Purification and Properties of a Phosphorylase (Phosphoprotein) Phosphatase Associated with an Alkaline Phosphatase of M_r 35000 from Bovine Adrenal Cortex

Abstract

A metal-ion-independent, nonspecific phosphoprotein phosphatase (MI = 35 000) which represents the major phosphorylase phosphatase activity in bovine adrenal cortex has been purified to apparent homogeneity. An alkaline phosphatase activity (p-nitrophenyl phosphate as a substrate) of the same molecular weight, which requires both a metal ion (Mg2+ > Mn2+ > Co2+) and a sulfhydryl compound for activity, has been found to co-purify with the phosphoprotein phosphatase throughout the purification procedures. Characterization of the phosphoprotein and the alkaline phosphatase activities with respect to their catalytic properties, substrate and metal ion specificities, relationship with large molecular forms of the enzymes and responses to various effectors has been carried out. The results indicate that the phosphoprotein phosphatase can be converted by pyrophosphoryl compounds (e.g. PPi and ATP) to a metal-ion-dependent form which, subsequently, can be reactivated by Co2+ > Mn2+ but not by Mg2+ or Zn2+. The results also indicate that, although the phosphoprotein and the alkaline phosphatase activities are closely associated, they exhibit distinct physical and catalytic properties. Discussions concerning whether these two activities represent two different forms of the same protein or two different yet very similar polypeptide chains have been presented.The covalent modification of enzymes and regulatory proteins through a cycle of phosphorylation and dephosphorylation reactions is an important control mechanism involved in the regulation of many biological processes [l -51. Evidence indicates that the effects of adrenocorticotropic hormone on steroidogenesis [6-91 and on glycogen phosphorylase [lo, 111 are mediated by cyclic AMP and protein phosphorylation. In the past years, several reports have appeared relative to purification, specificity, and regulation of adrenal cortex phosphoprotein phosphatase [lo-141. In spite of these efforts, a homogeneous preparation of the enzyme has not yet been obtained. In addition, the basic catalytic properties and mechanism of regulation of the adrenal cortex phosphorylase phosphatase remains unclear.

Site-directed mutagenesis of amino acid residues of protein phosphatase 1 involved in catalysis and inhibitor binding

“…Interaction with elements of the peptide in addition to phospho-serine or -threonine is clearly critical, since K m values for protein substrates are much lower than that of artificial substrates like paranitrophenyl phosphate (29). Unlike the protein kinases, where short peptides encompassing the phosphorylation site contain the structural elements necessary for efficient enzyme binding, similar studies with the PPases have been much less informative (30).…”

PP2A Activation by β2-Adrenergic Receptor Agonists

“…PP2A, a dual specificity phosphatase capable of dephosphorylating tyrosyl residues as well as serine/threonine residues (65,66), plays a role in a multitude of cellular functions (reviewed in Ref. 34).…”

Conversion of a phosphoseryl/threonyl phosphatase into a phosphotyrosyl phosphatase

“…It was observed that a major p-nitrophenyl phosphatase activity co-purified with this phosphorylase phosphatase activity [16]. It has been known for some time that p-nitrophenyl phosphatase [17,18] and low phosphotyrosyl phosphatase activities [19,20] are associated with this type of enzyme, and highly variable but so far unexplained p-nitrophenyl phosphate/phosphorylase phosphatase activity ratios have been reported [21,22]. The p-nitrophenyl phosphatase activity is specifically inactivated during purification, but can be restored by incubation with ATP, its analogues or PPi [16].…”