Kinetics and subunit interaction of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system

Purified mannitol-specific enzyme II (EIImtl), in the presence of the detergent Lubrol, catalyzes the phosphorylation of mannitol from P-HPr via a classical ping-pong mechanism involving the participation of a phosphorylated EIImtl intermediate. This intermediate has been demonstrated by using radioactive phosphoenolpyruvate. Upon addition of mannitol, at least 80% of the enzyme-bound phosphoryl groups can be converted to mannitol 1-phosphate. The EIImtl concentration dependence of the exchange reaction indicates that self-association is a prerequisite for catalytic activity. The self-association can be achieved by increasing the EIImtl concentration or at low concentrations of EIImtl by adding HPr or bovine serum albumin. The equilibrium is shifted toward the dissociated form by mannitol 1-phosphate, resulting in a mannitol 1-phosphate induced inhibition. Mannitol does not affect the association state of the enzyme. Both mannitol and mannitol 1-phosphate also act as classical substrate inhibitors. The apparent Ki of each compound, however, is approximately equal to its apparent Km, suggesting that mannitol and mannitol 1-phosphate bind at the same site on EIImtl. Due to strong inhibition provided by mannitol and mannitol 1-phosphate in the exchange reaction, the kinetics of this reaction cannot be used to determine whether the reaction proceeds via a ping-pong or an ordered reaction mechanism.     

Functional reconstitution of the purified phosphoenolpyruvate-dependent mannitol-specific transport system of Escherichia coli in phospholipid vesicles: coupling between transport and phosphorylation.

Purified mannitol-specific enzyme II (EII) from Escherichia coli was reconstituted into phospholipid vesicles with the aid of a detergent-dialysis procedure followed by a freeze-thaw sonication step. The orientation of EII in the proteoliposomes was random. The cytoplasmic moiety of the inverted EII could be removed with trypsin without effecting the integrity of the liposomal membrane. This enabled us to study the two different EII orientations independently. The population of inverted EII molecules was monitored by measuring active extrusion of mannitol after the addition of phosphoenolpyruvate, EI, and histidine-containing phosphocarrier protein (HPr) at the outside of the vesicles. The population of correctly oriented EII molecules was monitored by measuring active uptake of mannitol with internal phosphoenolpyruvate, EI, and HPr. A low rate of facilitated diffusion of mannitol via the unphosphorylated carrier could be measured. On the other hand, a high phosphorylation activity without translocation was observed at the outside of the liposomes. The kinetics of the phosphoenolpyruvate-dependent transport reaction and the nonvectorial phosphorylation reaction were compared. Transport of mannitol into the liposomes via the correctly oriented EII molecules occurred with a high affinity (Km, lower than 10 microM) and with a relatively low Vmax. Phosphorylation at the outside of the liposomes catalyzed by the inverted EII molecules occurred with a low affinity (Km of about 66 microM), while the maximal velocity was about 10 times faster than the transport reaction. The latter observation is kinetic proof for the lack of strict coupling between transport and phosphorylation in these enzymes.     

The redox state and the phosphorylation state of the mannitol-specific carrier of the E. coli phosphoenolpyruvate-dependent phosphotransferase system

This review summarizes the recent developments in identifying the activity-linked cysteine as one of the phosphorylation sites on the mannitol-specific EII of the E. coli phosphoenolpyruvate-dependent mannitol transport system. Two phosphorylation sites have been identified, one being the HPr/P-HPr exchange site, the other being the mannitol/mannitol-P exchange site. The activity-linked cysteine and the second phosphorylation site are located in the same 14 residue peptide. Phosphorylation of the second site and phosphoryl group transfer to mannitol do not occur as long as the activity-linked cysteine is oxidized or alkylated.A kinetic scheme has been developed which accounts for the relationships between the redox state, the phosphorylation state and the activity of the carrier. Kinetics of the individual reactions determine whether the enzyme cycles through an oxidized/reduced state during a cycle of phosphorylation/dephosphorylation.Abbreviations DTT Dithiothreitol - glc glucose - mtl mannitol - mtl-P mannitol Phosphate - frc fructose - bgl -glucoside - nag N-acetylglucosamine - PTS Phosphoenolpyruvate-dependent Phosphotransferase System - PEP Phosphoenolpyruvate - P-enolpyruvate Phosphoenolpyruvate     

Subunit structure and activity of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system solubilized in detergent

The original proposal of Saier stating that P-enolpyruvate-dependent mannitol phosphorylation is catalyzed by the monomeric form of the bacterial phosphotransferase enzyme IImtl, which would be the form predominantly existing in the phospholipid bilayer, whereas mannitol/mannitol-P exchange would depend on the transient formation of functional dimers, is refuted [Saier, M.H. (1980) J. Supramol. Struct. 14, 281-294]. The correct interpretation of the proportional relation between the rate of mannitol phosphorylation in the overall reaction and the enzyme concentration is that enzyme IImtl is dimeric under the conditions employed. Differences measured in the enzyme concentration dependency of the overall and exchange reactions were caused by different assay conditions. The dimer is favored over the monomer at high ionic strength and basic pH. Mg2+ ions bind specifically to enzyme IImtl, inducing dimerization. A complex formed by mixing inorganic phosphate, F-, and Mg2+ at sufficiently high concentrations inhibits enzyme IImtl, in part, by dissociation of the dimer. Enzyme IImtl was dimeric in 25 mM Tris, pH 7.6, and 5 mM Mg2+ over a large enzyme concentration range and under many different turnover conditions. The association/dissociation equilibrium was demonstrated in phosphate bufers, pH 6.3. The dimer was the most active form both in the overall and in the exchange reaction under the conditions assayed. The monomer was virtually inactive in mannitol/mannitol-P exchange but retained 25% of the activity in the overall reaction.     

Further characterization of the diacylglycerol-phosphatidylethanolamine exchange reaction catalyzed by cell-free extracts of Escherichia coli

The conditions for phosphatidylethanolamine (PE)-diacylglycerol (DAG) exchange catalysed by cell-free extracts of Escherichia coli were studied using 14C- or 3H-analogues of both these lipids. The reaction, examined with either labelled PE or labelled DAG, occurred without co-factor addition and was inhibited by Ca2+ and Mg2+. Detergents such as Triton X-100 greatly enhanced the activity; however, the optimal concentration of this agent depended on the lipid substrate concentration. The exchange-catalysing enzyme involved in these extracts appeared to be very specific for DAG and PE, since no other labelled phospholipid or acylglycerol derivative formed radioactive product under the assay conditions tested. Again, endogenous [3H]PE present in the enzyme source, but no other endogenous lipid, was converted to labelled DAG in the presence of added 1,2-dioleoyl-sn-glycerol. The Vmax value for the conversion of labelled PE to DAG was very similar to the Vmax value found for the conversion of labelled DAG to PE as would be expected in the case of an exchange reaction being responsible for both conversions. However, the Km value for PE was appreciably larger than that for DAG. The enzyme involved, displayed a broad acyl chain specificity as could be judged from: (1) the ability of various species of DAG and PE to stimulate the exchange; (2) the suitability of lipid substrates prepared from widely different biological sources; and (3) the interchange of acyl groups that occurred between dimyristoyl PE and dilauroylglycerol. As would be expected for an exchange reaction, the incorporation of lauroyl groups into PE occurred without an increase in the total fatty acid content of this phospholipid. The results of the present study confirm and further characterize the PE-DAG exchange reaction of E. coli.     

Serine base exchange enzyme in porcine lyophilised platelets: enzyme properties and modulation by AIF4-and different types of heparin

Phosphatidylserine is one of the PKC modulators and thus it may play an important role in signal transduction. Regulation of the synthesis of this phospholipid is not yet clarified. The contrasting reports are possibly related to the existence of different enzymes which, in mammalian tissues, catalyse the exchange between free serine and the nitrogen base of a membrane phospholipid. This study demonstrates that serine base exchange reactions of commercially available lyophilised porcine platelets exhibit similar pH optima, temperature and Ca²⁺ dependence as observed in fresh tissues. Analysis of fatty acids composition of the three phospholipid classes involved in base exchange reactions also demonstrated a similarity with fresh platelets. Serine and ethanolamine base exchange enzyme activities were assayed in parallel in platelet lysate subjected to preincubation at various temperatures (30-60°C). When dithioerithrol was omitted from the incubation medium, the two base exchange reactions were inhibited with a similar temperature-dependent pattern. Addition of the reducing agent enhanced the sensitivity to preincubation only for the serine base exchange reaction which was inhibited by 80% after preincubation at 45°C. With respect to its regulation, porcine platelet serine base exchange enzyme(s) was inhibited by fluoroalluminate, a widely used G-protein activator, and stimulated by unfractionated heparin. Low mol. wt. heparin did not influence enzyme activity. Unfractionated heparin greatly stimulated SBEE activity assayed at pH 7.4, a pH value far from the optimal pH.     

The enzymology of the bacterial phosphoenolpyruvate-dependent sugar transport systems

Summary The phosphoenolpyruvate-dependent sugar transport system (PTS) is present in a large variety of bacteria. It catalyzes transport and phosphorylation of hexoses and hexitols at the expense of phosphoenolpyruvate. Only three of four enzymes are required for this entire sequence. Each component has been isolated and purified to the homogeneity from one bacterial species or another allowing recent investigations intomechanistic aspects of energy coupling, energy conservation, transport and regulation using well-characterized enzymes. In each case the phosphorylation of the enzyme is a key element in that enzymes function.The initial step in the energy conversion process is the E_I catalyzed conversion of phosphoenolpyruvate to pyruvate and P-HPr. E_II is a metal requiring hydrophobic enzyme which is active only as a dimer. Kinetic and gel filtration data confirm that it forms functional ternary complexes with HPr or P-Hpr and phosphoenolpyruvate or pyruvate which influence both the degree of dimerization and the specific activity of the dimer. The dimer appears to carry only one phosphoryl group suggesting that negative cooperativity or a flip-flop mechanism may be involved in the sequence of phosphoryl group transfer.Many of the PTS phosphoenzyme intermediates carry the phosphoryl group as a phospho-histidine. A general mechanism for the transfer of the phosphoryl group to and from the active site histidine residue in each protein has been established with high resolution ¹H NMR data. At physiological pH the active site histidine is deprotonated, whereas the phosphohistidine is protonated. Consequently the histidine, as a strong nucleophile, can abstract the phosphoryl group from the donor while protonation destabilizes the phosphohistidine facilitating passage of the phosphoryl group to the following enzyme intermediate. The change in protonation state accompanies a phosphorylation induced conformational change in the carrier.The ability of the PTS to regulate the activity of other permeases and catabolic enzymes has been attributed to E_III ^Glc. Data obtained with mutants suggest that changes in the phosphorylation state alter the regulatory properties of the enzyme. The nonphosphorylated species blocks various permeases and suppresses adenylate cyclase activity thereby inhibiting the synthesis of catabolic enzyme systems. The phosphorylated species stimulates adenylate cyclase and permits the uptake of inducers leading to the initiation of catabolic enzyme synthesis. Experiments with the isolated E_III ^Glc confirm that a phosphoenzyme intermediate exists.Transport and phosphorylation of the sugar are catalyzed by a membrane-bound E_II via a phosphoenzyme intermediate which can be reached from P-HPr, P-E_III or sugar-P. The phosphorylation state controls the affinity of the enzyme for its substrates. E_II is high affinity for P-HPr or P-E_III and low affinity for sugar. P-E_II is high affinity for sugar and low affinity for P-HPr or P-E_III. The affinity of the enzyme for sugar substrates is controlled by the oxidation state of a dithiol. The reduced, dithiol form is high affinity for sugar substrates. The oxidized, disulfide form, is low affinity. Phosphorylation of the enzyme chould shift the affinity for substrates by altering the oxidation state of the enzyme.     

Regulatory aspects of mitochondrial phospholipase A2 from rat liver: effects of proteins, phospholipids and calcium ions

This paper deals with the search for specific inhibitors or activators of the mitochondrial phospholipase A2. Convincing evidence for the existence of proteins in the mitochondrial or cytosolic fraction that function as specific regulators of this enzyme was not obtained. The enzymatic activity appeared to be inhibited at low substrate concentrations by lipocortin isolated from human monocytes. However, at higher substrate concentrations, the inhibition disappeared, suggesting either that lipocortin sequestered the phospholipid substrate or that the putative inactive complex of enzyme and lipocortin dissociated in the presence of excess phospholipids. The hydrolysis of the neutral phospholipid phosphatidylethanolamine was stimulated by the presence of cardiolipin and phosphatidylglycerol. It is unlikely that this is caused merely by the negative charge of these phospholipids, since other negatively charged phospholipids did not show this effect. Using a phospholipid extract from mitochondria as substrate, the enzymatic activity as a function of the Ca2+ concentration was determined. Only one enzyme activity plateau was observed. The calculated KCa2+ value of 0.05 mM suggests that the mitochondrial phospholipase A2 could be regulated strictly by the modulation of the free Ca2+ concentration in vivo. The two activity plateaus observed previously upon variation of the Ca2+ concentration using phosphatidylethanolamine as substrate could be explained by a Ca2+-induced transition of the phospholipid structure.     

Modulation of (Na+,K+)-ATPase activity by the lipid bilayer examined with dansylated phosphatidylserine

The fluorescent probe 8-(dimethylamino)naphthalene-1-sulfonylphosphatidylserine (Dns-PS) was incorporated into purified lamb kidney Na+- and K+-stimulated adenosinetriphosphatase (EC 3.6.1.3) [(Na+,K+)-ATPase] by using a purified phospholipid exchange protein. Phospholipase C was used to reduce phospholipid content. Up to 40% of the phospholipid could be hydrolyzed with only 10% inhibition of the (Na+,K+)-ATPase, but when 67% of the phospholipid was hydrolyzed, the enzyme was inhibited 53%. To examine the effect of protein on the phospholipid bilayer, the fluorescent parameters of the probe incorporated into the enzyme preparation were contrasted with the same parameters for the probe incorporated into the total lipid extract of the preparation. The polarization of fluorescence of the probe in the lipid extract was 0.118 while in the enzyme preparation it was 0.218. This reflected a decrease in fluidity of the glycerol region of the phospholipid bilayer which was mediated by the protein. This effect increased as the phospholipid content of the (Na+,K+)-ATPase preparation was reduced so that with maximal phospholipid reduction the polarization of fluorescence was 0.262. The protein caused a decrease in the transition temperature from gel to fluid states of the bilayer detected by polarization of the probe. The midpoint temperature transition of the enzyme preparation decreased from 33 degrees C when all phospholipids were present to 20 degrees C when 67% of the phospholipids were hydrolyzed. This decrease was not observed for the lipid extract of these samples. A direct correlation between the (Na+,K+)-ATPase specific activity and the polarization of fluorescence of Dns-PS was found. The reduction in phospholipid content did not affect the steady-state level of phosphorylation of the enzyme by ATP but did affect the rate of dephosphorylation which would require conformational changes of the enzymes. The data showed that the fluidity of the phospholipid bilayer can modulate the activity of the (Na+,K+)-ATPase.     

Role of phospholipids in the calcium-dependent ATPase of the sarcoplasmic reticulum. Enzymatic and ESR studies with phospholipid-replaced membranes.

Three types of partially purified ATPase enzymes having different phospholipid contents and compositions have been prepared: (a) an enzyme whose phospholipid moiety has been replaced predominantly by dioleoyl lecithin (DOL-enzyme), with about the same phospholipid content as the original sarcoplasmic reticulum, (b) dipalmitoyl lecithin-replaced enzyme whose phospholipid content is 30% of that of DOL-enzyme (DPL-enzyme), and (c) a partially delipidated enzyme with about the same phospholipid content as DPL-enzyme but with the original sarcoplasmic reticulum phospholipid composition (del-enzyme). The temperature dependence of Ca2+-activated ATPase activity of these preparations showed clearcut differences; with DOL-enzyme there was no appreciable break in the Arrhenius plot in the 3-40 degrees range; DPL-enzyme showed a break at 29 degrees, and del-enzyme and sarcoplasmic reticulum one at 18 degrees. Transition temperatures obtained from ESR studies with the use of spin-labeled stearic acid incorporated into the membranes agreed with those derived from ATPase assays. Thermo-dynamic analysis of the ATP hydrolysis rates shows that DPL-enzyme has considerably larger values of activation enthalpy and activation entropy below the transition temperature (29 degrees) than those of the other preparations, while all enzyme preparations show similar free energies of activation. The ESR data show that below their transition temperatures DPL-enzyme, and to a lesser degree del-enzyme, have a strongly restricted motion of their phospholipid molecules as compared with either DOL-enzyme or sarcoplasmic reticulum. Studies on the formation and decomposition of phosphoenzyme have been carried out with the three types of ATPase preparations. At 0 degrees, the rate of inorganic phosphate liberation is 8 times lower in DPL-enzyme than in del-enzyme with little difference in the steady state level of phosphoenzyme. In DOL-enzyme, the level of phosphoenzyme and the rate of inorganic phosphate liberation are 1.8 and 3.5 times higher than the corresponding values obtained with del-enzyme. Addition of ADP to the phosphorylated intermediate of DPL-enzyme induces a fast reversal of the phosphorylation reaction. These results indicate that the physical state of the phospholipid molecules associated with the enzyme affects the decomposition of phosphoenzyme, with little effect on the phosphorylation reaction and its reversal.     

Sequential mechanism of calcium binding and translocation in sarcoplasmic reticulum adenosine triphosphatase

Cooperative calcium binding (apparent Kd = 1.04 X 10(-6) M) to the ATPase of sarcoplasmic reticulum vesicles occurs with a maximal stoichiometry of 2 mols of divalent cation/mol of enzyme in the absence of ATP. The bound calcium is distributed into two pools which undergo fast or slow isotopic exchange, respectively. The two pools retain a 1:1 molar ratio under various conditions and are both located within a protein crevice, as suggested by their cooperative interaction and exchange kinetics. Following enzyme phosphorylation by ATP, both pools of bound calcium are "internalized" (cannot be displaced by quench reagents). If following 45Ca2+ binding, isotopic dilution is obtained in the medium by adding 40Ca2+ with ATP, internalization of both pools of bound 45Ca2+ (2 mol/mol of phosphoenzyme) is still observed within the first enzyme cycle. When the cycle is reversed by addition of excess ADP soon after ATP, only half of the internalized 45Ca2+ is released from the enzyme into the medium outside the vesicles, while the other half remains with the vesicles. If half of the bound 45Ca2+ is exchanged (fast exchange) with 40Ca2+ previous to the addition of ATP, none of the remaining 45Ca2+ is released outside the vesicles upon reversal of the enzyme cycle. Therefore, the pool of bound calcium which undergoes slower exchange with the outside medium, is the first to be released inside the vesicles upon enzyme phosphorylation. A sequential mechanism of calcium binding and translocation is proposed, that accounts for binding cooperativity and exchange kinetics, presteady state transients following addition of ATP, and the Ca2+ concentration dependence of ATPase activity in steady state.     

Membrane integrity and phospholipid movement influence the base exchange reaction in rat liver microsomesý

Properties of Ca2+-stimulated incorporation of amincalcohols, serine and ethanolamine, into phospholipids, and factors regulating the reaction were studied in endoplasmic reticulum membranes isolated from rat liver. In contrast to apparent Km values for either aminoalcohol, maximal velocities of the reaction were significantly affected by Ca2+ concentration. No competition between these two soluble substrates used at equimolar concentrations close to their Km values was observed, suggesting the existence of two distinct phospholipid base exchange activities. The enzyme utilizing the electrically neutral serine was not sensitive to changes of membrane potential evoked by valinomycin in the presence of KCI. On the other hand, when positively charged ethanolamine served as a substrate, the enzyme activity was inhibited by 140 mM KCI and this effect was reversed by valinomycin. The rates of inhibition of phospholipid base exchange reactions by various thiol group modifying reagents were al so found to differ. Cd2+ and lipophylic p-chloromercuribenzoic acid at micromolar concentrations were most effective. It can be suggested that -SH groups located within the hydrophobic core of the enzymes molecules are essential for the recognition of membrane substrates. However, the influence of the -SH group modifying reagents on the protein-facilitated phospholipid motion across endoplasmic reticulum membranes can not be excluded, since an integral protein-mediated transverse movement of phospholipids within the membrane bilayer and Ca2+-mediated changes in configuration of the phospholipid polar head groups seem to be a regulatory step of the reaction. Indeed, when the membrane integrity was disordered by detergents or an organic solvent, the reaction was inhibited, although not due to the transport of its water-soluble substrates is affected, but due to modulation of physical state of the membrane bilayer and, in consequence, the accessibility of phospholipid molecules.     

Characteristics of lead ion-stimulated phosphorylation of Electrophorus electricus electroplax (Na+ + K+)-adenosine triphosphatase and inhibition of ATP-ADP exchange.

Pb2+-stimulated phosphorylation of Electrophorus electricus electroplax (Na+ + K+)-adenosine triphosphatase is prevented by stoichiometric quantities of 2,3-dimercaptopropanol. The chelator in the same low concentrations does not block Na+-dependent phosphorylation. Both Pb2+-and Na+-dependent phosphorylation reactions show the same dependence on MgCl2. Phosphorylation in the presence of both Na+ and Pb2+ is cumulative suggesting that Pb2+ and Na+ bind at separate, independent sites. The enthalpy change due to binding of Pb2+ is about -1.76 kcal/mol. 32P-phosphopeptides obtained from pronase or pepsin digests of Pb2+-and Na+-dependent phosphoproteins are electrophoretically identical. Pb2+ does not stimulate but does inhibit ATP-ADP exchange activity under the conditions in which this activity is stimulated by Na+. Since the phosphorylation sites are identical, it is concluded that the differences in reactivity of the Na+- and Pb2+-phosphoenzymes are due to different conformational changes produced by binding of Na+ and Pb2+. The Pb2+-sensitive conformation is critical for Na+ specificity of phosphorylation, reversibility of phosphorylation, and for phosphatase activity but not for acceptor site phosphorylation by ATP. These findings have implications for enzyme reaction models.     

The β-glucoside system of Escherichia coli III. Properties of A P–HPr: β-Glucoside phosphotransferase extracted from membranes with detergent

A P-HPr:β-glucoside phosphotransferase (enzyme II^bgl)

1 The nomenclautre of the enzymes II is that suggested by Lin (1)

has been extracted from membranes of a β-glucoside fermenting strain of Escherichia coli K 12 using the nonionic detergent Triton X–100. The extracted enzyme was rendered virtually free of both lipid and detergent by chromatography on DEAE-cellulose. At this stage, the partially purified enzyme had negligible activity, but activity was restored effectively by the addition of (1) nonionic detergents of the Tween or Triton series and (2) crude E. coli phospholipids or an anionic lipid enriched fraction, but not phosphatidylethanolamine. Detergent activators were most effective at or near the critical micelle concentration, but were inhibitory when added at concentrations above the critical micelle concentration. In order to obtain maximal initial rates of phosphotransferase activity, it was necessary to incubate the extracted, partially purified enzyme with detergent activator and HPr prior to the addition of the other assay system components. High detergent concentration inhibited the initial rate of phosphorylation by interfering with an essential step (or steps) that occur during this preliminary incubation. The activation occuring during the preliminary incubation was also highly temperature dependent; a precipitous decrease in activation was detected below 16° when Tween 40 was employed as the detergent activator. Phosphorylation mediated by the membrane associated form of the phosphotransferase was not influenced by the physical state of the lipid components of the membrane. This is in marked contrast to the properties of the phosphorylation reaction mediated by the phosphotransferase in intact cells.     

Activation of protein kinase in the bovine corpus luteum by phospholipid and Ca2+.

A new species of protein kinase has been identified in cytosol preparations from bovine corpora lutea. Enzyme activity required the simultaneous presence of Ca2+ and phospholipid, and was also enhanced by glyceryl dioleate. Phosphatidylserine was the most effective phospholipid for stimulating histone phosphorylation. Other phospholipids capable of supporting enzymic activity were, in order of decreasing activity, phosphatidylinositol, phosphatidic acid, cardiolipin and phosphatidylglycerol. Several other phospholipids tested were ineffective. A cyclic AMP-dependent protein kinase was also present in the luteal cytosol. This enzyme activity was eliminated by protein kinase inhibitor without affecting the Ca2+- and phospholipid-stimulated activity. Lysine-rich histone (IIIS) was a much better substrate than type-IIA histone for Ca2+- and phospholipid-dependent phosphorylation. Ca2+ and phospholipid also enhanced phosphorylation of endogenous luteal cytosol protein. Calmodulin, alone or in the presence of Ca2+, was unable to increase phosphorylation. Trifluoperazine inhibited protein kinase activity stimulated by Ca2+ and phospholipid. These data suggest that a phospholipid-sensitive, Ca2+-dependent protein kinase may provide an important link between hormonally-induced changes in phospholipid metabolism and corpus-luteum function.     

A new kinetic approach for studying phospholipase C (Clostridium perfringens alpha toxin) activity on phospholipid monolayers

The enzymatic activity of purified phospholipase C (alpha toxin) from Clostridium perfringens was investigated with various phospholipid monolayers. A two-step reaction was used. Enzymatic hydrolysis of insoluble lecithin films by phospholipase C, generating 1,2-diacylglycerol and water-soluble phosphocholine, was coupled with the action of pancreatic lipase in order to give rise to fatty acid and 2-monoacylglycerol, which are rapidly desorbed from the interface. With this new procedure, it is possible to obtain continuous and accurate kinetic measurements of the phospholipase C catalyzed reaction with phospholipid monolayers as the substrate. It is thus possible to avoid the use of radiolabeled substrates as necessary in previous studies, and the difficulties caused by diacylglycerol accumulation in the lipid film are minimized. No hydrolysis was detected when either phosphatidylethanolamine, phosphatidylserine, or phosphatidylglycerol films were used as substrates. By means of a film transfer technique, Ca2+ and Zn2+ ions were found to play a specific and critical role. The present study demonstrates clearly for the first time that Ca2+ is essential for enzyme binding to lipid films, whereas Zn2+ is specifically involved in the catalytic hydrolysis of the substrate.     

Interdependence of H+, Ca2+, and Pi (or vanadate) sites in sarcoplasmic reticulum ATPase

The phosphorylation of sarcoplasmic reticulum ATPase with Pi in the absence of Ca2+ was studied by equilibrium and kinetic experimentation. The combination of these measurements was then subjected to analysis without assumptions on the stoichiometry of the reactive sites. The analysis indicates that the species undergoing covalent interaction is the tertiary complex E X Pi X Mg formed by independent interaction of the two ligands with the enzyme. The binding constant of Pi or Mg2+ to either free or partially associated enzyme is approximately equal to 10(2) M-1, and no significant synergistic effect is produced by one ligand on the binding of the other; the equilibrium constant (Keq) for the covalent reaction E X Pi X Mg E-P X Mg is approximately equal to 16, with kphosph = 53 s-1, and khyd = 3-4 s-1 (25 degrees C, pH 6.0, no K+). The phosphorylation reaction of sarcoplasmic reticulum ATPase with Pi is highly H+ dependent. Such a pH dependence involves the affinity of enzyme for different ionization states of Pi, as well as protonation of two protein residues per enzyme unit in order to obtain optimal phosphorylation. The experimental data can then be fitted satisfactorily assuming pK values of 5.7 and 8.5 for the two residues in the nonphosphorylated enzyme (changing to 7.7 for one of the two residues, following phosphorylation) and values of 50.0 and 0.58 for the equilibrium constants of the H2(E X HPO4) in equilibrium with H(E-PO3) + H2O and H(E X HPO4) in equilibrium with E-PO3 + H2O reactions, respectively. In addition to the interdependence of H+ and phosphorylation sites, an interdependence of Ca2+ and phosphorylation sites is revealed by total inhibition of the Pi reaction when two high affinity calcium sites per enzyme unit are occupied by calcium. Conversely, occupancy of the phosphate site by vanadate (a stable transition state analogue of phosphate) inhibits high affinity calcium binding. The known binding competition between the two cations and their opposite effects on the phosphorylation reaction suggest that interdependence of phosphorylation site, H+ sites, and Ca2+ sites is a basic mechanistic feature of enzyme catalysis and cation transport.     

Binding of ethylenediamine to phosphatidylserine is inhibitory to Na+/K(+)-ATPase.

Covalent linkage of ethylenediamine with the Na+/K(+)-ATPase complex from rabbit kidney outer medulla by the use of the water-soluble carbodiimide, N-ethyl,N'-(3-dimethylaminopropyl)carbodiimide, resulted in a 73% reaction with phosphatidylserine and only 27% with carboxylic groups in the proteic component of the enzyme. Condensation products from the reaction between phosphatidylserine and ethylenediamine, N-(O-phosphatidylseryl)ethylenediamine, N,N'-bis(O-phosphatidylseryl)ethylenediamine and its intermediary product O-phosphatidyl-[N,N'-bis(seryl)]ethylenediamine, were synthesised. Symmetrically substituted ethylenediamine was the most likely condensation product of ethylenediamine with endogenous phosphatidylserine. The synthesised lipids were incorporated in proteoliposomes containing Na+/K(+)-ATPase and only the addition of the phospholipid phosphatidylcholine. The ratio of phospholipid to protein was 52 (w/w). These proteoliposomes were perforated by the addition of 0.5% cholate and both the Na(+)-dependent phosphorylation level and its dependence on Na+, Mg2+ and ATP were measured. Phosphatidylcholine alone increased the half-maximal activation concentration for Na+ ([Na+]0.5) from 0.2 to 1-2 mM, for Mg2+ from 0.1 to 0.8 microM and for ATP from 0.02 to 0.3 microM. The Ki for K+ (in the absence of Na+) was unaffected: 12.8 microM vs. 12.5 microM in the non-reconstituted system. Replacing 10 mol% of phosphatidylcholine by phosphatidylethanolamine: or phosphatidylserine had no significant effect on [Na+]0.5: 1.1 and 0.7 mM, respectively. Replacing 5 mol% phosphatidylcholine by the bis(phosphatidylseryl) substituent of ethylenediamine further increased [Na+]0.5 to 13.7 mM, while half-maximal activation concentrations for Mg2+ and ATP were unaltered. The mono-phosphatidylseryl derivatives of ethylenediamine, each 5 mol%, also increased [Na+]0.5, but to a lesser extent (3.2-3.8 mM). In addition to their competitive effects, the phosphatidylseryl-substituted ethylenediamine compounds exerted a slowly-increasing non-competitive inhibition, not only in phosphorylation, but also in overall ATPase activity, which was reduced, although not abolished, by exogenous protein (bovine serum albumin). A detergent-like action in the usual sense is unlikely since liposomes containing these lipids remained intact. These studies prove that phospholipids are not only required for optimal activity of this transport enzyme, but in excess or in compositions deviating from the normal, may also be inhibitory.     

Bimodal lipid substrate dependence of phosphatidylinositol kinase

Phosphatidylinositol (PI) kinase activity was solubilized from rat liver microsomes and partially purified by chromatography on hydroxyapatite and Reactive Green 19-Superose. Examination of the ATP dependence using a mixed micellar assay gave a Km of 120 microM. The dependence of reaction rate on PI was more complicated. PI kinase bound a large amount of Triton X-100, and as expected for a micelle-associated enzyme utilizing a micelle-associated lipid substrate, the reaction rate was dependent on the micellar mole fraction, PI/(PI + Triton X-100), with a Km of 0.02 (unitless). Activity showed an additional dependence on bulk PI concentration at high micelle dilution. These results demonstrated two kinetically distinguishable steps leading to formation of a productive PI/enzyme(/ATP) complex. The rate of the first step, which probably represents exchange of PI from the bulk micellar pool into enzyme-containing micelles, depends on bulk PI concentration. The rate of the second step, association of PI with enzyme within a single micelle, depends on the micellar mole fraction of PI. Depression of the apparent Vmax at low ionic strength suggested that electrostatic repulsion between negatively charged PI/Triton X-100 mixed micelles inhibits PI exchange, consistent with a model in which intermicellar PI exchange depends on micellar collisions.     

Reconstitution of (Na+ + K+)-ATPase proteoliposomes having the same turnover rate as the membranous enzyme

Membranous (Na+ + K+)-ATPase from the electric eel was solubilized with 3-[3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate (Chaps). 50 to 70% of the solubilized enzyme was reconstituted in egg phospholipid liposomes containing cholesterol by using Chaps. The obtained proteoliposomes consisted of large vesicles with a diameter of 134 +/- 24 nm as the major component, and their protein/lipid ratio was 1.25 +/- 0.07 g protein/mol phospholipid. The intravesicular volume of these proteoliposomes is too small to consistently sustain the intravesicular concentrations of ligands, especially K+, during the assay. The decrease in K+ concentration was cancelled by the addition of 20 microM valinomycin in the assay medium. The low value of the protein/lipid ratio suggests that these proteoliposomes contain one Na+/K+-pump particle with a molecular mass of 280 kDa per one vesicle as the major component. In these proteoliposomes, the specific activity of the (Na+ + K+)-ATPase reaction was 10 mumol Pi/mg protein per min, and the turnover rate of the ATP-hydrolysis was 3500 min-1, the same as the original enzyme under the same assay condition. The ratio of transported Na+ to hydrolyzed ATP was 3, the same as that in the red cell. The proteoliposomes could be disintegrated by 40-50 mM Chaps without any significant inactivation. This disintegration of proteoliposomes nearly tripled the ATPase activity compared to the original ones and doubled the specific ATPase activity compared to the membranous enzyme, but the turnover rate was the same as the original proteoliposomes and the membranous enzyme. This disintegration of proteoliposomes by Chaps suggests the selective incorporation of the (Na+ + K+)-ATPase particle into the liposomes and the asymmetric orientation of the (Na+ + K+)-ATPase particle in the vesicle.