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C Acebal

Publications and source records attributed to C Acebal.

At least 37 records · Page 2Linked to original sources
PubMed1990

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.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1990

Theoretical approach to the steady-state kinetics of a bi-substrate acyl-transfer enzyme reaction that follows a hydrolysable-acyl-enzyme-based mechanism. Application to the study of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase from rabbit lung.

A kinetic model is proposed for catalysis by an enzyme that has several special characteristics: (i) it catalyses an acyl-transfer bi-substrate reaction between two identical molecules of substrate, (ii) the substrate is an amphiphilic molecule that can be present in two physical forms, namely monomers and micelles, and (iii) the reaction progresses through an acyl-enzyme-based mechanism and the covalent intermediate can react also with water to yield a secondary hydrolytic reaction. The theoretical kinetic equations for both reactions were deduced according to steady-state assumptions and the theoretical plots were predicted. The experimental kinetics of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase from rabbit lung fitted the proposed equations with great accuracy. Also, kinetics of inhibition by products behaved as expected. It was concluded that the competition between two nucleophiles for the covalent acyl-enzyme intermediate, and not a different enzyme action depending on the physical state of the substrate, is responsible for the differences in kinetic pattern for the two activities of the enzyme. This conclusion, together with the fact that the kinetic equation for the transacylation is quadratic, generates a 'hysteretic' pattern that can provide the basis of self-regulatory properties for enzymes to which this model could be applied.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1989

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.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1989

Evidence of a pH-dependent conformational change at the active site of lysolecithin:lysolecithin acyltransferase from rabbit lung.

It has been shown that both activities, hydrolysis and transacylation, of lysolecithin:lysolecithin acyltransferase, as well as the conformation of the polypeptide are critically dependent on a pK around 5.8, but the question remains if the same residue(s) is responsible for the conformational change and the loss of activity. In this paper, ultrasonic cavitation is used to study the pH-dependent inactivation. The results show that there are two first-order inactivation constants which depend on pH and that the transition between them has a pK of 5.9. As the constants of ultrasonic inactivation are very dependent on the accessibility of the residues it is concluded that the conformational change modifies the accessibility of the active site.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1987

The kinetic mechanism of acyl-CoA:lysolecithin acyltransferase from rabbit lung.

Acyl-CoA:lysolecithin acyltransferase is a key enzyme in the deacylation-reacylation pathway of biosynthesis of molecular species of lecithin. However, the mechanism of the reaction has been little studied. In this paper, the kinetic mechanism of acyl-CoA:lysolecithin acyltransferase, partially purified from rabbit lung, is studied. The double-reciprocal plots of initial velocity vs substrate concentration gave two sets of parallel lines which fitted to a ping-pong equation with the following parameters: Km (palmitoyl-CoA) = 8.5 +/- 2 microM, Km (lysolecithin) = 61 +/- 16 microM, and V = 18 +/- 4 nmol/min/mg protein. Inhibition studies by substrates, alternate substrates, and products supported the ping-pong mechanism, although some nonclassical behavior was observed. Palmitoyl-CoA did not inhibit even at concentrations of 100 Km. In contrast, lysolecithin was a dead-end inhibitor with a dissociation constant of Ki = 930 +/- 40 microM. Alternate substrates and CoA showed alternate pathways for the reaction due to the formation of ternary complexes. Dipalmitoylphosphatidylcholine inhibition pointed to an isomerization of the free enzyme prior to the start of the reaction. From these results, an iso-ping-pong kinetic mechanism for lysolecithin acyltransferase is proposed. The kinetic steps of the reaction are correlated with previous chemical studies of the enzyme.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1985

Substrate selectivity of acyl-CoA:lysolecithin acyltransferase from rabbit lung.

The influence of both polar head and acyl chain of lysophospholipid on the activity of partially purified acyl-CoA:lysolecithin acyltransferase from rabbit lung was studied. It was concluded that the presence of methyl groups on the nitrogen of the base was essential for recognition of lysophospholipid as substrate by the enzyme. With respect to the acyl chain length and saturation, the activity followed the order: 16:0 approximately equal to 18:1 greater than 14:0 greater than greater than greater than 18:0 approximately equal to 12:0. Also, the effect on the activity of the acyl chain on acyl-CoA was studied. The activity showed great selectivity for saturated acyl-CoAs. The activity with polyunsaturated fatty acids was very low and in the case of arachidonoyl-CoA was almost negligible. The comparison between crude microsomal preparations and partially purified preparations allowed to suggest that it could exist two different acyl-CoA:lysolecithin acyltransferases differing in their selectivity towards saturated and unsaturated fatty acids.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1985

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.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1984

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

Lysolecithin:lysolecithin 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.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1984

Substrate selectivity of lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung.

The influence of both polar group and acyl chain of lysophospholipids on the lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung was studied. Both, transacylase and hydrolase activities of this enzyme, utilize selectively 1-[1-14C]palmitoyl-sn-glycero-3-phosphocholine when compared with 1-[9,10-3H2]palmitoyl-sn-glycero-3-phosphoethanolamine. Transacylase activity is more selective for lysophosphatidylcholine as acyl acceptor than as acyl donor. The amount of dipalmitoylphosphatidylcholine/min/mg protein synthesized from mixed lysophosphatidylcholine/lysophosphatidylethanolamine micelles does not change with increasing molar percentages of lysophosphatidylethanolamine in the mixture and is similar to that formed with pure lysophosphatidylcholine micelles. Transacylation reaction takes place preferentially with long and saturated acyl chains whereas hydrolysis reaction does more efficiently with longer acyl chains, independently of their insaturation degree.

Acyltransferases↗
PubMed1984

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.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1984

Influence of temperature on stability and activity of lysolecithin acyltransferase and acyl-CoA hydrolase from rabbit lung.

The effect of temperature on the activities of acyl-CoA:lysolecithin acyltransferase and acyl-CoA hydrolase has been studied in microsomal preparations. The enzymes had different thermal stabilities, the hydrolase being more stable. The temperature dependence of enzyme activities was studied either in 4 M NaCl-washed microsomes or NaCl-washed detergent-treated microsomes. Both preparations showed little increase in acylation rate when the temperature was increased, whereas hydrolase activity was increased markedly. This increase in hydrolytic activity was higher when lysolecithin was absent. Based on this behavior of the enzymes, the relative accuracies of the spectrophotometric and radioactive assay methods are discussed.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1983

Labelled lipids distribution following [3H]glycerol injection to pregnant rabbits.

1. The time dependent variation in specific activity of serum triglycerides and phospholipids has been studied following an intravenous pulse-labelling with 2-[3H]glycerol to pregnant rabbits. 2. The specific activity of triglycerides varied according to the gestation time and the maximal values of specific activity shifted to shorter times when gestation progressed to term. 3. The specific activity of serum phospholipids was higher in pregnant animals and increased with gestation time. 4. Concentrations and specific activities of both triglycerides and phospholipids were investigated in serum of pregnant and fetal rabbits at term. Concentrations of both lipid classes were notably higher in the fetal blood than in the maternal one. 5. The results were discussed on the basis of changes in hepatic biosynthesis of phosphatidic acid. Also, it was suggested a high drainage of either lipids or their degradation products by the feto-placental unit at the end of pregnancy. 6. Levels and specific activities of maternal and fetal lipids from liver and lung were also examined. The different metabolic roles of the main tissues involved in triglyceride and phospholipid metabolism were discussed.

Animals↗