Activation of PMCA by calmodulin or ethanol in plasma membrane vesicles from rat brain involves dissociation of the acetylated tubulin/PMCA complex

We have recently shown that acetylated tubulin interacts with plasma membrane Na(+),K(+)-ATPase and inhibits its enzyme activity in several types of cells. H(+)-ATPase of Saccharomyces cerevisiae is similarly inhibited by interaction with acetylated tubulin. The activities of both these ATPases are restored upon dissociation of the acetylated tubulin/ATPase complex. Here, we report that in plasma membrane vesicles isolated from brain synaptosomes, another P-type ATPase, plasma membrane Ca(2+)-ATPase (PMCA), undergoes enzyme activity regulation by its association/dissociation with acetylated tubulin. The presence of acetylated tubulin/PMCA complex in membrane vesicles was demonstrated by analyzing the behavior of acetylated tubulin in a detergent partition, and by immunoprecipitation experiments. PMCA is known to be stimulated by ethanol and calmodulin at physiological concentrations. We found that treatment of plasma membrane vesicles with these reagents induced dissociation of the complex, with a concomitant restoration of enzyme activity. Conversely, incubation of vesicles with exogenous tubulin induced the association of acetylated tubulin with PMCA, and the inhibition of enzyme activity. These findings indicate that activation of synaptosomal PMCA by ethanol and calmodulin involves dissociation of the acetylated tubulin/PMCA complex. This regulatory mechanism was shown to also operate in living cells.     

Plasma membrane calcium pump activity is affected by the membrane protein concentration: evidence for the involvement of the actin cytoskeleton

Plasma membrane calcium pumps (PMCAs) are integral membrane proteins that actively expel Ca(2+) from the cell. Specific Ca(2+)-ATPase activity of erythrocyte membranes increased steeply up to 1.5-5 times when the membrane protein concentration decreased from 50 microg/ml to 1 microg/ml. The activation by dilution was also observed for ATP-dependent Ca(2+) uptake into vesicles from Sf9 cells over-expressing the PMCA 4b isoform, confirming that it is a property of the PMCA. Dilution of the protein did not modify the activation by ATP, Ca(2+) or Ca(2+)-calmodulin. Treatment with non-ionic detergents did not abolish the dilution effect, suggesting that it was not due to resealing of the membrane vesicles. Pre-incubation of erythrocyte membranes with Cytochalasin D under conditions that promote actin polymerization abolished the dilution effect. Highly-purified, micellar PMCA showed no dilution effect and was not affected by Cytochalasin D. Taken together, these results suggest that the concentration-dependent behavior of the PMCA activity was due to interactions with cytoskeletal proteins. The dilution effect was also observed with different PMCA isoforms, indicating that this is a general phenomenon for all PMCAs.     

The superoxide-generating respiratory burst oxidase of human neutrophil plasma membrane. Phosphatidylserine as an effector of the activated enzyme

The superoxide-generating respiratory burst oxidase is an integral membrane enzyme found in the plasma membrane of polymorphonuclear leukocytes (neutrophils). NADPH-dependent superoxide generation is seen in isolated plasma membranes and in their detergent extracts following activation of the intact cells with phorbol myristate acetate. We have herein examined the effects of phospholipids on the activity of the solubilized oxidase. Solubilization of plasma membranes with 0.5% each of Tween 20 plus deoxycholate resulted in an approximately 2-fold enhancement of activity. Inclusion of phospholipids in the extraction medium resulted in further activation. At 1.0 mg/ml the order of effectiveness was phosphatidylserine (PS) greater than cardiolipin greater than phosphatidylethanolamine greater than phosphatidylinositol; phosphatidylcholine and phosphorylated inositol lipids were not effective. The concentrations required for half-maximal activation by PS and phosphatidylethanolamine were 85 and 200 micrograms/ml, respectively. When PS was used at a maximally activating concentration (0.5 mg/ml), the activity was enhanced 3-5-fold. Detergent solubilization alone elevated the Km of the oxidase for NADPH from 68 microM in intact plasma membranes to 123 microM, but inclusion of PS with detergent restored the Km to near or below that seen in intact membranes. PS also increased the Vmax by a factor of 2-3, but had no effect on the pH optimum. A plot of the activity versus enzyme concentration was linear when membranes were used, but activity showed a quadratic dependence on concentration in solubilized membrane, with lower than expected activity at lower enzyme concentration. PS restored linearity of the concentration-activity plot. The activation by PS was not influenced by the addition of Ca2+, EGTA, or dioctanoylglycerol, indicating that activation was not dependent on protein kinase C. These results implicate phosphatidylserine as a direct effector of the NADPH-oxidase.     

Regulation of plasma membrane Ca(2+)-ATPase activity by acetylated tubulin: influence of the lipid environment

We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca(2+)-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300μg/ml) in combination with acidic lipids at concentrations >10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50μg/ml tubulin, increased PMCA activity >12-fold, whereas tubulin alone at high concentration (≥300μg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca(2+) transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (<50μg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.     

Kinetic characterization of Na,K-ATPase from rabbit outer renal medulla: properties of the (alpha beta)(2) dimer

We describe and compare the main kinetic characteristics of the (alpha beta)(2) form of rabbit kidney Na,K-ATPase. The dependence of ATPase activity on ATP concentration revealed high (K(0.5)=4 microM) and low (K(0.5)=1.4 mM) affinity sites for ATP, exhibiting negative cooperativity and a specific activity of approximately 700 U/mg. For p-nitrophenylphosphate (PNPP) as substrate, a single saturation curve was found, with a smaller apparent affinity of the enzyme for this substrate (K(0.5)=0.5 mM) and a lower hydrolysis rate (V(M)=42 U/mg). Stimulation of ATPase activity by K(+) (K(0.5)=0.63 mM), Na(+) (K(0.5)=11 mM) and Mg(2+) (K(0.5)=0.60 mM) all showed V(M)'s of approximately 600 U/mg and negative cooperativity. K(+) (K(0.5)=0.69 mM) and Mg(2+) (K(0.5)=0.57 mM) also stimulated PNPPase activity of the (alpha beta)(2) form. Ouabain (K(0.5)=0.01 microM and K(0.5)=0.1 mM) and orthovanadate (K(0.5)=0.06 microM) completely inhibited the ATPase activity of the (alpha beta)(2) form. The kinetic characteristics obtained constitute reference values for diprotomeric (alpha beta)(2)-units of Na,K-ATPase, thus contributing to a better understanding of the biochemical mechanisms of the enzyme.     

Kinetics behaviors of Na,K-ATPase: comparison of solubilized and DPPC:DPPE-liposome reconstituted enzyme

We describe and compare the main kinetic characteristics of rabbit kidney Na,K-ATPase incorporated inside-out in DPPC:DPPE-liposomes with the C(12)E(8) solubilized and purified form. In proteoliposomes, we observed that the ATP hydrolysis of the enzyme is favored and also its affinity for Na(+)-binding sites increases, keeping the negative cooperativity with two classes of hydrolysis sites: one of high affinity (K(0.5)=6 microM and 4 microM for reconstituted enzyme and purified form, respectively) and another of low affinity (K(0.5)=0.4 mM and 1.4 mM for reconstituted enzyme and purified form, respectively). Our data showed a biphasic curve for ATP hydrolysis, suggesting the presence of (alphabeta)(2) oligomer in reconstituted Na,K-ATPase similar to the solubilized enzyme. The Mg(2+) concentration dependence in the proteoliposomes stimulated the Na,K-ATPase activity up to 476 U/mg with a K(0.5) value of 0.4 mM. The Na(+) ions also presented a single saturation curve with V(M)=551 U/mg and K(0.5)=0.2 mM with cooperative effects. The activity was also stimulated by K(+) ions through a single curve of saturation sites (K(0.5)=2.8 mM), with cooperative effects and V(M)=641 U/mg. The lipid microenvironment close to the proteic structure and the K(+) internal to the liposome has a key role in enzyme regulation, affecting its kinetic parameters while it can also modulate the enzyme's affinity for substrate and ions.     

Stimulation of Bacillus subtilis membrane adenosine triphosphatase by cationic bactericidal agents

The environmental Mg2+ used in preparation of Bacillus subtilis membranes was found to influence the responses of the associated ATPase to cetyltrimethylammonium bromide (CTAB). Membranes prepared using fluids containing higher Mg2+ levels exhibited lower control activity than was seen with low Mg2+ membranes. Increased environmental Mg2+ resulted in higher stimulations with lower doses of the agent. ATPase of all three membrane types was stimulated in two concentration ranges, but in the doses tested, CTAB inhibited the ATPase of only those membranes obtained using fluids containing high Mg2+ for every stage of the isolation. Sonication of membranes for 25 s at half maximum output yielded three fractions, consisting of a soluble form which was sensitive to CTAB stimulation at 25 microg/ml of assay mixture; small, 95-110 nm, vesicles, which were resistant to CTAB at 25, 75, and 150 microg/ml, and large vesicles, similar to untreated membranes both in morphology and responses to detergent. Combinations of detergent and protein (beta-lysin or arginine-rich histone) produced activity appearing to be additive when the protein level was present in a high concentration and the detergent was present at levels corresponding to the minimum influence. Mixtures of a maximally stimulating dose (75 or 100 microg/ ml) of detergent and a small amount of protein produced activities that were at least 92% or more of the expected sums of individual stimulations. Interference occurred with the following mixtures: high amounts of detergent and protein; high protein and 10 or 15 microg/ml CTAB; and beta-lysin and arginine-rich histone, both at high levels. These data are consistent with a hypothesis that the two peaks in CTAB stimulation reflect two adjacent ATPase sites, one of which is also susceptible to stimulation by cationic protein.     

Glutaraldehyde crosslinking analysis of the C12E8 solubilized H,K-ATPase

A soluble porcine H,K-ATPase preparation was obtained with the nonionic detergent, C12E8. ATP hydrolysis by the soluble H,K-ATPase was stimulated with respect to the native preparation at pH 6.1, while the K(+)-phosphatase activity was comparable to the native enzyme. The soluble enzyme demonstrated characteristic ligand-dependent effects on ATP hydrolysis, including ATP activation of K(+)-stimulated hydrolysis with a K0.5 of 28 +/- 4 microM ATP, and inhibition with an IC50 of 2.1 mM ATP. The activation and inhibition of ATP hydrolysis by K+ was also observed with a K0.5 for activation of 2.8 +/- 0.4 mM KCl at 2.0 mM ATP (pH 6.1) and inhibition with an IC50 of 135 mM KCl at 0.05 mM ATP. 2-Methyl-8-(phenylmethoxy)imidazo[1,2a]pyridine-3-acetonitrile (SCH 28080), a specific inhibitor of the native H,K-ATPase, competitively inhibited the K(+)-stimulated activity with a Ki of 0.035 microM. The soluble enzyme was stable with a t0.5 for ATPase activity of 6 h between 4 and 11 degrees C. The demonstration of these related ligand responses in the catalytic reactions of the soluble preparation indicates that it is an appropriate medium for investigation of the subunit associations of the functional H,K-ATPase. Subunit associations of the active soluble enzyme were assessed following treatment with the crosslinking reagent, glutaraldehyde. The distribution of crosslinked particles was independent of the soluble protein concentration in the crosslinking buffer within the protein range 0.3 to 2.0 mg/ml or the detergent to protein ratio varied from 1 to 15 (w/w). The crosslinked pattern was unaffected by the presence or absence of K during crosslinking or nucleotide concentration. These observations suggest that crosslinking occurs in associated subunits that do not undergo rapid associations dependent upon enzyme turnover. Phosphorylation of the soluble enzyme with 0.1 mM MgATP produced a phosphoprotein at 94 kDa. A phosphoprotein obtained after glutaraldehyde treatment exhibited identical electrophoretic mobility to the crosslinked particle identified by silver stain. Glutaraldehyde treatment of soluble protein fractions resolved on a linear 10-35% glycerol gradient revealed several smaller peptides partially resolved from the crosslinked pump particle, but no active fraction enriched in the monomeric H,K-ATPase. This data indicates that the functional porcine gastric H,K-ATPase is organized as a structural dimer.     

Enzyme concentration affects the allosteric behavior of yeast phosphofructokinase

The influence of enzyme concentration on the kinetic behavior of yeast phosphofructokinase has been examined. A marked decrease in the ATP inhibition was observed when the enzyme activity was studied in permeabilized cells (in situ) as well as when the kinetic study was carried out with the purified yeast enzyme at a concentration of 120 micrograms/ml as compared to a 100-fold diluted enzyme. A similar result was obtained by adding polyethylene glycol either to a cell free extract or to the diluted pure enzyme to increase the local protein concentration. However, enzyme concentration had no significant effect on the fructose-6-P saturation curve. These results provide evidence that the allosteric behavior of yeast phosphofructokinase is affected by enzyme concentration.     

Concanavalin A induced changes of lymphocyte p-nitrophenylphosphatase (author's transl)]

The effect of K+, Na+, Mg2+ and ATP on the p-nitrophenylphosphatase activity was investigated. As an enzyme preparation a microsomal fraction of sheep lymphocytes was used. Low concentrations of Mg2+, K+ and Na+ increased, whereas high concentrations decreased the enzyme activity. There was an inhibition of activity by ATP without Na+ in the incubation medium and an increase of enzyme activity at low K:Na-ratio. By concanavalin A in a concentration of 15 mug/ml the p-nitrophenylphosphatase activity was increased in intact cells and the microsomal fraction for 30-40%. The activation was not Na+, K+, Mg2+, p-nitrophenylphosphate or ATP dependent.     

Action of nonviral gene delivery vectors on human complement system: low anticomplementary activity of lipoplexes based on lacZ plasmid and phospholipid/oligocation liposomes

A simple test-system has been developed for the first time in order to detect the ability of effectors (lipoplexes) to activate the complement system in an antibody-independent manner to serve as acceptors of nascent C4b and to inhibit formation of the key enzyme of complement, C3-convertase. The effect of plasmid DNA (pCMV-SPORT-LacZ), negatively charged cardiolipin (CL), neutral phosphatidylcholine (PC) vesicles and their lipoplexes, on the complement system was studied using the method developed. It was revealed that PC vesicles did not affect the complement system, while CL vesicles manifested low activation. The influence of plasmid DNA and its lipoplex based on PC liposomes as well on the complement system was very low. PC/LacZ lipoplex (143 microg/ml) acted on the complement system like 5.36 microg/ml heat aggregated IgG (agg) (the level of no pathological ruptures), whereas CL/LacZ lipoplex (143 microg/ml) acted similar to 10.7 microg/ml IgG (agg). Thus, weak activation of the complement system with CL lipoplex, and even weaker for the PC lipoplex testified to the use of neutral and positively charged lipoplexes preferably in gene therapy protocols. The technique can also be used for testing the influence of injectable gene therapy vectors on the complement system.     

Stoichiometry of lipid-protein interaction assessed by hydrophobic photolabeling

Here we undertook a comparative study of the composition of the lipid annulus of three ATPases pertaining to the P-type family: plasma membrane calcium pump (PMCA), sarcoplasmic reticulum calcium pump (SERCA) and Na,K-ATPase. The photoactivatable phosphatidylcholine analogue [(125)I]TID-PC/16 was incorporated into mixtures of dimyristoyl phosphatidylcholine (DMPC) and each enzyme with the aid of the nonionic detergent C(12)E(10). After photolysis, the extent of the labeling reaction was assessed to determine the lipid:protein stoichiometry: 17 for PMCA, 18 for SERCA, 24 for the Na,K-ATPase (alpha-subunit) and 5.6 mol PC/mol protein for the Na,K-ATPase (beta-subunit).     

Site-site interaction in the phospholipid activation of D-beta-hydroxybutyrate dehydrogenase

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with absolute specificity for phosphatidylcholine (PC). The enzyme devoid of lipid, the apodehydrogenase, inserts spontaneously into phospholipid vesicles where it exists as a tetramer. We now find the lipid activation to be limited by the mole fraction of PC in the total phospholipid. These studies suggest that the concentration of the enzyme-PC complex, which is essential for enzymic activity, becomes diffusion limited at lower PC concentration. The lipid activation and the tryptophan fluorescence of purified D-beta-hydroxybutyrate dehydrogenase were studied in the presence of a constant "bilayer background" of approximately 100 nonactivating phospholipid molecules/enzyme monomer. Activation by PC was half-maximal at 20 PC molecules/enzyme monomer. This value was doubled when the amount of "background" phospholipid was doubled. Activation proceeded with positive cooperativity having a Hill coefficient of approximately 2.4. These data indicate interactions between at least three PC-binding sites. The quenching of tryptophan fluorescence by the phospholipid activator, 1-palmitoyl-2-(1-pyrenyl)-decanoyl-PC (2-pyrenyl-PC), gives a saturation curve with half-maximal quenching of 6 quencher molecules/enzyme monomer. This value is equivalent to an apparent phospholipid-protein dissociation constant in the two-dimensional membrane and corresponds to approximately 6 mol % of total phospholipid. In distinct contrast to the phospholipid activation curve, the fluorescence quenching saturation curve was hyperbolic and there was no specificity for PC. The fluorescence quenching by 2-pyrenyl-PC could be diminished by using a several-fold excess of PC or other phospholipids so as to reduce the mole fraction of quencher in the bilayer. It would appear that formation of enzyme-PC complex is a dynamic process consisting of at least two discernible steps: 1) a primary interaction, as measured by tryptophan quenching, which is hyperbolic and not specific for lecithin. This interaction is independent from and precedes 2) phospholipid activation of D-beta-hydroxybutyrate dehydrogenase, which is cooperative in nature and specific for lecithin.     

A continuous spectrophotometric assay for phospholipase A(2) activity

This paper describes a simple continuous spectrophotometric method for assaying phospholipase A(2) (PLA(2)) activity. The procedure is based on a coupled enzymatic assay, using dilinoleoyl phosphatidylcholine as phospholipase substrate and lipoxygenase as coupling enzyme. The linoleic acid released by phospholipase was oxidized by lipoxygenase and then phospholipase activity was followed spectrophotometrically by measuring the increase in absorbance at 234 nm due to the formation of the corresponding hydroperoxide from the linoleic acid. The optimal assay concentrations of hog pancreatic phospholipase A(2) and lipoxygenase were established. PLA(2) activity varied with pH, reaching its optimal value at pH 8.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (V(m)=1.8 microM/min, K(m)=4.5 microM, V(m)/K(m)=0.4 min(-1)). This assay also allows PLA(2) inhibitors, such as p-bromophenacyl bromide or dehydroabietylamine acetate, to be studied. This method was proved to be specific since there was no activity in the absence of phospholipase A(2). It also has the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase A(2).     

Kinetic studies on the regulation of rabbit liver pyruvate kinase

Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K(+) and optimum activity was recorded with 30mm-K(+), 4mm-MgADP(-), with a MgADP(-)/ADP(2-) ratio of 50:1, but inhibition occurred with K(+) concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg(2+) was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (n(H)=2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent K(m) for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis-Menten response was obtained when phosphoenolpyruvate was the variable substrate (K(m)=0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg(2+).     

Stimulation of rat renal Na+, K+-ATPase activity by thyroid hormones

The effect of thyroid hormones (T4, T3 and reverse T3) on rat renal Na+,K+-ATPase activity was investigated by a cytochemical technique. T3 caused stimulation of Na+,K+-ATPase activity in the renal medulla but not in the renal cortex. There was a peak in enzyme activity after cultured renal segments had been exposed to T3 for 11 min and this time of maximal stimulation did not vary with the concentration of T3. A rectilinear response in Na+,K+-ATPase activity was observed over T3 concentration range 10 pmol l-1 to 100 nmol l-1; at higher T3 concentrations, Na+,K+-ATPase activity was inhibited. The enzyme response was totally blocked by specific T3 antiserum. Addition of T4 and reverse T3 (100 fmol l-1 -1 mmol l-1) failed to stimulate Na+,K+-ATPase activity in any part of the kidney. Plasma (neat and diluted 1:10) stimulated the enzyme in parallel with the dose response curve and the stimulatory effect was abolished by prior addition of specific T3 antiserum.     

Rapid Activation of Tyrosine Hydroxylase in Response to Nerve Growth Factor

Abstract: Nerve growth factor protein (NGF) was found to rapidly promote the activation of tyrosine hydroxylase in cultured rat PC 12 pheochromocytoma cells. PC 12 cultures were exposed to NGF for periods of less than 1 h and the soluble contents of homogenates prepared from the cells were assayed for tyrosine hydroxylase activity. Under these conditions, the specific enzymatic activity was increased by 60 ± 10% (n = 13) in comparison with that in untreated sister cultures. The increase was half maximal by 2–5 min of exposure and at NGF concentrations of about 10 ng/ml (0.36 n M ). Antiserum against NGF blocked the effect. Tyrosine hydroxylase activity could also be rapidly increased by NGF in cultures of PC12 cells that had been treated with the factor for several weeks in order to produce a neuron-like phenotype. This was achieved by withdrawing NGF for about 4 h and then readding it for 30 min. The NGF-induced increase of tyrosine hydroxylase activity in PC12 cultures was not affected by inhibition of protein synthesis and therefore appeared to be due to activation of the enzyme. Kinetic experiments revealed that NGF brought about no change in the apparent K_m of the enzyme for tyrosine or for co-factor (6-methyltetrahydropteridine), but that it did significantly increase the apparent maximum specific activity of the enzyme. These observations suggest that NGF (perhaps released by target organs) could promote a rapid and local enhancement of noradrenergic transmission in the sympathetic nervous system.     

General trend of lipase to self-assemble giving bimolecular aggregates greatly modifies the enzyme functionality

Three microbial lipases (those from Candida rugosa, Humicola lanuginosa, and Mucor miehei) have been found to exhibit a tendency to form bimolecular aggregates in solution even at very low enzyme concentrations (44 microg/mL) in the absence of a detergent, as detected by gel filtration. The monomolecular form of the enzymes was found as unique only at low enzyme concentration and in the presence of detergents. However, in the case of the lipase B from Candida antarctica, no bimolecular form could be identified even at enzyme concentrations as high as 1.2 mg/mL in the absence of detergent. It has been stated that bimolecular and monomolecular structures display very different functional properties: (i) the enzyme specific activity decreased when the lipase concentration increased; (ii) the bimolecular form was much more stable than the monomeric one yielding a higher optimal T (increasing between 5 and 10 degrees C) and higher stability in inactivation experiments (the dimer half-life became several orders of magnitude higher than that of the monomer); (iii) the enantioselectivity depended on the enzyme concentration even after immobilization. For example, with use of the lipase from H. lanuginosa, the enantiomeric excess of the remaining ester in the hydrolysis of fully soluble ethyl ester of (R,S)-2-hydroxy-4-phenylbutanoic acid varied from 4 to 57 when the concentrated or diluted enzyme immobilized on PEI support, respectively, was used. It seems that the bimolecular structure of lipases might be formed by two open lipase molecules (interfacially activating each other) in very close contact and hence with a very altered active center.     

The phosphatase activity of the plasma membrane Ca2+ pump. Activation by acidic lipids in the absence of Ca2+ increases the apparent affinity for Mg2+

The purified PMCA supplemented with phosphatidylcholine was able to hydrolyze pNPP in a reaction media containing only Mg(2+) and K(+). Micromolar concentrations of Ca(2+) inhibited about 75% of the pNPPase activity while the inhibition of the remainder 25% required higher Ca(2+) concentrations. Acidic lipids increased 5-10 fold the pNPPase activity either in the presence or in the absence of Ca(2+). The activation by acidic lipids took place without a significant change in the apparent affinities for pNPP or K(+) but the apparent affinity of the enzyme for Mg(2+) increased about 10 fold. Thus, the stimulation of the pNPPase activity of the PMCA by acidic lipids was maximal at low concentrations of Mg(2+). Although with differing apparent affinities vanadate, phosphate, ATP and ADP were all inhibitors of the pNPPase activity and their effects were not significantly affected by acidic lipids. These results indicate that (a) the phosphatase function of the PMCA is optimal when the enzyme is in its activated Ca(2+) free conformation (E2) and (b) the PMCA can be activated by acidic lipids in the absence of Ca(2+) and the activation improves the interaction of the enzyme with Mg(2+).     

Overproduction and high-yield purification of phospholipase A from the outer membrane of Escherichia coli K-12

The cloned gene for the outer-membrane-bound phospholipase A from Escherichia coli was placed under control of the strong PL promoter of phage lambda. Induction of PL resulted in a 250-fold overexpression up to about 2% total cellular protein. This overproduced enzyme was indistinguishable from the wild-type enzyme. A homogeneous phospholiphase A preparation was obtained in high yield from overproducing bacteria, using the zwitterionic detergent C12-Sulfobetaine and anion-exchange chromatography. Detergent gradients were found to exert great influence on the elution characteristics. Considerations for the choice of optimal detergent gradients are discussed. The purified enzyme migrated as a single 29-kDa band in SDS/polyacrylamide gels, and required Ca(II) for activity. Maximum activity was displayed by enzyme samples taken from solutions with detergent concentrations near the critical micelle concentration. However, upon switching from high to optimal detergent concentration, maximum activity was restored in several hours, probably reflecting a slow conformational transition of the protein. Because the final pure protein was found to hydrolyze phospholipids in the intact erythrocyte membrane, a densely packed bilayer, we assume that this protein is in its biological native state.