The association of rat brain hexokinase with heterologous recombinant yeast mitochondria harborin... more The association of rat brain hexokinase with heterologous recombinant yeast mitochondria harboring human porin (Yh) is comparable to that with rat liver mitochondria in terms of cation requirements, cooperativity in binding, and the effect of amphipathic compounds. Mg2+, which is required for hexokinase binding to all mitochondria, can be replaced by other cations. The efficiency of hexokinases, however, depends on the valence of hydrophilic cations, or the partition of hydrophobic cations in the membrane, implying that these act by reducing a prohibitive negative surface charge density on the outer membrane rather than fulfilling a specific structural requirement. Macromolecular crowding (using dextran) has dual effects. Dextran added in excess increases hexokinase binding to yeast mitochondria, according to the porin molecule they harbor. This effect, significant with wild-type yeast mitochondria, is only marginal with Yh as well as rat mitochondria. On the other hand, an increase in the number of hexokinase binding sites on mitochondria is also observed. This increase, moderate in wild-type organelles, is more pronounced with Yh. Finally, dextran, which has no effect on the modulation of hexokinase binding by cations, abolishes the inhibitory effect of amphipathic compounds. Thus, while hexokinase binding to mitochondria is predetermined by the porin molecule, the organization of the latter in the membrane plays a critical role as well, indicative that porin must associate with other mitochondrial components to form competent binding sites on the outer membrane.
Studies on steady state kinetics of enzymes bound to energy-transducing membranes have generally ... more Studies on steady state kinetics of enzymes bound to energy-transducing membranes have generally tacitly assumed that their kinetic behavior does not depart greatly from that of enzymes in free solution. However, the microenvironment of these membranes, upon which electrochemical changes occur during catalysis, should be different than an ideal aqueous solution which allows rapid diffusion of solutes. Since different kinetic principles apply to enzymes immobilized on solid supports, due to impaired translocation of reactants near the support (Engasser, Horvath, 1976), the macroscopic behavior of such enzymes does not necessarily reflect intrinsic properties relevant to the molecular mechanism of catalysis (conformational changes, site-site cooperativity, etc.).
Publisher Summary This chapter presents experimental procedures, showing that the steady-state ra... more Publisher Summary This chapter presents experimental procedures, showing that the steady-state rate of phosphorylation may be limited by the mass transfer of nucleotides between the adenosine triphosphate (ATP) synthetase loci and the bulk medium. The data presented in the chapter indicates the limitation of catalysis by mass transfer of nucleotides through the environment of the membrane in normally prepared thylakoids. Most of the ATP, rapidly labeled in the light and retained on de-energized membranes, is not bound to the catalytic sites of ATP synthetase and probably originates from a free species of newly formed ATP, which diffuses slowly to the bulk medium. Hypotonic treatment and washing of normally prepared thylakoids, which could remove kinetic barriers to diffusion by opening grana stacks, increases the apparent affinity of the membrane-bound ATP synthetase for substrate. This effect seems to depend on the catalytic ability of the thylakoids and their efficiency of substrate utilization. The chapter proposes a model that introduces a new perspective to explain modulation of ATP synthetase activities and medium exchange reactions by energy input, substrate concentration, and, in general, any factor that could affect the microenvironment of the enzyme, provided that proper conditions for diffusion control are present.
The association of rat brain hexokinase with heterologous recombinant yeast mitochondria harborin... more The association of rat brain hexokinase with heterologous recombinant yeast mitochondria harboring human porin (Yh) is comparable to that with rat liver mitochondria in terms of cation requirements, cooperativity in binding, and the effect of amphipathic compounds. Mg2+, which is required for hexokinase binding to all mitochondria, can be replaced by other cations. The efficiency of hexokinases, however, depends on the valence of hydrophilic cations, or the partition of hydrophobic cations in the membrane, implying that these act by reducing a prohibitive negative surface charge density on the outer membrane rather than fulfilling a specific structural requirement. Macromolecular crowding (using dextran) has dual effects. Dextran added in excess increases hexokinase binding to yeast mitochondria, according to the porin molecule they harbor. This effect, significant with wild‐type yeast mitochondria, is only marginal with Yh as well as rat mitochondria. On the other hand, an increase in the number of hexokinase binding sites on mitochondria is also observed. This increase, moderate in wild‐type organelles, is more pronounced with Yh. Finally, dextran, which has no effect on the modulation of hexokinase binding by cations, abolishes the inhibitory effect of amphipathic compounds. Thus, while hexokinase binding to mitochondria is predetermined by the porin molecule, the organization of the latter in the membrane plays a critical role as well, indicative that porin must associate with other mitochondrial components to form competent binding sites on the outer membrane.
Classical biochemistry has provided a great deal of information about isolated catalytic units (f... more Classical biochemistry has provided a great deal of information about isolated catalytic units (from enzymes to organelles), by studying their properties in dilute solution or suspension. The results are often directly projected on their functionality in vivo, implicitly assuming a homogeneous behavior in situ. However, a critical look at the cellular environments1,2 strongly refutes the latter assumption and indicates that special kinetic treatments would rather be required to describe these situations, taking into account physical steps (diffusion, partition, etc.) inherent to heterogeneous systems, in addition to the intrinsic properties of the catalysts as observed in homogeneous phase3. Such treatment has been successfully applied for immobilized enzyme systems4, stressing that the activity of the enzymes is exclusively determined by the local concentration of reactants in their own microenvironment. However, in these systems only the bulk concentrations of reactants have been assessed. It is thus important to have means to monitor also local events which reflect more directly the activity of enzymes in situ.
A kinetic scheme for photophosphorylation catalyzed by the chloroplast ATP synthetase is describe... more A kinetic scheme for photophosphorylation catalyzed by the chloroplast ATP synthetase is described by Equation 1. This sequence of reactions assumes that binding of ADP occurs prior to that of Pi (Selman, Selman-Reimer, 1981). Thus, the Pi-ATP exchange, one of the so-called partial reactions of photophosphorylation, might occur without significant dissociation of the E•ADP complex and without a concomitant ADP-ATP exchange reaction (k-1 ≪ k1) In this mechanism, formation and hydrolysis of ATP and the accompanying exchange reactions are reversible reactions that occur on the same catalytic site(s) on the energized membrane-bound enzyme (CF1). It has been about ten years since it was reported that the synthesis of ATP and its hydrolysis or the Pi-ATP exchange reaction show a dissimilar substrate specificity. Modified antibodies against CF1 were also shown to inhibit preferentially the Pi-ATP exchange. These and more recent data support the view that different enzyme conformations or altered catalytic sites may participate in the catalysis of ADP (photophosphorylation) and ATP (hydrolysis and exchange) utilizing reactions (Shavit, 1980).
Insulin-producing pancreatic β cells are functionally impaired or destroyed in diabetes mellitus.... more Insulin-producing pancreatic β cells are functionally impaired or destroyed in diabetes mellitus. The onset of type 1 diabetes (T1D) represents the culmination of a prolonged prediabetic phase of immune-mediated β-cell destruction. To assess the in vivo metabolic status of these cells, we used the ATP-sensitive firefly luciferase bioluminescence imaging approach, as a noninvasive probe to monitor pathological alterations in β-cell function in the nonobese-diabetic (NOD) mouse model of T1D. Hence, we generated the ToIβ-NOD transgenic mice in which doxycycline-inducible luciferase gene is selectively expressed in β cells. A sharp reduction in bioluminescence emitted in vivo from β cells at the early stages, preceded by several weeks of a limited reduction in β-cell mass. Since this decline could be due to the ongoing inflammatory process occurring in vivo, we exposed control islets to inflammatory cytokines and observed a dramatic decrease in luciferase luminescence, which appears to be due in part to a decrease in protein levels and a drop in intracellular ATP levels. This is the first evidence that selective expression of the luciferase gene represents a sensitive method for noninvasive in vivo monitoring of early β-cell dysfunction, subtle metabolic changes, such as endogenous ATP levels, indicative of a pathological condition in a tissue at the cellular level.
The study of enzymes sequestered in artificial or biological systems is generally conducted by in... more The study of enzymes sequestered in artificial or biological systems is generally conducted by indirect methodology with macroscopic measurements of reactants in the bulk medium. This paper describes a new approach with firefly luciferase to monitor ATP concentration directly in the microenvironment of enzymes producing or consuming ATP. Upon addition of ATP to immobilized firefly luciferase, the onset of light production is slower than that observed with the soluble enzyme, due to a slower diffusion of ATP to the immobilized enzyme. With immobilized pyruvate kinase, a relative accumulation of ATP inside the beads is demonstrated, as measured with coimmobilized firefly luciferase. The accumulation of product (ATP) is enhanced when the bead suspension is not stirred. This ATP in the beads is relatively inaccessible to soluble hexokinase added to the bulk medium. Similarly, a rapid ATP depletion in the microenvironment of immobilized hexokinase is demonstrated. This microscopic event is kinetically distinguishable from the slower macroscopic depletion of substrate in the bulk medium. The rate of depletion in the microenvironment depends on the local activity of the immobilized enzyme but not on the total amount of enzyme in suspension, as does the macroscopic phenomenon. The theoretical principles for the interaction of diffusion and catalysis in these systems are briefly summarized and discussed. These results are relevant to various molecular mechanisms proposed for membrane-bound enzyme action and regulation, derived from macroscopic kinetic measurements assuming a negligible diffusion control.
a Microalgal Biotechnology Laboratory b Department of Solar Energy and Environmental Physics The ... more a Microalgal Biotechnology Laboratory b Department of Solar Energy and Environmental Physics The Blaustein Institutes for Desert Research, Ben Gurion University of the Negev, Sede Boqer, Israel SUMMARY: The dependence of the rate of photosynthesis on light intensity is routinely described by parameters derived from experimental P-I curves (model1, Fig.1) of the rate under continuous irradiation. A cyclic model serves as deterministic formulation of photosynthesis, in which a resting reaction center (RC) yields an activated, then an inhibited state by sequential photons absorption (kl, ki). Both states return (in the dark) to the resting state, the former after energy dissipation or transduction (productive, kp) and the latter after a repair process (kc). We applied the model above as well as a more elaborate one involving the sequential absorption of four photons to reach activation (model2, Fig.1) on two regimes of illumination: (i) continuous as a static reference state at steady ...
The association of rat brain hexokinase with heterologous recombinant yeast mitochondria harborin... more The association of rat brain hexokinase with heterologous recombinant yeast mitochondria harboring human porin (Yh) is comparable to that with rat liver mitochondria in terms of cation requirements, cooperativity in binding, and the effect of amphipathic compounds. Mg2+, which is required for hexokinase binding to all mitochondria, can be replaced by other cations. The efficiency of hexokinases, however, depends on the valence of hydrophilic cations, or the partition of hydrophobic cations in the membrane, implying that these act by reducing a prohibitive negative surface charge density on the outer membrane rather than fulfilling a specific structural requirement. Macromolecular crowding (using dextran) has dual effects. Dextran added in excess increases hexokinase binding to yeast mitochondria, according to the porin molecule they harbor. This effect, significant with wild-type yeast mitochondria, is only marginal with Yh as well as rat mitochondria. On the other hand, an increase in the number of hexokinase binding sites on mitochondria is also observed. This increase, moderate in wild-type organelles, is more pronounced with Yh. Finally, dextran, which has no effect on the modulation of hexokinase binding by cations, abolishes the inhibitory effect of amphipathic compounds. Thus, while hexokinase binding to mitochondria is predetermined by the porin molecule, the organization of the latter in the membrane plays a critical role as well, indicative that porin must associate with other mitochondrial components to form competent binding sites on the outer membrane.
Studies on steady state kinetics of enzymes bound to energy-transducing membranes have generally ... more Studies on steady state kinetics of enzymes bound to energy-transducing membranes have generally tacitly assumed that their kinetic behavior does not depart greatly from that of enzymes in free solution. However, the microenvironment of these membranes, upon which electrochemical changes occur during catalysis, should be different than an ideal aqueous solution which allows rapid diffusion of solutes. Since different kinetic principles apply to enzymes immobilized on solid supports, due to impaired translocation of reactants near the support (Engasser, Horvath, 1976), the macroscopic behavior of such enzymes does not necessarily reflect intrinsic properties relevant to the molecular mechanism of catalysis (conformational changes, site-site cooperativity, etc.).
Publisher Summary This chapter presents experimental procedures, showing that the steady-state ra... more Publisher Summary This chapter presents experimental procedures, showing that the steady-state rate of phosphorylation may be limited by the mass transfer of nucleotides between the adenosine triphosphate (ATP) synthetase loci and the bulk medium. The data presented in the chapter indicates the limitation of catalysis by mass transfer of nucleotides through the environment of the membrane in normally prepared thylakoids. Most of the ATP, rapidly labeled in the light and retained on de-energized membranes, is not bound to the catalytic sites of ATP synthetase and probably originates from a free species of newly formed ATP, which diffuses slowly to the bulk medium. Hypotonic treatment and washing of normally prepared thylakoids, which could remove kinetic barriers to diffusion by opening grana stacks, increases the apparent affinity of the membrane-bound ATP synthetase for substrate. This effect seems to depend on the catalytic ability of the thylakoids and their efficiency of substrate utilization. The chapter proposes a model that introduces a new perspective to explain modulation of ATP synthetase activities and medium exchange reactions by energy input, substrate concentration, and, in general, any factor that could affect the microenvironment of the enzyme, provided that proper conditions for diffusion control are present.
The association of rat brain hexokinase with heterologous recombinant yeast mitochondria harborin... more The association of rat brain hexokinase with heterologous recombinant yeast mitochondria harboring human porin (Yh) is comparable to that with rat liver mitochondria in terms of cation requirements, cooperativity in binding, and the effect of amphipathic compounds. Mg2+, which is required for hexokinase binding to all mitochondria, can be replaced by other cations. The efficiency of hexokinases, however, depends on the valence of hydrophilic cations, or the partition of hydrophobic cations in the membrane, implying that these act by reducing a prohibitive negative surface charge density on the outer membrane rather than fulfilling a specific structural requirement. Macromolecular crowding (using dextran) has dual effects. Dextran added in excess increases hexokinase binding to yeast mitochondria, according to the porin molecule they harbor. This effect, significant with wild‐type yeast mitochondria, is only marginal with Yh as well as rat mitochondria. On the other hand, an increase in the number of hexokinase binding sites on mitochondria is also observed. This increase, moderate in wild‐type organelles, is more pronounced with Yh. Finally, dextran, which has no effect on the modulation of hexokinase binding by cations, abolishes the inhibitory effect of amphipathic compounds. Thus, while hexokinase binding to mitochondria is predetermined by the porin molecule, the organization of the latter in the membrane plays a critical role as well, indicative that porin must associate with other mitochondrial components to form competent binding sites on the outer membrane.
Classical biochemistry has provided a great deal of information about isolated catalytic units (f... more Classical biochemistry has provided a great deal of information about isolated catalytic units (from enzymes to organelles), by studying their properties in dilute solution or suspension. The results are often directly projected on their functionality in vivo, implicitly assuming a homogeneous behavior in situ. However, a critical look at the cellular environments1,2 strongly refutes the latter assumption and indicates that special kinetic treatments would rather be required to describe these situations, taking into account physical steps (diffusion, partition, etc.) inherent to heterogeneous systems, in addition to the intrinsic properties of the catalysts as observed in homogeneous phase3. Such treatment has been successfully applied for immobilized enzyme systems4, stressing that the activity of the enzymes is exclusively determined by the local concentration of reactants in their own microenvironment. However, in these systems only the bulk concentrations of reactants have been assessed. It is thus important to have means to monitor also local events which reflect more directly the activity of enzymes in situ.
A kinetic scheme for photophosphorylation catalyzed by the chloroplast ATP synthetase is describe... more A kinetic scheme for photophosphorylation catalyzed by the chloroplast ATP synthetase is described by Equation 1. This sequence of reactions assumes that binding of ADP occurs prior to that of Pi (Selman, Selman-Reimer, 1981). Thus, the Pi-ATP exchange, one of the so-called partial reactions of photophosphorylation, might occur without significant dissociation of the E•ADP complex and without a concomitant ADP-ATP exchange reaction (k-1 ≪ k1) In this mechanism, formation and hydrolysis of ATP and the accompanying exchange reactions are reversible reactions that occur on the same catalytic site(s) on the energized membrane-bound enzyme (CF1). It has been about ten years since it was reported that the synthesis of ATP and its hydrolysis or the Pi-ATP exchange reaction show a dissimilar substrate specificity. Modified antibodies against CF1 were also shown to inhibit preferentially the Pi-ATP exchange. These and more recent data support the view that different enzyme conformations or altered catalytic sites may participate in the catalysis of ADP (photophosphorylation) and ATP (hydrolysis and exchange) utilizing reactions (Shavit, 1980).
Insulin-producing pancreatic β cells are functionally impaired or destroyed in diabetes mellitus.... more Insulin-producing pancreatic β cells are functionally impaired or destroyed in diabetes mellitus. The onset of type 1 diabetes (T1D) represents the culmination of a prolonged prediabetic phase of immune-mediated β-cell destruction. To assess the in vivo metabolic status of these cells, we used the ATP-sensitive firefly luciferase bioluminescence imaging approach, as a noninvasive probe to monitor pathological alterations in β-cell function in the nonobese-diabetic (NOD) mouse model of T1D. Hence, we generated the ToIβ-NOD transgenic mice in which doxycycline-inducible luciferase gene is selectively expressed in β cells. A sharp reduction in bioluminescence emitted in vivo from β cells at the early stages, preceded by several weeks of a limited reduction in β-cell mass. Since this decline could be due to the ongoing inflammatory process occurring in vivo, we exposed control islets to inflammatory cytokines and observed a dramatic decrease in luciferase luminescence, which appears to be due in part to a decrease in protein levels and a drop in intracellular ATP levels. This is the first evidence that selective expression of the luciferase gene represents a sensitive method for noninvasive in vivo monitoring of early β-cell dysfunction, subtle metabolic changes, such as endogenous ATP levels, indicative of a pathological condition in a tissue at the cellular level.
The study of enzymes sequestered in artificial or biological systems is generally conducted by in... more The study of enzymes sequestered in artificial or biological systems is generally conducted by indirect methodology with macroscopic measurements of reactants in the bulk medium. This paper describes a new approach with firefly luciferase to monitor ATP concentration directly in the microenvironment of enzymes producing or consuming ATP. Upon addition of ATP to immobilized firefly luciferase, the onset of light production is slower than that observed with the soluble enzyme, due to a slower diffusion of ATP to the immobilized enzyme. With immobilized pyruvate kinase, a relative accumulation of ATP inside the beads is demonstrated, as measured with coimmobilized firefly luciferase. The accumulation of product (ATP) is enhanced when the bead suspension is not stirred. This ATP in the beads is relatively inaccessible to soluble hexokinase added to the bulk medium. Similarly, a rapid ATP depletion in the microenvironment of immobilized hexokinase is demonstrated. This microscopic event is kinetically distinguishable from the slower macroscopic depletion of substrate in the bulk medium. The rate of depletion in the microenvironment depends on the local activity of the immobilized enzyme but not on the total amount of enzyme in suspension, as does the macroscopic phenomenon. The theoretical principles for the interaction of diffusion and catalysis in these systems are briefly summarized and discussed. These results are relevant to various molecular mechanisms proposed for membrane-bound enzyme action and regulation, derived from macroscopic kinetic measurements assuming a negligible diffusion control.
a Microalgal Biotechnology Laboratory b Department of Solar Energy and Environmental Physics The ... more a Microalgal Biotechnology Laboratory b Department of Solar Energy and Environmental Physics The Blaustein Institutes for Desert Research, Ben Gurion University of the Negev, Sede Boqer, Israel SUMMARY: The dependence of the rate of photosynthesis on light intensity is routinely described by parameters derived from experimental P-I curves (model1, Fig.1) of the rate under continuous irradiation. A cyclic model serves as deterministic formulation of photosynthesis, in which a resting reaction center (RC) yields an activated, then an inhibited state by sequential photons absorption (kl, ki). Both states return (in the dark) to the resting state, the former after energy dissipation or transduction (productive, kp) and the latter after a repair process (kc). We applied the model above as well as a more elaborate one involving the sequential absorption of four photons to reach activation (model2, Fig.1) on two regimes of illumination: (i) continuous as a static reference state at steady ...
Qualitative characteristics of biomass production in ultrahigh density algal bioreactors with a s... more Qualitative characteristics of biomass production in ultrahigh density algal bioreactors with a small optical path (specifically, thin flat-plate reactors) are analyzed and explained in terms of models, which combine the random motion of cells across the optical path with simple models for the photosynthetic process. Characteristics of different models at extreme densities are compared with existing data. An analogy between flashing light illumination and the light regime experienced by the randomly moving cells provides basic insight into the important role of timescales in reactor performance. The emergence of an optimal culture density (OCD), at which the volumetric and areal production rates are maximal, is understood in simple terms. While higher density implies an increase in the number of photosynthesizing cells, it leads to narrowing of the illuminated (photic) zone, hence to a decrease in the time spent by these cells in the photic zone. When the time spent by cells in the photic zone is longer than the time needed to collect the photons required for the photosynthetic process, the addition of cells increases the volumetric production rate. When the time spent by cells in the illuminated zone falls below the time needed for the collection of photons, the volumetric production rate is decreased. The combined effects of changes in density are the cause of the emergence of an OCD. At the OCD, the time spent by cells in the thin illuminated layer of the culture and the time needed for the collection of the photons required for the photosynthetic process coincide.
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Papers by Claude Aflalo