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The effects of polyamines on the oligomeric forms of protein phosphatase-1. (1G), protein phosphatase-2A (2A0, 2A1 and 2A2) and their free catalytic subunits (1C and 2Ac) has been studied using homogeneous enzymes isolated from rabbit skeletal muscle. Spermine increased the activity of protein phosphatase-2A towards eight of nine substrates tested. Half-maximal activation was observed at 0.2 mM with optimal effects at 1–2 mM. Above 2 mM, spermine became inhibitory. The most impressive activation of protein phosphatase-2A was obtained with glycogen synthase, especially when phosphorylated at sites-3 (8–15-fold with protein phosphatase-2A1) and phenylalanine hydroxylase (6–7-fold with protein phosphatase-2A1) as substrates. Activation of protein phosphatases 2A0 2A1 and 2A2 was greater than that observed with 2Ac. Spermine was a more potent activator than spermidine, while putrescine had only a small effect. Qualitatively similar results were obtained with five other substrates, although maximal activation was much less (1.3–3-fold with protein phosphatase+2A1). The rate of dephosphorylation of glycogen phosphorylase was decreased by spermine, inhibition being more pro- nounced with protein phosphatase-2Ac than with 2A0, 2A1 and 2A2. Spermine (I50 = 0.1 mM with protein phosphatase-2Ac) was a more potent inhibitor than spermidine (I50 = 0.9 mM) or putrescine (I50 = 8 mM). Partially purified preparations of protein phosphatases-2A0, 2A1 and 2A2 from from rat liver were affected by spermine in a similar mariner to the homogeneous enzymes from rabbit skeletal muscle. Spermine did not activate protein phosphatase-1 to the stone extent as protein phosphatase-2A. Greatest stimulation (2.5-fold) was again observed with glycogen synthase labelled in sites-3, with half-maximal activation at 0.2 mM and optimal effects at 1–2 mM spermine. Spermine was a much more effective stimulator than spermidine, while putrescine was ineffective. Very similar results were obtained with protein phosphatases 1G and 1c. With four other substrates maximal activation by spermine was < 1.5-fold, while the dephosphorylation of glycogen synthase (labelled in site-2), phosphorylase kinase, pyruvate kinase and glycogen phosphorylase were inhibited. Spermine (I50 = 0.04 mM) was a more potent inhibitor of the dephosphorylation of glycogen phosphorylase than spermidine (I50 = 0.9 mM) or putrescine (I50 = 9 mM). There was some resemblance between the effects of Mn2+ and spermine on the activity of protein phosphatase- 2A, although with the substrates inhibitor-1 and phenylalanine hydroxylase, activation by Mn2+ was greater than that observed with spermine, while with glycogen synthase the converse was true. Mn2+ could not mimic the activating effect of spermine on dephosphorylation of glycogen synthase (labelled in sites-3) by protein phosphatase 1. By contrast, Spermine hardly affected the dephosphorylation of inhibitor-1 by phosphatase-1, a reaction that was almost completely dependent on Mn2+. Physiological concentrations of Mg2+ (1.0 mM) neither mimicked, nor influenced, the response to spermine with either protein phosphatase-1 or 2A. The effects of spermine appear to result from interaction of the polyamine with both the protein phosphatases and their substrates. The possibility that activation of protein phosphatase-1 and/or 2A by spermine underlies the relatively specific dephosphorylation of sites-3 of glycogen synthase by insulin is discussed. [ABSTRACT FROM AUTHOR]

The identities of the protein phosphatases involved in the regulation of hepatic glycolysis, gluconeogenesis and aromatic amino acid breakdown were investigated using 6-phosphofructo-1-kinase, fructose-1,6-biphosphatase, L-pyruvate kinase, phenylalanine hydroxylase and the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates. Purified preparations of protein phosphatases-1, 2A, 2B and 2C exhibited activity towards all five substrates in vitro, although phosphatases-1 and 2B were only weakly active. Studies in liver extracts using inhibitor-2 and trifluoperazine, which inhibit protein phosphatase-1 and 2B, respectively, confirmed that these phosphatases are unlikely to be important in dephosphorylating these substrates in vivo. Sequential fractionation of rat liver extracts by anion-exchange chromatography and gel-filtration failed to resolve any protein phosphatases acting on each substrate, apart from protein phosphatases-2A and 2C. The present results, together with those described in the following paper (in this journal) indicate that under the assay conditions used, protein phosphatase-2A is the most powerful phosphatase acting on each substrate, although protein phosphatase-2C contributes a significant percentage of the activity towards 6-phophofructor-1-kinase. No clear evidence was obtained for a role of metabolites in the regulation of dephosphorylation of the five substrates. This study reinforces our contention that only a few serine-specific and threonine-specific protein phosphatase catalytic subunits participate in cellular regulation. [ABSTRACT FROM AUTHOR]

1. Aminooxyacetate is a potent inhibitor of glutamate oxaloacetate transaminases from both rat and guinea pig kidney cortex. 2. In guinea pig kidney cortex, phosphoenolpyruvate carboxykinase activity was found to be 59% cytoplasmic, 41% mitochondrial. 3. Mitochondrial phosphoenolpyruvate carboxykinase activity is expressed only after sonication indicating that the enzyme is located within the inner mitochondrial membrane. 4. Aminooxyacetate inhibits gluconeogenesis from lactate, but not from pyruvate, in both rat and guinea-pig kidney cortex. 5. It is concluded that gluconeogenesis in guinea-pig kidney cortex proceeds by a pathway involving cytoplasmic, rather than mitochondrial phosphoenolpyruvate carboxykinase. [ABSTRACT FROM AUTHOR]