PubMed Health⌕ Search

Biomedical subjects

P B Chock

Publications and source records attributed to P B Chock.

At least 91 records · Page 5Linked to original sources
PubMed1981

Fluorometric studies of aza-epsilon-adenylylated glutamine synthetase from Escherichia coli.

Glutamine synthetase in Escherichia coli is regulated by adenylation and deadenylation reactions. The adenylation reaction converts the divalent cation requirement of the enzyme from Mg2+ to Mn2+. Previously, the catalytic action of unadenylated glutamine synthetase was elucidated by monitoring the intrinsic tryptophan fluorescence change accompanying substrate binding. However, due to the lack of changes in the tryptophan fluorescence, a similar study could not be done with the adenylated enzyme. In this study, therefore, an extrinsic fluor is introduced into the adenylated glutamine synthetase by adenylating the enzyme with 2-aza-1,N6-ethenoadenosine triphosphate, a fluorescent analog of ATP. The modified enzyme (aza-epsilon-glutamine synthetase) exhibits catalytic and kinetic properties similar to those of the naturally adenylated enzyme. The results of fluorometric studies on this aza-epsilon-glutamine synthetase indicated that L-glutamate and ATP bind to both Mn2+ and Mg2+ forms of the enzyme in a random order, but only the Mn2+ form is capable of forming a highly reactive enzyme-bound intermediate which is a prerequisite for the reaction with NH4+ to form products. The extrinsic fluorescence changes are also used to determine the binding constants of various substrates and inhibitors of both the biosynthetic and gamma-glutamyl transfer reactions.

Adenosine Triphosphate↗
PubMed1981

Subunit interaction in unadenylylated glutamine synthetase from Escherichia coli. Evidence from methionine sulfoximine inhibition studies.

Although glutamine synthetase from Escherichia coli is composed of 12 identical subunits, there is no evidence that homologous subunit interactions occur in fully unadenylylated or fully adenylylated enzyme. Meister and co-workers (Manning, J. M., Moore, S., Rowe, W. B., and Meister, A. (1969) Biochemistry 8, 2681-2685) have shown that L-methionine-S-sulfoximine, one of the four diastereomers of methionine sulfoximine, preferentially inhibits glutamine synthetase irreversibly in the presence of ATP, due to the formation of tightly bound products, ADP, and methionine sulfoximine phosphate. Using highly purified unadenylylated glutamine synthetase and the two resolved diastereomers of L-methionine-S,R-sulfoximine, we have studied both the kinetics of glutamine synthetase inactivation in the presence of excess methionine sulfoximine and ATP, and the binding of methionine sulfoximine to the enzyme. The results reveal that (a) the apparent first order rate constant of irreversible inactivation by the S isomer decreases progressively from the expected first order rate, indicating that an inactivated subunit retards the reactivity of its neighboring subunits toward methionine sulfoximine and ATP; (b) the R isomer does not inactivate glutamine synthetase irreversibly in the presence of ATP; however, the R isomer is capable of protecting the enzyme temporarily from the irreversible inhibition by the S isomer; and (c) the binding of the S isomer monitored by changes in protein fluorescence exhibits an apparent negative cooperative binding isotherm, whereas the R isomer yields an apparent positive cooperative pattern.

Adenosine Diphosphate↗
PubMed1981

Mechanism of action of troponin . tropomyosin. Inhibition of actomyosin ATPase activity without inhibition of myosin binding to actin.

The regulation of vertebrate skeletal muscle contraction by the troponin . tropomyosin complex is generally thought to be the result of tropomyosin physically blocking the myosin binding site of actin in the absence of Ca2+. This mechanism was tested during steady state ATP hydrolysis by comparing the degree of association of myosin subfragment 1 (S-1) with the actin . troponin . tropomyosin complex in the absence and presence of Ca2+. Binding in the presence of ATP was determined by stopped flow absorbance measurements at 25 degrees C. Although the steady state ATPase rate was reduced 96% in the absence of Ca2+, the association constant of S-1 with regulated actin was virtually the same in the absence of Ca2+ (1.3 X 10(4) M-1) as in the presence of Ca2+ (2.3 X 10(4) M-1). The association constant of S-1 to regulated actin in the presence of Ca2+ was similar to the association constant of S-1 to unregulated actin. These results suggest that the troponin . tropomyosin complex does not inhibit the actin-activated ATPase activity by preventing the binding of S-1 . ATP or S-1 . ADP . Pi to actin; rather, it may act by blocking the release of Pi from the acto-S-1 . ADP . Pi complex.

Actins↗
PubMed1981

Fluorometric assay for adenosine 3',5'-cyclic monophosphate-dependent protein kinase and phosphoprotein phosphatase activities.

A novel peptide substrate for adenosine 3',5'-cyclic monophosphate-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37), Leu-Arg-Arg-Trp-Ser-Leu-Gly, was synthesized. Phosphorylation of the peptide causes a 20% increase in the peptide fluorescence intensity at 358 nm. Values of Km and kcat for the phosphorylation reaction at pH 7.0 (25 degrees C), were determined to be 2.7 +/- 0.5 microM and 5.5 +/- 0.4 sec-1, respectively. The phosphorylated peptide was shown to be an effective substrate for phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) with a Km of 113 +/- 10 microM and a kcat of 2.4 +/- 0.2 sec-1 in the presence of 2.5 mM MnCl2. Changes in the peptide fluorescence intensity as a function of its phosphorylation state provide a highly sensitive assay of cyclic AMP-dependent protein kinase and phosphoprotein phosphatase activities.

Amino Acid Sequence↗
PubMed1981

Mechanism of activation of cyclic nucleotide phosphodiesterase: requirement of the binding of four Ca2+ to calmodulin for activation.

Kinetic studies on the activation of cyclic nucleotide phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) as a function of calmodulin and Ca2+ concentrations have been carried out. A general approach to analyzing the mechanism of activation, which takes into consideration the various interactions among phosphodiesterase and calmodulin liganded with Ca2+ to differing degrees, is presented. The method is applicable to other calmodulin-regulated enzyme systems. Our kinetic analysis reveals that all four Ca2+ must be bound to calmodulin for the protein to form an activated complex with phosphodiesterase. The mechanistic and regulatory advantages of having four Ca2+ sites on calmodulin can be briefly stated as follows. (i) With the enzyme--calmodulin--Ca4(2+) complex as the dominant active species, the activation of phosphodiesterase as a function of Ca2+ concentration is highly cooperative. This phenomenon serves as an effective on/off switch for phosphodiesterase activation. (ii) At normal cellular levels of Ca2+ (less than 0.1 microM), phosphodiesterase and calmodulin do not form a complex. Thus, the distribution of calmodulin among its various target enzymes is reshuffled for each Ca2+ surge. (iii) The affinity between the enzyme and the fully liganded calmodulin (0.1-1 mM) is 10(4)-10(5) times better than that in the absence of Ca2+ (greater than or equal to 10 microM). The tremendous increase in affinity can be achieved rather easily through a 10- to 20-fold increase in the affinity of Ca2+ for the enzyme-calmodulin complex in each of the four binding steps.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

Mechanism of the actomyosin ATPase: effect of actin on the ATP hydrolysis step.

The Lymn-Taylor model for the actomyosin ATPase suggests that during each cycle of ATP hydrolysis the complex of myosin subfragment 1 (S-1) with actin must dissociate into S-1.ATP plus actin before ATP hydrolysis can occur. In the present study we tested whether such a mandatory detachment step occurs by measuring the effect of actin on the rate and magnitude of the ATP hydrolysis step (initial Pi burst) and on the steady-state ATPase rate. We find that the rate of the initial Pi burst markedly increases at high actin concentration although the Lymn-Taylor model predicts the rate should remain nearly constant or decrease. In addition, at high actin concentration, the magnitude of the initial Pi burst is much larger than is predicted by the Lymn-Taylor model. Finally, at 360 microM actin, at which more than 90% of the S-1.ATP is bound to actin, there is no inhibition of the steady-state ATPase activity although the Lymn-Taylor model predicts that 70% inhibition should occur. We conclude that the acto-S-1 complex is not dissociated by ATP during each cycle of ATP hydrolysis; in fact, the rate of the initial Pi burst appears to be even faster when S-1.ATP is bound to actin than when it is dissociated.

Actins↗
PubMed1980

Transient kinetic analysis of the catalytic cycle of alkaline phosphatase.

Transient kinetic studies were carried out to elucidate the catalytic cycle of Escherichia coli alkaline phosphatase at ph 8.3, 10 degrees C, and to evaluate the rate constants for the individual steps. Using a rapid mixing cell, we were able to detect the burst phase of the reaction, which could not be obtained at alkaline ph values with conventional mixing devices. Analysis of the burst phase revealed that the equilibrium of the initial binding of the substrate, 4-methylumbelliferyl phosphate, to the enzyme is attained rapidly, and that the production of the alcohol, 4-methylumbelliferone, is fast. Analysis of the steady state phase of the reaction yielded a phosphate release rate constant which agrees very well with the kcat determined by initial rate studies. Dephosphorylation of the phosphoryl enzyme prepared at pH 5.7 was studied by the pH-jump technique, using a three-syringe stopped flow apparatus. The results showed that the dephosphorylation step is not rate-limiting in the catalytic cycle and that the presence of substrates or inhibitor has no effect on this step. The lack of effect of substrates on the rate of dephosphorylation and on the rate of phosphate dissociation indicates that the flip-flop mechanism, in which the product release is supposedly facilitated by the binding of a 2nd molecule of substrate, is not valid for alkaline phosphatase. Kinetic constants for various steps in the catalytic cycle of alkaline phosphatase at pH 8.3, 10 degrees C, are reported and a reaction scheme which is in harmony with previous observations from other laboratories is described.

Alkaline Phosphatase↗
PubMed1980

Subunit interaction of adenylylated glutamine synthetase.

The glutamine synthetase (GS) activity in Escherichia coli is regulated by a bicyclic interconvertible enzyme cascade which involves the cyclic adenylylation (inactivation) and deadenylylation (activation) of GS on the one hand, and the modulation of these processes by the uridylylation and deuridylylation of Shapiro's regulatory protein on the other. The specific activity of GS in a given metabolic state is determined by the fraction of its subunits that are adenylylated, and this fraction is determined by the concentration of over 40 metabolites. Through allosteric and substrate interactions with one or more of the cascade enzymes, these metabolites alter the rates of the covalent modification and demodification reactions. By means of immunoprecipitation studies with anti-AMP specific antibodies, it has been established that the partially adenylylated glutamine synthetase, which is present in a given steady state, is a mixture of hybrid molecules containing different numbers and possibly distributions of adenylylated subunits. Partial separation of these hybrid mixtures has been achieved by affinity chromatography on Affi-Blue Sepharose columns. From immunochemical studies it is evident that anti-AMP antibodies can react with adenylylated subunits of all molecular species of GS, but that the capacities of these primary antigen-antibody reactions to yield precipitable aggregates is very dependent of the number of adenylylated subunits per molecule, and much less so upon the total concentration of adenylylated subunits present. the studies suggest that precipitability is a function either of the distribution of adenylylated subunits within hybrid species, or of the kinetics of intra- vs intermolecular bivalent interactions.

Glutamate-Ammonia Ligase↗
PubMed1979

Mechanism of actomyosin adenosine triphosphatase. Evidence that adenosine 5'-triphosphate hydrolysis can occur without dissociation of the actomyosin complex.

We have investigated the steps in the actomyosin ATPase cycle that determine the maximum ATPase rate (Vmax) and the binding between myosin subfragment one (S-1) and actin which occurs when the ATPase activity is close to Vmax. We find that the forward rate constant of the initial ATP hydrolysis (initial Pi burst) is about 5 times faster than the maximum turnover rate of the actin S-1 ATPase. Thus, another step in the cycle must be considerably slower than the forward rate of the initial Pi burst. If this slower step occurs only when S-1 is complexed with actin, as originally predicted by the Lymn-Taylor model, the ATPase activity and the fraction of S-1 bound to actin in the steady state should increase almost in parallel as the actin concentration is increased. As measured by turbidity determined in the stopped-flow apparatus, the fraction of S-1 bound to actin, like the ATPase activity, shows a hyperbolic dependence on actin concentration, approaching 100% asymptotically. However, the actin concentration required so that 50% of the S-1 is bound to actin is about 4 times greater than the actin concentration required for half-maximal ATPase activity. Thus, as previously found at 0 degrees C, at 15 degrees C much of the S-1 is dissociated from actin when the ATPase is close to Vmax, showing that a slow first-order transition which follows the initial Pi burst (the transition from the refractory to the nonrefractory state) must be the slowest step in the ATPase cycle. Stopped-flow studies also reveal that the steady-state turbidity level is reached almost instantaneously after the S-1, actin, and ATP are mixed, regardless of the order of mixing. Thus, the binding between S-1 and actin which is observed in the steady state is due to a rapid equilibrium between S-1--ATP and acto--S-1--ATP which is shifted toward acto-S-1--ATP at high actin concentration. Furthermore, both S-1--ATP and S-1--ADP.Pi (the state occurring immediately after the initial Pi burst) appear to have the same binding constant to actin. Thus, at high actin concentration both S-1--ATP and S-1--ADP.Pi are in rapid equilibrium with their respective actin complexes. Although at very high actin concentration almost complete binding of S-1--ATP and S-1--ADP.Pi to actin occurs, there is no inhibition of the ATPase activity at high actin concentration. This strongly suggests that both the initial Pi burst and the slow rate-limiting transition which follows (the transition from the refractory to the nonrefractory state) occur at about the same rates whether the S-1 is bound to or dissociated from actin. We, therefore, conclude that S-1 does not have to dissociate from actin each time an ATP molecule is hydrolyzed.

Actins↗
PubMed1979

The mechanism of the skeletal muscle myosin ATPase. II. Relationship between the fluorescence enhancement induced by ATP and the initial Pi burst.

A major question about the mechanism of the myosin ATPase is how much of the fluorescence change which accompanies the binding of ATP to myosin is due to the conformational change induced by ATP and how much is due to the subsequent hydrolysis of ATP in the initial Pi burst. Several laboratories have suggested that the maximal rate of the fluorescence change represents the rate of the irreversible conformational change induced by ATP. In the present study, the rate of irreversible ATP binding, the rate of the initial Pi burst, and the rate of the fluorescence enhancement were compared under varied conditions. The results show that: 1) the fluorescence enhancement is mainly due to the hydrolysis of ATP in the initial Pi burst rather than to the conformational change induced by the binding of ATP; 2) the rate of the initial Pi burst is considerably slower than the rate of irreversible ATP binding at high ATP concentration; 3) the rate of the initial Pi burst is almost the same as the rate of the fluorescence enhancement. Therefore, the maximum rate of the fluorescence enhancement represents the rate of the initial Pi burst rather than the rate of the conformational change induced by ATP binding.

Adenosine Triphosphatases↗