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R Dijkman

Publications and source records attributed to R Dijkman.

44 records · Page 3Linked to original sources
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↗
PubMed1982

Studies on prophospholipase A2 and its enzyme from human pancreatic juice. Catalytic properties and sequence of the N-terminal region.

Upon tryptic activation of pure human prophospholipase A2, a heptapeptide is released from the N-terminal part of the protein yielding active phospholipase A2 (EC 3.1.1.4). Both the kinetics of the activation process and the amino acid sequence of the activation peptide strongly resemble those of pancreatic zymogens of other mammalian sources. The kinetic properties of human phospholipase A2 and its zymogen are compared with those of the corresponding porcine enzyme using substrates present at micelles, molecular dispersed solutions or as monomolecular surface films. The most obvious difference between the human and porcine phospholipase A2 is the low enzyme activity of the former protein at pH 8.0 as compared to pH 6.0, both against micellar and monomeric substrates. Neither the Ca2+ binding properties nor the inhibition of the human enzyme using haloketones can easily explain this different pH optimum. The sequence analysis of the N-terminal region of the first 40 residues is reported.

Amino Acid Sequence↗
PubMed1981

Effects of temperature and lipid composition on the serum albumin-induced aggregation and fusion of small unilamellar vesicles.

Small unilamellar vesicles of egg phosphatidylcholine (PC) or dimyristoylphosphatidylcholine, mixed with small unilamellar vesicles labelled with 2-(10-(1-pyrene)decanoyl)phosphatidylcholine, exhibit a constant average size and excimer to monomer (E/M) ratio for several hours when incubated at pH 3.6 at a temperature higher than the phase transition temperature (Tc) of the lipids. Addition of bovine serum albumin to this system produces a transient turbidity increase, a fast decrease in the E/M ratio, a partial loss of vesicle-entrapped [14C]sucrose and a measurable leak-in of externally added sucrose. Sepharose 4B filtration of the system demonstrates that the E/M ratio decrease is strictly paralleled by the formation of liposomes which exhibit a low E/M ratio and a hydrodynamic radius larger than that of small unilamellar vesicles. These data demonstrate that the E/M ratio decrease can be unequivocally ascribed to a vesicle-vesicle fusion process induced by serum albumin. The rate of serum-albumin induced fusion of small unilamellar vesicles is: (a) maximal at a stoichiometric ratio of approx. 2 albumins per vesicle; (b) sensitive to the nature of the lipid and; (c) not altered when human serum albumin replaces bovine serum albumin. The rate of albumin-induced fusion of dimyristoylphosphatidylcholine small unilamellar vesicles is higher below the Tc of the lipid and increases with temperature above the Tc. The formation of protein-bound aggregates with defined stoichiometries and a high local vesicle concentration, as well as changes in the local degree of hydration, are proposed to be the driving forces for the protein-induced vesicle fusion in this system.

Animals↗
PubMed1981

Binding of porcine pancreatic phospholipase A2 to various micellar substrate analogues. Involvement of histidine-48 and aspartic acid-49 in the binding process.

The interaction of porcine pancreatic phospholipase A2 (PA2) with micelles of various single-chain phospholipid analogues was studied by ultraviolet absorption difference spectroscopy and light-scattering measurements. The phospholipids used were either substrate analogues or products, varying in hydrocarbon chain lengths and polar head groups. The results indicate that the enzyme forms a stable complex over a wide range of enzyme and lipid concentrations. From the equivalent "molecular weight" and from the lipid to enzyme molar ratio (N) of the micelle--enzyme complex, it can be calculated that complexes containing saturated hydrocarbon chain lipids generally consist of two enzyme molecules and half of the number of lipid monomers present in free micelles. The interaction forces between the enzyme and lipid monomers bound in the complex are mainly hydrophobic. Stronger binding is found when the essential cofactor Ca2+ is bound to the enzyme. pH-titration studies on the binding of native PA2 to aggregated lipid structures showed that at least one group with a pKA value of 6.25 is involved in the interaction with lipid micelles. At acidic pH, micelle binding is stronger than at neutral or alkaline pH. Alkylation of the active site residue His48 resulted in a shift of the pKA value to 4.6, while addition of Ca2+ appears to stabilize the micelle-binding conformation of both native and modified enzymes over a broad pH range (pH 4--9.5). From these observations it is suggested that both the Ca2+ binding residue Asp49 [Fleer, E. A. M., Verheij, H. M., & de Haas, G. H. (1980) Eur. J. Biochem. 113, 283--288] and His48 control micelle binding of the native enzyme. For optimal binding in the absence of Ca2+, a long-distance hydrogen bond between these two residues is required; this can be established via a water molecule. It is assumed that it is a proton of this "H bond" which is titrated with a pKA value of 6.25. When the "H bond" is absent, as in the alkylated enzymes, Asp49 alone controls micelle binding with a pKA of 4.6. These results, together with the effect of Ca2+ on micelle binding, indicate that it is not the "hydrogen bridge" between His48 and Asp49 which is of main importance for an optimum binding conformation of the enzyme but the effective charge in the microenvironment of Asp49. It is proposed that a negative charge on this carboxylate causes a conformational change of the enzyme which leads to a protein conformation lacking an active micelle binding site. Binding of Ca2+ or reprotonation neutralizes this negative charge and restores the enzyme's ability to bind micelles.

Animals↗
PubMed1978

Immunological studies on pancreatic phospholipase A2. Antigenic characterization of the NH2-terminal region.

Rabbit antisera elicited against pure pig, horse, ox, and sheep pancreatic phospholipase A2 revealed considerable immunological differences when tested by double immunodiffusion and microcomplement fixation assays. Snake venom phospholipases did not show any detectable cross-reactions with the pancreatic enzymes. Microcomplement fixation also clearly demonstrated conformational differences between porcine phospholipase A2 and its zymogen. NH2 terminally modified analogs of porcine phospholipase A2 could be clearly distinguished using the same assay. Moreover, strong evidence was obtained that Ala1-Arg6 is a part of an antigenic determinant. Radioimmune assay, using monovalent phospholipase-specific Fab fragments revealed a maximum number of three antigenic sites of phospholipase that can simultaneously be occupied by antibody. The Fab fragments were separated into three fractions, using three immunoadsorbent columns in series. These Fab fractions showed different inhibitory properties toward micellar binding of phospholipase A2. They also exhibited different protective effects against active center modification.

Animals↗
PubMed1975

Studies on lysophospholipases, V. The action of lysolecithin-hydrolyzing enzymes on lecithins and 1-acyl lysolecithins with varying fatty acid chain-length.

The activity of two purified lysolecithin-hydrolyzing enzymes on homologous series of synthetic lecithins containing two identical fatty acyl chains and of 1-acyl-lysolecithins has been measured as a function of substrate concentration. In general, enzymatic activity toward lecithins decreased with increasing chain length. Maximal hydrolysis rates for the lysolecithin series were measured with 1-dodecanoyllysolecithin. In this series increased affinities for substrates with increasing acyl-chain length was noticed. In the substrate concentration versus enzymatic velocity curves no breaks were observed at the critical micelle concentration of the various substrates. The initial site of attack during hydrolysis of short-chain lecithins was determined using 1-octanoyl-2pentanoyl-lecithin, 1-hexanoyl-2-hexyllecithin and 1 -hexyl-2-hexanoyllecithin. Both enzymes exhibited a pronounced preference for hydrolysis of the acyl ester bond at the 1-position. Especially the enzyme from beef pancreas seems to be suitable for the enzymatic preparation of 2-acyl lysolecithins from the corresponding short-chain lecithins.

Animals↗