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Structure and evolution of protein interaction networks: a statistical model for link dynamics and gene duplications.

BACKGROUND: The structure of molecular networks derives from dynamical processes on evolutionary time scales. For protein interaction networks, global statistical features of their structure can now be inferred consistently from several large-throughput datasets. Understanding the underlying evolutionary dynamics is crucial for discerning random parts of the network from biologically important properties shaped by natural selection. RESULTS: We present a detailed statistical analysis of the protein interactions in Saccharomyces cerevisiae based on several large-throughput datasets. Protein pairs resulting from gene duplications are used as tracers into the evolutionary past of the network. From this analysis, we infer rate estimates for two key evolutionary processes shaping the network: (i) gene duplications and (ii) gain and loss of interactions through mutations in existing proteins, which are referred to as link dynamics. Importantly, the link dynamics is asymmetric, i.e., the evolutionary steps are mutations in just one of the binding parters. The link turnover is shown to be much faster than gene duplications. Both processes are assembled into an empirically grounded, quantitative model for the evolution of protein interaction networks. CONCLUSIONS: According to this model, the link dynamics is the dominant evolutionary force shaping the statistical structure of the network, while the slower gene duplication dynamics mainly affects its size. Specifically, the model predicts (i) a broad distribution of the connectivities (i.e., the number of binding partners of a protein) and (ii) correlations between the connectivities of interacting proteins, a specific consequence of the asymmetry of the link dynamics. Both features have been observed in the protein interaction network of S. cerevisiae.

Biological Evolution↗

Synthesis, in vitro skin permeation studies, and PLS-analysis of new naproxen derivatives.

PURPOSE: To synthesize new naproxen (01) derivatives with amide or ester structures or with a combination of the two (02-15). To compare their physicochemical properties with naproxen esters (16-22) and their respective skin permeation behavior. To study structure-permeation relationships via partial least squares (PLS)-analysis. METHODS: Stability, aqueous, and octanol solubility were determined. Lipophilicity and further 53 chemical descriptors were computed. A suitable in-vitro skin permeation model was developed to compare maximal flux (Jmax) of derivatives. Based on these flux data, PLS-analysis was performed to derive structure-permeation relationships. RESULTS: None of the new derivatives showed an improved flux in comparison to naproxen. This result can be explained by PLS-analysis: skin permeation increases with the solubility both in water and in octanol. For a good permeation, an optimized molecule should exhibit a small volume with a spherical shape. The surface area should be large in relation to volume, as indicated by the rugosity parameter. A clear separation between the hydrophobic and the hydrophilic domain (= high amphiphilic moment) is favorable. Lipophilicity is inversely correlated with skin permeation. CONCLUSIONS: PLS-analysis is a valuable tool to derive significant, internally predictive quantitative models for structure-permeation relationships of naproxen derivatives in the above described skin permeation assay.

Animals↗

Principal causes of hot spots for cytosine to thymine mutations at sites of cytosine methylation in growing cells. A model, its experimental support and implications.

In Escherichia coli and human cells, many sites of cytosine methylation in DNA are hot spots for C to T mutations. It is generally believed that T.G mismatches created by the hydrolytic deamination of 5-methylcytosines (5meC) are intermediates in the mutagenic pathway. A number of hypotheses have been proposed regarding the source of the mispaired thymine and how the cells deal with the mispairs. We have constructed a genetic reversion assay that utilizes a gene on a mini-F to compare the frequency of occurrence of C to T mutations in different genetic backgrounds in exponentially growing E. coli. The results identify at least two causes for the hot spot at a 5meC: (1) the higher rate of deamination of 5meC compared to C generates more T.G than uracil.G (U.G) mismatches, and (2) inefficient repair of T.G mismatches by the very short-patch (VSP) repair system compared to the repair of U. G mismatches by the uracil-DNA glycosylase (Ung). This combination of increased DNA damage when the cytosines are methylated coupled with the relative inefficiency in the post-replicative repair of T.G mismatches can be quantitatively modeled to explain the occurrence of the hot spot at 5meC. This model has implications for mutational hot and cold spots in all organisms.

5-Methylcytosine↗

Kinetics of Na+-ATPase: influence of Na+ and K+ on substrate binding and hydrolysis.

An analysis of the influence of Na+ and K+ on the kinetics of Na+-ATPase in broken membrane preparations from bovine brain is presented with particular emphasis on the effect of the cations on the binding and splitting of the substrate MgATP and on the derivation of a detailed kinetic model for that interaction. It was found that the enzyme in the absence of Na+ and K+, but in the presence of 7 mM free Mg2+, at pH 7.4 (37 degrees C) exhibits an ouabain-sensitive ATPase activity. The simplest model quantitatively compatible with all the data involves two different, interconvertible (conformational) forms of the enzyme, E1 and E'1, with the following properties: The E1 form does not bind K+ but has three independent and equivalent high-affinity sites (Kd = 5.6 mM) for Na+. It binds and hydrolyzes substrate only when two or three sodium ions are bound to it. The E'1 form binds and hydrolyzes the substrate only in the absence of monovalent cations. It is competitively inhibited by K+ (Kd = 0.23 mM), and this inhibition is further enhanced by binding of Na+ to the K+-bound form at two equivalent, independent sites (Kd = 12 mM). It is suggested that the E'1 form is the Mg2+-induced conformational state of the enzyme observed by others, which differs from the usually encountered E1 and E2 forms. The model allows the calculation of ATP-binding and ADP-releasing rate constants for the E1-form for later comparison with corresponding rate constants for the (na+ + K+)-ATPase (following paper).

Adenosine Triphosphatases↗

Liposomal formulations of cyclosporin A: influence of lipid type and dose on pharmacokinetics.

PURPOSE: Liposomal formulations of Cyclosporin A (CyA)3 have been described in more than 30 publications to substitute Cremophor EL (CrEL), a triricinoleate ester of ethoxylated glycerol, as drug carrier. However, conflicting reports did not allow to draw consistent conclusions about the influence of liposomes on CyA pharmacokinetics (PK) and pharmacodynamics. METHODS: A series of liposomal CyA-formulations with varying liposome composition and lipid dose but constant CyA dose was compared in rats. Data were analysed with a PK-model taking into account the varying volume of distribution with the varying lipid concentration in blood. RESULTS: Surface properties and lipid type of liposomes are not important PK predictors of liposomal CyA, at least for small dosages of liposomes. Rather, the absolute lipid amount and the lipophilicity of cyclosporins are critical factors influencing the PK of liposomal CyA. The higher the concentration of lipid in blood and the greater the lipophilicity of cyclosporin is, the higher are the concentrations of CyA in blood. CONCLUSIONS: These relations may explain the inconsistent literature results. Together with earlier observations from our group the above findings indicate, that CyA is not caged in the liposomal membranes. Reports in literature, which claim lower clearance and a lower volume of distribution of CyA in obese rats compared to lean rats, support our assumption about the involved mechanisms. A semi-quantitative model of CyA distribution is presented, which points to the variable free fraction of CyA in plasma as the crucial factor for all previously reported phenomena in liposomal CyA formulations.

Animals↗

Different HPMC viscosity grades as coating agents for an oral time and/or site-controlled delivery system: an investigation into the mechanisms governing drug release.

When used as release-controlling coating agents for tableted core-based pulsatile delivery systems, three different hydroxypropyl methylcellulose (HPMC) grades, Methocel E5, E50, and K4M, provided lag phases of varying duration (Methocel K4M > E50 > E5) and a prompt and quantitative model drug release. Dissolution/mechanical erosion, permeability increase and disruption of the hydrated polymeric layer were assumed to participate in the definition of the overall release pattern. Based on these premises, we investigated what process(es) might prevail in the release-controlling mechanism for each HPMC grade. The polymers were evaluated for dissolution and swelling, while the finished systems were concomitantly evaluated for drug release and polymer dissolution. The obtained results indicated likely similarities between Methocel E5 and E50 performances, which we hypothesized to be mainly dissolution/erosion-controlled, and a clearly different behavior for Methocel K4M. This polymer indeed proved to yield higher viscosity and slower dissolving gel layer, which was able to withstand extensive dissolution/erosion for periods that exceeded the observed lag phases. The particular characteristics of swollen Methocel K4M were shown to be associated with possible drug diffusion phenomena, which might impair the prompt and quantitative release phase that is typical of pulsatile delivery.

Administration, Oral↗

A fully coupled binary biochemical reactive-diffusion model with analytic solution.

Coupled multicomponent biochemical reactive diffusion underlies a variety of biological signalling processes and pharmacokinetic applications, such as paracrine signalling involving "cocktails" comprised of growth promoter/inhibitor factors and proteases associated with tumor angiogenesis, invasion and metastasis, extravascular drug delivery, and polymeric controlled-release drug codelivery design. Here, we present a model and develop a new analytic solution to illustrate the spatiotemporal behavior associated with fully coupled binary biochemical reactive diffusion. The complete coupling renders the solution appreciably more complex in structure and behavior than solutions for unicomponent or partially coupled models. Concentration behavior is illustrated by the computational simulation of binary-species tumor angiogenesis factor reactive-diffusion in the extravascular tissue matrix. The computational results indicate that (a) steady-state concentration profiles are achieved within 1 h of a change in factor production; (b) in the steady state, the spatial profiles of the two components tend to be similar; (c) exceedingly steep concentration gradients, involving several orders-of-magnitude differences in concentration over a few tenths of a millimeter, can occur in the vicinity of boundary sources due to inter-species reaction; (d) the concentration profiles of the two species differ from unicomponent predictions due to the simultaneous mass interchange between the two species. The analytic solution predictions are also used to provide a first-ever validation of a time-dependent, binary-component Crank-Nicholson numerical solution. The ability to quantitatively model interacting and often strongly varying concentration levels as a function of time and position can serve as a powerful complementary tool to experimental analyses for assessing disease state and interventional pharmacological efficacy, especially when the spatial scales on which in vivo behavior occurs taxes the limits of imaging capabilities.

Angiogenesis Inducing Agents↗

Modeling birthweight and gestational age distributions: additive vs. multiplicative processes.

Researchers have traditionally employed Gaussian distributions to model quantitative biological traits. Recently, mixtures of Gaussian distributions have begun to be used as well. However, there are many alternatives to the Gaussian distribution. From a theoretical perspective, the lognormal distribution is as applicable as the Gaussian (both are justified on the basis of the Central Limit Theorem). Here, the utility of mixtures of Gaussians and lognormals for describing birthweight and gestational age distributions are compared. This is carried out within the context of the hybrid-lognormal distribution, in which the Gaussian and lognormal are special cases. The data consists of African American births (1985-1988) and European American births (1988) in the state of New York. The results suggest that of the conventional distributions, a mixture of two Gaussians generally provides the best fit to birthweight and gestational age. However, in the case of birthweight a two-component hybrid-lognormal fits better than any of the simpler models. This may be due to a feature of the hybrid-lognormal distribution that can be interpreted as maternal constraints on fetal development.

Birth Weight↗

Bayesian comparison of cost-effectiveness of different clinical approaches to diagnose coronary artery disease.

The objective of this study was to compare the cost-effectiveness of four clinical policies (policies I to IV) in the diagnosis of the presence or absence of coronary artery disease. A model based on Bayes' theorem and published clinical data was constructed to make these comparisons. Effectiveness was defined as either the number of patients with coronary disease diagnosed or as the number of quality-adjusted life years extended by therapy after the diagnosis of coronary disease. The following conclusions arise strictly from analysis of the model and may not necessarily be applicable to all situations. As prevalence of coronary disease in the population increased, it caused a linear increase in cost per patient tested, but a hyperbolic decrease in cost per effect, that is, increased cost-effectiveness. Thus, cost-effectiveness of all policies (I to IV) was poor in populations with a prevalence of disease below 10%, for example, asymptomatic people with no risk factors. Analysis of the model also indicates that at prevalences less than 80%, exercise thallium scintigraphy alone as a first test (policy II) is a more cost-effective initial test than is exercise electrocardiography alone as a first test (policy I) or exercise electrocardiography first combined with thallium imaging as a second test (policy IV). Exercise electrocardiography before thallium imaging (policy IV) is more cost-effective than exercise electrocardiography alone (policy I) at prevalences less than 80%. 4) Noninvasive exercise testing before angiography (policies I, II and IV) is more cost-effective than using coronary angiography as the first and only test (policy III) at prevalences less than 80%. 5) Above a threshold value of prevalence of 80% (for example patients with typical angina), proceeding to angiography as the first test (policy III) was more cost-effective than initial noninvasive exercise tests (policies I, II and IV). One advantage of this quantitative model is that it estimates a threshold value of prevalence (80%) at which the rank order of policies changes. The model also allows substitution of different values for any variable as a way of accounting for the uncertainty inherent in the data. In conclusion, it is essential to consider the prevalence of disease when selecting the most cost-effective clinical approach to making a diagnosis.

Angiography↗

In retinal cones, membrane depolarization in darkness activates the cGMP-dependent conductance. A model of Ca homeostasis and the regulation of guanylate cyclase.

We measured outer segment currents under voltage clamp in solitary, single cone photoreceptors isolated from the retina of striped bass. In darkness, changes in membrane voltage to values more positive than 10 mV activate a time- and voltage-dependent outward current in the outer segment. This dark, voltage-activated current (DVAC) increases in amplitude with a sigmoidal time course up to a steady-state value, reached in 0.75-1.5 s. DVAC is entirely suppressed by light, and its current-voltage characteristics and reversal potential are the same as those of the light-sensitive currents. DVAC, therefore, arises from the activation by voltage in the dark of the light-sensitive, cGMP-gated channels of the cone outer segment. Since these channels are not directly gated by voltage, we explain DVAC as arising from a voltage-dependent decrease in cytoplasmic Ca concentration that, in turn, activates only guanylate cyclase and results in net synthesis of cGMP. This explanation is supported by the finding that the Ca buffer BAPTA, loaded into the cytoplasm of the cone outer segment, blocks DVAC. To link a decrease in cytoplasmic Ca concentration to the synthesis of cGMP and the characteristics of DVAC, we develop a quantitative model that assumes cytoplasmic Ca concentration can be continuously calculated from the balance between passive Ca influx via the cGMP-gated channel and its active efflux via a Na/Ca,K exchanger, and that further assumes that guanylate cyclase is activated by decreasing cytoplasmic Ca concentration with characteristics identical to those described for the enzyme in rods. The model successfully simulates experimental data by adjusting the Ca conductance of the cGMP-gated channels as a function of voltage and the Ca buffering power of the cytoplasm. This success suggests that the activity of guanylate cyclase in cone outer segments is indistinguishable from that in rods.

Animals↗

An inwardly rectifying potassium channel in apical membrane of Calu-3 cells.

Patch clamp methods and reverse transcription-polymerase chain reaction (RT-PCR) were used to characterize an apical K+ channel in Calu-3 cells, a widely used model of human airway gland serous cells. In cell-attached and excised apical membrane patches, we found an inwardly rectifying K+ channel (Kir). The permeability ratio was PNa/PK = 0.058. In 30 patches with both cystic fibrosis transmembrane conductance regulator and Kir present, we observed 79 cystic fibrosis transmembrane conductance regulator and 58 Kir channels. The average chord conductance was 24.4 +/- 0.5 pS (n = 11), between 0 and -200 mV, and was 9.6 +/- 0.7 pS (n = 8), between 0 and 50 mV; these magnitudes and their ratio of approximately 2.5 are most similar to values for rectifying K+ channels of the Kir4.x subfamilies. We attempted to amplify transcripts for Kir4.1, Kir4.2, and Kir5.1; of these only Kir4.2 was present in Calu-3 lysates. The channel was only weakly activated by ATP and was relatively insensitive to internal pH. External Cs+ and Ba2+ blocked the channel with Kd values in the millimolar range. Quantitative modeling of Cl- secreting epithelia suggests that secretion rates will be highest and luminal K+ will rise to 16-28 mm if 11-25% of the total cellular K+ conductance is placed in the apical membrane (Cook, D. I., and Young, J. A. (1989) J. Membr. Biol. 110, 139-146). Thus, we hypothesize that the K+ channel described here optimizes the rate of secretion and is involved in K+ recycling for the recently proposed apical H+ -K+ -ATPase in Calu-3 cells.

Adenosine Triphosphate↗

Quantitative analysis of NF-kappaB-expression in cartilage and synovium of rat knee induced by intra-articular injection of synthetic lipid A.

OBJECTIVE: NF-kappaB expression in cartilage and synovium of rat knee after intra-articular injection of lipid A was investigated. METHODS: Inflammation was evaluated in histological sections stained with hematoxylin and eosin, toluidine blue and immunostained with monoclonal anti-NF-kappaB antibody or monoclonal anti-TNF-alpha antibody. RESULTS: Inflammatory cell infiltration into the synovium, hyperplasia of synovial lining layers, loss of metachromasia of cartilage, TNF-alpha-positive cells and NF-kappaB-positive cells in the synovium and cartilage were observed in all 26 rat knees intra-articularly injected with lipid A. The numbers of TNF-alpha-positive and NF-kappaB-positive synovial cells and chondrocytes after the injection were significantly greater than those in knees injected with triethylamine (p < 0.05). The amount of inflammatory cell infiltration into the synovium, synovial lining layers, TNF-alpha-positive synovial cells and NF-kappaB-positive synovial cells 12 h after the injection was significantly smaller than those after 1 day (p < 0.05). CONCLUSION: Intra-articular injection of lipid A induced NF-kappaB expression in cartilage and synovium. Cartilage degeneration started earlier than synovitis in this model. Quantitative analysis of NF-kappaB in cartilage might be useful to evaluate the extreme acute phase of cartilage degeneration.

Animals↗

Greenbottle (Lucilia sericata) larval secretions delivered from a prototype hydrogel wound dressing accelerate the closure of model wounds.

The resurgence of larval biotherapy as a debridement tool in wound management has been accompanied by several clinical reports highlighting concomitant tissue regeneration. Studies employing in vitro cell motility assays have found that purified excretory/secretory (ES) products from Greenbottle larvae (blowfly, Lucilia sericata) are motogenic for human dermal fibroblasts when used as a supplement in culture media. The objective of the present study was to determine whether ES delivered using a prototype hydrogel wound dressing induced similar motogenic effects on fibroblastic (3T3) and epithelial cells (HaCaTs) comprising a scratched-monolayer wound model. Quantitative analysis by MTT assay failed to detect significant mitogenic effects of ES on either cell type. Quantitative image analysis revealed that ES exposure markedly accelerated wound closure through a motogenic effect on both fibroblasts and keratinocytes. Quantitative histochemical analysis detected significantly higher phosphotyrosine (pTyr) expression in ES-exposed cell cultures than in controls; moreover immunocytochemistry revealed conspicuously raised levels of pTyr expression in cells located at the wound margin. By attenuation with a panel of enzyme inhibitors these effects were attributed to the protease components of ES. The present results suggest that controlled delivery of ES as a follow-up to maggot debridement therapy may be an effective therapeutic option for stimulation of tissue regeneration in wound management.

3T3 Cells↗

Suppression of stimulus frequency otoacoustic emissions.

Observations of the suppression of stimulus frequency evoked otoacoustic emissions (SFOAEs) by a second tone were made in human subjects. Measurements were made with the suppressor tone at frequencies and levels at, above, and below the stimulus tone generating the SFOAE. Data were collected with the stimulus tone at a range of levels (20-60 dB SPL), over a range of frequencies and in six different ears, in three subjects. The results were fitted to a phenomenological model, which allowed the data to be expressed as two parameters against the frequency of the suppressor tone. Characteristics of the suppression were examined, in particular the asymmetry between suppression by tones of higher and lower frequency than the tone evoking the SFOAE. At relatively low levels of suppressor tone, suppressors with frequencies higher than the stimulus tone were more effective suppressors than lower frequencies. At higher levels of suppressor tone, the situation was reversed, with lower frequencies being more effective than higher frequencies. These results were discussed in terms of nonlinear interaction between waves in the cochlea. This interpretation was used to estimate the shape of the traveling wave envelope produced by the stimulus tone, from the results of the suppression experiments. It was shown that the estimates of the shapes of the traveling wave envelope were nonlinear, the peak of the envelope becoming sharper at lower levels of stimulus. A simple quantitative model of SFOAE suppression was formulated using concepts of energy flow within the cochlea. This model produced SFOAE suppression results with all the major characteristics of SFOAE suppression from a real, human ear.

Acoustic Stimulation↗

A mathematical model of glioma growth: the effect of extent of surgical resection.

We have developed a mathematical model based on proliferation and infiltration of neoplastic cells that allows predictions to be made concerning the life expectancies following various extents of surgical resection of gliomas of all grades of malignancy. The key model parameters are the growth rate and the diffusion rate. These rates were initially derived from analysis of a case of recurrent anaplastic astrocytoma treated by chemotherapies. Numerical simulations allow us to estimate what would have happened to that patient if various extents of surgical resection, rather than chemotherapies, had been used. In each case, the shell of the infiltrating tumour that remains after 'gross total removal' or even a maximal excision continues to grow and regenerates the tumour mass remarkably rapidly. By developing a model that allows the growth and diffusion rates to define the distribution of cells at the time of diagnosis, and then varying these rates by about 50%, we created a hypothetical tumour patient population whose survival times show good agreement with the results recently reported by Kreth for treatments of glioblastomas. Tenfold decreases in the rates of growth and diffusion mimic the results reported by many other investigators with more slowly growing gliomas. Thus, the model quantitatively supports the ideas that (i) gliomas infiltrate so diffusely that they cannot be cured by resection alone, surgical or radiological, no matter how extensive that may be; (ii) the more extensive the resection, regardless of the degree of malignancy of the glioma, the greater the life expectancy; and (iii) measurements of the two rates, growth and diffusion, may be able to predict survival rates better than the current histological estimates of the type and grade of gliomas.

Cell Division↗

A ligand-induced conformational change in apolipoprotein(a) enhances covalent Lp(a) formation.

Lipoprotein(a) (Lp(a)) assembly proceeds via a two-step mechanism in which initial non-covalent interactions between apolipoprotein(a) (apo(a)) and low density lipoprotein precede disulfide bond formation. In this study, we used analytical ultracentrifugation, differential scanning calorimetry, and intrinsic fluorescence to demonstrate that in the presence of the lysine analog epsilon-aminocaproic acid, apo(a) undergoes a substantial conformational change from a "closed" to an "open" structure that is characterized by an increase in the hydrodynamic radius (approximately 10%), an alteration in domain stability, as well as a decrease in tryptophan fluorescence. Although epsilon-aminocaproic acid is a well characterized inhibitor of the non-covalent interaction between apo(a) and low density lipoprotein, we report the novel observation that this ligand at low concentrations (100 microm-1 mm) significantly enhances covalent Lp(a) assembly by altering the conformation of apo(a). We developed a model for the kinetics of Lp(a) assembly that incorporates the conformational change as a determinant of the efficiency of the process; this model quantitatively explains our experimental observations. Interestingly, an analogous conformational change has been previously described for plasminogen resulting in an increase in the hydrodynamic radius, an increase in tryptophan fluorescence, and an acceleration of the rate of plasminogen activation. Although the functions of apo(a) and plasminogen have diverged considerably, elements of structural and conformational homology have been retained leading to similar regulation of two unrelated biological processes.

Aminocaproic Acid↗

Time-course of experimental choroidal neovascularization in Dutch-Belted rabbit: clinical and histological evaluation.

In order to study the pathogenesis of choroidal neovascularization (CNV) and explore a suitable animal model for assessing anti-angiogenic agents, rabbit CNV was induced by subretinal injection of a cocktail containing endotoxin and growth-factor, incorporated in Heparin-sepharose beads. The presence and development of CNV lesions was visualized by fluorescein angiography and quantified by image analysis. The time-course of experimental CNV was evaluated clinically and histologically, with some lesions followed up to 3-years. The suitability of this model for drug evaluation was appraised by the systemic administration of dexamethasone. The experimental results suggest two subsets of CNV, primary and secondary, observed in all treated eyes. Primary CNV, defined as neovascularization extending into the subretinal space and associated with injury to Bruch's membrane at the time of injection, was visible in 100% of eyes by 2-weeks and stable by 3-months. Secondary CNV, defined as neovascularization extending into the sub RPE space away from the initial injection related injury, became visible as early as 2-weeks in some of eyes and developed in 100% of eyes by 8-months. Both primary and secondary CNV were maintained and demonstrated leakage throughout the entire observation period. Atrophy of primary retinal pigmented epithelium (RPE) cells and hyper-proliferation of secondary RPE cells were observed in tissue sections with CNV lesions. The formation and growth of primary and secondary CNV were significantly inhibited by dexamethasone. This study indicates that a reproducible and quantitative model of rabbit CNV has been established utilizing subretinal administration of endotoxin and growth-factor. Studies of the stages of experimental CNV both clinically and histologically indicated an intimate relationship between CNV, macrophages and RPE. Furthermore, the inhibition observed with dexamethasone points to the possibility of being able to evaluate effective means of pharmacological intervention.

Animals↗

Enzymology below 200 K: the kinetics and thermodynamics of the photochemistry catalyzed by protochlorophyllide oxidoreductase.

The chlorophyll biosynthesis enzyme protochlorophyllide reductase (POR) catalyzes the light-dependent reduction of protochlorophyllide (Pchlide) into chlorophyllide in the presence of NADPH. As POR is light-dependent, catalysis can be initiated by illumination of the enzyme-substrate complex at low temperatures, making it an attractive model for studying aspects of biological proton and hydride transfers. The early stages in the photoreduction, involving Pchlide binding and an initial photochemical reaction, have been studied in vitro by using low-temperature fluorescence and absorbance measurements. Formation of the ternary POR-NADPH-Pchlide complex produces red shifts in the fluorescence and absorbance maxima of Pchlide, allowing the dissociation constant for Pchlide binding to be measured. We demonstrate that the product of an initial photochemical reaction, which can occur below 200 K, is a nonfluorescent intermediate with a broad absorbance band at 696 nm (A696) that is suggested to represent an ion radical complex. The temperature dependence of the rate of A696 formation has allowed the activation energy for the photochemical step to be calculated and has shown that POR catalysis can proceed at much lower temperatures than previously thought. Calculations of differences in free energy between various reaction intermediates have been calculated; these, together with the quantum efficiency for Pchlide conversion, suggest a quantitative model for the thermodynamics of the light-driven step of Pchlide reduction.

Binding Sites↗