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H M Verheij

Publications and source records attributed to H M Verheij.

At least 109 records · Page 6Linked to original sources
PubMed1987

Lipoprotein nature of the colicin A lysis protein: effect of amino acid substitutions at the site of modification and processing.

The colicin A lysis protein (Cal) is required for the release of colicin A to the medium by producing bacteria. This protein is produced in a precursor form that contains a cysteine at the cleavage site (-Leu-Ala-Ala-Cys). The precursor must be modified by the addition of lipid before it can be processed. The maturation is prevented by globomycin, an inhibitor of signal peptidase II. Using oligonucleotide-directed mutagenesis, the alanine and cystein residues in the -1 and +1 positions of the cleavage site were replaced by proline and threonine residues, respectively, in two different constructs. Both substitutions prevented the normal modification and cleavage of the protein. The marked activation of the outer membrane detergent-resistant phospholipase A observed with wild-type Cal was not observed with the Cal mutants. Both Cal mutants were also defective for the secretion of colicin A. In one mutant, the signal peptide appeared to be cleaved off by an alternative pathway involving signal peptidase I. Electron microscope studies with immunogold labeling of colicin A on cryosections of pldA and cal mutant cells indicated that the colicin remains in the cytoplasm and is not transferred to the periplasmic space. These results demonstrate that Cal must be modified and processed to activate the detergent-resistant phospholipase A and to promote release of colicin A.

Amino Acid Sequence↗
PubMed1986

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.

Bacterial Outer Membrane Proteins↗
PubMed1984

Interaction of native and modified Naja melanoleuca phospholipases A2 with the fluorescent probe 8-anilinonaphtalene-1-sulfonate.

The fluorescent probe 8-anilinonaphtalene-1-sulfonate (ANS) binds at the active site of the Naja melanoleuca snake venom phospholipase A2, thus protecting the enzyme against active-site-directed chemical modification. Both hydrophobic and electrostatic interactions are involved in the binding. At pH 7.5, a binding constant of 100 microM was determined, which improved twofold upon addition of the enzymatic cofactor Ca2+. The pH dependence of the ANS binding in the absence and presence of Ca2+ ions showed a perturbation of a group with a pKa value of 5.2, which could be assigned to the carboxylate group of the Ca2+-binding ligand Asp49 at the active site of the protein. Monomeric concentrations of the substrate analog n-decylphosphocholine displace ANS from the protein, indicating again that both ligands bind at the active site. Binding studies with several modified N. melanoleuca enzymes showed that a loss of enzymatic activity on aggregated substrates was correlated with a loss of affinity for the active site bound ANS molecule. It is suggested therefore, that the fluorescent ANS probe can detect structural rearrangements at the active site, which are important for enzymatic activity.

Anilino Naphthalenesulfonates↗
PubMed1984

Characterization of phospholipases A2 of Naja melanoleuca snake venom modified at the N-terminal region.

In phospholipase A2 from Naja melanoleuca snake venom all four lysines were converted into the epsilon-amidinated derivatives without reaction of the alpha-amino group. The amidinated phospholipase (AMPA) showed high enzymatic activity. Starting from AMPA, chemical modification reactions were carried out at the alpha-amino function. This group was blocked with a tert-butyloxycarbonyl or a phenylthiocarbamyl group. Furthermore the polypeptide chain was shortened by one residue by removing the N-terminal asparagine, resulting in the formation of des-Asn1-AMPA. The native enzyme was shortened by eight residues by cyanogen bromide cleavage at the single methionine residue. Although all modified proteins show a reduced affinity for monomeric lipids, they are easily saturated with micellar substrate analogs. Whereas the removal of the N-terminal octapeptide abolished all enzymatic activity the other modified enzymes possess a low (1%), but measurable enzymatic activity. It is concluded that chemical modifications in the N-terminal region give rise to a distortion of the active site, thus reducing the activity of the lipid-bound enzyme.

Animals↗
PubMed1984

The pro- and mature forms of the E. coli K-12 outer membrane phospholipase A are identical.

The nucleotide sequence of the pldA gene, coding for the outer membrane (OM) phospholipase A of Escherichia coli K-12, and flanking sequences, was determined. Data were obtained from sequences of overlapping deletions which had been generated in vitro from both ends of the gene, using DNase I in the presence of Mn2+ and Bal31 nuclease. The deduced amino acid sequence of the pldA gene product is the first primary sequence of a membrane-bound phospholipase. The complete PldA protein contains 260 amino acids, which include a putative signal sequence, and has a calculated mol. wt. of 29 946 similar to that of the purified protein. Furthermore we found the N terminus of the purified protein to be blocked and the overall amino acid composition to be consistent with the one deduced from the complete pldA gene. Analysis of proteins synthesized in minicells with a pldA coding plasmid in the presence of 8% ethanol did not reveal any new bands on polyacrylamide gels, whereas the control beta-lactamase clearly showed its unprocessed form under the same conditions. These data are consistent with the empirical prediction from the primary sequence, that the PldA protein lacks any signal peptidase 'target' site. We therefore conclude that the PldA protein is exported to the OM without proteolytic removal of the signal peptide.

Amino Acid Sequence↗
PubMed1983

The complete primary structure of phospholipase A2 from human pancreas.

The complete amino acid sequence of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) from human pancreas was determined. The protein consists of a single polypeptide chain of 125 amino acids and has a molecular weight of 14003. The chain is cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digestion of the reduced and thialaminated derivative of the protein with clostripain, yielding three fragments. The largest fragment (residues 7-100) was further degraded both with staphylococcal proteinase and chymotrypsin. The sequence was determined by automated Edman degradation of the intact protein and of several large peptide fragments. Phospholipase A2 from human pancreas contains the same number of amino acids (125) as the enzyme from horse, while the enzymes from pig and ox contain 124 and 123 residues, respectively. The enzymes show a high degree of homology; human phospholipase differs from the other enzymes by substitutions of 26 (porcine), 28 (bovine) and 32 (equine) residues, respectively.

Amino Acid Sequence↗
PubMed1983

Chemical modification of Lys-6 in phospholipase A2 from Naja melanoleuca snake venom.

Phospholipase A2 isolated from Naja melanoleuca snake venom was modified with 4-chloro-3,5-dinitrobenzoate. The modification reaction was accelerated by the cofactor Ca2+. Micellar concentrations of the substrate analog n-tetradecylphosphocholine retarded the inactivation of the enzyme. Modification resulted in the incorporation of one mole dinitrobenzoate/mole of protein. The modified residue could be identified as Lys-6 in the amino acid sequence of the enzyme. Although the modified enzyme still retains a high affinity for micellar substrate analogs and the cofactor Ca2+, it had lost nearly all its activity towards micellar substrates. Upon reduction of the nitro groups in the modified protein the enzymatic activity could be restored by more than 50%. Therefore Lys-6 cannot be directly involved in enzymatic catalysis. It is concluded that introduction of the negatively charged dinitrobenzoate group on Lys-6 gives rise to a distortion of the active site of the enzyme.

Animals↗
PubMed1983

Interaction of phospholipase A2 from Naja melanoleuca snake venom with monomeric substrate analogs. Activation of the enzyme by protein-protein or lipid-protein interactions?

Unlike porcine pancreatic phospholipase A2, the enzyme from Naja melanoleuca does not display biphasic kinetic behaviour at substrate concentrations around the critical micelle concentration. This snake venom enzyme was further investigated by direct binding studies using n-tridecylphosphocholine. Binding of this substrate analog to the enzyme was monitored by using equilibrium gel filtration, equilibrium dialysis and ultraviolet difference spectroscopy. It is concluded that, in the presence of submicellar concentrations of n-tridecylphosphocholine, a lipid-protein complex is formed consisting of about 4 protein and 36 lipid molecules. Ca2+ ions are required for the formation of this complex. A model is proposed which describes the formation of this type of complex. These lipid-protein aggregates are held responsible for the non-hyperbolic kinetic behaviour of the snake venom enzyme towards monomeric substrates.

Animals↗
PubMed1982

Interaction of phospholipase A2 and phospholipid bilayers.

Binding of phospholipase A2 from porcine pancreas and from Naja melanoleuca venom to vesicles of 1,2-di(tetradecyl)-rac-glycero-3-phosphocholine (diether-PC14) is studied in the presence and absence of 1-tetradecanoyl-sn-glycero-3-phosphocholine and myristic acid. The bound enzyme coelutes with the vesicles during gel filtration through a nonequilibrated Sephadex G-100 column, modifies the phase transition behavior of bilayers, and exhibits an increase in fluorescence intensity accompanied by a blue shift. Using these criteria it is demonstrated that the snake-venom enzyme binds to bilayers of the diether-PC14 alone. In contrast, the porcine enzyme binds only to ternary codispersions of dialkyl (or diacyl) phosphatidylcholine, lysophosphatidylcholine and fatty acid. Binding of pig-pancreatic enzyme to vesicles of the diether-PC14 could not be detected even after long incubation (up to 24 H) below, at, or above the phase-transition temperature, whereas the binding in the presence of products is almost instantaneous and observed over a wide temperature range. Thus incorporation of the products in substrate dispersions increases the binding affinity rather than increase the rate of binding. The results are consistent with the hypothesis that the pancreatic enzyme binds to defect sites at the phase boundaries in substrate bilayers induced by the products. The spectroscopically obtained hyperbolic binding curves can be adequately described by a single equilibrium by assuming that the enzyme interacts with discrete sites. The binding experiments are supported by kinetic studies.

Animals↗
PubMed1981

Modification of carboxylate groups in bovine pancreatic phospholipase A2. Identification of aspartate-49 as Ca2+-binding ligand.

Carboxylase groups in bovine pancreatic phospholipase A2 were modified using a water-soluble carbodiimide and semicarbazide. At pH 5.5 rapid modification of 13 out of the total of 15 carboxylates occurred, leaving Asp-39 and Asp-99 unmodified. Subsequent modification with radioactive semicarbazide at pH 3.5 resulted in the labelling of Asp-39. In the modified proteins both Ca2+ binding and enzymatic activity are completely lost. Modification of the enzyme at pH 5.5 in the presence of Ca2+ ions yielded a protein with three unmodified carboxylates which is capable of binding Ca2+ ions, while 15% residual activity was found. A second reaction, using [14C]semicarbazide at pH 5.5 in the absence of Ca2+, leads to the incorporation of one mole of [14C]semicarbazide/mole of protein with loss of enzymatic activity and Ca2+ binding. The label was found to be attached to Asp-49, demonstrating the essential role of Asp-49 in Ca2+ binding. Studies on the pH dependence of the Ca2+ binding to the protein suggested that Asp-49 has an apparent pK of 5.2.

Amino Acid Sequence↗
PubMed1980

The action of cobra venom phospholipase A2 isoenzymes towards intact human erythrocytes.

1. Cobra venom phospholipase A2 from three different sources has been fractionated into different isoenzymes by DEAE ion-exchange chromatography. 2. Treatment of intact human erythrocytes with the various isoenzymes revealed significant differences in the degree of phospatidylcholine hydrolysis in those cells. 3. It is argued that the plateaus observed in dose-response curves for such treatments may be caused by an increase in lateral surface presure within the outer half of the membrane due to the production of free fatty acids and lyso-compounds.

Animals↗
PubMed1980

Methylation of histidine-48 in pancreatic phospholipase A2. Role of histidine and calcium ion in the catalytic mechanism.

It is known that His-48 is part of the active center in pancreatic phospholipase. To further elucidate the role of histidine-48 in the active center of pancreatic phospholipase A2, we have modified the enzyme with a number of bromo ketones and methyl benzenesulfonates. Rapid methylation occurred with methyl p-nitrobenzenesulfonate. Methylated phospholipase shows total loss of enzymatic activity whereas binding of substrate and the cofactor Ca2+ remains intact. Amino acid analysis of methylated equine phospholipase showed the loss of the single molecule of histidine and the formation of one molecule of 2-amino-3-(1-methyl-5-imidazolyl)propanoic acid (1-methylhistidine). Equine phospholipase was also modified by [13C]methyl p-nitrobenzenesulfonate and the methylated enzyme was studied by 13C NMR. The results indicate that the proton on the nitrogen in position 3 of the imidazole ring is involved in a strong interaction with a buried carboxylate group, thereby hindering rotation of the imidazole ring, and that the nitrogen in position 1 is involved in catalysis. These data are in full agreement with the three-dimensional structure at 1.7-A resolution of bovine pancreatic phospholipase. A catalytic mechanism is proposed in which a water molecule which is close to the nitrogen at position 1 of the imidazole ring of the Asp-99-His-48 couple acts as the nucleophile. A comparison is made between phospholipase A2 and the serine esterases.

Animals↗
PubMed1980

Correlation of enzymatic activity and anticoagulant properties of phospholipase A2.

Some highly purified phospholipases A from the venom of viperidae, crotalidae and elapidae were found to hve anticoagulant properties. All phospholipases which exhibited anticoagulant properties are characterized by a high isoelectric point, but not all strongly basic phospholipases are anticoagulant. Anticoagulant phospholipases hydrolyse highly packed monomolecular films of phospholipids without any lag time while non-anticoagulant phospholipases present considerable induction times indicative of a low penetrating power. When the ester linkages in the procoagulant lipids were replaced by the non-hydrolysable ether bonds, the mixture retained its clotting ability even in the presence of phospholipases, thus suggesting that anticoagulant phospholipases prevent clot formation by hydrolysis of phospholipids. This was confirmed by chemical modification of phospholipases, viz. alkylation of the active-centre histidine with 1-bromo-octan-2-one. This modification yielded proteins which had lost their anticoagulant properties but which retained a high affinity for phospholipids.

Animals↗
PubMed1979

The amino acid sequence of the phospholipase A2 isoenzyme from porcine pancreas.

The primary structure of porcine pancreatic isophospholipase A2 (EC 3.1.1.4) has been investigated. The sequence of procine isophospholipase differs from the sequence of porcine phospholipasy by four substitutions; viz. Ala12 leads to Thr; His17 leads to Asp leads to; Met20 leads to Leu and Glu71 leads to Asn.

Amino Acid Sequence↗