Kd = 0.38 +/- 0.18 nM; the second, 50.6% of the population, has a Kd = 6.8 +/- 2.2 nM; and the third, 31.9% of the population, has a Kd of 410 +/- 110 nM. Thus, in the presence of sodium, a high affinity complex between receptor (R), GTP binding component (Ni), and ligand (L) was formed which was different from that formed in the absence of sodium. These multiple affinity states of receptor in the hybrid cells are agonist-specific, and the percentage of total opiate receptor in high affinity state is relatively constant in various concentrations of Na+. Multiple affinity states of opiate receptor can be demonstrated further by Scatchard analysis of saturation binding studies with [3H]DADLE. In the presence of Mg2+, or Gpp(NH)p, analysis of [3H]DADLE binding demonstrates that opiate receptor can exist in a single affinity state, with apparent Kd values of [3H]DADLE in 10 mM Mg2+ = 1.75 +/- 0.28 nM and in 10 microM Gpp(NH)p = 0.85 +/- 0.12 nM. There is a reduction of Bmax value from 0.19 +/- 0.02 nM in the presence of Mg2+ to 0.14 +/- 0.03 nM in the presence of Gpp(NH)p. In the presence of 100 mM Na+, Scatchard analysis of saturation binding of [3H]DADLE reveals nonlinear plots; two-site analysis of the curves yields Kd = 0.43 +/- 0.09 and 7.9 +/- 3.2 nM. These Kd values are analogous to that obtained with competition binding studies. Again, this conversion of single site binding Scatchard plots to multiple sites binding plots in the presence of Na+ is restricted to 3H-agonist binding only.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro phosphorylation and inactivation of rat liver acetyl-CoA carboxylase purified by avidin affinity chromatography

Acetyl-CoA carboxylase (EC 6.4.1.2) has been isolated from rat liver by an avidin-affinity chromatography technique. This preparation has a specific activity of 1.17 +/- 0.06 U/mg and appears as a major (240,000 dalton) and minor (140,000 dalton) band on SDS-polyacrylamide gel electrophoresis. Enzyme isolated by this technique can incorporate 1.09 +/- 0.07 mol phosphate per mol enzyme (Mr = 480,000) when incubated with the catalytic subunit of the cyclic AMP-dependent protein kinase at 30 degrees C for 1 h. The associated activity loss under these conditions is 57 +/- 4.0% when the enzyme is assayed in the presence of 2.0 mM citrate. Less inactivation is observed when the enzyme is assayed in the presence of 5.0 mM citrate. The specific protein inhibitor of the cyclic AMP-dependent protein kinase blocks both the protein kinase stimulated phosphorylation and inactivation of acetyl-CoA carboxylase. The phosphorylated, inactivated rat liver carboxylase can be partially dephosphorylated and reactivated by incubation with a partially purified protein phosphatase. Preparations of acetyl-CoA carboxylase also contained an endogenous protein kinase(s) which incorporated 0.26 +/- 0.11 mol phosphate per mol carboxylase (Mr = 480,000) accompanied by a 26 +/- 9% decline in activity. We have additionally confirmed that the rat mammary gland enzyme, also isolated by avidin affinity chromatography, can be both phosphorylated and inactivated upon incubation with the cyclic AMP-dependent kinase.     

The kinetic mechanism of yeast inorganic pyrophosphatase

The kinetic mechanism of yeast inorganic pyrophosphatase (PPase) was examined by carrying out initial velocity studies. Ca2+ and Rh(H2O)4(methylenediphosphonate) (Rh(H2O)4PCP) were used as dead-end inhibitors to study the order of binding of Cr(H2O)4PP to the substrate site and Mg2+ to the "low affinity" activator site on the enzyme. Competitive inhibition was observed for Ca2+ vs Mg2+ (Kis = 0.93 +/- 0.03 mM), for Rh(H2O)4PCP vs Cr(H2O)4PP (Kis = 0.25 +/- 0.07 mM), and for RH(H2O)4PCP vs Mg2+ (Kis = 0.38 +/- 0.03 mM). Uncompetitive inhibition was observed for Ca2+ vs Cr(H2O)4PP (Kii = 0.49 +/- 0.01). On the basis of these results a rapid equilibrium ordered mechanism in which Cr(H2O)4PP binding precedes Mg2+ ion binding is proposed. The inert substrate analog, Mg(imidodiphosphate) (MgPNP) was shown to induce Mg2+ inhibition of the PPase-catalyzed hydrolysis of MgPP. The Mg2+ inhibition observed was competitive vs MgPP and partial. These results suggest that Mg2+/MgPNP release from the enzyme occurs in preferred rather than strict order and that the Mg2+/MgPP-binding steps are at steady state. Zn2+, Co2+, and Mn2+ (but not Mg2+) displayed activator inhibition of the PPase-catalyzed hydrolysis of PPi (this study) and of Cr(H2O)4PP (W.B. Knight, S. Fitts, and D. Dunaway-Mariano, (1981) Biochemistry 20, 4079). These findings suggest that cofactor release from the low affinity cofactor site on the enzyme must precede product release and that Zn2+, Mn2+, and Co2+ (but not Mg2+) have high affinities for the cofactor sites on both the PPase.M.MPP and PPase.M.M(P)2 complexes. The role of the metal cofactor in determining PPase substrate specificity was briefly explored by testing the ability of the Mg2+ complex of tripolyphosphate (PPPi) (a substrate for the Zn2+-activated enzyme but not the Mg2+-activated enzyme) to induce Mg2+ inhibition of PPase-catalyzed hydrolysis of MgPP. MgPPP was shown to be as effective as MgPNP in inducing competitive Mg2+ inhibition (vs MgPP). This result suggests that the low affinity Mg2+ cofactor-binding site present in the enzyme-MgPP complex is maintained in the enzyme-MgPPP complex. Thus, failure of Mg2+ to bind to the enzyme-MgPPP complex was ruled out as a possible explanation for the failure of the Mg2+-activated enzyme to catalyze the hydrolysis of MgPPP.     

Divalent cations regulate glucagon binding. Evidence for actions on receptor-Ns complexes and on receptors uncoupled from Ns

The effects of Mg2+ or ethylenediaminetetraacetic acid (EDTA) on 125I-glucagon binding to rat liver plasma membranes have been characterized. In the absence of guanosine 5'-triphosphate (GTP), maximal binding of 125I-glucagon occurs in the absence of added Mg2+. Addition of EDTA or Mg2+ diminishes binding in a dose-dependent manner. In the presence of GTP, maximal binding occurs in the presence of 2.5 mM Mg2+ (EC50 = 0.3 mM) while EDTA or higher concentrations of Mg2+ diminish binding. Response to exogenous Mg2+ or EDTA depends on the concentration of Mg2+ in the membranes and may vary with the method used for membrane isolation. Solubilized 125I-glucagon-receptor complexes fractionate on gel filtration columns as high molecular weight, GTP-sensitive complexes in which receptors are coupled to regulatory proteins and lower molecular weight, GTP-insensitive complexes in which receptors are not coupled to other components of the adenylyl cyclase system. In the absence of GTP, 40 mM Mg2+ or 5 mM EDTA diminishes receptor affinity for hormone (from KD = 1.2 +/- 0.1 nM to KD = 2.6 +/- 0.3 nM) and the fraction of 125I-glucagon in high molecular weight receptor-Ns complexes without affecting site number (Bmax = 1.8 +/- 0.1 pmol/mg of protein). Thus, while GTP promotes disaggregation of receptor-Ns complexes, Mg2+ or EDTA diminishes the affinity with which these species bind hormone. In the presence of GTP, hormone binds to lower affinity (KD = 9.0 +/- 3.0 nM), low molecular weight receptors uncoupled from Ns.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Mn2+ on the exchange reaction of phosphoenolpyruvate carboxykinase in the presence of high concentrations of Mg2+

The mitochondrial phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32), purified from chick embryo liver, was synergistically activated by a combination of Mn2+ and Mg2+ in the oxaloacetate ---- H14CO-3 exchange reaction. Increases in the Mg2+ concentration caused decreases in the K0.5 value of Mn2+ in line with the earlier finding that the enzyme was markedly activated by low Mn2+ (microM) plus high Mg2+ (mM). In the presence of 2.5 mM Mg2+, increases in the Mn2+ level first enhanced the activity of phosphoenolpyruvate carboxykinase, and then suppressed it to the maximal velocity shown in the presence of Mn2+ alone. Kinetic studies showed that high Mn2+ inhibited the activity of Mg2+ noncompetitively, and those of GTP and oxaloacetate uncompetitively. The inhibition constant for oxaloacetate (K'i = 550 microM) was lower than that of Mg2+ (Ki = K'i = 860 microM) or GTP (K'i = 1.6 mM), and was nearly equal to the apparent half-maximal inhibition concentration of Mn2+. These results suggested that Mn2+ can play two roles, of activating and suppressing phosphoenolpyruvate carboxykinase activity in the presence of high Mg2+.     

Membrane bound and soluble adenosine triphosphatase of Escherichia coli K 12. kinetic properties of the basal and trypsin-stimulated activities

Basal and trypsin-stimulated adenosine triphosphatase activities of Escherichia coli K 12 have been characterized at pH 7.5 in the membrane-bound state and in a soluble form of the enzyme. The saturation curve for Mg2+/ATP = 1/2 was hyperbolic with the membrane-bound enzyme and sigmoidal with the soluble enzyme. Trypsin did not modify the shape of the curves. The kinetic parameters were for the membrane-bound ATPase: apparent Km = 2.5 mM, Vmax (minus trypsin) = 1.6 mumol-min-1-mg protein-1, Vmax (plus trypsin) = 2.44 mumol-min-1-mg protein-1; for the soluble ATPase: [S0.5] = 1.2 mM, Vmax (-trypsin) = 4 mumol-min-1-mg protein-1; Vmax (+ trypsin) = 6.6 mumol-min-1-mg protein-1. Hill plot analysis showed a single slope for the membrane-bound ATPase (n = 0.92) but two slopes were obtained for the soluble enzyme (n = 0.98 and 1.87). It may suggest the existence of an initial positive cooperativity at low substrate concentrations followed by a lack of cooperativity at high ATP concentrations. Excess of free ATP and Mg2+ inhibited the ATPase but excess of Mg/ATP (1/2) did not. Saturation for ATP at constant Mg2+ concentration (4 mM) showed two sites (groups) with different Kms: at low ATP the values were 0.38 and 1.4 mM for the membrane-bound and soluble enzyme; at high ATP concentrations they were 17 and 20 mM, respectively. Mg2+ saturation at constant ATP (8 mM) revealed michealian kinetics for the membrane-bound ATPase and sigmoid one for the protein in soluble state. When the ATPase was assayed in presence of trypsin we obtained higher Km values for Mg2+. These results might suggest that trypsin stimulates E. coli ATPase by acting on some site(s) involved in Mg2+ binding. Adenosine diphosphate and inorganic phosphate (Pi) act as competitive inhibitors of Escherichia coli ATPase. The Ki values for Pi were 1.6 +/- 0.1 mM for the membrane-bound ATPase and 1.3 +/- 0.1 mM for the enzyme in soluble form, the Ki values for ADP being 1.7 mM and 0.75 mM for the membrane-bound and soluble ATPase, respectively. Hill plots of the activity of the soluble enzyme in presence of ADP showed that ADP decreased the interaction coefficient at ATP concentrations below its Km value. Trypsin did not modify the mechanism of inhibition or the inhibition constants. Dicyclohexylcarbodiimide (0.4 mM) inhibited the membrane-bound enzyme by 60-70% but concentrations 100 times higher did not affect the residual activity nor the soluble ATPase. This inhibition was independent of trypsin. Sodium azide (20 muM) inhibited both states of E. coli ATPase by 50%. Concentrations 25-fold higher were required for complete inhibition. Ouabain, atebrin and oligomycin did not affect the bacterial ATPase.