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Biomedical subjects

O G Berg

Publications and source records attributed to O G Berg.

At least 37 records · Page 2Linked to original sources
PubMed1997

Codon bias in Escherichia coli: the influence of codon context on mutation and selection.

The codon bias in Escherichia coli for all two-fold degenerate amino acids was studied as dependent on the context from the six bases in the nearest surrounding codons. By comparing the results in genes at different expression levels, effects that are due to differences in mutation rates can be distinguished from those that are due to selection. Selective effects on the codon bias is found mostly from the first neighbouring base in the 3'direction, while neighbouring bases further away influence mostly the mutational bias. In some cases it is also possible to identify specific molecular processes, repair or avoidance of frame shift, that lead to the context dependence of the bias.

Amino Acids↗
PubMed1996

Intermembrane molecular contacts by polymyxin B mediate exchange of phospholipids.

Direct intermembrane exchange of dimyristoylphosphatidylmethanol is mediated by polymyxin B (PxB), a cationic amphipathic cyclic decapeptide. The possibility that the phospholipid exchange is mediated by solubilization of phospholipids or by fusion of vesicles is ruled out. By kinetic and spectroscopic methods it is shown that the exchange occurs directly through vesicle-vesicle contacts formed by a few PxB molecules. The contact is stable on the time scale of several minutes such that neither PxB nor the vesicles in the pair forming a contact exchange with excess vesicles. Several contacts may be formed on a vesicle, which leads to the formation of a cluster of vesicles, and the lipid molecules on the outer monolayers of vesicles exchange throughout the cluster. Kinetics of substrate replenishment during processive interfacial catalysis suggests that the exchange of anionic lipids over the contact occurs at a rate considerably faster than 300 s-1. The exchange through the contact is specific for certain lipids, and phospholipids with a modified head group or phospholipase A2 bound to a vesicle are not transferred to the other vesicle in contact. Since this phenomenon has not been described before, possible implications of direct vesicle-vesicle exchange of phospholipids through peptide-mediated molecular contacts are discussed. Such a mechanism for intermembrane transfer of phospholipids could be responsible for intracellular trafficking and sorting of phospholipids; it could be a necessary first step for the sequence of events leading to budding, vesiculation, and secretion; and PxB-mediated transfer between the inner and outer membranes of Gram-negative bacteria could also account for its antibiotic action.

Cell Membrane↗
PubMed1996

Selection intensity for codon bias and the effective population size of Escherichia coli.

The selection intensity for codon bias and the synonymous diversity have been used in the recent literature to estimate the effective population size of Escherichia coli. The results have varied between (10)5 and (10)8. It is suggested here that most of this disparity can be explained by a model that accounts for the population structure of the species. Thus it is assumed that weakly selected characters, like synonymous substitutions, are selectively fixed within individual lines or colonies but spread throughout the population in an essentially neutral way when colonies replace one another. In this way, the effective population size that enters expressions for the codon bias will be that of an individual colony, which, if hitchhiking effects are considered, can be a very small number. The effective population size that appears together with the mutation rate in expressions for the synonymous diversity, on the other hand, will be related to the total number of colonies that make up the species and can be a very large number.

Codon↗
PubMed1995

Direct vesicle-vesicle exchange of phospholipids mediated by polymyxin B.

Direct and rapid intermembrane exchange of phospholipids without fusion is shown to occur across stable vesicle-vesicle contacts formed by stoichiometric amounts of polymyxin B. The exchange is selective for monoanionic, but not dianionic, glycerophospholipids irrespective of their chain length or phase properties. Selective transfer mediated by protein contacts between membranes could serve as a model for stable fusion intermediates and provide a structural and organizational basis for direct phospholipid transfer as implicated in entry of enveloped viruses, secretion, endocytosis, and intracellular transport and targeting of lipids. It is also proposed that the loss of the specificity of phospholipid composition in the inner and outer membranes of gram negative bacteria mediated by polymyxin B could be the basis for its bactericidal action.

Amino Acid Sequence↗
PubMed1995

Periodic selection and hitchhiking in a bacterial population.

The accumulation of a neutral marker in a bacterial population under balanced growth in a chemostat follows a jagged curve as adaptive variants continuously appear and sweep the population. Such periodic selection curves are simulated in the present work using deterministic equations that, in contrast to previous models, take full account of the stochastic character of the process. The uncertainties due to the appearance time, the survival probability, and the extent of early growth at small numbers are included as stochastic initial conditions for every new variant--adaptive or neutral--that appears. The model is used to calculate the substitution rate via hitchhiking where a neutral or weakly selected mutation is carried along when a new adaptive one takes over the population. The expected ratio for the probabilities of the presence or absence of a weakly selected or counterselected mutation in the population is also calculated. This can be related to the standard result without hitchhiking if the average time between adaptive shifts is interpreted as an effective population size.

Bacteria↗
PubMed1995

Synonymous substitution-rate constants in Escherichia coli and Salmonella typhimurium and their relationship to gene expression and selection pressure.

Based on the differences in synonymous codon use between E. coli and S. typhimurium, the synonymous substitution rates can be estimated. In contrast to previous studies on the substitution rates in these two organisms, we use a kinetic model that explicitly takes the selection bias into account. The selection pressure on synonymous codons for a particular amino acid can be calculated from the observed codon bias. This offers a unique opportunity to study systematically the relationship between substitution-rate constants and selection pressure. The results indicate that the codon bias in these organisms is determined by a mutation-selection balance rather than by stabilizing selection. A best fit to the data implies that the mutation rate constant increases about threefold in genes at low expression levels relative to those that are highly expressed.

Base Sequence↗
PubMed1995

Kinetics of synonymous codon change for an amino acid of arbitrary degeneracy.

The kinetics of synonymous codon change and species divergence is described in a matrix formalism that is equally applicable to all levels of codon degeneracy and all levels of codon or nucleotide bias. Based on the formalism it is possible to calculate the sum of all synonymous substitution rate constants from the observed sequence differences between two species. This sum, the relaxation rate, is equivalent to the Log Det transformation that has recently been proposed as a new measure of evolutionary distance (Lockhardt et al. Mol. Biol. Evol. 11(4):605-612, 1994). The relationship between this measure and the average number of base changes per site (Ks) is discussed. The formalism is tested on some sets of simulated sequence divergence data.

Amino Acids↗
PubMed1995

Interfacial enzymology of glycerolipid hydrolases: lessons from secreted phospholipases A2.

Interfacial enzymes operate at an organized interface such as lipid aggregates in contact with the aqueous phase. The enzyme phospholipase A2 is a well studied interfacial enzyme, and a discussion of its behavior at interfaces is the topic of this review. Knowledge gained from studies of phospholipases A2 can be applied toward the quantitative analysis of other interfacial enzymes. The kinetic analysis of these enzymes is greatly simplified if one establishes certain experimental conditions that limit the exchange of enzyme and substrate between different substrate aggregates. With such constraints, the kinetics can be analyzed in terms of classical Michaelis-Menten theory adopted for the action of enzymes at interfaces. It is also possible to describe other enzyme properties such as inhibition and substrate preferences in a meaningful way using formalism that is well known in solution-phase enzymology.

Animals↗
PubMed1994

Inhibition of phospholipase A2.

Phospholipases A2 are involved in inflammatory processes such as the liberation of free arachidonic acid from the membrane pool for the biosynthesis of eicosanoids. Inhibitors of these enzymes are proving useful in determining the biological roles of phospholipases A2 in complex cellular processes and may also have therapeutic potential. Inhibition of these lipolytic enzymes is more difficult to characterize as the enzymatic reaction occurs at a lipid/water interface. This review focuses on the description of a number of classes of rationally designed phospholipase A2 inhibitors. The development of a theoretical framework for the proper analysis of inhibitors is presented. Structural studies of phospholipase A2-inhibitor complexes suggest how the lipolysis reaction is catalyzed. Finally, some recent results on the use of phospholipase A2 inhibitors in living cells and tissues are revealed.

Animals↗
PubMed1993

Relationship of interfacial equilibria to interfacial activation of phospholipase A2.

The equilibrium dissociation constants for the distribution of pig pancreatic phospholipase A2, its competitive inhibitors, and their complexes at the interface of a neutral diluent are determined. The relationship between these parameters and their significance for interfacial catalysis is elaborated in terms of a model based on the relationship between the underlying equilibria. By using a combination of spectroscopic and chemical modification methods, it was possible to determine the equilibrium dissociation constant of an inhibitor bound to the interface (K') or of the inhibitor bound to the enzyme in the aqueous phase (KI) or the interface (KI*). The equilibrium dissociation constant for the free enzyme (Kd) or for the enzyme-inhibitor complex (KdI) from the interface were also obtained. These constants are shown to be thermodynamically related, i.e., K'KdKI* = KIKdI, as predicted on the basis of the cyclic equilibrium scheme (thermodynamic box) describing the distribution of the enzyme and the inhibitor between the aqueous phase and the interface at constant calcium concentration. Results show that (i) calcium is required for the binding of a substrate or inhibitor molecule to the catalytic site; (ii) the effective dissociation constant of the inhibitor-enzyme complex in the aqueous phase is considerably larger than that for the enzyme at the interface, i.e., KI >> effective KI*; (iii) KdI does not depend on the structure of the inhibitor and Kd >> KdI; and (iv) structure-activity correlations suggest that ionic interactions between a ligand and the interfacial recognition site of the enzyme are important for K', which controls the concentration of the bound inhibitor that the enzyme "sees" in the interface. These observations demonstrate that the binding of the enzyme to the interface and the binding of the inhibitor to the active site of the enzyme at the interface are two distinguishable processes. Therefore, binding of a ligand to the active site of the enzyme promotes binding of the enzyme-inhibitor complex to other amphiphiles and the interface with higher affinity. It is suggested that the primary effect of binding the enzyme to the interface is to increase its intrinsic affinity toward the active-site-directed ligands, i.e., the interfacial activation of PLA2 is of K-type.

Animals↗
PubMed1993

The chemical step is not rate-limiting during the hydrolysis by phospholipase A2 of mixed micelles of phospholipid and detergent.

The effect of detergents on the overall catalytic turnover by secreted phospholipase A2 (PLA2) on codispersions of the substrate phospholipid is characterized. The overall rate of interfacial catalytic turnover depends on the effective substrate "concentration" (mole fraction) that the bound enzyme "sees" at the interface. Therefore, besides the intrinsic catalytic turnover rate determined by the Michaelis-Menten cycle in the interface [Berg et al. (1991) Biochemistry 30, 7283], two other interfacial processes significantly alter the overall effective rate of hydrolysis: first, the fraction of the total enzyme at the interface; second, the rate of replenishment of the substrate. At low mole fractions (< 0.3), bile salts promote the binding of pig pancreatic PLA2 to zwitterionic vesicles, and the rate of hydrolysis increases with the fraction of the enzyme in the interface. At higher (> 0.3) mole fractions of the detergent, the bilayer is disrupted, and the rate of hydrolysis decreases by more than a factor of 10. The detergent-dependent decrease in the rate of hydrolysis of the sn-2-oxyphospholipids is much larger than that of sn-2-thiophospholipid, and therefore the element effect (O/S ratio) decreases from about 10 in bilayers to less than 2 in mixed micelles. This loss of the element effect in mixed micelles shows that the chemical step is no longer rate-limiting during the hydrolysis of mixed micelles formed by the disruption of vesicles by the detergent. Such effects were observed with phospholipase A2 from several sources acting on substrates dispersed in a variety of detergents including bile salts, 2-deoxylysophosphatidylcholine, and Triton X-100.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

The divalent cation is obligatory for the binding of ligands to the catalytic site of secreted phospholipase A2.

The divalent cation requirement for partial reactions of the catalytic turnover cycle during interfacial catalysis by pig pancreatic phospholipase A2 (PLA2) is investigated. Results show that the specific role of calcium in all the events of the catalytic cycle at the active site is not shared by other divalent cations. Cations such as calcium, barium, and cadmium bind to the enzyme in the aqueous phase. The active-site-directed ligands (substrate, products, and transition-state mimics) do not bind to the enzyme in the absence of a divalent cation. The synergistic binding of such ligands to the active site of PLA2 bound to the interface is, however, observed only in the presence of isosteric ions like calcium and cadmium, but not with larger ions like strontium or barium. The equilibrium constants for ligands bound to the enzyme in the presence of calcium and cadmium are virtually the same. However, only calcium supports the catalytic turnover; the rate of hydrolysis in the presence of cadmium is less than 1% of that observed with calcium. The role of divalent ions on the interfacial catalytic turnover cycle of PLA2 is not only due to the cation-assisted binding of the substrate but also due to its participation in the chemical step. Other roles of divalent ions in the events of interfacial catalytic turnover are also identified. For example, the binding of the enzyme to the interface is apparently promoted because the divalent cation is required for the sequential step, i.e., the binding of the substrate to the active site of PLA2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The evolutionary selection of DNA base pairs in gene-regulatory binding sites.

The DNA base-pair sequences that serve as gene-regulatory sites have been selected during evolution to provide an appropriate functional binding for a particular protein. In most cases, the function depends on the binding probability, which can be influenced both by the binding strength and by the abundance of the protein in the cell. As a consequence, the same function can be achieved with strong binding sites and a small amount of protein as with weak binding sites and a large amount of protein. However, increasing the protein burden will decrease the growth rate of the cells, even when all functions remain the same. Thus, for maximal growth, the protein levels should be as low as possible and the binding correspondingly strong. On the other hand, sequences with a weaker binding can be formed in many more ways and are, therefore, more probable, and random mutations are more likely to produce them. Thus, the selection pressure against an increased protein burden can be balanced against the random mutational drift in the recognition sequences, thereby tying together the statistics of base-pair choice, the binding strength, and the protein burden. In terms of this model, the selection pressure can be estimated from the properties of a gene-regulatory protein and its recognition sites. A key feature is the mutational randomization pressure that appears as a fundamental force shaping the optimal solutions that provide maximal growth. The model is tested on a number of gene-regulatory systems in Escherichia coli. The same principles should hold for all proteins for which overall activity in the cell is proportional to abundance; then the selective pressure to increase the efficiency of an individual protein cannot be larger than the selective pressure to decrease the total protein burden.

Base Composition↗
PubMed1991

Interfacial catalysis by phospholipase A2: determination of the interfacial kinetic rate constants.

Hydrolysis of vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) by pig pancreatic phospholipase A2 (PLA2) occurs in a highly processive "scooting" mode, and the rate is comparable to or exceeds the rates observed with detergent-dispersed mixed micelles under optimal conditions. A complete kinetic description of the steady-state time course of the hydrolysis is developed. The analysis covers the whole Michaelis-Menten space: it emphasizes the key features of interfacial catalysis by a detailed theoretical analysis, describes the experimental protocols to determine the values of the kinetic and equilibrium constants for interfacial catalysis, and provides an interpretation of the effect of calcium, substrate, products, apparent activators, and competitive inhibitors on the reaction progress curve by a single set of rate and equilibrium parameters. In this paper, the integrated reaction progress curve was rigorously interpreted in terms of a minimal model involving the Michaelis-Menten reaction sequence in the interface: E* + S in equilibrium E*S----E*P in equilibrium E* + P, and most of the individual rate and equilibrium constants for the catalytic cycle were determined. This rigorous description of interfacial catalysis was made experimentally possible by examining the action of PLA2 in the scooting mode under conditions of at most one enzyme per vesicle, where it hydrolyzed all of the substrate in the outer monolayer of vesicles without leaving the surface. Other experimentally verified constraints for this analysis include the following: all enzyme was bound to vesicles; the integrity of vesicles was maintained during the course of hydrolysis; and the substrate, enzyme, and products did not exchange between vesicles nor did they exchange across the bilayer. The mechanistic significance of the rate constants is discussed in the accompanying papers.

Animals↗
PubMed1991

Interfacial catalysis by phospholipase A2: dissociation constants for calcium, substrate, products, and competitive inhibitors.

Interpretation of the kinetics of interfacial catalysis in the scooting mode as developed in the first paper of this series [Berg et al. (1991) Biochemistry 30 (first paper of six in this issue)], was based on the binding equilibrium for a ligand to the catalytic site of phospholipase A2. In this paper, we describe direct methods to determine the value of the Michaelis-Menten constant (KMS) for the substrate, as well as the equilibrium dissociation constants for ligands (KL) such as inhibitors (KI), products (KP), calcium (KCa), and substrate analogues (KS) bound to the catalytic site of phospholipase A2 at the interface. The KL values were obtained by monitoring the susceptibility to alkylation of His-48 at the catalytic site of pig pancreatic PLA2 bound to micellar dispersions of the neutral diluent 2-hexadecyl-sn-glycero-3-phosphocholine. The binding of the enzyme to dispersions of this amphiphile alone had little effect on the inactivation rate. The half-time for inactivation of the enzyme bound to micelles of the neutral diluent depended not only on the nature of the alkylating agent but also on the structure and the mole fraction of other ligands at the interface. The KL values for ligands obtained from the protection studies were in excellent accord with those obtained by monitoring the activation or inhibition of hydrolysis of vesicles of 1,2-dimyristoyl-sn-glycerophosphomethanol. Since only calcium, competitive inhibitors, and substrate analogues protected phospholipase A2 from alkylation, this protocol offered an unequivocal method to discern active-site-directed inhibitors from nonspecific inhibitors of PLA2, such as local anesthetics, phenothiazines, mepacrine, peptides related to lipocortin, 7,7-dimethyleicosadienoic acid, quinacrine, and aristolochic acid, all of which did not have any effect on the kinetics of alkylation nor did they inhibit the catalysis in the scooting mode.

Animals↗
PubMed1990

The influence of macromolecular crowding on thermodynamic activity: solubility and dimerization constants for spherical and dumbbell-shaped molecules in a hard-sphere mixture.

Macromolecules in solution can have large effects on the properties of other solutes through nonideal excluded-volume (crowding) interactions. Minton has calculated such effects by treating the macromolecules as a hard-sphere fluid in a background of an inert structureless solvent. In the present paper these calculations are extended by including the primary solvent as a separate component in a hard-sphere mixture. The results are in good agreement with experimental data. However, some predictions of this model differ drastically from those based on Minton's approach. Thus, much smaller effects from macromolecular crowding, particularly by smaller molecules, are expected. The present results also predict a much larger dependence on the shape of the molecules under study; notably for a dimerization reaction, it is found that the excluded-volume effects actually can destabilize side-by-side binding of two spherical molecules, while a dimerization to a spherical complex is stabilized. Therefore there will exist intermediate shapes of complexes whose stability is insensitive to crowded-volume effects. The consequences for crowding effects inside the living cell are also discussed.

Macromolecular Substances↗
PubMed1990

Base-pair specificity of protein-DNA recognition: a statistical-mechanical model.

The statistics of base-pair choice in individual recognition sites on DNA is shown to be determined by the functional binding requirements for recognition and a selection parameter. This selection parameter can be identified as a generalized external force required to deform a random-choice base-pair distribution into the observed specific-choice distribution. This external force is balanced by the randomization pressure which--driven by mutations--always tends to increase randomness in the base-pair choices. The model makes it possible to predict relative binding constants of particular recognition sequences based primarily on the statistics of base-pair usage. A further consequence of this formulation is that the randomization pressure appears explicitly as an important force shaping the evolutionary selection not only of DNA sites, but also of other properties involving macromolecular design.

Base Composition↗