Phosphatases inactivation

Alkaline phosphomonoesterase (EC 3.1.3.1). The existence of a phosphatase in milk was first recognized in 1925. Subsequently characterized as an alkaline phosphatase, it became significant when it was shown that the time-temperature combinations required for the thermal inactivation of alkaline phosphatase were slightly more severe than those required to destroy Mycobacterium tuberculosis, then the target micro-organism for pasteurization. The enzyme is readily assayed, and a test procedure based on alkaline phosphatase inactivation was developed for routine quality control of milk pasteurization. Several major modifications of the test have been developed. The usual substrates are phenyl phosphate, p-nitrophenyl-phosphate or phenolphthalein phosphate which are hydrolysed to inorganic phosphate and phenol, p-nitrophenol or phenolphthalein, respectively ... 

Other signaling pathways activate Rho kinase, which can stimulate myosin activity in two ways. First, Rho kinase can phosphorylate myosin LC phosphatase (see Figure 19-25b), thereby Inhibiting Its activity. With the phosphatase inactivated, the level of myosin LC phosphorylation and thus myosin activity Increase. In addition, Rho kinase direcdy activates myosin by phosphorylating the regulatory light chain. Note that Ca plays no role in the regulation of myosin activity by Rho kinase. 

A phosphatase-inactivating system is found in the soluble fraction of liver (Beaufay et al., 1954). Such inactivation may be inhibited by epinephrine in excess of the substrate and by chelating agents such as Versene. The products of reaction tend to inhibit slightly. In this respect phosphate has a more pronounced effect than does glucose. 

Rapid inactivation of added lincomycin was found to result from the growth of Streptomjces rochei in a synthetic medium the antibiotic was converted into lincomycin 3-phosphate [23670-99-7] (5, R = CH3, R = PO3H2, R = SCH3), C2gH33N202PS, readily cleaved back to the antibiotic upon treating with alkaline phosphatase (53). 

Dephosphorylation of glycogen phosphorylase is carried out by phospho-protein phosphatase 1. The action of phosphoprotein phosphatase 1 inactivates glycogen phosphorylase. 

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. 

Phosphorylation by cAMP-dependent protein kinases inactivates the reductase. This inactivation can be reversed by two specific phosphatases (Figure 25.33). 

A phosphatase that dephosphoiylates a conserved carboxyl-terminal site on PKC and Akt, thus inactivating these kinases and terminating their signaling pathways. 

For in vitro studies there are a number of compounds available to block protein phosphatase activity. Phosphate buffers inactivate all of these enzymes. Several naturally occurring toxins are potent inhibitors of PPPs, e.g., okadaic acid or microcystin, and are frequently used tools. PPM and PTP family members are not affected by these toxins. Vanadate containing solutions are competitive inhibitors of PTPs, pervanadate is an irreversible inhibitor of PTPs. 

A subfamily of Rho proteins, the Rnd family of small GTPases, are always GTP-bound and seem to be regulated by expression and localization rather than by nucleotide exchange and hydrolysis. Many Rho GTPase effectors have been identified, including protein and lipid kinases, phospholipase D and numerous adaptor proteins. One of the best characterized effector of RhoA is Rho kinase, which phosphorylates and inactivates myosin phosphatase thereby RhoA causes activation of actomyosin complexes. Rho proteins are preferred targets of bacterial protein toxins ( bacterial toxins). 

Figure 2. Mechanism of PDH. The three different subunits of the PDH complex in the mitochondrial matrix (E, pyruvate decarboxylase E2, dihydrolipoamide acyltrans-ferase Ej, dihydrolipoamide dehydrogenase) catalyze the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2. E, decarboxylates pyruvate and transfers the acetyl-group to lipoamide. Lipoamide is linked to the group of a lysine residue to E2 to form a flexible chain which rotates between the active sites of E, E2, and E3. E2 then transfers the acetyl-group from lipoamide to CoASH leaving the lipoamide in the reduced form. This in turn is oxidized by E3, which is an NAD-dependent (low potential) flavoprotein, completing the catalytic cycle. PDH activity is controlled in two ways by product inhibition by NADH and acetyl-CoA formed from pyruvate (or by P-oxidation), and by inactivation by phosphorylation of Ej by a specific ATP-de-pendent protein kinase associated with the complex, or activation by dephosphorylation by a specific phosphoprotein phosphatase. The phosphatase is activated by increases in the concentration of Ca in the matrix. The combination of insulin with its cell surface receptor activates PDH by activating the phosphatase by an unknown mechanism.

Phosphorylation of HSFl is rapid, and one or more protein kinases are likely to be activated upon HS. An alternative explanation is that heat inactivates a phosphatase which is more active than the HSF kinase at 37 °C. Inactivation of the phosphatase by heat would allow the presumptive heat stable HSF kinase activity to predominate, thus increasing the phosphorylation of HSFl. 

Both phosphorylase a and phosphorylase kinase a are dephosphorylated and inactivated by protein phos-phatase-1. Protein phosphatase-1 is inhibited by a protein, inhibitor-1, which is active only after it has been phosphorylated by cAMP-dependent protein kinase. Thus, cAMP controls both the activation and inactivation of phosphorylase (Figure 18-6). Insulin reinforces this effect by inhibiting the activation of phosphorylase b. It does this indirectly by increasing uptake of glucose, leading to increased formation of glucose 6-phosphate, which is an inhibitor of phosphorylase kinase. 

Definition of Ej and E2 eonformations of the a subunit of Na,K-ATPase involves identification of cleavage points in the protein as well as association of cleavage with different rates of inactivation of Na,K-ATPase and K-phosphatase activities [104,105]. In the Ei form of Na,K-ATPase the cleavage patterns of the two serine proteases are clearly distinct. Chymotrypsin cleaves at Leu (C3), Fig. 3A, and both Na,K-ATPase and K-phosphatase are inactivated in a monoexponential pattern [33,106]. Trypsin cleaves the E form rapidly at Lys ° (T2) and more slowly at Arg (T3) to produce the characteristie biphasic pattern of inactivation. Localization of these splits was determined by sequencing N-termini of fragments after isolation on high resolution gel filtration columns [107]. 

The E2 form is not cleaved by chymotrypsin, but trypsin cleaves at Arg (T1) and subsequently at Lys (T2) and tryptic inactivation of E2K or E2P forms is linear and associated with cleavage at Arg" (Ti) [104,108], Inactivation of K-phosphatase is delayed because cleavage of T1 and T2 in sequence is required for inactivation of K-phosphatase activity [105],... 

In soil, the chances that any enzyme will retain its activity are very slim indeed, because inactivation can occur by denaturation, microbial degradation, and sorption (61,62), although it is possible that sorption may protect an enzyme from microbial degradation or chemical hydrolysis and retain its activity. The nature of most enzymes, particularly size and charge characteristics, is such that they would have very low mobility in soils, so that if a secreted enzyme is to have any effect, it must operate close to the point of secretion and its substrate must be able to diffuse to the enzyme. Secretory acid phosphatase was found to be produced in response to P-deficiency stress by epidermal cells of the main tap roots of white lupin and in the cell walls and intercellular spaces of lateral roots (63). Such apoplastic phosphatase is safe from soil but can be effective only when presented with soluble organophosphates, which are often present in the soil. solution (64). However, because the phosphatase activity in the rhizo-sphere originates from a number of sources (65), mostly microbial, and is much higher in the rhizosphere than in bulk soil (66), it seems curious that plants would have a need to secrete phosphatase at all. 

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