formycin-diphosphate and formycin-triphosphate

The kinetics of nucleotide binding to pyruvate carboxylase have been studied by measuring the fluorescence changes that occur on the binding and release of FTP and FDP, which are fluorescent formycin analogues of ATP and ADP. The rate constants and equilibrium binding constants for both MgFTP and MgFDP binding to pyruvate carboxylase have been determined. From the kinetics of displacement of MgFTP by MgATP and binding of MgFTP in the presence of MgATP, the rate constants of MgATP binding were estimated. A slow component to the fluorescence changes was seen to occur after the initial rapid, bimolecular binding step, when formycin nucleotides were mixed with the enzyme. HCO3- and pyruvate were shown to quench the fluorescence of enzyme-bound MgFTP, but did not affect the affinity of the enzyme for the nucleotide. Acetyl CoA reduced the affinity of the enzyme for both MgFDP and MgFTP by about 3-fold by decreasing the association rate constants (by 25%) and increasing the dissociation rate constants (by 2-fold). In the absence of Mg2+ a very rapid component to FTP binding was observed that was complete within about 3 ms, but no fast component was observed comparable to that seen in the presence of 4.5 mM MgCl2. Increasing the [Mg2+] gradually abolished this very fast component of the binding, while the amplitude of the fast component increased, although the rate constant for this component did not appear to be strongly dependent on [Mg2+]. The rate constants of the slow component of Mg.formycin nucleotide binding did not appear to be dependent on nucleotide concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetyl Coenzyme A; Adenosine Triphosphate; Animals; Bicarbonates; Binding Sites; Binding, Competitive; Chickens; Fluorescent Dyes; Formycins; In Vitro Techniques; Kinetics; Liver; Magnesium; Models, Biological; Pyruvate Carboxylase; Pyruvates; Pyruvic Acid; Ribonucleotides; Spectrometry, Fluorescence

The purpose of this study was to determine the quantitative relationship between the number of myosin molecules that increase their ATPase activity and the degree of myosin light chain phosphorylation in smooth muscle. Single turnover experiments on the nucleotide bound to myosin were performed in the permeabilized rabbit portal vein. In the resting muscle, the rate of exchange of bound nucleoside diphosphate was biphasic and complete in approximately 30 min. When approximately 80% of the myosin light chain was thiophosphorylated, the nucleoside diphosphate exchange occurred at a much faster rate and was almost complete in 2 min. Thiophosphorylation of 10% of the myosin light chains caused an increase in the rate of ADP exchange from much more than 10% of the myosin subfragment-1. Less than 20% thiophosphorylation of the total myosin light chains resulted in the maximum increase in ADP exchanged in 2 min. It appears that a small degree of myosin light chain phosphorylation cooperatively turns on the maximum number of myosin molecules. Interestingly, even though less than 20% thiophosphorylation of the myosin light chain caused the maximum exchange of ADP within 2 min, higher degrees of thiophosphorylation were associated with further increases in the ATPase rates. We conclude that a small degree of myosin light chain thiophosphorylation cooperatively activates the maximum number of myosin molecules, and a higher degree of thiophosphorylation makes the myosin cycle faster. A kinetic model is proposed in which the rate constant for attachment of unphosphorylated cross bridges varies as a function of myosin light chain phosphorylation.

Topics: Adenosine Diphosphate; Adenosine Triphosphatases; Animals; Enzyme Activation; Female; Fluoresceins; Formycins; Muscle, Smooth; Myosins; Nucleoside Diphosphate Sugars; Nucleosides; Permeability; Phosphates; Phosphorylation; Rabbits; Ribonucleotides

The kinetics of interaction of formycin nucleotides with scallop myosin subfragments were investigated by exploiting the fluorescence signal of the ligand. Formycin triphosphate gives a 5-fold enhancement of the emission intensity on binding to heavy meromyosin, and the profile indicates that the kinetics of binding are Ca2+-insensitive. In contrast, the subsequent product-release steps show a marked degree of regulation by Ca2+. In the absence of Ca2+ formycin triphosphate turnover by the unregulated and the regulated heavy meromyosin fractions are clearly resolved, the latter showing a fluorescence decay rate of 0.002 s-1, corresponding to the Pi-release step. In the presence of Ca2+ this step is activated 50-fold. Formycin diphosphate release is also regulated by Ca2+, being activated from 0.008 s-1 to 5 s-1. In contrast with protein tryptophan fluorescence [Jackson & Bagshaw (1988) Biochem. J. 251, 515-526], formycin fluorescence is sensitive to conformational changes that occur subsequent to the binding step and demonstrate, directly, an effect of Ca2+ on both forward and reverse rate constants. Apart from a decrease in the apparent second-order association rate constants, formycin derivatives appear to mimic adenosine nucleotides closely in their interaction with scallop heavy meromyosin and provide a spectroscopic handle on steps that are optically silent with respect to protein fluorescence. A novel mechanism is discussed in which regulation of the formycin triphosphate activity by Ca2+ involves kinetic trapping of product complexes.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Antibiotics, Antineoplastic; Calcium; Formycins; Kinetics; Mollusca; Myosin Subfragments; Ribonucleotides; Sodium Chloride; Spectrometry, Fluorescence