Methods were developed for quantifying protein phosphatases-1, 2B and 2C in cell extracts, and thse 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 metabolis, glycolysis/gluconeogenesais, fatty acid synthesis and cholesterol synthesis was assessed.
Each protein phosphatase was present at significant concentrations in skeletal muscle, heart muscle, liverr, brain and adipose tissue, although the relative amounts differed considerably. In skeletal muscle, protein phosphatase-1 was the major enzyme acting on phosphorylase, glycogen synthase and phosphorylase kinase (β-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-1 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-1 accounted for a significant fraction of the total potenitial activity towards phosphorylase and glycogen synthase, and was the major phosphorylase kinase (β-subunit) 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 (β-subunit).
In the absence of Ca2+, protein phosphatase 2A was the major phosphorylase kinase (αsubunit) 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 hydroxymethyglutaryl-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-1 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-1 in dilute skeletal muscle and liver extracts was just as sensitive to inhibitor-1 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 reports that phosphorylase phosphatase in skeletal muscle and liver extracts is insensitive to inhibitor-1 and inhibitor-2. Possible reasons for the decreased sensitivity of protein phosphatase-1 to the inhibitor proteins in concentrated tissue extracts are considered and the metabolic roles of inhibitor-1 and inhibitor-2 are discussed.