Pheromone deactivation catalyzed by receptor molecules: a quantitative kinetic model

A quantitative model of pheromone-receptor interaction and pheromonedeactivation, the supposed rate-limiting processes underlying the receptorpotential kinetics, is worked out for the moth Antheraea polyphemus. Inthis model, the pheromone interacts with the receptor molecule while boundto the reduced form of the pheromone binding protein. The receptormolecules--besides their receptor function-- catalyze the observed shift ofthe pheromone-binding protein from the reduced to the oxidized form(Ziegelberger, G., Eur. J. Biochem., 232, 706-711, 1995), which deactivatesthe pheromone bound to pheromone binding protein. With the followingparameters, the model fits morphological, radiometric, electrophysiologicaland biochemical data: a maximum estimate of 1.7 x 10(7) receptormolecules/cell (with 40,000 units/micron 2 of receptor cell membrane), rateconstants k1 = 0.2/(s.microM) for the association, k2 = 10/s for thedissociation of the ternary complex of binding protein, pheromone andreceptor, and k3 = 10/s for the deactivation via the redox shift. Withthese parameters, the duration of elementary receptor potentials elicitedby single pheromone molecules (approximately 50 ms) reflects the lifetimeof the ternary complex, tau = 1/(k2 + k3). The receptor occupancy producedby the model for threshold stimuli fits the sensitivity of the receptorcell to single pheromone molecules.     

Modular evolution and increase of functional complexity in replicating RNA molecules

At early stages of biochemical evolution, the complexity of replicating molecules was limited by unavoidably high mutation rates. In an RNA world, prior to the appearance of cellular life, an increase in molecular length, and thus in functional complexity, could have been mediated by modular evolution. We describe here a scenario in which short, replicating RNA sequences are selected to perform a simple function. Molecular function is represented through the secondary structure corresponding to each sequence, and a given target secondary structure yields the optimal function in the environment where the population evolves. The combination of independently evolved populations may have facilitated the emergence of larger molecules able to perform more complex functions (including RNA replication) that could arise as a combination of simpler ones. We quantitatively show that modular evolution has relevant advantages with respect to the direct evolution of large functional molecules, among them the allowance of higher mutation rates, the shortening of evolutionary times, and the very possibility of finding complex structures that could not be otherwise directly selected.     

Restriction analysis of autonomously replicating molecules containing exogenous DNA in a transgenic silkworm line]

Early embryos of the silkworm, Bombyx mori, were injected with plasmid p1,5LTR, harboring DNA copies of the Rous sarcoma long terminal repeats (LTR). Three generations of insects were obtained whose total DNA carried DNA sequences of the injected plasmid. Plasmid rescue method revealed that exogenous DNA was present in extrachromosomal molecules that harbored also cellular DNA sequences. A restriction map of the plasmid rescued from the F2 generator was constructed.     

Persistence of freely replicating SV40 recombinant molecules carrying a selectable marker in permissive simian cells

We have demonstrated that a SV40-pBR322 recombinant vector (pSV2-gpt) carrying a bacterial gene of selectable phenotype (Eco-gpt) may persist extrachromosomally in COS1 cells, a simian cell line that endogenously produces SV40 large T antigen. The amount of circular (supercoiled) recombinant DNA was estimated to be between 5 and 2000 copies per cell among several pSV2-transformed COS1 clonal lines examined. Complete pSV2 molecules were found in the majority of the transformants, although some of the pSV2 DNAs recovered were shown to have deletions in the pBR322 region. Our results indicate that removal of the pBR322 "inhibitory sequence" in pSV2 is not necessary for stable maintenance of these recombinant molecules in COS1 cells. In addition, large amounts of pSV2-related high molecular weight DNAs, probably concatemers of pSV2, were detected in the transformed lines.     

Anomalous electrophoretic behavior of creatine kinase: A thiol-dependent isomeric system with migration subject to kinetic control

Moving boundary electrophoresis of creatine kinase in 0.1 I diethylbarbiturate buffer, pH 8.9, has yielded anomalous migration behavior that indicates intereonversion between two coexisting states of the enzyme at a rate comparable with the rate at which the two enzymic forms tend to separate by differential migration. Whereas a single, symmetrical boundary is observed in the ascending limb, distinct bimodality of the descending pattern is evident in electrophoresis of enzyme isolated from skeletal muscle of either rabbit or fish (Mugil cephalus): pronounced changes in the nature of this bimodality with time are observed in the case of fish muscle creatine kinase. The abnormal migration behavior is eliminated by inclusion of dithiothreitol in the electrophoresis medium or by covalent modification of the enzyme with 5,5′-dithiobis(2-nitrobenzoic acid). Velocity and equilibrium sedimentation studies have been used to identify the macromolecular event as an isomerization, and studies of sulfhydryl content to implicate either reversible sulfhydryl oxidation or thiol-disulfide interchange in the isomerization mechanism.     

Sticking coefficient-orchestrated selection in a kinetic model for the first origin

The mathematical formalism of the steady-state Poisson equation is applied to a variant of Freeman Dyson's "Toy Model" for a first origin. Our kinetic approach allows for an examination of the requisite conditions under which metabolism is quantized into discrete eigenstates (e.g. Dyson's disordered, saddle point, and metabolically active toy cell states). The surface reaction machinery additionally allows for more realistic modeling, whence the crucial role of sticking coefficients (catalyst precursors) as prebiotic selectors emerges. In our interior source model, a steady influx of vent nutrients fuels the intracellular synthesis of (impermeable) monomers within a rock-encradled cavity. Random adsorptions and desorptions occur at inactive "cell" wall sites (where the inert monomers remain impermeable until their eventual return to the intracellular metabolite pool). Occasionally, metabolizing reactions also occur due to endogeneous source monomers adsorbing at their "active" sites. Dyson's mean field approach is used to simplify the species-specific sticking coefficients at empty active (substrate) sites to functions of the fraction x of sites occupied by (catalytically) active monomers. In short, our work suggests that disorder-order transition models based on random drift between discrete metabolic eigenstates (Dyson's Toy Model) do not, in general, extend to more realistic metabolisms. From a perspective based on quasi-random feedback kinetics, the contraindication for discretization (spontaneous generation) in non-autocatalytic metabolisms is consistent with the emergence of ordered metabolism under hydrothermal driving forces, a provisio the occurrence of each period of vent dormancy coincides with a discrete zero-source (dormant) metabolic state. Cell drift to higher order is induced by the random reactions which happen to enhance the substrate specificity (chemical selectivity) of the sticking coefficients for active monomers. The result is stronger sink effects for metabolizing species, whence active adsorptions are promoted in favor of inactive adsorptions at substrate sites. Positive feedback plays a crucial role in preserving ("propagating") order in the cell wall reaction kinetics and is held in check by negative feedback inhibition of excessive cell growth. Finally, the eventual desorption of randomly growing dysfunctional proteins is postulated as a deterrent to deterioration catastrophes.     

Catfish liver acid phosphatases: differently glycosylated enzyme molecules with altered kinetic properties

Two forms of catfish liver acid phosphatase (AcPase I and II) were separated and purified to homogeneity and their carbohydrate compositions and some biochemical properties were studied. Evidence is given that AcPase I and II are differently glycosylated forms of the same enzyme. The enzyme forms differ significantly in the size and the composition of their carbohydrate components, sensitivity towards sulfhydryl-blocking and protecting reagents, sensitivity to ferric and ferrous ions, thermostability and ability to hydrolyze some nucleotides. The more highly glycosylated form is more sensitive to thermal denaturation. AcPase I and II behave differently towards ascorbate and changes in its concentration and it is suggested that the concentration of reducing modifiers may regulate AcPase activity at the cellular level. It is hypothesized that the differing extents of glycosylation influence the structure of the enzyme forms. This is expressed in altered conformations of two enzyme forms and results in a different exposure of the essential cysteine residues.     

The origin of kinetic cooperativity in prehiotic catalysts

A polyallylamine carrying long hydrophobic dodecyl groups and adenine residues as side chains (PALAD C₁₂) may be able to catalyze the hydrolysis ofN-carbobenzoxy-l-alaninep-nitrophenyl ester (N-Cbz-Ala) as well asp-nitrophenyl acetate (pNPA). The progress curve of hydrolysis of the former displays a long lag and apparently no steady state. After this transient the rate falls off due to the accumulation of the products. Conversely, the hydrolysis ofp-nitrophenyl acetate displays classical burst kinetics followed by a slow decline of the reaction rate. Theoretical considerations show that a steady state may be expected to occur only if the concentration of the free catalyst is very small during the reaction. This condition is sufficient to allow the rate of disappearance of the substrate to be equal to the rate of appearance of the products, which is precisely a condition for the existence of a steady state. If the catalyst is poorly active and has a loose affinity for its substrate and product, the measurement of a significant reaction rate will require a much larger concentration of the catalyst. Therefore, under these conditions, one cannot expect a steady state to occur. The mathematical expression of the error made in the steady-state assumption has been derived. This error increases with the catalyst concentration and decreases if the affinity of the substrate for the catalyst is high. Therefore the lack of steady state is associated with the affinity (or the dissociation) of the substrate and the product for the catalyst. When this affinity is low, the free concentration of the catalyst during the reaction is high and one cannot expect a steady state to occur. This is precisely what takes place with N-Cbz-Ala. A mathematical expression of the rate of hydrolysis of N-Cbz-Ala and of any reactant that displays this type of kinetics may be derived at the end of the transient when the rate is close to its maximum value. Under these conditions the rate cannot follow classical Michaelis-Menten kinetics and displays positive cooperativity. It may therefore be speculated that primordial template-like catalysts that were displaying a poor affinity for their substrates and products were already exhibiting apparent positive cooperativity in the kinetic reactions they were able to catalyze. Correspondence to: J. Ricard     

On the origin of avian flight: Compromise and system approaches

Based on evolutionary morphological analysis of the fore and hind limbs of extinct and extant birds, a new compromise hypothesis of the origin of flight in birds and theropod dinosaurs is proposed. The bipedalism and anisodactylous foot suitable for various functions were key adaptations for the development of flight. The bipedalism freed forelimbs from the supporting function and promoted transformation into wings, as animals moved from one tree branch to another and descended from trees. At the initial stage, the strong hind limbs provided the opportunity to climb and leap onto trees, bushes, or eminence, while the anisodactylous foot provided a firm support on both dry land and trees. The support provided by this foot allowed the reduction of the tail, which was initially composed of a long row of vertebrae. Thus, a stage of gliding flight was not necessarily passed by early birds. In the other lineages of feathered creatures, functional changes in forelimbs that resulted in the formation of wings developed in parallel and followed almost the same scenario.     

Reversion of autonomously replicating sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic replication origin within procaryotic vector DNA.

To investigate how a defective replicon might acquire replication competence, we have studied the reversion of autonomously replicating sequence (ARS) mutations. By mutagenesis of a Saccharomyces cerevisiae plasmid lacking a functional origin of replication, we have obtained a series of cis-acting mutations which confer ARS activity on the plasmid. The original plasmid contained an ARS element inactivated by point mutation, but surprisingly only 1 of the 10 independent Ars+ revertants obtained shows a back mutation in this element. In the remainder of the revertants, sequence changes in the M13 vector DNA generate new ARSs. In two cases, a single nucleotide change results in an improved match to the ARS consensus, while six other cases show small duplications of vector sequence creating additional matches to the ARS consensus. These results suggest that changes in replication origin distribution may arise de novo by point mutation rather than by transposition of preexisting origin sequences.     

DNA作催化剂:小分子体外不对称催化合成

综述了以插入非手性二价铜配合物的鲑鱼精DNA(st-DNA)作为催化剂,于体外催化包括不对称DielsAlder反应、Friedel-Crafts反应与Michael加成等一系列小分子反应的研究进展。这些反应在天然产物与其他具生物活性物质合成途径设计中有重要作用。反应通常可以取得大于80%的对映体过量百分数(ee)值,并于优化后可进一步提高到90%至99%。DNA的碱基序列对对映选择性将产生影响,且因配体结构而异,有较多G或C相连的DNA序列通常可以产生较好的对映选择性。     

On the origin of mitochondria: a reexamination of the molecular structure and kinetic properties of pyruvate dehydrogenase complex from brewer''s yeast

45Ca2+ incorporated in response to glucose was selectively mobilized from the beta-cell-rich pancreatic islets of ob/ob-mice after raising the intracellular Na+ by removal of K+ or addition of ouabain or veratridine. Also studies of insulin release indicated opposite effects of glucose and Na+ on the intracellular sequestration of calcium. The fact that glucose inhibits insulin release induced by raised intracellular Na+ indicates that this sugar can lower the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+] might well explain previous observations of an inhibitory component in the glucose action on the 45Ca2+ efflux.     

On the origin of mitochondria: a genomics perspective

The availability of complete genome sequence data from both bacteria and eukaryotes provides information about the contribution of bacterial genes to the origin and evolution of mitochondria. Phylogenetic analyses based on genes located in the mitochondrial genome indicate that these genes originated from within the alpha-proteobacteria. A number of ancestral bacterial genes have also been transferred from the mitochondrial to the nuclear genome, as evidenced by the presence of orthologous genes in the mitochondrial genome in some species and in the nuclear genome of other species. However, a multitude of mitochondrial proteins encoded in the nucleus display no homology to bacterial proteins, indicating that these originated within the eukaryotic cell subsequent to the acquisition of the endosymbiont. An analysis of the expression patterns of yeast nuclear genes coding for mitochondrial proteins has shown that genes predicted to be of eukaryotic origin are mainly translated on polysomes that are free in the cytosol whereas those of putative bacterial origin are translated on polysomes attached to the mitochondrion. The strong relationship with alpha-proteobacterial genes observed for some mitochondrial genes, combined with the lack of such a relationship for others, indicates that the modern mitochondrial proteome is the product of both reductive and expansive processes.     

Ligand recombination and a hierarchy of solvent slaved dynamics: the origin of kinetic phases in hemeproteins

Ligand recombination studies play a central role both for characterizing different hemeproteins and their conformational states but also for probing fundamental biophysical processes. Consequently, there is great importance to providing a foundation from which one can understand the physical processes that give rise to and modulate the large range of kinetic patterns associated with ligand recombination in myoglobins and hemoglobins. In this work, an overview of cryogenic and solution phase recombination phenomena for COMb is first reviewed and then a new paradigm is presented for analyzing the temperature and viscosity dependent features of kinetic traces in terms of multiple phases that reflect which tier(s) of solvent slaved protein dynamics is (are) operative on the photoproduct population during the time course of the measurement. This approach allows for facile inclusion of both ligand diffusion among accessible cavities and conformational relaxation effects. The concepts are illustrated using kinetic traces and MEM populations derived from the CO recombination process for wild type and mutant myoglobins either in sol-gel matrices bathed in glycerol or in trehalose-derived glassy matrices.     

Synchronization in a pool of mutually coupled oscillators with random frequencies

An exact solution to a model of mutually interacting sinusoidal oscillators is found. Limits on the variation of the native frequencies are determined in order for synchronization to occur. These limits are computed for different distributions of native frequencies.This research was supported by NSF Award No. MCS8300885 and the Alfred Sloan Foundation.     

Functional multiplicity of motor molecules revealed by a simple kinetic analysis.

We present a simple analytical solution for a kinetic model of motor molecule function with multiple arms. This model has a rate of motion proportional to the probability that all arms in a complex are detached from the cytoskeleton and, therefore, we refer to it as obligate cooperativity. The model has the form: v = Vmax/(1 + q/S)n, where Vmax is the maximum velocity, the product nq is the effective Michaelis constant at high [ATP], and n is the number of arms. Values of n = 2 and n = 1 give good fits to the heavy meromyosin and myosin S1 sliding velocity data, respectively, consistent with the number of active sites. Despite the complexity of the eukaryotic axoneme, beat frequency data from Chlamydomonas wild-type and oda mutants are also fit by this model.