Phosphatase binding

Zinc can be removed165 and the resulting apoalkaline phosphatase binds no phosphate with a dissociation constant smaller than 5 x 10-5 M. Upon addition of metal ions to the apoenzyme, a high affinity binding site may be reactivated. Two metals are needed to form one highly specific phosphate binding site. 

P-myosin is higher than that of the isolated catalytic subunit. Thus dissociation of the trimeric phosphatase could be a regulatory mechanism. The factors involved in dissociation of PPlc are not established, although there is the suggestion that arachidonic acid is implicated. It is possible that dissociation of the trimeric phosphatase is the end result of the G-protein-linked inhibition of phosphatase activity, observed in several systems. Another possibility is that the trimeric phosphatase binds only to phosphorylated myosin and has a lower affinity for the dephosphory-lated form. Whatever the scenario imagined, it is clear that much remains to be learned about regulation of myosin phosphatase activity. 

The preseiice of iron in these hydrolases came as something of a surprise since, with few exceptions iron-requiring enzymes function primarily in electron-transfer reactions. Even more curious was the demonstration that each molecule of the purple add phosphatases binds two iron atoms in a binuclear cluster As is now appredated, the redox activity of iron in the purple add phosphatases plays a central role in regulating their enzymic activities, while the binuclear configuration confers their unique spectroscopic properties. In this review we will consider what has been learned of the chemistry, spectroscopic features, and enzymic properties of the purple add phosphatases, emphasizing the problems and paradoxes still evading explanation. An historical perspective is provided in Ref. 13. 

The ability to identify and quantify cyanobacterial toxins in animal and human clinical material following (suspected) intoxications or illnesses associated with contact with toxic cyanobacteria is an increasing requirement. The recoveries of anatoxin-a from animal stomach material and of microcystins from sheep rumen contents are relatively straightforward. However, the recovery of microcystin from liver and tissue samples cannot be expected to be complete without the application of proteolytic digestion and extraction procedures. This is likely because microcystins bind covalently to a cysteine residue in protein phosphatase. Unless an effective procedure is applied for the extraction of covalently bound microcystins (and nodiilarins), then a negative result in analysis cannot be taken to indicate the absence of toxins in clinical specimens. Furthermore, any positive result may be an underestimate of the true amount of microcystin in the material and would only represent free toxin, not bound to the protein phosphatases. Optimized procedures for the extraction of bound microcystins and nodiilarins from organ and tissue samples are needed. 

Smooth muscle contractions are subject to the actions of hormones and related agents. As shown in Figure 17.32, binding of the hormone epinephrine to smooth muscle receptors activates an intracellular adenylyl cyclase reaction that produces cyclic AMP (cAMP). The cAMP serves to activate a protein kinase that phosphorylates the myosin light chain kinase. The phosphorylated MLCK has a lower affinity for the Ca -calmodulin complex and thus is physiologically inactive. Reversal of this inactivation occurs via myosin light chain kinase phosphatase. 

R. W. MacKintosh, K. N. Dalby, D. G. Campbell, P. T. W. Cohen, P. Cohen and C. MacKintosh, The cyanobacterial toxin microcystin binds covalently to cysteine-273 on protein phosphatase 1 , FEBS Lett. 371 236-240 (1995). 

A secondary metabolite produced by Tolypocladium inflation. This fungus was initially isolated in a soil sample collected in Norway. Cyclosporin A is a cyclic undecapeptide. Inside cells, cyclosporine A binds its immunophillin receptor known as cyclophillin. Like the FK506-FKBP12 complex, cyclosporin A-cyclophillin binds and inhibits the protein phosphatase calcineurin. 

Motor proteins move along MTs in an ATP-dependent manner. Members of the superfamily of kinesin motors move only to the plus ends and dynein motors only to the minus ends. The respective motor domains are linked via adaptor proteins to their cargoes. The binding activity of the motors to MTs is regulated by kinases and phosphatases. When motors are immobilized at their cargo-binding area, they can move MTs. 

FKBP12 is a member of immunophilin family that has prolyl isomerase activity and is related to the cyclophi-lins in function. FKBP12 binds immunosuppressant molecule FK506 (tacrolimus). The FBKP-FK506 complex inhibits calcineurin, a protein phosphatase, thus blocking signal transduction in the T-lymphocyte... 

Cyclosporine A (CsA) is a water-insoluble cyclic peptide from a fungus composed of 11 amino acids. CsA binds to its cytosolic receptor cyclophilin. The CsA/cyclophilin complex reduces the activity of the protein phosphatase calcineurin. Inhibition of this enzyme activity interrupts antigen receptor-induced activation and translocation of the transcription factor NEAT to the nucleus which is essential for the induction of cytokine synthesis in T-lymphocytes. 

STAT signaling occurs within minutes following activation but also underlies rapid deactivation by cytoplasmic phosphatases or antagonistic factors such as members of the SOCS-proteins themselves induced by activated STATs. Another group of proteins called PIAS (proteins that inhibit active STATs) inhibit binding of activated STATs to their target DNA sequences... 

STAT binding site in the promoter region of cytokine-responsive genes. It is nonameric palindrome with relaxed sequence specificity.) sites, to regulate gene expression. Tyrosine phosphatases located in the nucleus then dephosphorylate STAT molecules (Fig. 1). 

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