Essential histidine residues in lysolecithin:lysolecithin acetyltransferase from rabbit lung

Both activities of rabbit lung lysolecithin:lysolecithin acyltransferase (EC 3.1.1.5), hydrolysis and transacylation, are inactivated by diethylpyrocarbonate. The reaction follows pseudo-first-order kinetics, and second-order rate constants of 1.17 mM-1min-1 for hydrolysis and 0.56 mM-1 min-1 for transacylation were obtained at pH 6.5 and 37 degrees C. The rate of inactivation is dependent on pH, showing the involvement of a group with a pK of 6.5. The difference spectra showed an increase in absorbance at 242 nm, indicating the modification of histidine residues. The activity lost by diethylpyrocarbonate modification can be partially recovered by hydroxylamine treatment. The statistical analysis of residual fractional activity versus the number of modified histidine residues leads to the conclusion that two histidine residues are essential for the hydrolytic activity, whereas transacylation activity depends on only one essential histidine. The substrate and substrate analogs protected the enzyme against inactivation by diethylpyrocarbonate, suggesting that the essential residues are located at or near the active site of the enzyme.     

Lysolecithin:lysolecithin acyltransferase from rabbit lung. A conformational study

The enzyme lysolecithin:lysolecithin acyltransferase from rabbit lung has been found to have a relatively disordered conformation in solutions of high ionic strength. The protein exhibited an ordering of structure when salt was suppressed. This conformational change was concomitant with the loss of transacylase activity, the hydrolytic reaction remaining unchanged. Addition of NaCl caused a progressive disordering of structure with a parallel increase of transacylase activity. The acid denaturation of the protein, at low and high ionic strengths, showed that the ionization of groups with pK in the range 5.9-6.4 was essential for denaturation. The structure was stable at basic pH. The addition of lipids resulted in a non-specific stabilization of the disordered conformation, in the same manner as the addition of NaCl. From these results, it is suggested that there are two conformations for this protein which differ in their ability to bind lysolecithin molecules in the enzyme deacylation step of the reaction. This hypothesis agrees with previously published properties of the enzyme, concerning aggregation with other proteins and kinetic data. From the amino acid composition and conformational properties, the authors suggest that this enzyme could be a peripheral membrane protein.     

Effect of lipids on activity and conformation of lysolecithin:lysolecithin acyltransferase from rabbit lung

Summary Lysolecithin:lysolecithin acyltranferase is an enzyme which in several previous studies has shown a dual behavior catalyzing two types of reaction, transacylation or hydrolysis, with the same substrate. Both activities have shown to be dependent on several environmental conditions and among them, the presence of lipids.The addition of several classes of lipids activated in all the cases the enzyme, decreasing the hydrolysis/transacylation molar ratio. This effect was higher for PC/PE/Chol mixture than for other lipids assayed. Circular dichroism spectra of the enzyme did not show any change with the addition of lipids, concluding that the effect of lipids was not due to any structural change in the protein. The hypothesis has been made of an influence of lipids on the physical state of the substrate as well as, possibly, on the enzyme-substrate interaction.The significance of these effects on the physiological role of lysolecithin:lysolecithin acyltransferase from soluble fraction of rabbit lung is discussed.Abbreviations Chol cholesterol - CMC critical micellar concentration - DPPC dipalmitoylphosphatidylcholine - FA fatty acid - H/T hydrolysis/transacylation molar ratio - LPC lysophosphatidylcholine - PC phosphatidylcholine - PE phosphatidylethanolamine - TG triglyceride - UV ultraviolet     

Accessibility of lysolecithin in catecholamine secretory vesicles to acyl CoA:lysolecithin acyl transferase

Lysolecithin (monoacylglycerophosphorylcholine) accounts for 13 to 20% of the lipid phosphorous of the bovine adrenal catecholamine secretory vesicles (chromaffin granules). We have incubated purified vesicles with [1-¹⁴C] oleyl coenzyme A and rat liver microsomes containing acyl coenzyme A: monoacylglycerophosphorylcholine acyl transferase to determine the accessibility of the granule membrane lysolecithin to another membrane. No acylation of lysolecithin occurs when the chromaffin granules are intact. The accessibility of the granule membrane lysolecithin increases markedly when the vesicles are broken.     

Mechanism of the reaction catalyzed by acyl-CoA: lysolecithin acyltransferase from rabbit lung. pH studies and chemical modification

This paper deals with the first attempt to elucidate the chemical mechanism of acyl-CoA: lysolecithin acyltransferase from rabbit lung, a key enzyme in the metabolism of lung surfactant. For this purpose, the pH dependence of kinetic constants as well as the chemical modification of the protein have been studied on a partially-purified preparation. From these experiments, the pKs on which the activity of the enzyme relies have been calculated, giving values of pK1 congruent to 5.5 and pK2 congruent to 10. Analysis of the effect of organic solvents on these pKs and the calculation of the enthalpies of ionization, together with the chemical modification experiments, lead to the conclusion that pK1 is due to an histidine residue, whereas pK2 arises from the amino group of the adenine ring of palmitoyl-CoA. Moreover, chemical modification demonstrated an essential cysteine. A tentative chemical mechanism, in accordance with these results, is proposed and it is hypothesized, in view of other results obtained in our laboratory and from the literature, that the chemical mechanism of acyl transfer to sn-2 position may be common to other enzymes of glycerolipid metabolism.     

Activity of cholinephosphotransferase, lysolecithin: lysolecithin acyltransferase and lysolecithin acyltransferase in the developing mouse lung.

1. The present study presents the activity profiles of cholinephosphotransferase, lysolecithin:lysolecithin acyltransferase and lysolecithin acyltransferase at different stages of development of the mouse lung. 2. The specific activity of cholinephosphotransferase, a key enzyme in the de novo synthesis of phosphatidylcholine, increases during the later stages of fetal development until it reaches a maximal value at a gestational age of 17 days, i.e. 2 days before term. Thereafter, the activity of the enzyme declines again until around term. 2. The specific activity of lysolecithin:lysolecithin acyltransferase which catalyzes the transesterification between two molecules of 1-acyl-sn-glycero-3-phosphocholine, appears to be much lower than that of cholinephosphotransferase at gestational ages below 18 days. However, around day 18, the specific activity of lysolecithin:lysolecithin acyltransferase increases dramatically until it almost equals the maximal activity of cholinephosphotransferase measured on day 17. 4. The specific activity of lysolecithin acyltransferase, which catalyzes the direct acylation of 1-acyl-sn-glycero-3-phosphocholine, does not change significantly during the prenatal development and is lower than that of either lysolecithin:lysolecithin acyltransferase or cholinephosphotransferase at all stages of development. 5. These results are discussed in view of the possible role of these enzymes in the biosynthesis of pulmonary 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.     

Essential residues in lysolecithin:lysolecithin acyltransferase from rabbit lung: assessment by chemical modification

The inhibition of lysolecithin:lysolecithin acyltransferase by several specific reagents was studied. Diisopropyl fluorophosphate (DFP) completely inhibited both activities at a concentration of 4 mM. Activity was not protected by substrate and the enzyme showed a change in circular dichroism spectrum upon treatment with inhibitor. Phenylmethanesulfonyl fluoride, another serine-specific reagent, did not inhibit either hydrolysis or transacylation. Therefore, we suggest that DFP does not modify an active serine in the catalytic site. p-Hydroxymercury benzoate and N-ethylmaleimide (NEM) abolished both activities of the enzyme. The presence of substrate partially protected against inactivation. Far-uv CD spectrum of NEM-modified enzyme revealed no changes in protein structure. The existence of two classes of essential cysteine residues was deduced from kinetics of NEM inactivation. Both classes differ in NEM reactivity and also in their participation in the catalytic mechanism. A tyrosine-specific reagent, tetranitromethane, also inhibited hydrolysis and transacylation, following first-order kinetics. The partial protection by substrate suggested the possible existence of essential tyrosines near the active site. At pH 5.0 N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline inactivated hydrolysis but not transacylation. However, both of them remained unchanged at pH 6.5. The substrate prevented the loss of hydrolytic ability. Therefore, a carboxyl residue participating just in the catalytic mechanism of hydrolysis is proposed.     

Effect of albumin on acyl-CoA: lysolecithin acyltransferase,lysolecithin : lysolecithin acyltransferase and acyl-CoA hydrolase from rabbit lung

Acyl-CoA : lysolecithin and lysolecithin : lysolecithin acyltransferases, as well as acyl-CoA hydrolase are important enzymes in lung lipid metabolism. They use amphiphylic lipids as substrates and differ in subcellular localization. In this sense, lipid-protein interactions can be an essential factor in their activity. We have studied the effect of albumin, as lipid-binding protein model, in the activities of these enzymes. Acyl-CoA hydrolase was inhibited in the presence of albumin, whereas acyl-CoA : lysolecithin acyltransferase showed a complex effect of activation depending on both albumin concentration and palmitoyl-CoA/lysolecithin molar ratio. Lysolecithin : lysolecithin acyltransferase was affected differentially on its two activities. Hydrolysis remained unaffected and transacylation was inhibited by albumin. These results are consequence of the interaction of albumin with both lipidic substrates that changes their critical micellar concentration.Abbreviations TNS 6-(p-toluidino)-2-naphthalene-sulfonic acid - CMC Critical Micellar Concentration - LP Lysolecithin (1-acyl-sn-glycero-3-phosphocholine) - PalmCoA palmitoyl-CoA     

Synthesis and micellar formation of spin-labeled lysolecithin.

A spin-labeled lysolecithin, 1-[12'-(N-oxyl-4",4"-dimethyloxazolidine)-stearoyl]-sn-glycero-3-phosphorylcholine, has been synthesized in which the spin is covalently attached to its fatty acyl chain. The electron spin resonance spectra of this lysolecithin is an aqueous solution generally showed sharp three resonance lines superposed on a broad resonance line. Investigation of changes in the signal intensity of these spectra against the concentration of lysolecithin led to the inference that the sharp lines are due to monomers of lysolecithin while the broad one to micelles. The critical micellar concentration was consistent with that evaluated from the spectral shift of a dye. In the electron spin resonance spectra obtained from spin-labeled lysolecithin solutions with various amounts of dimyristoyllecithin, the line width of broad signal arised from micellar spin-labeled lysolecithin broadened as increase of dimyristoyllecithin. The line-broadening thus observed was briefly discussed.     

Mechanism of bactericidal activity of lysolecithin and its biological implication

Lysolecithin exhibited bactericidal effect on mycobacteria and staphylococci, but not on E. coli. The effect on staphylococci was manifested shortly after exposure, but that on mycobacteria was of the delayed type. The mycobactericidal activity was dependent on the fatty acid moiety of the chemical structure reflecting the cytotoxicity of the free form. The activity on staphylococci was not, however, of such fatty acid dependency and showed the same pattern of molecular species in hemolytic activity. These and other collateral findings suggest that the mycobactericidal effect of lysolecithin is due to the free fatty acids released therefrom by the enzymatic activity of the exposed bacterial cells, but that staphylococci are killed by the detergent effect of the whole lysolecithin molecule.     

Effects of zinc on creatine kinase: activity changes, conformational changes, and aggregation

The effects of zinc on creatine kinase (CK) are very distinctive compared with other bivalent metal ions. Zinc up to 0.1 mM induced increases in CK activity, accompanied by significant hydrophobic surface exposure and increase in a-helix content of CK. Zinc over 0.1 mM denatured and inactived CK. In the presence of 0.1 mM zinc, the CK activity was very close to that of the native CK, but its conformation changed greatly. The kinetic courses of CK inactivation and conformational change in the presence of 1 mM zinc were measured to determine apparent rate constants of inactivation and conformational change. Zinc over 0.05 mM induced CK aggregation at 37°C, and the aggregation was dependent on zinc concentration, CK concentration, and temperature. The inactivation and aggregation can be reversed by EDTA. An explanation for CK aggregation induced by zinc is proposed, as well as a mechanism for CK abnormality in Alzheimer's disease.To whom correspondence should be addressed.     

The interaction of apoA-I from human high density lipoprotein with lysolecithin.

The binding of apoA-I to lysolecithin has been studied by fluorescence and circular dichroism. The influence of the conformation of apoA-I on its interaction with lysolecithin has also been evaluated. ApoA-I is bound to lysolecithin with an association greater than 10(7) whether apoA-I is native or highly unfolded in 1.8 M guanidinium hydrochloride. The association of apoA-I with lysolecithin results in an increase in secondary structure. A 25-residue fragment of apoA-I binds to lysolecithin equally strongly as the native molecule.     

Probing the dynamics of a His73-heme alkaline transition in a destabilized variant of yeast iso-1-cytochrome c with conformationally gated electron transfer methods

The alkaline transition of cytochrome c involves substitution of the Met80 heme ligand of the native state with a lysine ligand from a surface Ω-loop (residues 70 to 85). The standard mechanism for the alkaline transition involves a rapid deprotonation equilibrium followed by the conformational change. However, recent work implicates multiple ionization equilibria and stable intermediates. In previous work, we showed that the kinetics of formation of a His73-heme alkaline conformer of yeast iso-1-cytochrome c requires ionization of the histidine ligand (pK(HL) ~ 6.5). Furthermore, the forward and backward rate constants, k(f) and k(b), respectively, for the conformational change are modulated by two auxiliary ionizations (pK(H1) ~ 5.5, and pK(H2) ~ 9). A possible candidate for pK(H1) is His26, which has a strongly shifted pK(a) in native cytochrome c. Here, we use the AcH73 iso-1-cytochrome c variant, which contains an H26N mutation, to test this hypothesis. pH jump experiments on the AcH73 variant show no change in k(obs) for the His73-heme alkaline transition from pH 5 to 8, suggesting that pK(H1) has disappeared. However, direct measurement of k(f) and k(b) using conformationally gated electron transfer methods shows that the pH independence of k(obs) results from coincidental compensation between the decrease in k(b) due to pK(H1) and the increase in k(f) due to pK(HL). Thus, His26 is not the source of pK(H1). The data also show that the H26N mutation enhances the dynamics of this conformational transition from pH 5 to 10, likely as a result of destabilization of the protein.     

Stimulation of galactosyltransferase in liver microsomes by lysolecithin

Lysolecithin markedly stimulated membrane-bound UDP-galactose:glycoprotein galactosyltransferase. The parent molecule lecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidic acid, lysophosphatidic acid or glycerophosphorylcholine did not activate the enzyme suggesting that both fatty acyl- and phosphorylcholine groups are required for the enzyme activation. The dose-effect of lysolecithin showed sigmoidal kinetics and the Vmax of the enzyme was increased several-fold by lysolecithin. Saturating amounts of Triton masked the effect of lysolecithin. Pre-incubation with phospholipase A also activated the enzyme. A possible role of membrane lysolecithin is indicated in regulating the enzymes of glycoprotein synthesis.     

Intrinsic fluorescence of a hydrophobic myelin protein and some complexes with phospholipids

The fluorescence characteristics of lipophilin, a proteolipid apoprotein from human myelin, were determined in aqueous and lipid environments. In all cases the tryptophan residues were located in buried hydrophobic sites of uniform, but limited, accessibility to the permeant quenching agent acrylamide; only in the helicogenic solvent 2-chloroethanol were the protein fluorophores exposed to the medium. Quantum yields were dependent on the state of aggregation of the protein in aqueous solution and increased considerably on treatment with lysolecithin micelles, or when the protein was combined with phosphatidylcholine by codialysis from 2-chloroethanol into water. Fluorescence titrations indicated that lipophilin bound to lysolecithin with an association constant greater than 10(6) L/mol. Radiationless singlet excitation energy transfer from tyrosine to tryptophan residues was found to decrease markedly when the protein was combined with lipids. When the protein was introduced into dimyristoylphosphatidylcholine vesicles, the tryptophan fluorescence did not detect any solid-liquid phase change. These results were consistent with strong hydrophobic interactions between lipophilin and phospholipids, which lead to conformational adjustments in the protein, and to establishment of an immobilized layer of boundary lipid in bilayer systems.     

Production of egg yolk lysolecithin with immobilized phospholipase A2

Production of egg yolk lysolecithin was compared using free phospholipase A₂ (PLA₂) and immobilized PLA₂ in alginate-silicate sol-gel matrix. Choice of solvent, water content, calcium, and temperatures changed the activity of the free and immobilized PLA₂ a lot, owing to their effects on the catalytic properties of the enzyme as well as the conformational change of lecithin in ethanol-buffer mixture. Free PLA₂ shows typical microemulsion kinetics in ethanol-buffer system. The effect of the water content on the enzyme reaction was greatly influenced by the presence of calcium ion. In the absence of calcium ion, certain optimal water content for the production of lysolecithin always exists in the free PLA₂ reaction. However, with calcium ion, three distinctive regions were observed with free PLA₂ reactions. Initially, in the micro-aqueous region of the ethanol-buffer system with calcium ion, the hydrolysis activity of PLA₂ was proportional to the water content. Beyond the region, concave type of activity profiles were observed as the water content increases. As the water content increases further, the hydrolysis rate of the PLA₂ abruptly decreased by the phase separation. On the contrary, in case of immobilized enzyme, optimal water content for the production of lysolecithin exists regardless of the presence of calcium ion. The calcium ion was essential for achieving the maximum activity of both free and immobilized PLA₂. The addition of calcium ion not only affected the catalytic activity of the enzyme but also was necessary to improve the enzyme stability. As the immobilization of the enzyme remarkably increased thermal stability of the free enzyme, the immobilized PLA₂ is more desirable to be used in the production of various lysophospholipids. It was successfully reused over 250 h.     

Inhibition of prostaglandin synthesis by lysolecithin

Exogenous lysolecithin inhibits prostaglandin E₂ synthesis from arachidonic acid in bovine seminal vesicle microsomes at plausible physiological levels (lysolecithin-to-protein ratios ? 0.03 [w/w]) by inhibiting fatty acid cyclo-oxygenase activity. Structurally defined lysolecithins with varying fatty acid chain length exhibit varying effectiveness as inhibitors. Addition of equimolar quantities of free fatty acid lowers the lysolecithin concetration required for inhibition. Exogenous lysolecithin inhibits unstimulated and thrombin-stimulated prostaglandin E₂ synthesis from endogenous substrate in SVBalb/3T3 cells. Serum treatment of SVBalb/3T3 cells, which generates endogenous lysolecithin and free fatty acids, decreases the efficiency of conversion of free arachidonic acid to prostaglandins. These results suggest a possible role for the products of phospholipase A2 action in the regulation of prostaglandin synthesis.     

The alkaline transition of swine pepsinogen.

At alkaline pH, swine pepsinogen is reversibly inactivated in a transition which involves the cooperative release of two protons from the molecule and is governed by a pK = 9. Stopped flow kinetic studies on the absorbance changes accompanying this reaction show that it can be resolved into two steps, with increasing pH; a slow conformational change, whose amplitude follows the ionisation curve of one group of pK = 9.9, followed by a rapid pH dependent conformational change, linked to a group of pK = 8.2. The pH dependence of the rate of the slow step is interpreted to show the presence of a protonated group which cannot ionise in the neutral form of the zymogen, but is in slow equilibrium with a form where it titrates with a pK 6.8. At the same time, a histidine in the amino terminal region of the protein becomes reactive to diethyl pyrocarbonate, suggesting this to be the group which triggers the reaction.     

Slow inactivation does not block the aqueous accessibility to the outer pore of voltage-gated Na channels

Slow inactivation of voltage-gated Na channels is kinetically and structurally distinct from fast inactivation. Whereas structures that participate in fast inactivation are well described and include the cytoplasmic III-IV linker, the nature and location of the slow inactivation gating mechanism remains poorly understood. Several lines of evidence suggest that the pore regions (P-regions) are important contributors to slow inactivation gating. This has led to the proposal that a collapse of the pore impedes Na current during slow inactivation. We sought to determine whether such a slow inactivation-coupled conformational change could be detected in the outer pore. To accomplish this, we used a rapid perfusion technique to measure reaction rates between cysteine-substituted side chains lining the aqueous pore and the charged sulfhydryl-modifying reagent MTS-ET. A pattern of incrementally slower reaction rates was observed at substituted sites at increasing depth in the pore. We found no state-dependent change in modification rates of P-region residues located in all four domains, and thus no change in aqueous accessibility, between slow- and nonslow-inactivated states. In domains I and IV, it was possible to measure modification rates at residues adjacent to the narrow DEKA selectivity filter (Y401C and G1530C), and yet no change was observed in accessibility in either slow- or nonslow-inactivated states. We interpret these results as evidence that the outer mouth of the Na pore remains open while the channel is slow inactivated.     

The alkaline transition of swine pepsinogen

At alkaline pH, swine pepsinogen is reversibly inactivated in a transition which involves the cooperative release of two protons from the molecule and is governed by a pK = 9. Stopped flow kinetic studies on the absorbance changes accompanying this reaction show that it can be resolved into two steps, with increasing pH; a slow conformational change, whose amplitude follows the ionisation curve of one group of pK = 9.9, followed by a rapid pH dependent conformational change, linked to a group of pK = 8.2. The pH dependence of the rate of the slow step is interpreted to show the presence of a protonated group which cannot ionise in the neutral form of the zymogen, but is in slow equilibrium with a form where it titrates with a pK = 6.8. At the same time, a histidine in the amino terminal region of the protein becomes reactive to diethyl pyrocarbonate, suggesting this to be the group which triggers the reaction.