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C Frieden

Publications and source records attributed to C Frieden.

At least 109 records · Page 6Linked to original sources
PubMed1979

Binding of regulatory ligands to rabbit muscle phosphofructokinase. A model for nucleotide binding as a function of temperature and pH.

The binding of nucleoside triphosphates to rabbit muscle phosphofructokinase has been determined in 0.05 M phosphate buffers by changes in intrinsic protein fluorescence and by direct binding measurements. These experiments have been performed over a wide range of pH, temperature, and effector concentration. Quenching of protein fluorescence is shown to measure binding of nucleotides to a site which is not the active site but rather a site responsible for inhibition of the kinetic activity. This site is relatively specific for either ATP or MgATP with free ATP binding about 10-fold more tightly than MgATP. A model to describe binding to this site as a function of pH and temperature is proposed. This model assumes that the apparent affinity for ATP is determined by protonation of two ionizable groups (per subunit) and that ATP binds exclusively to protonated enzyme forms. Several ligands which affect the apparent affinity for nucleotide binding at the inhibitory site act by shifting the apparent pK of the ionizable groups. NH4+ and citrate do not influence nucleotide binding to the inhibitory site. At pH 6.9 in 0.05 M phosphate, low concentrations of MgATP or MgGTP enhance the protein fluorescence due to binding at the active site. The fluorescence studies and direct binding studies show that there is one active site and one inhibitory site per subunit. As described elsewhere (Pettigrew, D. W., and Frieden, C. (1978) J. Biol. Chem. 253, 3623-3627), there is a third nucleotide binding site on each subunit which is specific for cAMP, AMP, and ADP.

Adenine Nucleotides↗
PubMed1979

Immunofluorescent and histochemical localization of AMP deaminase in skeletal muscle.

Fluorescent antibody staining experiments with both isolated myofibrils and muscle fibers grown in culture show that AMP deaminase is bound to the myofibril in the A band. The strongest staining occurs at each end of the A band. The approximate width of the fluorescent stripes and their relation to the A band remains constant as a function of sarcomere length. Removal of enzyme from the myofibrils leads to loss of staining, and readdition of purified enzyme restores the original staining pattern. A histoenzymatic method for the detection of AMP deaminase activity in cultured fibers gives comparable localization. The results are consistent with the previous observation (Ashby, B. and C. Frieden. 1977.J. Biol. Chem. 252:1869--1872) that AMP deaminase forms a tight complex in solution with subfragment-2 (S-2) of myosin or with heavy meromyosin (HMM).

AMP Deaminase↗
PubMed1978

Adenylate deaminase. Kinetic and binding studies on the rabbit muscle enzyme.

Kinetic studies with adenylate deaminase have been performed by stopped flow methods at 20 degrees C in 0.01 M imidazole/HCl, pH 6.5. The data were analyzed using either the whole time course of the reaction or the initial portion of the full time course. At low KCl concentrations, activation by the product IMP complicates any interpretation. In the presence of 0.15 M KCl, the results are interpreted in terms of three types of purine nucleotide binding sites: an active site, an inhibitory site which appears to be relatively specific for nucleoside triphosphates, and an activating site which shows relatively little specificity for nucleoside phosphates. Nucleotide binding to the activating site weakens binding to the inhibitory site. Sigmoidal kinetic data observed as a function of AMP in the presence of the inhibitor GTP are interpreted in terms of AMP binding to the activating site and weakening GTP binding. A fragment of myosin, subfragement-2, which has previously been shown to form a tight complex with adenylate deaminase (Ashby, B., and Frieden, C. (1977) J. Biol. Chem. 252, 1869--1875) activates the deaminase reaction only slightly. Complex formation, however, makes the reaction less susceptible to inhibition by GTP, although high levels of this nucleotide will disrupt the complex. In the presence of GTP or GTP plus subfragment-2, hysteretic effects are observed.

AMP Deaminase↗
PubMed1978

Rabbit muscle phosphofructokinase. Modification of molecular and regulatory kinetic properties with the affinity label 5'-p-(fluorosulfonyl)benzoyl adenosine.

The affinity label 5'-p-(fluorosulfonyl)benzoyl adenosine modifies rabbit muscle phosphofructokinase to the extent of one group/subunit. Modification appears to occur at a binding site specific for AMP, cyclic AMP, and ADP, i.e. those adenine nucleotides which are activators under conditions where regulatory kinetic behavior is obtained. The consequences of the modification are consistent with the model proposed previously for correlation between the pK of specific ionizable groups, regulatory kinetic behavior, ligand binding, and the reversible cold inactivation of the enzyme (Frieden, C., Gilbert. H. R., and Bock, P. E. (1976) J. Biol. Chem. 251, 5644-5647). Thus, the modification shifts the apparent pK of the essential ionizable groups from 6.9 to 6.4 at 25 degrees C, with the result that regulatory kinetic behavior at pH 6.9 and 25 degrees C is lost. Furthermore, the apparent affinity of a site (other than the active site) for ATP, as measured by ATP-dependent quenching of intrinsic protein fluorescence at pH 6.9 and 25 degrees C, is decreased by the modification. Regulatory kinetic behavior for both substrates is obtained with the modified enzyme at a lower pH, consistent with the downward shift in the pK of the ionizable groups, but sensitivity to cAMP activation is abolished by the modification. The loss of regulatory kinetic behavior upon modification of sulfhydryl groups does not appear to be the same as that due to modification by the affinity label.

Adenosine↗
PubMed1978

Malate dehydrogenases. The lack of evidence for dissociation of the dimeric enzyme in kinetic analyses.

The kinetic parameters of beef heart cytoplasmic and pig heart mitochondrial malate dehydrogenases have been examined over a wide range of enzyme concentration. No significant changes are observed in these properties. In conjunction with active enzyme sedimentation and sedimentation equilibrium experiments, it is concluded that there is no evidence for dissociation of the dimeric enzyme at any enyzme level in the kinetic analyses. Thus, if dissociation occurs, it must be too slow to be of significance in determining the kinetic properties of the enzyme. It is shown that unless a subunit and its dimeric form have identical kinetic and substrate binding characteristics, the kinetic parameters should change as a function of enzyme concentration.

Animals↗
PubMed1977

Interaction of AMP-aminohydrolase with myosin and its subfragments.

We have shown that purified rabbit skeletal muscle AMP-aminohydrolase binds to rabbit muscle myosin, heavy meromyosin, and Subfragment 2 but does not bind to light meromyosin nor to Subfragment 1. The dissociation constant for binding to myosin was determined to be 0.14 muM. A new sedimentation boundary, presumably reflecting formation of a complex between AMP-aminohydrolase and heavy meromyosin or Subfragment 2, can be observed using the analytical ultracentrifuge. Binding of AMP-aminohydrolase to myosin, heavy meromyosin, or Subfragment 2 is abolished by phosphate (less than 10 mM), an inhibitor of AMP-aminohydrolase. No other rabbit muscle enzyme tested showed any interaction with myosin under the same conditions and there was no indication of complex formation between AMP-aminohydrolase and phosphofructokinase or phosphocreatine kinase in the analytical ultracentrifuge.

AMP Deaminase↗
PubMed1976

Phosphofructokinase. I. Mechanism of the pH-dependent inactivation and reactivation of the rabbit muscle enzyme.

The kinetics of inactivation and reactivation of rabbit skeletal muscle phosphofructokinase have been studied as a function of pH and enzyme concentration at constant temperature in phosphate buffer. From the enzyme concentration dependence, we conclude that the minimal mechanism for inactivation involves a protonation step followed by isomerization to an inactive form and then dissociation to a species of one-half the molecular weight. Other data indicate a subsequent isomerization of the dissociated form. The pH and temperature dependence of the inactivation process shows that it is controlled by ionizable groups, and that the apparent pK for these groups is temperature-dependent in such a way as to make the enzyme show the characteristic of cold lability below pH 7. Reactivation of the inactive enzyme occurs by a kinetically different pathway involving deprotonation of an inactive, dissociated form to a form which may either isomerize to another inactive form, or dimerize to the active enzyme. A general mechanism is postulated in which the inactivation and reactivation processes are different aspects of the same mechanism. This mechanism assumes four species (two containing four subunits and two containing two subunits) each of which can exist in a protonated and unprotonated form. Inactivation or reactivation induced by changes in pH or temperature reflect the kinetic establishment of a new steady state between these forms. How the apparent pK values which control the distribution of the enzyme between protonated and unprotonated forms describe the pH-dependent characteristics of the enzyme is discussed in terms of the proposed mechanism.

Animals↗
PubMed1976

Phosphofructokinase. II. Role of ligands in pH-dependent structural changes of the rabbit muscle enzyme.

The effect of ligands, including substrates and allosteric effectors, on the pH-dependent inactivation and reactivation of rabbit muscle phosphofructokinase has been examined in terms of the mechanism proposed previously (Bock, P.E. and Fireden, C. (1976) J. Biol. Chem. 251, 5630-5636). It is concluded thatt many ligands exert their effect by binding preferentially to either protonated or unprotonated forms of the enzyme and thus shifting an apparent pK for the inactivation or reactivation process. ATP and fructose 6-phosphate influence the apparent pK to different extents and in different directions, with ATP binding preferentially to the protonated forms and fructose 6-phosphate to the unprotonated forms. Enzyme inactivated by ATP can be reactivated by the addition of fructose 6-phosphate. The experiments indicate that inactivation and reactivation in the presence of these ligands can occur by kinetically different pathways as has been found for these processes in the absence of ligands. The results are discussed in relation to what might be expected for ligand binding properties of the enzyme as a function of pH, temperature, and enzyme concentration. The effect of ATP and MgATP is complex, perhaps representing more than one site of binding. Citrate appears to bind preferentially to protonated forms of the enzyme while fructose 1,6-bisphosphate and AMP bind preferentially to the unprotonated forms. ADP, K+, and NH4+ appear to have little or no preference in binding to different enzyme forms.

Adenosine Triphosphate↗
PubMed1976

Phosphofructokinase. III. Correlation of the regulatory kinetic and molecular properties of the rabbit muscle enzyme.

It is shown that the degree of regulatory kinetic behavior of rabbit muscle phosphofructokinase increases at a given pH and lower temperatures, as well as at a given temperature and lower pH values. It is also shown that the regulatory kinetic behavior which appears at lower pH values is inherent in the tetrameric (active) form of the enzyme. We conclude that a portion of the mechanism proposed previously (Bock, P.E., and Frieden, C. (1976) J. Biol. Chem. 251, 5630-5636) to describe the pH and temperature-dependent inactivation or reactivation may also be used to explain the pH and temperature-dependent regulatory kinetic behavior. According to this proposal, two rapidly equilibrating forms of the enzyme, which differ in the degree of protonation of specific residues, differ in their ability to bind substrates. While the protonated form of the enzyme subsequently becomes inactive by isomerization and dissociation, this process is too slow to affect the kinetic results, making direct comparisons between the association-dissociation behavior and regulatory kinetic behavior invalid. The time dependence of the processes of inactivation or reactivation in the presence or absence of ligands and of the appearance of regulatory kinetic behavior is discussed in relation to their possible role in metabolic regulation.

Allosteric Regulation↗
PubMed1975

Kinetic studies on pig heart cytoplasmic malate dehydrogenase.

Kinetic studies on the pig heart cytoplasmic malate dehydrogenase have been performed over a wide range of conditions using the full time course of the reaction and computer simulation to obtain the kinetic parameters. The maximum velocity and Michaelis constants for the oxidation of reduced coenzyme have been determined as a fundtion of pH in 0.05 M phosphate buffer at 15 degrees. At pH 7.5 and at low substrate concentrations, the kinetic data are consistent with a sequential addition of substrates, coenzyme binding first, and involving the formation of at least one ternary complex. No oxalacetate binding to the enzyme was observed. The rate constants for the dissociation of coenzyme from the enzyme-coenzyme complex are small enough to define the maximum velocity in either direction of the reaction. These data, plus data using deuterated reduced coenzyme, indicate that the chemical transformation step is not rate determining. It is also shown that DPNH binding can be tight enough to practically exclude the possibility of obtaining initial velocities when measuring the reduction of DPN. Kinetic abnormalities do appear at higher substrate or product concentrations, but these do not appear to be related to the formation of inactive abortice, complexes.

Animals↗