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C Frieden

Publications and source records attributed to C Frieden.

At least 73 records · Page 4Linked to original sources
PubMed1987

Adenosine deaminase converts purine riboside into an analogue of a reactive intermediate: a 13C NMR and kinetic study.

The 13C NMR spectra of [2-13C]- and [6-13C]purine ribosides have been obtained free in solution and bound to the active site of adenosine deaminase. The positions of the resonances of the bound ligand are shifted relative to those of the free ligand as follows: C-2, -3.7 ppm; C-6, -73.1 ppm. The binary complexes are in slow exchange with free purine riboside on the NMR time scale, and the dissociation rate constant is estimated to be 13.5 s-1 from the slow exchange broadening of the free signal. In aqueous solution, protonation of purine riboside at N-1 results in changes in 13C chemical shift relative to those of the free base as follows: C-2, -4.9 ppm; C-6, -7.9 ppm. The changes in chemical shift that occur when purine riboside binds to the enzyme indicate that the hybridization of C-6 changes from sp2 to sp3 in the binary complex with formation of a new bond to oxygen or sulfur. A change in C-2 hybridization can be eliminated as can protonation at N-1 as the sole cause of the chemical shift changes. The kinetic constants for the adenosine deaminase catalyzed hydrolysis of 6-chloro- and 6-fluoropurine riboside have been compared, and the reactivity order implies that carbon-halogen bond breaking does not occur in the rate-determining step. These observations support a mechanism for the enzyme in which formation of a tetrahedral intermediate is the most difficult chemical step. Enzymic stabilization of this intermediate may be an important catalytic strategy used by the enzyme to lower the standard free energy of the preceding transition state.

Adenosine Deaminase↗
PubMed1987

Kinetic analysis of the mechanism of Escherichia coli dihydrofolate reductase.

A kinetic mechanism is presented for Escherichia coli dihydrofolate reductase which describes the full time course of the enzymatic reaction over a wide range of substrate and enzyme concentrations at pH 7.2 and 20 degrees C. Specific rate constants were estimated by computer simulation of the full time course of single turnover, burst, and steady-state experiments using both nondeuterated and deuterated NADPH. The mechanism involves the random addition of substrates, but the substrates and enzyme are not at equilibrium prior to the chemical transformation step. The rate-limiting step follows the chemical transformation, and the maximum velocity of the reaction is limited by the release of the product tetrahydrofolate. The full time course of the reaction is markedly affected by the formation of the enzyme-NADPH-tetrahydrofolate abortive complex, but not by the enzyme-NADP-dihydrofolate abortive complex.

Escherichia coli↗
PubMed1987

Divalent cation binding to the high- and low-affinity sites on G-actin.

Metal binding to skeletal muscle G-actin has been assessed by equilibrium dialysis using 45Ca2+ and by kinetic measurements of the increase in the fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine-labeled actin. Two classes of cation binding sites were found on G-actin which could be separated on the basis of their Ca2+ affinity: a single high-affinity site with a Kd considerably less than 1 microM and three identical moderate-affinity binding sites with a Kd of 18 microM. The data for the Mg2+-induced fluorescence enhancement of actin labeled with N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine support a previously suggested mechanism [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886] in which Ca2+ is replaced by Mg2+ at the moderate affinity site(s), followed by a slow actin isomerization. This isomerization occurs independently of Ca2+ release from the high-affinity site. The fluorescence data do not support a mechanism in which this isomerization is directly related to Ca2+ release from the high-affinity site. Fluorescence changes of labeled actin associated with adding metal chelators are complex and do not reflect the same change induced by Mg2+ addition. Fluorescence changes in the labeled actin have also been observed for the addition of Cd2+ or Mn2+ instead of Mg2+. It is proposed actin may undergo a host of subtle conformational changes dependent on the divalent cation bound. We have also developed a method by which progress curves of a given reaction can be analyzed by nonlinear regression fitting of kinetic simulations to experimental reaction time courses.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1987

Adenosine deaminase: viscosity studies and the mechanism of binding of substrate and of ground- and transition-state analogue inhibitors.

We have studied the effects of viscosogenic agents, sucrose and ficoll, on (1) the hydrolysis of adenosine and of 6-methoxypurine riboside catalyzed by adenosine deaminase and (2) the rates of association and dissociation of ground-state and transition-state analogue inhibitors. For adenosine, Vmax/Km is found to be inversely proportional to the relative viscosity with sucrose, an agent affecting the microscopic viscosity, while no effect is found with ficoll, an agent affecting the macroscopic viscosity. Viscosogenic agents have no effect on the kinetic constants for 6-methoxypurine riboside. Thus, the bimolecular rate constant, Vmax/Km = 11.2 +/- 0.8 microM-1 s-1, for the reaction with adenosine is found to be at the encounter-controlled limit while that for the reaction with the poor substrate 6-methoxypurine riboside, 0.040 +/- 0.004 microM-1 s-1, is limited by some other process. Viscosity-dependent processes do not make a significant (less than 10%) contribution to Vmax. The dissociation constants for inhibitors are unaffected by viscosity. The ground-state analogue inhibitor purine riboside appears to bind at a rate comparable to that of adenosine. However, the slower rates of association (0.16-2.5 microM-1 s-1) and dissociation (5 X 10(-6) to 12 s-1) of transition-state analogue inhibitors are affected by the viscosity of the medium to approximately the same extent as the encounter-controlled rates of association and dissociation of adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Deaminase↗
PubMed1987

Microinjection of gelsolin into living cells.

Gelsolins are actin-binding proteins that cap, nucleate, and sever actin filaments. Microinjection of cytoplasmic or plasma gelsolin into living fibroblasts and macrophages did not affect the shape, actin distribution, deformability, or ruffling activity of the cells. Gelsolin requires calcium for activity, but the NH2-terminal half is active without calcium. Microinjection of this proteolytic fragment had marked effects: the cells rounded up, stopped ruffling, became soft, and stress fibers disappeared. These changes are similar to those seen with cytochalasin, which also caps barbed ends of actin filaments. Attempts to raise the cytoplasmic calcium concentration and thereby activate the injected gelsolin were unsuccessful, but the increases in calcium concentration were minimal or transient and may not have been sufficient. Our interpretation of these results is that at the low calcium concentrations normally found in cells, gelsolin does not express the activities observed in vitro at higher calcium concentrations. We presume that gelsolin may be active at certain times or places if the calcium concentration is elevated to a sufficient level, but we cannot exclude the existence of another molecule that inhibits gelsolin. Microinjection of a 1:1 gelsolin/actin complex had no effect on the cells. This complex is stable in the absence of calcium and has capping activity but no severing and less nucleation activity as compared with either gelsolin in calcium or the NH2-terminal fragment. The NH2-terminal fragment-actin complex also has capping and nucleating activity but no severing activity. On microinjection it had the same effects as the fragment alone. The basis for the difference between the two complexes is unknown. The native molecular weight of rabbit plasma gelsolin is 82,500, and the extinction coefficient at 280 nm is 1.68 cm2/mg. A new simple procedure for purification of plasma gelsolin is described.

Animals↗
PubMed1986

Actin polymerization. The mechanism of action of cytochalasin D.

Fluorescence changes using actin covalently labeled with N-(1-pyrenyl)iodoacetamide have been used to determine the effect of cytochalasin D on actin polymerization. A mechanism for the effect of cytochalasin D on actin polymerization is presented, which explains the experimental observation of a cytochalasin D-induced increase in the initial rate of polymerization and a decrease in the final extent of the reaction. Central to this mechanism is the Mg2+-dependent formation of cytochalasin D-induced dimers. The dimers serve as nuclei to enhance the polymerization rate. Binding of Mg2+ to a low affinity site on the dimer induces a conformational change which can be observed as a rapid fluorescence increase. A subsequent time-dependent fluorescence decrease observed prior to polymerization appears to represent ATP hydrolysis resulting in dissociation of the dimer and release of actin monomers containing ADP. We postulate that a slow rate of exchange of ATP for bound ADP relative to hydrolysis results in the accumulation of monomers containing ADP. As these monomers have a high critical concentration, the final extent of polymerization is reduced dramatically. The Mg2+ dependence of the final extent of polymerization in the presence of cytochalasin D is also explained in the context of this mechanism.

Actins↗
PubMed1986

The kinetics of cytochalasin D binding to monomeric actin.

It has been shown previously, using G-actin labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene-diamine, that Mg2+ induces a conformational change in monomeric G-actin as a consequence of binding to a tight divalent cation binding site (Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886). Using the same fluorescent probe, we show that, subsequent to the Mg2+-induced conformational change, cytochalasin D induces a fluorescence decrease. The data are consistent with a mechanism which proposes that, after Mg2+ binding, cytochalasin D binds and induces a second conformational change which results in overall tight binding of the cytochalasin. The initial binding of cytochalasin D to monomeric actin labeled with the fluorescent probe was found to be 200 microM, and the forward and reverse rate constants for the subsequent conformational change were 350 s-1 and 8 s-1, respectively, with an overall dissociation constant to the Mg2+-induced form of 4.6 microM. The conformational change does not occur in monomeric actin in the presence of Ca2+ rather than Mg2+, but Ca2+ competes with Mg2+ for the tight binding site on the G-actin molecule. Direct binding studies show that actin which has not been labeled with the fluorophore binds cytochalasin D more tightly. The conformational change induced by Mg2+ and cytochalasin D precedes the formation of an actin dimer.

Actins↗
PubMed1986

Effect of temperature on the mechanism of actin polymerization.

The rate of the Mg2+-induced polymerization of rabbit skeletal muscle G-actin has been measured as as function of temperature at pH 8 by using various concentrations of Mg2+, Ca2+, and G-actin. A polymerization mechanism similar to that proposed at this pH [Frieden, C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6513-6517] was found to fit the data from 10 to 35 degrees C. From the kinetic data, no evidence for actin filament fragmentation was found at any temperature. Dimer formation is the most temperature-sensitive step, with the ratio of forward and reverse rate constants changing 4 orders of magnitude from 10 to 35 degrees C. Over this temperature change, all other ratios of forward and reverse rate constants change 7-fold or less, and the critical concentration remains nearly constant. The reversible Mg2+-induced isomerization of G-actin monomer occurs to a greater extent with increasing temperature, measured either by using N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled actin or by simulation of the full-time course of the polymerization reaction. This is partially due to Mg2+ binding becoming tighter, and Ca2+ binding becoming weaker, with increasing temperature. Elongation rates from the filament-pointed end, determined by using actin nucleated by plasma gelsolin, show a temperature dependence slightly larger than that expected for a diffusion-limited reaction.

Actins↗
PubMed1986

Formation of actin dimers as studied by small angle neutron scattering.

Small angle neutron scattering has been used to study the dimensions of G-actin and the formation of low molecular weight actin oligomers under conditions where rapid polymerization does not take place. In the presence of 200 microM Ca2+, actin in solution consists of a single component with a radius of gyration (Rg) of 19.9 +/- 0.4 A, consistent with the known molecular dimensions of the G-actin molecule. In the presence of 50 microM Mg2+, however, formation of an actin species with a larger Rg occurs over a 4-h period. Multicomponent fits were tried and the data were best fit assuming two components, the monomer and a species with an Rg of 29 +/- 1 A. This latter value is consistent with the dimensions expected for certain actin dimers. The apparent dissociation constant for dimer formation is approximately 150 microM with forward and reverse rate constants of 6.0 X 10(-7) microM-1 s-1 and 8.8 X 10(-5) s-1, respectively. Kinetic fluorescence experiments show that the dimer formed in the presence of low levels of Mg2+ is a nonproductive complex which does not participate in the polymerization process. However, the addition of cytochalasin D to actin in the presence of 50 microM Mg2+ rapidly induces the formation of dimers, presumably related to cytochalasin's ability to nucleate actin polymerization.

Actins↗
PubMed1985

Differences in G-actin containing bound ATP or ADP: the Mg2+-induced conformational change requires ATP.

The role of adenosine 5'-triphosphate (ATP) in the Mg2+-induced conformational change of rabbit skeletal muscle G-actin has been investigated by comparing actin containing bound ADP with actin containing bound ATP. As previously described [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886], N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled G-actin containing ATP undergoes a time-dependent Mg2+-induced fluorescence change that reflects a conformational change in the actin. Addition of Mg2+ to labeled G-actin containing ADP gives no fluorescence change, suggesting that the conformational change does not occur. The fluorescence change can be restored on the addition of ATP. Examination of the time courses of these experiments suggests that ATP must replace ADP prior to the Mg2+-induced change. The Mg2+-induced polymerization of actin containing ADP is extraordinarily slow compared to that of actin containing ATP. The lack of the Mg2+-induced conformational change, which is an essential step in the Mg2+-induced polymerization, is probably the cause for the very slow polymerization of actin containing ADP. On the other hand, at 20 degrees C, at pH 8, and in 2 mM Mg2+, the elongation rate from the slow growing end of an actin filament, measured by using the protein brevin to block growth at the fast growing end, is only 4 times slower for actin containing ADP than for actin containing ATP.

Actins↗
PubMed1985

The binding of cytochalasin D to monomeric actin.

The binding of cytochalasin D to monomeric actin has been measured directly. In the presence of 200 microM Ca2+, actin binds cytochalasin D in a 1:1 molar ratio with a KD of 18 microM. After incubation with 250 microM Mg2+ for 10 minutes, actin binds cytochalasin D with a KD of 2.6 microM but with one mole of cytochalasin D per 2 moles of actin. This suggests that cytochalasin D induces dimerization of Mg2+-induced actin monomers.

Actins↗
PubMed1985

Substrate-induced hysteresis in the activity of Escherichia coli dihydrofolate reductase.

Full time course studies of the kinetic activity of Escherichia coli dihydrofolate reductase show that there is an increase in activity with time. The half-time for this hysteretic behavior is about 9 s. Preincubation of the enzyme with either of the substrates abolishes the lag and results in initial velocities which are 2-2.3-fold faster than those observed for the non-preincubated enzyme. The kinetic properties of the activated and nonactivated forms of the enzyme appear to be similar as measured by the full time course of the reaction. The results are consistent with observations for NADPH binding studies that the enzyme exists in two interconvertible forms, one of which is incapable of binding NADPH (Cayley, P. J., Dunn, S. M. J., and King, R. W. (1981) Biochemistry 20, 874-879).

Binding Sites↗
PubMed1985

Protein structural changes accompanying formation of enzymatic transition states: tryptophan environment in ground-state and transition-state analogue complexes of adenosine deaminase.

The accessibility of protein tryptophan fluorescence to the quenching agent acrylamide has been studied in adenosine deaminase and in binary complexes of the enzyme with ground-state or transition-state analogues. Although the enzyme contains three tryptophan residues, Stern-Volmer plots are linear with all the fluorescence quenchable at high acrylamide concentrations. Tryptophan fluorescence is less easily quenched in the binary complexes than in the free enzyme, indicating a decrease in the accessibility of these residues. The greatest decrease in accessibility is found for the transition-state analogue complexes. Although the affinities of the transition-state analogues studied span a range of 10(6), the Stern-Volmer constants of the complexes are the same within experimental error. Thus, as measured by this technique, changes in enzyme conformation accompanying formation of these complexes are similar for all transition-state analogues. Resonance energy transfer from tryptophan as donor to ligand as acceptor successfully explains the differing abilities of ligands to quench the enzyme's intrinsic fluorescence upon formation of complexes in the absence of acrylamide. On the basis of Forster distance calculations, it is likely that the residues partially quenched upon formation of transition-state analogue complexes are distant from the active site.

Adenosine Deaminase↗
PubMed1985

The use of the fluorescence photobleaching recovery technique to study the self-assembly of tubulin.

Fluorescently labeled microtubule-associated proteins or poly-L-lysine (13,000 MW) were prepared by reaction with fluorescein isothiocyanate. The labeled compounds were used as probes of the assembly of calf brain tubulin using fluorescence photobleaching recovery techniques which allow measurement of the diffusion coefficient and percentage mobility of the fluorescent probe. When unfractionated tubulin (defined as material containing tubulin and microtubule-associated proteins) was polymerized at room temperature or 37 degrees C, either probe could be incorporated into microtubules, since the observed diffusion coefficient (approximately 1.7 X 10(-8) cm2/s) was much slower than that for either probe free in solution. The microtubules formed in the presence of labeled microtubule-associated proteins were free to diffuse while those formed in the presence of labeled polylysine were partially immobilized. Thus the fluorescence photobleaching recovery technique can be used to measure crosslinking of microtubules as well as assembly or interactions with other structures. When unfractionated tubulin was incubated with labeled polylysine in the presence of Ca2+ at room temperature, the observed diffusion coefficient (approximately 5.1 X 10(-8) cm2/s) probably represents the formation of rings of tubulin. The effect of mild and vigorous shearing, of cholchicine, and of different Mg2+ concentrations on the properties of the system were examined.

Animals↗
PubMed1984

Interaction of microtubule-associated proteins with actin filaments. Studies using the fluorescence-photobleaching recovery technique.

The interaction of microtubule-associated proteins with actin filaments has been investigated by measuring the diffusion coefficient of either the filament or the microtubule-associated proteins. Experiments were performed using the technique of fluorescence photobleaching recovery with actin labeled with iodoacetamidotetramethyl rhodamine or microtubule-associated proteins labeled with iodoacetamidofluorescein. Actin filaments composed of pure rhodamine-labeled actin are not immobilized under a variety of conditions (Tait, J. F., and Frieden, C. (1982c) Biochemistry 21, 6046-6053). We find that addition of microtubule-associated proteins to rhodamine-labeled actin in a ratio as low as 1:1000 can cause immobilization, presumably cross-linking actin into a network of nondiffusible filaments. Immobilization occurs after polymerization is complete, suggesting either a length redistribution of actin filaments, a redistribution of the cross-links between filaments, or the slow addition of actin filaments to other filaments via the microtubule-associated protein. Experiments using fluorescein-labeled microtubule-associated proteins show that these proteins are bound to actin filaments as they are formed and that binding depended on actin concentration, indicating that there are a number of binding sites on the actin filaments. However, while the actin filaments become completely immobilized, the microtubule-associated proteins become only partially immobilized suggesting at least two different classes of binding affinities. The large peptide obtained from trypsin-treated fluorescein-labeled microtubule-associated proteins is not able to immobilize actin filaments since it does not bind to the filaments.

Actins↗
PubMed1984

Actin polymerization. The effect of brevin on filament size and rate of polymerization.

Fluorescent probes covalently bound to actin or to the actin binding protein, brevin, have been utilized to provide information about actin filaments formed in the presence of brevin as well as about the effect of brevin on the rate of polymerization. At actin to brevin ratios of 10:1 to 100:1, the observed diffusion coefficients of filaments, as measured by fluorescence photobleaching recovery using rhodamine-labeled actin or fluorescein-labeled brevin, are similar to those calculated from theoretical considerations for rigid rods. At lower brevin concentrations, the observed diffusion coefficients for actin filaments are lower than predicted, indicating that the filament structure is closer to that observed in the absence of brevin where filaments are immobilized due to interactions between them. The fluorescein-labeled brevin was found to be about as effective in influencing actin polymerization as unlabeled brevin. Using pyrene-labeled actin, we show that brevin binds 2 mol of monomeric actin. We conclude that at sufficiently high brevin concentration there is one brevin molecule per actin filament. From measurements of the initial rate of polymerization at 5.9 microM actin in the presence of brevin, we calculate both the apparent elongation rate constant and dissociation rate constant from one end (presumably the slow-growing end) of the actin filament. The former is highly dependent on Mg2+ concentration while the latter is not.

Actins↗
PubMed1984

Analysis of the interaction of rabbit skeletal muscle adenylate deaminase with myosin subfragments. A kinetically regulated system.

The interaction of rabbit skeletal muscle adenylate deaminase with myosin fragments (heavy meromyosin and subfragment-2) has been studied by analytical centrifugation, gel chromatography, and stopped flow light scattering. Formation of the complex is highly cooperative with respect to addition of two molecules of adenylate deaminase/molecule of myosin fragment to form a ternary complex. Ternary complex formation is also highly pH-dependent with less complex formed at higher pH values, and the pH dependence is steeper with heavy meromyosin than with subfragment-2. At pH 6.5, the dissociation constant for the heavy meromyosin-deaminase complex is approximately 1.2 X 10(-15) M2. Over the pH range 6.5-7.0, rate constants for the formation and dissociation of both the ternary and binary complexes of adenylate deaminase with heavy meromyosin have been determined. From analysis of the time course of stopped flow light scattering, the association steps are found to be extremely rapid, while the rate constant for dissociation of the first molecule of adenylate deaminase from the ternary complex is quite slow. This rate constant increases as the pH increased, but is sufficiently low that the interacting system does not equilibrate on the time scale of mass transport experiments (sedimentation velocity and gel chromatography), and thus displays apparent "slow" behavior. The kinetic regulatory properties of adenylate deaminase are influenced by heavy meromyosin and subfragment-2, particularly with respect to inhibition by GTP. The association and dissociation of adenylate deaminase and myosin fragments and the resultant changes in kinetic properties of the adenylate deaminase can markedly alter the time course of the enzymatic reaction. The time scale over which this interaction is modulated by changes in pH may have significance in the metabolism of exercising muscle.

AMP Deaminase↗