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P B Chock

Publications and source records attributed to P B Chock.

At least 73 records · Page 4Linked to original sources
PubMed1986

How enzymes can capture and transmit free energy from an oscillating electric field.

Recently, it has been demonstrated that free energy from an alternating electric field can drive the active transport of Rb+ by way of the Na+, K+-ATPase. In the present work, it is shown why many transmembrane enzymes can be expected to absorb free energy from an oscillating electric field and transduce that to chemical or transport work. In the theoretical analysis it turned out to be sufficient that (i) the catalytic process be accompanied by either net or cyclic charge translocation across the membrane and (ii) the stability of the enzyme states involved be asymmetric. Calculations based on a four-state model reveal that free-energy transduction occurs with sinusoidal, square-wave, and positive-only oscillating electric fields and for cases that exhibit either linear or exponential field-dependent rate constants. The results suggest that in addition to oscillating electric field-driven transport, the proposed mechanism can also be used to explain, in part, the "missing" free energy term in the cases in which ATP synthesis has been observed with insufficient transmembrane proton electrochemical potential difference.

Adenosine Triphosphatases↗
PubMed1985

Immunological characterization of phosphoprotein phosphatases.

Phosphoprotein phosphatases regulate the biological activities of proteins through their involvement in cyclic phosphorylation/dephosphorylation cascades. A variety of multimeric phosphatases have been isolated and grouped into several classes, termed type 1 and types 2A, 2B, and 2C. To elucidate the relationship between the different phosphoprotein phosphatases, highly purified enzymes from soil amoebae, turkey gizzards, bovine heart and brain, and rabbit skeletal muscle and reticulocytes were tested for immunological antigenic relatedness. Two heterologous antibody preparations were employed for this purpose. One was made against an Acanthamoeba type 2A phosphatase and the other was made to bovine brain phosphatase type 2B (calcineurin, holoenzyme). Specific subunit cross-reactivity was examined by protein blot ("Western") analysis. The antibody to the type 2A phosphatase reacted with the catalytic subunits of every type 2 enzyme tested, including both the catalytic and Ca2+-binding subunits of the Ca2+/calmodulin-dependent type 2B phosphatase (calcineurin), bovine cardiac type 2A phosphatase, and turkey gizzard smooth muscle phosphatase-1 (type 2A1). It did not react with any type 1 phosphatase (catalytic subunit or ATP-Mg-dependent). The antigenic relatedness of calcineurin and the bovine cardiac type 2A phosphatase (Mr 38,000) was demonstrated further by protein blot analysis showing that the anti-calcineurin antibody cross-reacted with both enzymes. The mutual cross-reactivity poses an intriguing problem because these enzymes are so different in their molecular structures and modes of regulation. The degree of evolutionary conservation exhibited by the antigenic cross-reactivity of the type 2 enzymes from a broad range of species and tissues suggests a strong selective pressure on maintaining one or more features of these important regulatory enzymes.

Animals↗
PubMed1985

Kinase FA-mediated regulation of rabbit skeletal muscle protein phosphatase. Reversible phosphorylation of the modulator subunit.

A mechanism of activation of the ATP.Mg-dependent protein phosphatase (FC.M) has been proposed (Jurgensen, S., Shacter, E., Huang, C. Y., Chock, P. B., Yang, S.-D., Vandenheede, J. R., and Merlevede, W. (1984) J. Biol. Chem. 259, 5864-5870) in which a transient phosphorylation by the kinase FA of the modulator subunit (M) is the driving force for the transition of the inactive catalytic subunit (FC) into its active conformation. Incubation of FC.M with kinase FA and Mg2+ and adenosine 5'-(gamma-thio)triphosphate results in thiophosphorylation of M and also a conformational change in the phosphatase catalytic subunit; however, the enzyme remains inactive. Proteolysis of this inactive, thiophosphorylated complex causes proteolytic destruction of the modulator subunit and yields an active phosphorylase phosphatase species. Similar treatment of the native inactive enzyme does not yield active phosphatase. Evidence is presented, suggesting that a molecule of modulator is bound at an "inhibitory site" on the native enzyme. This modulator does not prevent the conformational change in the phosphatase catalytic subunit upon incubation with kinase FA and ATP.Mg but does partially inhibit the expression of the phosphorylase phosphatase activity.

Adenosine Triphosphate↗
PubMed1985

Rate-limiting step in the actomyosin adenosinetriphosphatase cycle: studies with myosin subfragment 1 cross-linked to actin.

Although there is agreement that actomyosin can hydrolyze ATP without dissociation of the actin from myosin, there is still controversy about the nature of the rate-limiting step in the ATPase cycle. Two models, which differ in their rate-limiting step, can account for the kinetic data. In the four-state model, which has four states containing bound ATP or ADP . Pi, the rate-limiting step is ATP hydrolysis (A . M . ATP in equilibrium A . M . ADP . Pi). In the six-state model, which we previously proposed, the rate-limiting step is a conformational change which occurs before Pi release but after ATP hydrolysis. A difference between these models is that only the four-state model predicts that almost no acto-subfragment 1 (S-1) . ADP . Pi complex will be formed when ATP is mixed with acto . S-1. In the present study, we determined the amount of acto . S-1 . ADP . Pi formed when ATP is mixed with S-1 cross-linked to actin [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Nature (London) 292, 301-306]. The amount of acto . S-1 . ADP . Pi was determined both from intrinsic fluorescence enhancement and from direct measurement of Pi. We found that at mu = 0.013 M, the fluorescence magnitude in the presence of ATP of the cross-linked actin . S-1 preparation was about 50% of the value obtained with S-1, while at mu = 0.053 M the fluorescence magnitude was about 70% of that obtained with S-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1985

Interaction of Cibacron Blue F3GA with glutamine synthetase: use of the dye as a conformational probe. 1. Studies using unfractionated dye samples.

Cibacron Blue F3GA dye has been used to probe subtle conformational changes in protein structure associated with the conversion of Escherichia coli glutamine synthetase (GS) between relaxed, taut, oxidized, and dissociated forms. Binding of the dye to each form of the enzyme elicits a different spectral perturbation of the dye which can be detected by difference spectroscopy. By following time-dependent changes in the difference spectrum associated with the binding of dye to the enzyme, it was demonstrated that dissociation of subunits provoked either by urea or by relaxation of the enzyme at pH 8.5 is a multiphasic process. In the presence of 3-4 M urea, dissociation of taut GS is associated with an almost instantaneous, transient increase in absorbancy of the difference spectrum at 638 nm and, after a lag, by a progressive decrease in absorbancy at 585 nm and an increase at 700 nm. The kinetics of these changes vary as a function of temperature, pH, and the concentrations of KCl, MnCl2, and urea, probably reflecting differences in the rates of GS relaxation and in the formation of aggregates of intermediate sizes. Results of direct binding measurements show that the taut and relaxed forms of GS can bind only 1-1.3 equiv of dye per subunit, whereas dissociated subunits bind up to 3.0 equiv per subunit. The Kd of the dye-taut GS complex as calculated from binding data was 0.55 microM. The binding of dye to taut GS was inhibited by its substrate, ADP, and by the allosteric effectors AMP and tryptophan. On the basis of the abilities of ADP, AMP, and tryptophan to inhibit the binding of dye to GS, dissociation constants of the respective GS-ligand complexes were 2.4, 121, and 1170 microM, respectively, in good agreement with previously determined values. From the difference spectra obtained between a given concentration of dye in a 5.0-cm cell and 10 times that concentration in a 0.5-cm cell, it was established that at concentrations greater than 5 microM a significant fraction of the dye is present as stacked aggregates. Because only the dye monomer binds to GS, the difference spectrum between dye and dye bound to GS is due in part to GS-promoted shifts in the equilibrium between stacked and unstacked dye molecules. Consequently, with increasing dye concentrations, the amplitude of the dye vs. dye + GS difference spectrum can continue to increase, even after the GS becomes saturated with dye.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine Nucleotides↗
PubMed1985

Inhibition of the Mg(II).ATP-dependent phosphoprotein phosphatase by the regulatory subunit of cAMP-dependent protein kinase.

We report potent inhibition of the Mg(II).ATP-dependent protein phosphatase, Fc.M, by the regulatory subunit dimer of type II cAMP-dependent protein kinase, RII2. The protein kinase catalytic subunit has no effect on phosphatase activity and is unable to substitute for kinase FA in the kinase FA- and Mg(II).ATP-mediated phosphatase activation reaction. Phosphatase inhibition was investigated as a function of RII2 concentration. The results suggest that RII2 both inhibits the active phosphatase and inhibits phosphatase activation. The inhibition is shown to be noncompetitive with respect to substrate (phosphorylase a). The potential physiological significance of this inhibition is discussed in terms of phosphorylation/dephosphorylation cascade systems involving this kinase and phosphatase.

Adenosine Triphosphate↗
PubMed1984

Regulation through phosphorylation/dephosphorylation cascade systems.

The cyclic interconversion of enzymes between phosphorylated and unphosphorylated forms comprises a major mechanism of cellular regulation. A theoretical analysis of reversible covalent modification systems (Stadtman, E.R., and Chock, P.B. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2761-2765) revealed that they are endowed with extraordinary regulatory capacities; they may exhibit smooth, flexible responses to changes in single and multiple metabolite levels, signal amplification, and apparent positive cooperativity. To test qualitatively and quantitatively the theories and equations involved in this analysis, a model in vitro phosphorylation/dephosphorylation cyclic cascade was developed in which the converter enzymes catalyzing the covalent modifications were cAMP-dependent protein kinase (EC 2.7.1.37; type II) and phosphoprotein phosphatase (EC 3.1.3.16; Mr = 38,000), both purified to near homogeneity from bovine heart. The kinetic constants for both enzymes were fully characterized using the nanopeptide Leu-Arg-Arg-Ala-Ser-Val-Ala-Gln-Leu as the interconvertible substrate, cAMP as an activator for the kinase, and Pi as an inhibitor for the phosphatase. In the presence of a nearly constant concentration of ATP, a steady-state level of phosphorylation of the peptide was attained which was determined by the relative concentrations of the kinase, phosphatase, and effectors. As predicted by the cyclic cascade model, this monocyclic cascade exhibited both signal amplification and an increase in sensitivity to variations in multiple effector concentrations. In addition, the data show that the steady-state level of phosphorylation obtained in the presence of an activator of the kinase (e.g. cAMP) and an inhibitor of the phosphatase (e.g. Pi) is a function of the product of the relative effector concentrations. Finally, the results reveal that when the concentration of enzyme-substrate complex is not negligible, cyclic cascades are potentially more sensitive to variations in effector concentrations and can achieve even greater signal amplification than predicted previously.

Adenosine Triphosphate↗
PubMed1984

Energy consumption in a cyclic phosphorylation/dephosphorylation cascade.

Cyclic phosphorylation/dephosphorylation cascade systems are responsible for regulating numerous metabolic pathways. The capacity of a cyclic cascade system to maintain a steady-state level of phosphorylation and, hence, a specific biological activity of a phosphorylatable protein is dependent upon a constant supply of metabolic energy (ATP). Quantification of the extent of ATP consumption in a cyclic cascade was examined experimentally with the model in vitro phosphorylation/dephosphorylation system described in detail in the previous paper (Shacter, E., Chock, P. B., and Stadtman, E. R. (1984) J. Biol. Chem. 259, 12252-12259). The results indicate that (a) when the concentrations of converter enzymes and interconvertible substrate are held constant and the fractional phosphorylation of the substrate is varied by changing the allosteric effector concentrations, the rate of ATP consumption in the monocyclic cascade is directly proportional to the steady-state level of phosphorylation being maintained. (b) Attainment of a particular steady-state level of phosphorylation is determined by the net ratio of the protein kinase and phosphatase activities and is independent of the absolute concentrations of these enzymes. (c) Whereas the time required to reach a given steady state is inversely proportional to the converter enzyme concentrations, the amount of ATP consumed in maintaining that steady state is directly proportional to the kinase and phosphatase concentrations. In addition, a theoretical analysis based upon experimentally determined parameters for two in vivo cyclic cascade systems (pyruvate kinase and glycogen phosphorylase) revealed that under normal conditions, cyclic phosphorylation/dephosphorylation cascades consume only a small proportion (less than 0.02%) of the total cellular energy flux.

Adenosine Triphosphate↗
PubMed1984

Mammalian brain phosphoproteins as substrates for calcineurin.

Calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase found in several tissues, is highly concentrated in mammalian brain. In an attempt to identify endogenous brain substrates for calcineurin, kinetic analyses of the dephosphorylation of several well-characterized phosphoproteins purified from brain were performed. The proteins studied were: G-substrate, a substrate for cyclic GMP-dependent protein kinase; DARPP-32, a substrate for cyclic AMP-dependent protein kinase; Protein K.-F., a substrate for a cyclic nucleotide- and Ca2+-independent protein kinase; and synapsin I, a substrate for cyclic AMP-dependent (site I) and a Ca2+/calmodulin-dependent protein kinase (site II). Calcineurin dephosphorylated each of these proteins in a Ca2+/calmodulin-dependent manner. Similar Km values were obtained for each substrate: G-substrate, 3.8 microM; DARPP-32, 1.6 microM; Protein K.-F., approximately 3 microM (S0.5); synapsin I (site I), 7.0 microM; synapsin I (site II), 4.4 microM. However, significant differences were obtained for the maximal rates of dephosphorylation. The kcat values were: G-substrate, 0.41 s-1; DARPP-32, 0.20 s-1; Protein K.-F., 0.7 s-1; synapsin I (site I), 0.053 s-1; synapsin I (site II), 0.040 s-1. Comparisons of the catalytic efficiency (kcat/Km) for each substrate indicated that DARPP-32, G-substrate, and Protein K.-F. are all potential substrates for calcineurin in vivo.

Animals↗
PubMed1984

On the mechanism of activation of the ATP X Mg(II)-dependent phosphoprotein phosphatase by kinase FA.

The mechanism of activation of the Mg(II) X ATP-dependent phosphatase by the kinase FA has been investigated. The inactive protein phosphatase can be represented as FC X M where FC is the inactive catalytic component and M is the heat-stable modulator protein (also known as inhibitor-2). Phosphorylation of the modulator protein is demonstrated during activation of FC X M. In addition, continuous ATP hydrolysis during the activation is observed. This suggests that a cyclic phosphorylation-dephosphorylation reaction is continuously occurring during the activation. It is proposed that phosphorylation of the modulator protein causes an isomerization in FC to generate an active phosphatase. The activated phosphatase is capable of dephosphorylating the phosphorylated modulator. Upon dephosphorylation of modulator, the active phosphatase returns to its inactive form via a slow isomerization.

Adenosine Triphosphatases↗
PubMed1984

The rate-limiting step in the actomyosin adenosinetriphosphatase cycle.

We have previously shown that myosin does not have to detach from actin during each cycle of ATP hydrolysis. In the present study, using the A-1 isoenzyme of myosin subfragment 1, we have investigated the nature of the rate-limiting steps in the ATPase cycle. Our results show that, at 15 degrees C, at very low ionic strength, KATPase determined from the double-reciprocal plot of ATPase activity vs. actin concentration is more than 6-fold stronger than KBINDING determined by directly measuring the binding of A-1 myosin subfragment 1 to actin during steady-state ATP hydrolysis. Computer modeling shows that this large difference between KATPase and KBINDING is not compatible with Pi release being the rate-limiting step in the ATPase cycle. If Pi release is not rate limiting, it is possible that the ATP hydrolysis step, itself, is rate limiting. However, this predicts that, at high actin concentration, the value of the initial Pi burst should be close to zero. Therefore, we measured the magnitude of the initial Pi burst in the presence of actin, using both direct measurement and measurement of relative fluorescence magnitude. Our results suggest that the magnitude of the initial Pi burst in the presence of actin is considerably higher than would be expected if the ATP hydrolysis step were the rate-limiting step in the ATPase cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1983

The apparently negatively cooperative phosphorylation of smooth muscle myosin at low ionic strength is related to its filamentous state.

The correlation curve between phosphorylation and MgATPase activity suggests that the 20,000-dalton light chain of both heads of a smooth muscle myosin or heavy meromyosin (HMM) molecule must be phosphorylated before the MgATPase activity of either head can be activated by actin. The two heads of HMM appear to be phosphorylated randomly at equal rates, while those of myosin are phosphorylated in a negatively cooperative manner (Persechini, A., and Hartshorne, D.J. (1981) Science, 213, 1383-1385; Ikebe, M., Ogihara, S., and Tonomura, Y. (1982) J. Biochem. 91, 1809-1812). We have investigated the cause of this difference between HMM and myosin. We find that if myosin is first phosphorylated at high ionic strength (0.6 M KCl), where it is monomeric, and then assayed for MgATPase activity (in 0.05 M KCl), the data support a model where the two heads are phosphorylated randomly with equal rates (i.e. similarly to HMM). The correlation curves between MgATPase activity and dephosphorylation of fully phosphorylated myosin, both in a filamentous and monomeric state, are also best explained by a model where dephosphorylation of one head is sufficient to deactivate the entire molecule. With monomeric myosin, the dephosphorylation appears to occur randomly with equal rates, whereas with filamentous myosin the dephosphorylation appears to be negatively cooperative. The correlation between dephosphorylation of HMM and its MgATPase activity is more complex and is consistent with a positively cooperative dephosphorylation. Direct analyses of the time courses of phosphorylation of HMM and monomeric myosin show that a single exponential is sufficient to fit the data through greater than 90% of the reaction. However, when phosphorylation is carried out at low ionic strength (0.02 M KCl), where myosin is present as filaments, the time course consists of two exponential functions where the rate constant for the phosphorylation of one myosin head is 6-10 times greater than that for the other head which is located on the same molecule. This suggests that when myosin is polymerized into filaments the two previously indistinguishable heads either become nonequivalent or are subject to head-head interactions leading to a negatively cooperative phosphorylation reaction.

Actins↗
PubMed1983

Protein phosphorylation as a regulatory device.

Phosphorylation-dephosphorylation cascade systems represent a major mechanism of cellular regulation. A theoretical analysis of such systems (Stadtman & Chock 1977) revealed that they are endowed with extraordinary regulatory capacities; they may exhibit smooth, flexible responses to changes in single and multiple metabolite levels, signal amplification, and apparent positive cooperativity. To test the theories and equations involved in this analysis, an in vitro phosphorylation-dephosphorylation model system was developed. The system consists of a cyclic-AMP-dependent protein kinase and a phosphoprotein phosphatase, both isolated to near-homogeneity from bovine heart, and a nanopeptide that serves as the interconvertible substrate. Experiments with the model system confirm the predictions about the behaviour of a monocyclic cascade. They also reveal that when the concentration of enzyme-substrate complex is not negligible, cyclic cascades are potentially more sensitive to variations in effector concentrations and can achieve even greater signal amplification than previously predicted.

Animals↗
PubMed1983

Properties of a Mr = 38,000 phosphoprotein phosphatase. Modulation by divalent cations, ATP, and fluoride.

A Mr = 38,000 phosphoprotein phosphatase (EC 3.1.3.16) was purified to near homogeneity from bovine cardiac muscle. The enzyme, classified as a type 2 phosphatase, was not inhibited by the heat-stable protein, inhibitor-2. Activity on peptide substrates was stimulated considerably by Mn2+ ions. The individual and combined effects of divalent cations, ATP, and fluoride were studied in detail employing the phosphonanopeptide Leu-Arg-Arg-Ala-Ser(P)-Val-Ala-Gln-Leu as the substrate. ATP and fluoride inhibited enzyme activity completely in the absence of divalent cations. Mg2+ either reduced or completely prevented this inhibition depending upon whether Mn2+ was present. Quantitative analysis of the results revealed that ATP and fluoride do not inhibit by chelation of an essential metal (e.g. Mn2+). Rather, a plausible model for the combined effects of Mg2+, Mn2+, ATP, and fluoride on phosphatase activity must assume that each of these factors acts by binding to individual sites on the enzyme and not to each other.

Adenosine Triphosphate↗
PubMed1982

Catalytic cycle of the biosynthetic reaction catalyzed by adenylylated glutamine synthetase from Escherichia coli.

The covalently attached AMP moiety of adenylylated glutamine synthetase from Escherichia coli has been replaced by its fluorescent analog, 2-aza-1,N6-etheno-AMP (aza-epsilon-AMP). The modified glutamine synthetase (aza-epsilon-GS) exhibits divalent cation requirement (Mn2+, rather than Mg2+), pH profile, Vmax, and Km similar to those of naturally adenylylated glutamine synthetase. Whereas naturally adenylylated glutamine synthetase exhibits only negligible fluorescence changes upon the binding of substrates, aza-epsilon-GS exhibits large fluorescence changes. The fluorescence changes have been used by means of a stopped flow technique to reveal the involvement of five fluorometrically distinct intermediates in the catalytic cycle for the biosynthesis of glutamine catalyzed by the adenylylated glutamine synthetase. The mechanism is very similar to that previously established for the unadenylylated enzyme, using intrinsic tryptophan fluorescence. Substrates bind via a rapid equilibrium random mechanism, but the reaction proceeds in a stepwise manner. The formation of an enzyme-bound intermediate (probably gamma-glutamyl phosphate + ADP) from ATP and L-glutamate is the rate-limiting step, with the subsequent reaction of the enzyme-bound intermediate occurring very rapidly. The success in elucidating this complex mechanism is due largely to the vastly different amplitudes of the fluorescence changes at the two excitation maxima (300 nm and 360 nm) of the aza-epsilon-AMP moiety which accompany the formation of the various intermediates.

Adenosine Monophosphate↗