salicylates and 4-iodoacetamidosalicylic-acid

Equilibrium measurements of the rate of binding of caldesmon and myosin S1 to actin-tropomyosin from different laboratories have yielded different results and have led to different models of caldesmon function. An alternate approach to answering these questions is to study the kinetics of binding of both caldesmon and S1 to actin. We observed that caldesmon decreased the rate of binding of S1 to actin in a concentration-dependent manner. The inhibition of the rate of S1 binding was enhanced by tropomyosin, but the effect of tropomyosin on the binding was small. Premixing actin with S1 reduced the amplitude (extent) of caldesmon binding in proportion to the fraction of actin that contained bound S1, but the rate of binding of caldesmon to free sites was not greatly altered. No evidence for a stable caldesmon-actin-tropomyosin-S1 complex was observed, although S1 did apparently bind to gaps between caldesmon molecules. These results indicate that experiments involving caldesmon, actin, tropomyosin, and myosin are inherently complex. When the concentration of either S1 or caldesmon is varied, the amount of the other component bound to actin-tropomyosin cannot be assumed to remain fixed. The results are not readily explained by a mechanism in which caldesmon acts only by stabilizing an inactive state of actin-tropomyosin. The results support regulatory mechanisms that involve changes in the actin-S1 interaction.

Topics: Actins; Animals; Calmodulin-Binding Proteins; Fluorescein; Fluoresceins; Fluorescent Dyes; Iodoacetamide; Kinetics; Light; Myosin Subfragments; Oxadiazoles; Protein Binding; Pyrenes; Rabbits; Salicylates; Scattering, Radiation; Spectrometry, Fluorescence; Tropomyosin; Turkeys

The distance between the catalytic site on bovine liver glutamate dehydrogenase labeled with 4-(iodoacetamido)salicylic acid (ISA) and the adenosine 5'-diphosphate (ADP) activatory site occupied by the analogue 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-diphosphate (TNP-ADP) was evaluated by energy transfer. Native enzyme and enzyme containing about 1 mol of acetamidosalicylate/mol of subunit bind about 0.5 mol of TNP-ADP/mol of subunit, and TNP-ADP competes for binding with ADP to native and modified enzyme, indicating that the analogue is a satisfactory probe of the ADP site. From the quenching of acetamidosalicylate donor fluorescence upon addition of TNP-ADP, an average distance of 33 A was determined between the catalytic and ADP sites. The fluorescent nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]-2-aza-1,N6-ethenoadenosine (5'-FSBa epsilon A) reacts covalently with glutamate dehydrogenase to about 1 mol/peptide chain. As compared to native enzyme, the SBa epsilon A-enzyme exhibits decreased sensitivity to GTP inhibition but retains its catalytic activity as well as its ability to be activated by ADP and inhibited by high concentrations of NADH. Complete protection against decreased sensitivity to GTP inhibition is provided by GTP in the presence of NADH. It is concluded that 5'-FSBa epsilon A modifies a GTP site on glutamate dehydrogenase. The distance of 23 A between the catalytic site labeled with ISA and a GTP site labeled with 5'-FSBa epsilon A was measured from the quenching of salicylate donor fluorescence in the presence of the SBa epsilon A acceptor on a doubly labeled enzyme. The average distance between the ADP and GTP sites was previously measured as 18 A [Jacobson, M. A., & Colman, R. F. (1983) Biochemistry 22, 4247-4257], indicating that the regulatory sites of glutamate dehydrogenase are closer to each other than to the catalytic site.

Topics: Adenosine; Adenosine Diphosphate; Allosteric Site; Amino Acids; Animals; Binding Sites; Cattle; Energy Transfer; Enzyme Activation; Fluorescent Dyes; Glutamate Dehydrogenase; Guanosine Triphosphate; Iodoacetamide; Kinetics; Liver; Salicylates; Spectrometry, Fluorescence

Topics: Animals; Carboxy-Lyases; Iodoacetamide; Isocitrate Dehydrogenase; Kinetics; Ligands; Myocardium; Oxaloacetates; Salicylates; Swine

Topics: Adenosine Diphosphate; Affinity Labels; Animals; In Vitro Techniques; Iodoacetamide; Kinetics; Muscles; Phosphoenolpyruvate; Pyruvate Kinase; Rabbits; Salicylates; Salicylic Acid

1. Bovine, porcine and chicken liver glutamate dehydrogenases were irreversibly inhibited by a tenfold excess of radioactive 4-iodoacetamidosalicylic acid at pH7.5. 2. Inhibition was accompanied by the covalent incorporation of 1.1 mol of labelled inhibitor/mol of polypeptide chain. Acid hydrolysis yielded N(epsilon)-carboxymethyl-lysine as the sole labelled amino acid. No labelled S-carboxymethylcysteine was recovered from the bovine or porcine enzymes. 3. The labelled bovine enzyme was hydrolysed with trypsin. The radioactivity was found at lysine-126 in a peptide comprising residues 119-130 of the sequence. 4. The amino acid compositions of the tryptic peptides containing labelled lysine from the porcine and chicken enzymes were similar to that of the bovine peptide.

Topics: Amides; Amino Acid Sequence; Amino Acids; Animals; Binding Sites; Carbon Isotopes; Cattle; Chickens; Chromatography, Gel; Electrophoresis, Paper; Glutamate Dehydrogenase; Iodoacetamide; Iodoacetates; Kinetics; Liver; Lysine; Peptides; Protein Binding; Salicylates; Species Specificity; Spectrophotometry, Ultraviolet; Swine