Vanadate phosphorylation

The T2 site also became protected from tryptic hydrolysis after phosphorylation of the native or solubilized sarcoplasmic reticulum vesicles with inorganic phosphate in a calcium free medium in the presence of dimethylsulfoxide or glycerol [121,252]. Under these conditions the Ca -ATPase is converted into a covalent E2-P intermediate, that is analogous in conformation to the E2V intermediate formed in the presence of vanadate. In contrast to this, the T2 site in the stable phosphorylated Ca2E P intermediate generated by the reaction of the Ca -ATPase with chro-mium-ATP in the presence of Ca [178,253] was fully exposed to trypsin, just as it was in the nonphosphorylated Ca2Ei form. Therefore the phosphorylated intermediates show the same sensitivity to trypsin at the T2 site as the corresponding nonphosphorylated enzyme forms. 

Insulin binding to the extracellular side of cell membranes initiates the insulin cascade , a series of phosphorylation/dephosphorylation steps. A postulated mechanism for vanadium is substitution of vanadate for phosphate in the transition state structure of protein tyrosine phosphatases (PTP).267,268 In normal physiological conditions, the attainable oxidation states of vanadium are V111, Viv and Vv. Relevant species in solution are vanadate, (a mixture of HV042-/ H2VOO and vanadyl V02+. Vanadyl is not a strong inhibitor of PTPs, suggesting other potential mechanisms for insulin mimesis for this cation. 

It is not clear whether V(V) or V(IV) (or both) is the active insulin-mimetic redox state of vanadium. In the body, endogenous reducing agents such as glutathione and ascorbic acid may inhibit the oxidation of V(IV). The mechanism of action of insulin mimetics is unclear. Insulin receptors are membrane-spanning tyrosine-specific protein kinases activated by insulin on the extracellular side to catalyze intracellular protein tyrosine phosphorylation. Vanadates can act as phosphate analogs, and there is evidence for potent inhibition of phosphotyrosine phosphatases (526). Peroxovanadate complexes, for example, can induce autophosphorylation at tyrosine residues and inhibit the insulin-receptor-associated phosphotyrosine phosphatase, and these in turn activate insulin-receptor kinase. 

Vanderkooi et al.(m) examined the phosphorescence from tryptophan in sarcoplasmic reticulum vesicles and the purified Ca transport ATPase at room temperature in deoxygenated solutions. The phosphorescence decay is multiexponential the lifetime of the long-lived component of phosphorescence is 22 ms. Addition of ATP or vanadate decreased the phosphorescence yield. The Ca2+-ATPase of the sarcoplasmic reticulum alternates between two conformations, called Ei and E2, during Ca2+ transport. The observations were interpreted to indicate that either the binding of vanadate or phosphate to the phosphorylation site of the ATPase or the induced shift in the conformation from the i to the E2 state produced the phosphorescence quenching. 

Ribonuclease A hydrolyzes RNA adjacent to pyrimidine bases. The reaction proceeds through a 2, 3 -phosphate cyclic diester intermediate. Formation and breakdown of the cyclic diester appear to be promoted by concerted general-base and general-acid catalysis by two histidine residues, and by electrostatic interactions with two lysines. These reactions proceed through pentavalent phosphoryl intermediates. The geometry of these intermediates resembles the geometry of vanadate compounds that act as inhibitors of the enzyme. 

The activating effect of Mg2+ upon the cleavage of the phosphoryl group from the ATP could reflect the enhancement of an SN2 reaction at phosphorus by electron withdrawal and charge neutralization via coordination to the metal (equation 1). Support for an SN2 mechanism comes from a consideration57 of the inhibition by vanadate. Coordination of the transferable phosphoryl group would inhibit the SN1 mechanism. 

Acid and alkaline phosphatases with phosphorylated intermediates are inhibited by vanadate. This has been exploited in the study of the role of alkaline phosphatase in mineralization.1069 Vanadate also inhibits the ATP-dependent degradation of proteins in reticulocytes.1070... 

Vanadate stimulates protein kinases in the cytosol, as demonstrated in adipose cells and extracts. The activation of a membrane and cytosolic protein tyrosine kinase have been demonstrated in adipocytes, and the membranous enzyme has been postulated to be a way to involve PI-3K actions without activation of insulin receptor substrate-1 (IRS-1) in the insulin signal transduction pathway [140], It is always difficult to determine if protein kinase activation is direct or the result of stimulation of a protein phosphatase. The fact that kinase stimulation was seen in isolated extracts after cell disintegration in this adipocyte cell system supports the idea that vanadium addition to cells could directly stimulate kinases via an as-yet-undetermined mechanism. In other experiments with 3T3-L1 adipocytes bis(acetylacetonato)oxovana-dium (IV) BMOV and bis(l-N-oxide-pyridine-2thiolato)oxovanadium (TV) caused increased tyrosine phosphorylation of both the insulin receptor and IRS-1 in a synergistic way with insulin, as measured by antibodies to phosphotyrosine residues [141]. 

Messmore, J.M. and R.T. Raines. 2000. Pentavalent organo-vanadates as transition state analogues for phosphoryl transfer reactions. J. Am. Chem. Soc. 122 9911-9916. 

The phosphorylated receptor appears to be capable of being dephosphorylated by the action of endogenous cytosolic phosphatases, as incubation of cells with vanadate, which inhibits the action of tyrosine-phosphate-specific phosphatases, increased the phosphorylation state of the receptor [60,61]. The significance of this observation has yet to be ascertained. 

Acid and alkaline phosphatases which are believed to pass through a phosphorylated intermediate during the hydrolysis of p-nitrophenolphosphate are inhibited by vanadate )68-69. In the case of alkaline phosphatase from E. Coli, V02+ inhibits better than vanadate(V) (Table 2)68. Typical inhibitor plots are shown in Fig. 10. The reduction of the effectiveness of VOz+ as an inhibitor by about a factor of five in tris buffer probably stems from the complexation of the metal ion by tris. Such complexation is known to occur21. ... 

Acylphosphate formation is characteristic for P-type ATPases and involves the transfer of the y-phosphate of ATP to an aspartic acid residue to form a high-energy enzyme intermediate. The phosphorylated aspartic acid residue is located in the sequence DKTGT, which is universally conserved in all members of the P-type superfamily. By this criterion, CopA and CopB of En. hirae are clearly members of the P-type superfamily of ATPases and probably function by the same underlying mechanism. Vanadate sensitivity is another hallmark of P-type ATPases. CopA and CopB were inhibited by vanadate with /50 values of around 0.1 mM. This is a low vanadate sensitivity compared to /50 values in the micromolar to submicromolar range observed for non-heavy metal P-type ATPases. 

An important characteristic of P-type ATPases is their ability to be transiently phosphorylated at the invariant aspartate residue by inorganic orthophosphate in the absence of the cation (Fig. 2, step 5). A diagnostic inhibitor of P-type ATPases, ortho vanadate, can also bind to the invariant aspartate but, unlike orthophosphate, cannot participate in the reaction cycle (Carafoli, 1991 MacLennan ei a/., 1997). 

P-type ATPase. Methanococcus voltae, a marine organism growing on H2/CO2, contains high activities of a membrane-bound ATPase, which was sensitive towards vanadate rather than to DCCD [147], suggesting the presence of a P-type ATPase (see ref [141]). Accordingly the purified enzyme, composed of one 74 kD subunit, could be phosphorylated in a vanadate-sensitive fashion [148], a characteristic property for P-type ATPases, which involve a phosphoprotein as intermediate in the catalytic cycle. 

Not so much is known about the significance of protein tyrosine phosphatases (PTP). hihibition of PTPs (by vanadate) leads to increase in tyrosine phosphorylation, secretion and aggregation (biazuer ail, 1990 Pumigliae/a/., 1992). At least two PTPs appear to be involved in platelet aggregatioa PTPIB is proteolytically cleaved and activated by calpain and released into the cytosol during aggregation. SH-PTPl associates with the... 

Vanadate (VOj or H2VO4 ) was first recognized in 1979 as having insulin mimetic properties [258]. Since then, vanadate and vanadyl (V ) have been shown to mimic most but not all biological actions of insulin in vitro and to lower blood glucose in streptozotocin-treated rats [259, 260]. Vanadate is a potent inhibitor of phosphotyrosine phosphatases, an interesting activity since the insulin receptor is a tyrosine kinase, and some of the actions of insulin have been proposed to take place via autophosphorylation of the insulin receptor and phosphorylation of cellular substrates on tyrosine residues [261]. Some recent developments on the mechanism and the in vivo activity of vanadate and its derivatives are presented here. 

Evidence has been provided showing that some of the insulin-mimicking actions of vanadate could be elicited without any effect on protein phosphorylation [262, 263]. Eriksson et al. showed that vanadate in rat adipocytes increases insulin binding by increasing the receptor number, without changing the affinity of insulin, and stimulates glucose transport into the cell. Whether this effect is linked to tyrosine phosphorylation is unclear [264]. 

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