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C Frieden

Publications and source records attributed to C Frieden.

At least 91 records · Page 5Linked to original sources
PubMed1983

Polymerization of actin and actin-like systems: evaluation of the time course of polymerization in relation to the mechanism.

The time course of protein polymerization of the nucleation--elongation type is examined by using a general computer-simulation solution. For a simple nucleation--elongation scheme, it is shown that the half-time of polymerization is not necessarily a good measure of the nucleus size as has been previously suggested [Oosawa, F., & Kasai, M. (1962) J. Mol. Biol. 4, 10-21] since, depending on the mechanism, the apparent nucleus size, measured by a ratio of half-times at two actin concentrations, may be either larger or smaller than the real size. Steady-state equations developed by Wegner and Engel [Wegner, A., & Engel, J. (1975) Biophys. Chem. 3, 215-225] present a good description of the time course of polymerization although they are somewhat inflexible with regard to allowing for different mechanisms. Some of the assumptions implicit in the development of these equations are discussed in terms of the effect of changing individual rate constants or dissociation constants on the time course of polymerization. In addition, these steady-state equations have been expanded to include the consequences of a reversible first-order conformational change prior to polymerization. It is shown that a conformational change as a prerequisite to polymerization lengthens the lag time of polymerization and, depending on the conditions, may slow the rate of polymerization. The question of fragmentation and of reannealling is examined, and it is noted that simple relationships to describe these processes may not be possible.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1983

Analysis of numerical methods for computer simulation of kinetic processes: development of KINSIM--a flexible, portable system.

A flexible and convenient computational method for the simulation of kinetic progress curves has been developed. A mechanism is represented in conventional chemical format with either kinetic or rapid equilibrium steps separating chemical species. A table describing the differential equations of the mechanism is generated and a direct numerical integration is performed. The same program can be used to simulate any number of mechanisms. The user may interactively set kinetic parameters to seek the optimal fit for a set of experiments, as determined by graphical superimposition of simulated curves with experimental data. Standard error analysis and automatic optimization may also be included. The program is computationally efficient and its interactive nature makes it a good teaching tool. The source code is written in FORTRAN IV and adheres closely with the ANSI 1966 standard, so as to make it maximally portable and machine independent.

Chemistry Techniques, Analytical↗
PubMed1983

Preparation, purification and properties of a crosslinked trimer of G-actin.

Phenylenebismaleimide has been used to form crosslinks between actin monomers [Knight, P. and Offer, G. (1978) Biochem. J. 175, 1023-1032]. We have purified a trimer of actin monomers as well as a dimer and a mixture of higher molecular weight oligomers. The trimer is much more effective than the dimer in enhancing the rate of polymerization while higher oligomers do not appear to be any more effective than the trimer. A lag in the polymerization process, as measured fluorescence enhancement of trace pyrene-actin, still occurs in the presence of trimers serving as the nuclei, suggesting that the mechanism for polymerization is more complex than nucleation followed by elongation.

Actins↗
PubMed1983

Polymerization of actin: mechanism of the Mg2+-induced process at pH 8 and 20 degrees C.

A detailed mechanism that fully accounts for the Mg2+-induced polymerization of actin in the presence or absence of Ca2+ at 20 degrees C and pH 8 is presented. In the absence of Ca2+, the mechanism of the Mg2+-induced polymerization is as follows: Mg2+ binds to a metal-binding site on G-actin and induces a conformational change, which is required for eventual polymerization. The overall dissociation constant for this binding is about 30 microM. This actin species then binds a second molecule of Mg2+ (Kd = 5 mM), which yields a species capable of polymerization. Dimer formation from this monomeric species is quite unfavorable, but trimer formation from dimer and monomer is much more favorable. The trimer may then elongate to give filaments. Ca2+, when present, binds at the same site as the tightly bound Mg2+ and must be displaced by Mg2+ before the conformational change can occur. The rate and dissociation constants for tight binding of Ca2+ and Mg2+ and for the conformational change are consistent with those observed previously by using a fluorescently labeled G-actin. With the mechanism proposed, it is possible to fit the full time course of polymerization over a wide range of actin concentrations, Mg2+ concentrations, and Ca2+ concentrations.

Actins↗
PubMed1982

Chemical modification of actin. Acceleration of polymerization and reduction of network formation by reaction with N-ethylmaleimide, (iodoacetamido)tetramethylrhodamine, or 7-chloro-4-nitro-2,1,3-benzoxadiazole.

We examined the properties of rabbit skeletal muscle actin labeled at Cys-373 with N-ethylmaleimide or with (iodoacetamido)tetramethylrhodamine, and of N-ethylmaleimide-actin further modified with 7-chloro-4-nitro-2,1,3-benzoxadiazole (which primarily labels Lys-372). All three derivatives polymerize more rapidly than unlabeled actin. As measured by fluorescence photobleaching recovery and low-shear viscometry, all three also show a lower extent of network formation relative to native actin. N-Ethylmaleimide has a much smaller effect on the rate of polymerization and on network formation than do the other two derivatives. We suggest that chemical modification of actin with these compounds may stabilize nuclei, accounting for the acceleration of polymerization. Stabilization of nuclei also reduces the average filament length at equilibrium, thereby reducing the extent of network formation. We note a parallel between these results and the effects that cytochalasin and capping proteins have on the polymerization of actin.

4-Chloro-7-nitrobenzofurazan↗
PubMed1982

The Mg2+-induced conformational change in rabbit skeletal muscle G-actin.

The divalent cation Mg2+ binds specifically and tightly to a specific site on rabbit muscle G-actin, and it has been shown previously that such binding involves a conformational change in the monomeric actin as measured by a time-dependent change in the fluorescence of G-actin labeled with the fluorescent probe N-iodoacetyl N'-(5-sulfo-1-naphthyl)ethylenediamine (Frieden, C., Lieberman, D., and Gilbert, H. R. (1980) J. Biol. Chem. 255, 8991-8993). The characteristics of specific Mg2+-induced conformational changes using this labeled G-actin have been examined. The data are consistent with an initial poor binding of Mg2+ followed by an isomerization process resulting in tight binding. Mg2+ and Ca2+ compete for this site and the isomerization induced by Mg2+ can be reversed with high Ca2+ concentrations. The overall dissociation constant for Mg2+ binding is highly pH-sensitive, becoming larger with decreasing pH, implying that ionizable groups may control Mg2+ and Ca2+ binding and that these cations bind preferentially to the unprotonated form. Mg2+ binding to this site increases the off rate constant of the tightly bound ATP relative to Ca2+ by about 6-fold. It is suggested that Mg2+ binding to this site may be related to hydrolysis of the ATP.

Actins↗
PubMed1980

A fluorescent probe for conformational changes in skeletal muscle G-actin.

Actin from rabbit skeletal muscle has been modified with the fluorescent label N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS). Under conditions where the actin is in the unpolymerized form (G-actin), the addition of Mg2+ or KCl results in enhancement of the fluorescence. Titration of the labeled G-actin with Mg2+ at varying concentrations of CaCl2 gives, by extrapolation, a value for the dissociation constant for Mg2+ of 35 microM in the absence of Ca2+ and a calculated value of 10 microM for Ca2+ in the absence of Mg2+. The two metal ions compete with each other. The fluorescence enhancement induced by Mg2+ is reversed by the addition of Ca2+ and both processes are time-dependent, indicating a reversible conformational change of G-actin as a consequence of addition of divalent metal. KCl also enhances the fluorescence of the labeled G-actin but does not appear to compete with the divalent metal ion. The enhancement of the fluorescence is very rapid and any conformational change induced by KCl is probably different from that induced by divalent metal ions. Finally, it is shown that loss of fluorescence of the labeled G-actin may be associated with inactivation of the actin.

Actins↗
PubMed1980

Adenylate deaminase binding to synthetic thick filaments of myosin.

Adenylate deaminase (AMP deaminase; AMP aminohydrolase, EC 3.5.4.6), a tetrameric enzyme found at particularly high concentrations in skeletal muscle, has previously been shown to bind strongly to the subfragment-2-portion of myosin in vitro and to the ends of the A band in vivo. It is shown here that when adenylate deaminase is dialyzed with skeletal myosin during formation of synthetic filaments at pH 7.0 it decorates the filament at 14.3-nm intervals, presumably in the region of exposed backbone between crossbridge levels. Optical diffraction of the aggregates reveals both enhancement of reflections arising from underlying myosin organization and other reflections arising from adenylate deaminase arrangement on the filament surface. Adenylate deaminase can thus be used as a specific label in the study of myosin presence and organization.

AMP Deaminase↗
PubMed1979

Crystallographic studies of glutamate dehydrogenase. Preliminary crystal data.

Native and pyridoxal phosphate modified rat liver glutamate dehydrogenase crystals have been obtained and used for a preliminary x-ray crystallographic analysis. The space group is P6222 (P6422) having unit cell dimensions a = b = 101 A, c = 724 A and gamma = 120 A. The unit cell contains 36 subunits (six hexameric molecules) of molecular weight 56,000 and there is one half-molecule, i.e. three subunits, in the asymmetric unit. Packing considerations suggest that the glutamate dehydrogenase molecule has the point group symmetry 32 and that each subunit can be represented as a particle with approximate dimensions of 45 x 45 x 60 A.

Animals↗