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R L Biltonen

Publications and source records attributed to R L Biltonen.

At least 37 records · Page 2Linked to original sources
PubMed1995

Modulation of phospholipase A2: identification of an inactive membrane-bound state.

Phospholipase A2-catalyzed hydrolysis of vesicular phospholipid has been used to model the modulation of an enzyme's function by membrane properties. Phospholipase A2's (PLA2) kinetics toward large unilamellar vesicles (LUV) composed of dipalmitoylphosphatidylcholine (DPPC) are anomalous; these is a slow initial phase of catalysis (a lag) which ends abruptly with a sudden increase in the catalytic rate (a burst). The sudden increase in activity due to the accumulation of a critical mole fraction of reaction products and substrate undergoes compositional phase separation. In this work, we address the molecular details of the coupling between compositional phase separation and activation of PLA2. A prominent model for this coupling is that compositional phase separation leads to a surface for which PLA2 has increased affinity, resulting in the recruitment of PLA2 from solution to the surface. Here, we show that the bulk of PLA2 is associated with the membrane at a time well before the abrupt increase in catalytic rate. This finding indicates that there must be a relatively inactive, membrane-bound state. Furthermore, PLA2's kinetics are anomalous even when the substrate comprises a surface to which PLA2 is bound throughout the time course. With DPPC LUV as the substrate, detailed time courses show that the description of the time course as a lag and a burst is inadequate. Instead, the time course consists of multiple phases of acceleration and deceleration. The data presented here suggest that all these various changes in catalytic rate may be due to product-induced changes in membrane properties. In particular, we suggest that nonequilibrium, microheterogeneities of lipid composition may underlie these very complicated kinetics.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Lipid bilayer heterogeneities and modulation of phospholipase A2 activity.

The regulation of phospholipase A2 (PLA2) activity toward synthetic vesicular substrates is a model for the modulation c enzyme function by biological membranes. PLA2's catalytic rate toward membrane phospholipids can be modified by several order of magnitude by altering the membrane's composition and structure. The physical basis of this sensitivity is the subject of thi report. The results described here imply that the salient features of membrane-structure which modulate PLA2 activity include compositional phase separation; membrane curvature and, possibly, curvature-associated defects; and dynamic product inhibition due to limitations imposed by the rate of lateral diffusion of lipid in the membrane. Furthermore, it is shown that the effects of membrane structure on the catalytic rate are not exerted merely by enhancing association of PLA2 with the membrane surface: a membrane-bound inactive state is spectroscopically identified. Finally, these results are discussed in the context of some published models for the role of membrane structure in the regulation of membrane-bound enzymes.

Enzyme Activation↗
PubMed1994

Thermotropic behavior of dimyristoylphosphatidylglycerol and its interaction with cytochrome c.

The thermotropic behavior of dimyristoylphosphatidylglycerol (DMPG) in the absence and presence of cytochrome c under low-salt conditions has been investigated using differential scanning calorimetry (DSC), 31P nuclear magnetic resonance (31P NMR), electron spin resonance (ESR), viscosity, light scattering, and electron microscopy. In the absence of protein, the lipid undergoes a sequence of transitions over the temperature range of 7-40 degrees C. ESR studies demonstrate increased acyl chain mobility associated with these transitions. 31P NMR indicates that the lipid, in the absence of protein, retains a lamellar structure throughout the temperature range investigated. At high lipid concentration the DSC curves exhibit a pronounced maximum in the excess heat capacity (Cp) function at about 23 degrees C with a shoulder on the high-temperature side. As the lipid concentration is reduced to 10 mM, the Cp curves broaden, retaining a sharp maximum at about 20 degrees C and a broader transition with a maximum at 27 degrees C. The overall enthalpy change of 6 kcal/mol is independent of lipid concentration. Most interestingly, the lipid dispersion becomes highly viscous and optically isotropic in the main transition range (20-28 degrees C), suggesting long-range order even at lipid concentrations as low as 10 mM. The existence of long-range order is confirmed by negative stain electron microscopy. The heat capacity curve in the presence of protein is broad, with a single Cp maximum and an overall enthalpy change of 1.7 kcal/mol. Similarly, the temperature dependence of the ESR spectra shows none of the detail observed in the absence of the protein. Of specific interest is that partially saturating amounts of protein prevented the large increase in the viscosity of the dispersion in the main transition range. This result suggested that the protein prevented development of long-range order. However, under saturating conditions the viscosity of the protein-lipid complex increased with increasing temperature even beyond the transition range. This increase does not appear to be the result of formation of an extended lipid structure but is the result, according to electron microscopic evaluation, of aggregation of small protein-containing lipid vesicles. The 31P NMR spectra of the lipid in the presence of protein are isotropic, consistent with the formation of highly curved particles. Calorimetric titration studies of the binding of cytochrome c to DMPG indicate that protein binding is coupled cooperatively to changes in the state of the lipid.(ABSTRACT TRUNCATED AT 400 WORDS)

Calorimetry, Differential Scanning↗
PubMed1993

Role of lateral phase separation in the modulation of phospholipase A2 activity.

Phospholipase A2-catalyzed hydrolysis of phosphatidylcholine large unilamellar vesicles is characterized by a period of slow hydrolysis followed by a rapid increase in the rate of hydrolysis. The temporal relationship between the burst of PLA2 activity and the lateral distribution of substrate and product lipids was examined by simultaneously recording product accumulation and the fluorescence of 1-pyrenyldecanoate, a fatty acid derivative sensitive to lipid distribution and lateral diffusion. The excimer: monomer ratio of the probe changes slowly prior to the burst in activity and then abruptly at the time of the burst. A partial phase diagram for the ternary codispersion of substrate and products (dipalmitoylphosphatidylcholine and 1:1 monopalmitoylphosphatidylcholine/palmitic acid) was constructed by differential scanning calorimetry and suggests gel/gel immiscibility in this system. Thus, the changes in pyrene fluorescence during the time course of hydrolysis appear to be due to lateral phase separation. The critical mole fraction of product both for lateral phase separation in the gel state and for elimination of the lag phase is approximately 0.083. The simultaneous recordings of PLA2 activity and pyrene fluorescence show that the lateral rearrangement of lipids begins prior to and continues during the rapid activation process of PLA2. Two possible effects of lateral phase separation are that concentration of the protein in the product-rich regions promotes putative dimerization or that formation of phase interface regions promotes enzyme activation.

Enzyme Activation↗
PubMed1992

Calcium and magnesium dependence of phospholipase A2-catalyzed hydrolysis of phosphatidylcholine small unilamellar vesicles.

The Ca2+ requirement for lipid hydrolysis catalyzed by phospholipase A2 from Agkistrodon piscivorus piscivorus (App-D49) and porcine pancreas has been examined using small, unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC SUV). Hydrolysis was affected by product inhibition even at early times, and the extent of this inhibition depended on the concentration of divalent cations. The Ca2+ requirement for half-maximal rates of hydrolysis reflected, in part, this non-catalytic role of divalent cations. The presence of 10 mM Mg2+, a cation which does not support catalysis, reduced the Ca2+ required for half-maximal rates of hydrolysis from millimolar concentrations to 40 microM for App-D49. Since the dissociation constant of the enzyme for Ca2+ in solution is 2 mM, these results indicate a change in the interaction of the enzyme with Ca2+ under catalytic conditions. The kinetic dissociation constant of Ca2+ for the pancreatic enzyme was 20 microM which is substantially lower than the dissociation constant in solution, 0.35 mM. The similarity of apparent kinetic dissociation constants for these enzymes suggests that structurally similar features determine the affinity for Ca2+ under catalytic conditions. Evidence is presented that the affinity of phospholipase A2 for Ca2+ changes subsequent to the initial interaction of the enzyme with the substrate interface. However, the apparent Michaelis constant, KMapp, for App-D49, 0.03-0.06 mM, is independent of [Ca2+] and is about the same as the equilibrium dissociation constant for DPPC SUV, 0.14 mM. We thus suggest that KMapp is a steady-state constant.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Molecular details of the activation of soluble phospholipase A2 on lipid bilayers. Comparison of computer simulations with experimental results.

The initial rate of hydrolysis of large unilamellar vesicles of dipalmitoylphosphatidylcholine by phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus is small and elevates gradually until it suddenly increases by a factor of 10 to 1000 depending on the experimental conditions. This abrupt onset of high enzyme activity appears to be correlated to a specific mole fraction of reaction product at which point a cooperative compositional phase transition in the bilayer occurs. Five models that describe the activation process in terms of its being coupled to the putative product-induced lipid transition are presented. These models include one in which the lipid structure enhances the affinity of enzyme binding to the bilayer surface, two in which the equilibrium position between an active and an inactive form of the enzyme-substrate complex is altered, and two in which the rate of a quasi-irreversible spontaneous activation process is increased. Whether the active form of the enzyme is a monomer or dimer is also considered in the last two pairs of models. Computer simulations of time courses for the different models show how a set of four experimental observables distinguishes qualitatively among them. Comparison of the experimental behavior with the computer-simulated behavior of the observables for each model indicates that activation of phospholipase A2 on the lipid surface involves formation of an enzyme dimer which spontaneously converts to an active form. The active enzyme persists in the active state as it exchanges between vesicles. This model of activation is similar to that proposed previously for activation of porcine pancreatic phospholipase A2.

Computer Simulation↗
PubMed1992

Expression of a group II phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus in Escherichia coli: recovery and renaturation from bacterial inclusion bodies.

A synthetic gene encoding the Group II phospholipase A2 (PLA2) from the venom of Agkistrodon piscivorus piscivorus has been constructed and expressed with high efficiency in Escherichia coli. No enzymatic activity was recovered when the polypeptide contained the initiator Met residue. Replacement of an Asn residue penultimate to the initiator Met with Ser or Gly permitted removal of the initiator Met by the endogenous methionine aminopeptidase. The amino-terminal serine (N-Ser) and amino-terminal glycine PLA2's were isolated from intracellular inclusion bodies and were renatured with 25% recovery. Automated Edman degradation confirmed the removal of the initiator Met and confirmed the sequence of the first 40 residues of N-Ser PLA2. The recombinant proteins were purified to apparent homogeneity and showed the same specific activity as the wild-type protein. N-Ser PLA2 demonstrated the same kinetics of activation as the wild type enzyme on large vesicles of zwitterionic lipid.

Amino Acid Sequence↗
PubMed1992

Effects of the anesthetic dibucaine on the kinetics of the gel-liquid crystalline transition of dipalmitoylphosphatidylcholine multilamellar vesicles.

The effects of the anesthetic dibucaine on the relaxation kinetics of the gel-liquid crystalline transition of dipalmitoylphosphatidylcholine (DC16PC) multilamellar vesicles have been investigated using volume-perturbation calorimetry. The temperature and pressure responses to a periodic volume perturbation were measured in real time. Data collected in the time domain were subsequently converted into and analyzed in the frequency domain using Fourier series representations of the perturbation and response functions. The Laplace transform of the classical Kolmogorov-Avrami kinetic relation was employed to describe the relaxation dynamics in the frequency domain. The relaxation time of anesthetic-lipid mixtures, as a function of the fractional degree of melting, appears to be qualitatively similar to that of pure lipid systems, with a pronounced maximum, tau max, observed at a temperature corresponding to greater than 75% melting. The tau max decreases by a factor of approximately 2 as the nominal anesthetic/lipid mole ratio increases from 0 to 0.013 and exhibits no further change as the nominal anesthetic/lipid mole ratio is increased. However, the fractional dimensionality of the relaxation process decreases monotonically from slightly less than two to approximately one as the anesthetic/lipid mole ratio increases from 0 to 0.027. At higher ratios, the dimensionality appears to be less than one. These results are interpreted in terms of the classical kinetic theory and related to those obtained from Monte Carlo simulations. Specifically, low concentrations of dibucaine appear to reduce the average cluster size and cause the fluctuating lipid clusters to become more ramified. At the highest concentration of dibucaine, where n < 1, the system must be kinetically heterogeneous.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

Thermodynamics of phospholipase A2-ligand interactions.

Future investigations into the role of the structure of phospholipid substrates and the interrelationships between substrate, calcium, and enzyme conformation in the activation process are clearly needed. Enzyme dimerization in the activation of phospholipase A2 has been indicated, and a complex equilibrium between calcium, substrate, and monomer and dimer enzyme apparently exists. The incorporation of proton binding further complicates the scheme, and one is quickly faced with obtaining a large number of equilibrium constants in order to describe the system explicitly. Nevertheless, similarly complex systems have been well characterized using thermodynamic approaches such as those described herein. An excellent example is the complex equilibrium involving the protonation of the histidine residues and the binding of a mononucleotide to ribonuclease A. Achieving a complete thermodynamic description of that system allowed the investigators to make strong mechanistic statements about models for the catalytic mechanism of ribonuclease A. Since phospholipase A2 is available for study at the same level of detail, one can anticipate a similar degree of quantitative detail regarding the important interactions of this enzyme to be forthcoming.

Animals↗
PubMed1991

Activation of phospholipase A2 on lipid bilayers.

So far, three phospholipases A2 that display activation kinetics during the time course of hydrolysis of DPPC LUV have been found to undergo a fluorescence change coincident with the activation: the monomer (AppD49) and the dimer enzymes from A. piscivorus piscivorus and the dimer enzyme from C. atrox. The porcine pancreatic enzyme produces similar time courses of hydrolysis but does not display a concurrent fluorescence change. It is assumed that other phospholipases A2 will behave similarly in terms of the hydrolysis reaction. Which enzymes respond with a similar change in intrinsic fluorescence during the time course may well depend on the position of tryptophan residues and the amino acid sequence. Even though a given phospholipase A2 may not change its fluorescent properties on activation, the simultaneous monitoring of the hydrolysis reaction and the fluorescence of probes of the bilayer structure can be done with any phospholipase A2. A variety of probes exist which are sensitive to slightly different membrane properties and could be used as described here for TMA-DPH. For example, 1,3-dipyrenylpropane is sensitive to the apparent microviscosity of the bilayer is terms of the ability of molecules to translationally diffuse in the membrane. 6-Palmitoyl-2-[[2-(trimethylammonio)ethyl]methylamino]naphthalene chloride is sensitive to the ability of a molecule to rotate in the bilayer and displays large changes in its steady-state fluorescence as the anisotropy of the bilayer changes. 6-Propionyl-2-(dimethylamino)naphthalene is sensitive to the polarity and degree of hydration of its environment. Finally, a compound titled NK-529 has recently been introduced that apparently monitors the lateral phase separation of fatty acids in the bilayer. The fact that activation of phospholipase A2 can be monitored during the time course of hydrolysis of DPPCLUV makes this system an excellent choice for studying the mechanisms of activation and possible effects of various activators and inhibitors. The experimental system described here provides a way to determine whether such regulators exert their effects through alterations of the properties of the membrane and/or the enzyme. Importantly, this system allows one to seek temporal correlations of the various events in the process.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

Gel-liquid crystalline transition of some multilamellar lipid bilayers follows classical kinetics with a fractional dimensionality of approximately two.

The relaxation kinetics of the gel-liquid crystalline transition of phosphatidylcholine (DC14PC, DC16PC, and DC18PC) multilamellar vesicles have been examined using volume-perturbation calorimetry. The time-dependent temperature and pressure changes associated with a periodic volume perturbation are monitored in real time. Data collected in the time domain are transformed to the frequency domain using Fourier series representations of the perturbation and response functions. Because a very small perturbation is imposed during the experiment, linear response theory is suitable for analysis of the relaxation process. The Laplace transform of the classical Kolmogorov-Avrami relation of transition kinetics is used to describe the dynamic response in the frequency domain. For DC14PC and DC16PC, the relaxation process is better fit with an effective dimensionality of n = 2 rather than n = 1. For DC18PC, we estimate that an effective dimensionality of approximately 1.5 will best fit the data. These results indicate that the gel-liquid crystalline transition of these lipid bilayers follows the classical Kolmogorov-Avrami kinetic model with an effective dimensionality greater than 1 and the assumption of simple exponential decay (n = 1) commonly used in data analysis may not always be valid for lipid transitions. Insofar as the dimensionality of the relaxation reflects the geometry of fluctuating lipid clusters, this parameter may be useful in connecting experimental thermodynamic and kinetic results with those obtained from Monte Carlo simulations.

Biophysical Phenomena↗
PubMed1991

Relaxation dynamics of the gel to liquid-crystalline transition of phosphatidylcholine bilayers. Effects of chainlength and vesicle size.

The relaxation kinetics of the gel to liquid-crystalline transition of five phosphatidylcholine (DC14PC to DC18PC) bilayer dispersions have been investigated using volume perturbation calorimetry, a steady-state technique which subjects a sample to sinusoidal changes in volume. Temperature and pressure responses to the volume perturbation are measured to monitor the relaxation to a new equilibrium position. The amplitude demodulation and phase shift of these observables are analyzed with respect to the perturbation frequency to yield relaxation times and amplitudes. In the limit of low perturbation frequency, the temperature and pressure responses are proportional to the equilibrium excess heat capacity and bulk modulus, respectively. At all temperatures, the thermal response data are consistent with a single primary relaxation process of the lipid. The less accurate bulk modulus data exhibit two relaxation times, but it is not clear whether they reflect lipid processes or are characteristic of the instrument. The observed thermal relaxation behavior of all multilamellar vesicles are quantitatively similar. The relaxation times vary from approximately 50 ms to 4 s, with a pronounced maximum at a temperature just greater than Tm, the temperature of the excess heat capacity maximum. Large unilamellar vesicles also exhibit a single relaxation process, but without a pronounced maximum in the relaxation time. Their relaxation time is approximately 80 ms over most of the transition range.

Fourier Analysis↗
PubMed1990

Non-steady state kinetic analysis of the regulation of adenylate cyclase by GTP-binding proteins.

The time course of cAMP production by S49 cell membranes in the presence of forskolin and a nonhydrolyzable GTP analog can yield information about the regulation of adenylate cyclase by both the inhibitory and stimulatory GTP-binding proteins (Gi and Gs). The time courses are complex and interpretation in terms of the activities of G1 and Gs requires a quantitative hypothesis. We present a general quantitative hypothesis that defines adenylate cyclase as existing in a distribution of two states, active and inactive. Gi and Gs, in their active states, alter the equilibrium of this distribution. Two distinct models are derived based on this hypothesis to accommodate two different proposed mechanisms for the action of Gi to inhibit adenylate cyclase: 1) a direct interaction between Gi and the catalytic subunit of adenylate cyclase and 2) a direct interaction between Gi and Gs. Perturbations of the regulation of adenylate cyclase by pertussis toxin and phorbol ester are simulated and interpreted using the models. The effect of pertussis toxin is quantitatively reconciled by decreases in the guanine nucleotide-independent adenylate cyclase activity and in the apparent rate of activation of Gi from 2.0/min to 0.01/min. The effect of phorbol ester is best accommodated by the model as a change in the distribution of active and inactive adenylate cyclase from 36% initially active to 47% active after phorbol ester treatment, without postulating any effect of phorbol ester on Gi or Gs. Both of these interpretations are independent of the model used. The effect of forskolin is also examined within the context of the two models. The results of this examination suggest an experimental approach for testing the models. These examples illustrate the usefulness of quantitative analysis of time course data using a model for the regulation of adenylate cyclase. We propose that, with this combined experimental and theoretical approach, one can address the relevance of hypotheses generated from experimental studies with isolated components to the molecular mechanisms of adenylate cyclase regulation in cellular membranes.

Adenylate Cyclase Toxin↗
PubMed1989

The temporal sequence of events in the activation of phospholipase A2 by lipid vesicles. Studies with the monomeric enzyme from Agkistrodon piscivorus piscivorus.

The substrate dependence of the time courses of hydrolysis of both small and large unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) by Agkistrodon piscivorus piscivorus monomeric phospholipase A2 is consistent with an activation process involving enzyme aggregation on the vesicle surface. The time course of hydrolysis of large unilamellar vesicles is particularly complex; a slow initial rate of hydrolysis is followed by an extremely abrupt increase in enzyme activity. The length of this slow phase is a minimum at the phase transition temperature of the vesicles. The intrinsic fluorescence intensity of the phospholipase A2 also abruptly increases (50-60%) after a latency period revealing a strong temporal correlation between enzyme activity and the increase in fluorescence intensity. The length of the latency period before the sudden increase in fluorescence intensity is directly proportional to substrate concentration at DPPC concentrations above 20-100 microM. At lower concentrations, the length of the latency period is inversely proportional to the DPPC concentration. Such biphasic substrate dependence is predicted by a previously proposed enzyme activation model involving dimerization on the surface vesicle. Simultaneous monitoring of the protein fluorescence and hydrolysis demonstrates that the magnitude of the fluorescence change and the rate of hydrolysis are in exact temporal correlation. Furthermore, simultaneous monitoring of the fluorescence of the protein and that of a lipid probe, trimethylammonium diphenylhexatriene, indicates a change in lipid vesicle structure prior to, or coincident with, the abrupt change in protein activation. These results are consistent with the hypothesis that the monomeric phospholipase A2 from A. piscivorus piscivorus initially possesses a low level of intrinsic activity toward large unilamellar DPPC vesicles and that the enzyme slowly becomes further activated on the vesicle surface via dimerization. Eventually, the vesicles undergo an abrupt transition in internal structure leading to sudden rapid activation of the enzyme.

1,2-Dipalmitoylphosphatidylcholine↗