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D Lombardo

Publications and source records attributed to D Lombardo.

At least 91 records · Page 5Linked to original sources
PubMed1987

The inhibition of phospholipase A2 by manoalide and manoalide analogues.

Manoalide, a natural product from sponge, displays anti-inflammatory activity in vivo. Previous work has shown that manoalide is also a potent covalent inhibitor of the extracellular phospholipase A2 from cobra venom and that the inhibition correlated with a pH-dependent change in manoalide (Lombardo and Dennis (1985) J. Biol. Chem. 260, 7234-7240). Manoalide contains two rings and the opening of either would produce an alpha,beta-unsaturated aldehyde. The cobra venom phospholipase A2 may be able to catalyze the opening or isomerization of one of these rings, raising the possibility that manoalide is acting as a suicide substrate. To ascertain the role of the gamma-lactone ring in the inhibition, we have now investigated a synthetic manoalide analogue, 3(cis,cis-7,10)-hexadecadienyl-4-hydroxy-2-butenolide (HDHB) which contains only the alpha,beta-unsaturated gamma-lactone ring. We have found that the closed and open forms are in rapid equilibrium between pH 4 and 9 with the cyclic form being preferred at acidic pH values and the open cis form preferred at pH 9.5. When the pH is raised above 12, the alpha,beta double bond isomerizes to form trans-HDHB. Once the trans compound is formed, it is stable at all pH values and does not recyclize to the gamma-lactone ring. The observed pKa of 7.7 found for the inhibition of manoalide agrees well with the transition of the closed to the cis form of the gamma-lactone ring. Kinetic experiments with the HDHB compound show that under conditions in which the cis and closed form of the inhibitor are present in equal molar ratios, HDHB is not an irreversible inhibitor, but reversibly competes with substrate. However, the kinetics of this inhibition are complex and do not follow either pure competitive or non-competitive inhibition. The trans-HDHB exhibits similar complex kinetic but is several times more potent. The distinct differences between the behavior of manoalide and HDHB clearly indicate that while the gamma-lactone ring may play an important role in manoalide inhibition, it alone does not produce irreversible inhibition.

Hydrogen-Ion Concentration↗
PubMed1986

Rate-determining step in phospholipase A2 mechanism. 18O isotope exchange determined by 13C NMR.

H2(18)O isotope exchange into specifically 13C-labeled substrate was used to obtain information on the rate-limiting step in the action of the phospholipase A2 from the venom of the Indian cobra (Naja naja naja). Incorporation of 18O was detected by the effect of 18O on 13C chemical shifts in 13C NMR. The enzymatic hydrolysis of a micellar phosphatidylcholine analogue of platelet-activating factor 1-alkyl-2-[1-13C]lauroyl-sn-glycero-3-phosphorylcholine proceeds by an O-acyl cleavage of the sn-2 ester bond. The reaction was examined for simultaneous 18O incorporation into the substrate. No exchange was found, suggesting that the hydrolytic step is not followed by a higher energy transition state and that it or a step before it appears to be rate-limiting. Previous experiments on phosphatidylethanolamine activation indicate that kcat is altered but that the km remains the same upon activation, suggesting that the binding steps occurring before the hydrolytic step are not affected. This strongly suggests that the hydrolytic step is in fact the rate-limiting step under these conditions. The 13C, 18O NMR technique should be generally applicable to mechanistic questions of this type.

Carbon Isotopes↗
PubMed1986

Immunocytochemical localization of pancreatic carboxylic ester hydrolase in human paneth cells.

The protein-A gold method using specific rabbit sera directed against pure human pancreatic chymotrypsinogen and carboxylic ester hydrolase was applied to locate these (pro)enzymes in human pancreatic acinar cells and intestinal Paneth cells. Quantitative evaluation of the labelling indicated that both (pro)enzymes are present in pancreatic acinar secretory granules. In Paneth cell secretory granules, only carboxylic ester hydrolase was present in significant amounts, although the labelling for this enzyme was less intense than that observed in pancreatic zymogen granules. The results obtained support the view that Paneth cells represent a "diffuse exocrine gland" scattered along the intestine, whose role is either to act as a substitute in the event of a deficient pancreas or to regulate the intestinal flora.

Carboxylesterase↗
PubMed1985

Immobilized phospholipase A2 from cobra venom. Prevention of substrate interfacial and activator effects.

The activation of cobra venom phospholipase A2 by activators (containing phosphorylcholine moieties) appears to depend upon the aggregation state of the enzyme, and the presence of a lipid-water interface. The characteristics of this activation were studied by comparing the behavior of the enzyme immobilized on an agarose gel to that of the soluble enzyme. The immobilized enzyme displays only a few per cent of the soluble enzyme activity toward micellar dipalmitoyl-phosphatidylcholine (PC). However, the relative loss of activity is much less with micellar dipalmitoylphosphatidylethanolamine or soluble diheptanoyl-PC. The affinity for Ca2+ is increased about 10-fold by immobilization while the apparent pKa of the enzyme is decreased by 0.5-0.8 pH units. Activation energies are similar for the two enzyme forms and are independent of the physical state of the substrate used. Catalytic constants of the enzyme toward monomeric PC are not changed by immobilization. Yet, activators of the soluble enzyme have negligible effect on the immobilized enzyme, either in the presence or absence of an interface. Monomeric activators promote the binding of the soluble enzyme to the immobilized form. Apparently, immobilization mainly produces monomerically constrained enzyme which cannot be activated under any condition, whereas normally, activators in the presence of lipid-water interfaces induce the formation of enzyme dimers or possibly higher order aggregates.

Animals↗
PubMed1985

Cobra venom phospholipase A2 inhibition by manoalide. A novel type of phospholipase inhibitor.

Manoalide, an unusual nonsteroidal sesterterpenoid recently isolated from sponge, antagonizes phorbol-induced inflammation but not that induced by arachidonic acid, suggesting that manoalide acts prior to the cyclooxygenase step in prostaglandin synthesis, possibly by inhibiting phospholipase A2. We have now studied the inhibitory effect of manoalide on a homogeneous preparation of phospholipase A2 from cobra venom. For a given concentration of manoalide, the inhibition of phospholipase A2 activity toward dipalmitoylphosphatidylcholine/Triton X-100 mixed micelles is time-dependent and plateaus at about 85% inhibition of the initial velocity even after extensive preincubation. Metal ions (Ca2+, Ba2+, Mn2+) increase the inhibition, while lysophosphatidylcholine and substrate micelles protect. Increasing manoalide concentration shows increasing inhibition of the initial velocity until a plateau is reached, giving a typical saturation curve with a linear double-reciprocal plot. Under typical conditions (20-min preincubation, 40 degrees C, pH 7.1), 50% inhibition is achieved at a manoalide concentration of about 2 X 10(-6) M. The data indicate that manoalide is a potent inhibitor of the cobra venom phospholipase A2. Manoalide is now shown to react irreversibly with lysine residues in the enzyme. Surprisingly, the cobra venom phospholipase normally acts poorly on phosphatidylethanolamine as substrate, but after reaction with manoalide, the enzyme is somewhat more active toward this substrate rather than being inhibited. This suggests that a lysine residue may be important in understanding the substrate specificity of phospholipase A2.

Amino Acids↗
PubMed1985

Pancreatic stone protein. II. Implication in stone formation during the course of chronic calcifying pancreatitis.

Pancreatic stone protein, a novel protein isolated from pancreatic stones of patients suffering from chronic calcifying pancreatitis and secreted in normal human pancreatic juice, was measured by radial immunodiffusion in pure pancreatic juice. Patients with chronic calcifying pancreatitis of different etiologies had significantly lower levels of pancreatic stone protein when compared with other pancreatic diseases and controls. Pancreatic stone protein suppresses in vitro calcium carbonate precipitation and therefore stabilizes normally supersaturated pancreatic juice. The decreased pancreatic stone protein levels observed could be a key factor in the growth of calcium carbonate crystals and stone development during the course of chronic calcifying pancreatitis.

Adolescent↗
PubMed1984

The molecular characteristics of a human pancreatic acidic phosphoprotein that inhibits calcium carbonate crystal growth.

A CaCO3-crystal-growth inhibitor was isolated from human pancreatic stones by using EDTA demineralization, followed by DEAE-Trisacryl chromatography. The isolated inhibitor was found to be a phosphoglycoprotein with Mr 14017 and having an unusual chemical composition. It is characterized by a high (42%) acidic amino acid content, but lacks methionine and gamma-carboxyglutamic acid. The protein contains 2.65 mol of P/mol of protein, as phosphoserine (2 mol) and phosphothreonine (0.5 mol). Isoelectric focusing of the protein yields one major band corresponding to an isoelectric point of 4.2. Immunochemical quantification of the crystal-growth inhibitor in pure pancreatic juice reveals that it constitutes 14% of the normal exocrine secretion. Our findings demonstrate that this is a novel secretory protein, which has no enzymic activity and which maintains pancreatic juice in a supersaturated state with respect to CaCO3.

Amino Acids↗
PubMed1984

Implication of a tyrosine residue in the unspecific bile salt binding site of human pancreatic carboxylic ester hydrolase.

Tyrosine residues of the human pancreatic carboxylic-ester hydrolase (EC 3.1.1.1) (also referred to as cholesterol-ester hydrolase, EC 3.1.1.13) were nitrated in the ortho-position by the use of tetranitromethane. The specificity of the reaction has been verified and the inhibition observed was shown to be unrelated to the weak polymerization of the protein. Among the 27 tyrosines present in the enzyme, seven or eight were nitrated but only one residue, with a pK of 8.3, seems to be responsible for the loss of activity. This decrease in enzyme activity appears only in assays which were performed in the presence of bile salts, suggesting that of the two bile salt binding sites postulated on the enzyme, only one, referred to the as the 'unspecific site' (Lombardo, D. and Guy, O. (1980) Biochim. Act 611, 147-155), was modified. This is in agreement with the similar loss of enzyme activity observed on emulsified and soluble substrate. The most important result is the difference observed in experiments of the protective effects of bile salts. The protection with sodium taurodeoxycholate is independent of its critical micellar concentration, showing that monomers protect this site, whereas the protection observed in experiments with sodium cholate appears only for supramicellar concentrations of bile salt. Since this latter bile salt promotes the dimerization of the enzyme, we can conclude that a premicellar bile salt binding site (protected by monomers) is transformed in a functional micellar binding site (protected by micelles). This conformational transformation seems to be consecutive to the dimerization, as has been recently proposed.

Bile Acids and Salts↗
PubMed1983

On the probable involvement of arginine residues in the bile-salt-binding site of human pancreatic carboxylic ester hydrolase.

Modification of arginine residues with 2,3-butanedione inhibits the carboxylic-ester hydrolase activity on soluble and emulsified substrates when assayed with bile salts. The alpha-dicarbonyl reagent modifies seven of the nineteen arginine residues present per enzyme molecule. Nevertheless the inactivation with butanedione is greatly diminished when the protein is in the presence of negatively charged micellar bile salt. In these conditions we observe the protection of one arginine residue by sodium taurodeoxycholate and of two arginine residues by sodium cholate. This suggests that the carboxylic-ester hydrolase from human pancreatic juice contains at least two arginine residues essential for the activation by bile salts. All our data confirm the presence of two bile-salt-binding sites on the enzyme in which one arginine per site is involved and plays the general role of an anionic binding site. This study provides evidence that arginine residues may play an essential role in the interaction between bile salts and protein.

Amino Acids↗
PubMed1982

Modification of the essential amino acids of human pancreatic carboxylic-ester hydrolase.

Chemical modification of human pancreatic carboxylic-ester hydrolase (EC 3.1.1.1) were performed using organophosphorus compounds, ethoxyformic anhydride and Woodward's K reagent. It has been shown that: (1) the inhibition of the enzyme activity by organophosphorus compounds is due to the phosphorylation of only one alcohol residue, probably a serine residue which may represent the acylable group of the enzyme: (2) 35-36 free carboxyl groups are modified by Woodward's K reagent but only one is responsible for the loss of enzyme activity. This carboxyl group has a pKa of 5.2.: (3) carbethoxylation of histidine residues leads to the inhibition of the enzyme activity. All nine histidine residues are reactive but only one is essential for activity. Taking into account the probable formation of an acyl-enzyme intermediate (Lombardo, D. and Guy, O. (1981) Biochim. Biophys. Acta 657, 425-437) during substrate hydrolysis and the present data we discuss a possible mechanism for carboxylic-ester hydrolase catalysis, a mechanism similar to that described for chymotrypsin.

Carboxylesterase↗
PubMed1982

Catalytic properties of modified human pancreatic carboxylic-ester hydrolase.

Human pancreatic carboxylic-ester hydrolase (EC 3.1.1.1) modified by specific reagents (diisopropyl phosphorofluoridate, diethyl p-nitrophenyl phosphate, Woodward's K reagent and ethoxyformic anhydride) was studied for its activity. The three residues probably implicated in the active site acting on soluble substrate are shown to be essential for the activity on emulsified substrates such as tributyrin or cholesterol esters solubilized by bile salts (hydrolyzing and synthetizing activities). The modified enzyme is still able to bind to interfaces. Nevertheless, one supplementary carboxylic acid group is responsible for the stability of the enzyme bound to interfaces.

Binding Sites↗
PubMed1981

Binding of human pancreatic carboxylic ester hydrolase to lipid interfaces.

Human pancreatic carboxylic ester hydrolase (EC 3.1.1.1), usually characterized by its activity on water-soluble substrates, is shown to catalyze reactions taking place at a lipid/water interface. The inhibition of tributyrin hydrolysis by 1-alcohols follows the pattern of a Langmuir adsorption isotherm. Experiments performed with siliconized glass beads show that human pancreatic carboxylic ester hydrolase is adsorbed on this artificial (or substitute) interface with a dissociation constant for the enzyme-glass beads complex equal to 1.5 . 10(-8) M. The average molecular area at saturation is 4375 A2. Tripropionin hydrolysis is increased by the presence of glass beads. These results are strong arguments in favor of the interfacial activity of pancreatic carboxylic ester hydrolase. The activation of the enzyme bound to the interface is very weak. Bile salts do not prevent the adsorption of carboxylic ester hydrolase on siliconized glass beads and increase strongly the hydrolysis rate of emulsified tributyrin.

Alcohols↗
PubMed1981

Effect of alcohols on the hydrolysis catalyzed by human pancreatic carboxylic-ester hydrolase.

Transfer reactions catalyzed by human pancreatic carboxylic-ester hydrolase (EC 3.1.1.1) were studied in the presence of methanol and butanol as nucleophiles. The addition of alcohols produced an increase in the total rate of 4-nitrophenyl acetate and n-propylthiol acetate disappearance and a concomitant slow decrease of the hydrolysis rate. These results indicate a competitive partitioning of an acyl-enzyme intermediate between water and nucleophile. Moreover, a strong inhibition of the rates of hydrolysis of methyl butyrate and triacetin by nucleophiles is in agreement with a rate-limiting acylation step. The kinetic data and a trans-ester characterization argue in favor of the formation of an acyl-enzyme intermediate and a two-step reaction mechanism, acylation and deacylation both being rate-limiting. The experiments performed with 4-nitrophenyl acetate show the existence of a nucleophile binding site.

1-Butanol↗
PubMed1981

Structure and conformation of human pancreatic carboxyl-ester hydrolase.

Human pancreatic carboxyl-ester hydrolase is a glycoprotein with a molecular weight of 100 000 and a high content in carbohydrate, 20%. Sedimentation studies indicate that the molecule resembles an ellipsoid. The results of hydrodynamic and vacuum-ultraviolet circular dichroism investigation allow one to propose a model of the carboxyl-ester hydrolase three-dimensional structure. The enzyme belongs to the "all beta" class of proteins; about 54-60% of its residues are in beta-sheets and in beta-turns, most probably forming the surface of an ellipsoid. A similarity with prealbumin structure is proposed and a comparison with structure of other pancreatic enzymes is presented.

Amino Acids↗