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Methods were developed for quantifying protein phosphatases-I, 2A, 2B and 2C in cell extracts, and these procedures were exploited to determine their tissue and subcellular distributions. In addition, the contribution of each enzyme to the total protein phosphatase activity in skeletal muscle and liver extracts towards nine proteins involved in the control of glycogen metabolism, glycolysis/gluconeogenesis, fatty acid synthesis and cholesterol synthesis was assessed.Each protein phosphatase was present at significant concentrations in skeletal muscle, heart muscle, liver, brain and adipose tissue, although the relative amounts differed considerably. In skeletal muscle, protein phosphatase-I was the major enzyme acting on phosphorylase, glycogen synthase and phosphorylase kinase (B-subunit), and thus was the major protein phosphatase responsible for the inactivation of glycogenolysis and stimulation of glycogen synthesis. This idea was reinforced by the observation that 50 ' %, of the protein phosphatase-I activity was associated with the protein-glycogen complex.In the liver, protein phosphatases-1, 2A and 2C each appear to play a role in the regulation of glycogen metabolism. Protein phosphatase-I accounted for a significant fraction of the total potential activity towards phosphorylase and glycogen synthase, and was the major phosphorylase kinase ([hubunit) phosphatase of this tissue. In addition, it was the only protein phosphatase present in the protein-glycogen complex. Protein phosphatase 2A was also a major phosphorylase phosphatase and glycogen synthase phosphatase in this tissue. Protein phosphatase 2C was a significant glycogen synthase phosphatase in the liver, but had negligible activity toward phosphorylase or phosphorylase kinase (p-subunit).In the absence of Ca2+, protein phosphatase 2A was the major phosphorylase kinase (a-subunit) phosphatase and the only inhibitor-1 phosphatase, in skeletal muscle or liver. In the presence of Ca2+, protein phosphatase 2B accounted for most of the activity towards these substrates.Protein phosphatase 2A was the major enzyme acting on L-pyruvate kinase, ATP-citrate lyase and acetyl-CoA carboxylase in rat liver, suggesting an important role in the regulation of glycolysis/gluconeogenesis and fatty acid synthesis. Protein phosphatase 2C was the major enzyme acting on hydroxymethylglutaryl-CoA (HMG-CoA) reductase and HMG-CoA reductase kinase, suggesting an important role in the regulation of cholesterol synthesis. However, the observation that 20% of the protein phosphatase-I in liver was associated with the microsomal fraction suggests that this enzyme may also be involved in regulating HMG-CoA reductase, which is tightly associated with microsomes.The activity of protein phosphatase-I in dilute skeletal muscle and liver extracts was just as sensitive to inhibitor-I and inhibitor-2 as the purified enzyme. In concentrated extracts, higher concentrations of the inhibitor proteins were required and the inhibition was time-dependent. The results explain previous report...

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Protein phosphatase-2B was purified from extracts of rabbit skeletal muscle by a procedure that involved fractionation with ammonium sulphate, chromatography on DEAE-Sepharose, fractionation with poly(ethy1ene glycol), gel filtration on Sephadex G-200 ( M , = 98000 +-4000), chromatography on Affi-Gel Blue and affinity chromatography on calmodulin-Sepharose. The enzyme was purified 3500-fold in seven days with an overall yield of 0.5 %. / "The a-subunit of phosphorylase kinase, protein phosphatase inhibitor-1 and the myosin P-light chain from rabbit skeletal muscle were dephosphorylated by protein phosphatase-2B with similar kinetic constants. The cr-subunit of phosphorylase kinase was dephosphorylated at least 100-fold more rapidly than the P-subunit, while glycogen phosphorylase, glycogen synthase, histones HI and H2B, ATP-citrate lyase, acetyl-CoA carboxylase, L-pyruvate kinase and protein synthesis initiation factor eIF-2 were not dephosphorylated at significant rates.= 6 nM) after chromatography on DEAE-Sepharose and this degree of activation was maintained throughout the remainder of the purification. Calmodulin increased the V,,, of the reaction without altering the K , for inhibitor-1.The activity of protein phosphatase-2B was completely dependent on Ca2+ in the presence or absence of calmodulin. Half-maximal activation was observed at 1.0 pM Ca2' in the absence, and at 0.5 pM Ca2+ in the presence, of 0.03 pM calmodulin. Protein phosphatase-2B was inhibited completely by trifluoperazine ; halfmaximal inhibition occurred at 45 pM in the absence and 35 pM in the presence of 0.03 pM calmodulin.The metabolic role of protein phosphatase-2B

Increased exposure to light pollution perturbs physiological processes through misalignment of daily rhythms at the cellular and tissue levels. Effects of artificial light-at-night (ALAN) on diel properties of immunity are currently unknown. We therefore tested the effects of ALAN on diel patterns of cytokine gene expression, as well as key hormones involved with the regulation of immunity, in zebra finches (Taeniopygia guttata). Circulating melatonin and corticosterone, and mRNA expression levels of pro- (IL-1β, IL-6) and anti-inflammatory (IL-10) cytokines were measured at six time points across 24-h day in brain (nidopallium, hippocampus, and hypothalamus) and peripheral tissues (liver, spleen, and fat) of zebra finches exposed to 12 h light:12 h darkness (LD), dim light-at-night (DLAN) or constant bright light (LLbright). Melatonin and corticosterone concentrations were significantly rhythmic under LD, but not under LLbright and DLAN. Genes coding for cytokines showed tissue-specific diurnal rhythms under LD and were lost with exposure to LLbright, except IL-6 in hypothalamus and liver. In comparison to LLbright, effects of DLAN were less adverse with persistence of some diurnal rhythms, albeit with significant waveform alterations. These results underscore the circadian regulation of biosynthesis of immune effectors and imply the susceptibility of daily immune and endocrine patterns to ALAN.