Calmodulins dissociation

Taylor A naive question on the one hand your inability to wash out the calmodulin made it look like it was held by high-affinity binding, yet presumably the TFP, which seemed to work rather quickly, depends on calmodulin dissociating to prevent it from rebinding. Does this imply it is dissociating quite quickly ... 

Relaxation follows the drop in cytosolic calcium below the activation threshold, which initiates release of calcium from calmodulin, whereupon calmodulin dissociates from the kinase catalytic subunit. This results in loss of myosin light chain kinase activity. Under these conditions, the dephosphorylation of myosin light chains will be the predominant reaction. Consequently, actin and myosin will no longer interact and the muscle relaxes. 

McCabe, TJ., Fulton, D., Roman, LJ., and Sessa, W.C. (2(XX)). Enhanced electron flux and reduced calmodulin dissociation may explain calcium-independent eNOS activation by phosphorylation. J. Biol. Chem. 275(9), 6123-6128. 

Myosin-I molecules have several IQ sequences on or near the head and have light chains associated with them (Cheney and Mooseker, 1992 Cheney et al., 1993). Frequently, the light chains appear to be calmodulin molecules and some myosin-I molecules can bind three to four molecules of calmodulin at one time. Brush-border and adrenal myosin-I also bind calmodulin. Acanthamoeba myosin-I has a light chain that can be removed, in vitro, without adversely affecting the ATPase activity or the heavy chain phosphorylation (Korn and Hammer, 1988). The role of these calmodulin molecules in regulating myosin-I is complex and poorly understood. One possibility is that the calmodulin molecules dissociate from the heavy chains when calcium binds to the calmodulin, thereby imparting greater flexibility to the head of the myosin-I molecules. 

It has been shown that the activity of NO synthases is regulated by cofactors calcium binding protein calmodulin and tetrahydrobiopterin (H4B). Abu-Soud et al. [149] have studied the effect of H4B on the activity of neuronal nNOS I, using the isolated heme-containing oxygenase domain nNOSoxy. It was found that nNOSoxy rapidly formed an oxygenated complex in the reaction with dioxygen, which dissociated to produce superoxide (Reaction (6)) ... 

Most protein serine-threonine kinases undergo autophosphorylation. The autophosphorylation of most protein kinases is associated with an increase in kinase activity [4, 10]. In some instances, such as with the RII subunit of PKA, autophosphorylation represents a positive feedback mechanism for kinase activation, in this case by enhancing the rate of dissociation of the RII and C subunits. In the case of CaMKII, autophosphorylation causes the catalytic activity of the enzyme to become independent of Ca2+ and calmodulin. This means that the enzyme, activated originally in response to elevated cellular Ca2+, remains active after Ca2+ concentrations have returned to baseline. By this mechanism, neurotransmitters that activate CaMKII can produce relatively long-lived alterations in neuronal function. In other instances, such as with the receptor-associated protein tyrosine kinases (discussed in Ch. 24), autophosphorylation is an obligatory step in the sequence of molecular events through which those kinases are activated and produce physiological effects. 

Nelson Yes, it is different, because you remove calmodulin. Calmodulin cannot be readily dissociated from the SK channel. 

Walsh I would say, however, that, while you can dissociate calmodulin from MLCK with TFP, you probably can t get it off under physiological conditions. 

Sorensen, S. D., Macek, T. A., Cai, Z Saugstad, J. A., and Conn, P. J. (2002) Dissociation of protein kinase-mediated regulation of metabotropic glutamate receptor 7 (mGluR7) interactions with calmodulin and regulation of mGluR7 function. Mol. Pharmacol. 61,1303-1312. 

Any reactant that remains unchanged (or is rapidly regenerated) before and after (but not necessarily during) an enzymic reaction. These could include a metal ion, an activator (e.g., calmodulin), a dissociable regulatory... 

Fig. 7.14. Regulation of CaM kinase II. Scheme of regulation of CaM kinase II by Ca Vcalmodu-lin and by autophosphorylation. CaM kinase II is inactive in the unphosphorylated form and in the absence of Ca calmodulin. Binding of Ca Vcalmodulin activates the kinase for phosphorylation of protein substrates. In the process, autophosphorylation takes place at a conserved Thr residue that stabilizes the active state of the enzyme. In this state, significant residual activity is still present after dissociation of Ca Vcalmodulin and the enzyme remains in an active state for a longer time after the Ca signal has died away. The active state is only terminated when the activating phosphate residue is cleaved off by a protein phosphatase.

The affinity for Ca Vcahnodulin is increased by close to three orders of magnitude. Ca /calmodulin only dissociates very slowly from this high affinity complex. The activated state is thus preserved over a longer period of time. Even when the Ca signal has died away and the Ca concentration has fallen to a level of lO M, the enzyme... 

After dissociation of calmodulin, the phosphorylated enzyme stiU has 20—80 % of the activity of the Ca Vcalmodulin bound form. This ensures that significant activity remains after the Ca /calmodulin signal has died away. In the phosphorylated form, CaM kinase is in an autonomous, Ca Vcalmodulin independent state. This is only terminated when phosphatases cleave off the activating phosphate residue and thus lead the enzyme back into the inactive state. 

Fig. 7. (A) Aligned, partial sequences of a number of calmodulin-binding peptides. The boxes indicate residues that are generally occupied by apolar residues. Reported dissociation constants for interaction with calmodulin are given on the right. LK2, A mode peptide VIP, vasoactive intestinal peptide GIP, gastric inhibitory peptide. (B) The mean hydropho-bicities for the residues at a given position were plotted versus their position in the aligned sequence. The horizontal bar indicates the period of an a helix. From Cox et al. (1985).

Reversible Ligand Binding The protein calcineurin binds to the protein calmodulin with an association rate of 8.9 X 10s M-1s-1 and an overall dissociation constant, Kd, of 10 nM. Calculate the dissociation rate, kd, including appropriate units. 

Surface plastnon resonance Association constant Dissociation constant Hexaethylene glycol spacer Sambucus nigra agglutinin High-mobility-group transcriptional factor Soluble green fluorescent protein Calmodulin... 

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