Mechanism of palmityl coenzyme A inhibition of liver glycogen synthase.

Palmityl-CoA inhibits free liver glycogen synthase; the concentration required for half-maximum inhibition is 3 to 4 micrometer. Almost complete inhibition was observed at 50 micrometer. Palmityl-CoA inhibition is associated with dissociation of the tetrameric enzyme into monomers, and binding of palmityl-CoA to the monomers. Glycogen-bound enzyme is also inhibited by palmityl-CoA, resulting in dissociation of the enzyme into monomers and concomitant release of the enzyme from the primer glycogen. Palmityl-CoA inhibition of the enzyme is partially reversed by the glycogen synthase activator, glucose-6-P, whereas sodium lauryl sulfate-inhibited enzyme is not reactivated by glucose-6-P. Sodium lauryl sulfate inhibition results in the dissociation of the tetramer into the monomers. Bovine serum albumin and cyclodextrin can prevent palmityl-CoA inhibition only when they are added prior to palmityl-CoA addition. The possible physiological role of palmityl-CoA in glucose homeostasis is discussed.

to the glycogen synthase activity. One single protein band was also detected in the presence of sodium lauryl sulfate. Assay of Glycogen Synthase D ~ The enzyme activity was assayed as described previously by determining the incorporation of radioactive glucose into glycogen from UDP-[i4Clglucose (9). The standard assay mixture contained 0.67 pmol of UDP-['"Clglucose (8,000 to 9,000 cpm), 1.2 mg of shellfish glycogen, 50 pmol of glycylglycine (pH 7.41, and 10 pmol of glucose-6-P in a final volume of 0.5 ml. Reconstituted Glycogen-bound Glycogen Synthase D ~ High molecular weight protein-free glycogen was prepared from rat liver by a modification of the method of Bueding and Orrell (11). Glycogen synthase D was incubated with the glycogen in 50 rnM glycylglycine buffer, pH 7.4, in the presence of 20 rnM glucose-6-P at 37" for 10 min. The glycogen-bound enzyme was collected by centrifugation at 78,000 x g for 60 min. The pellet was resuspended in glycylglycine buffer.
Homogeneous glycogen synthase D was used in all of the experiments reported, unless otherwise indicated.
Assay of Glycogen F'hosphorylase ~ Glycogen phosphorylase activity was assayed by determining the incorporation of radioactive glucose into glycogen from YClglucose-1-P. The 'standard assay system contained 2 mg of glycogen, 2 +mol of ['"Clglucose-1-P (8,500 cpm/assay), 1 pmol of EDTA, and 50 pmol of P-glycerophosphate, pH 6.8, in a final volume of 0.5 ml. The reaction was run at 30" for 5 min after the addition of enzyme preparation. Labeled glycogen was assayed as described. protein-free glycogen prepared from rat liver in 50 rnM glycylglycine, pH 7.4, for 10 min at 37" in the presence of 20 rnM glucose-6-P.

Properties of
The complex was separated as a pellet by centrifugation at 78,000 x g for 60 min. The pellet thus obtained was dissolved in 50 rnM glycylglycine, pH 7.4, and incubated with [l-14Clpalmityl CoA (0.20 mM, 200,000 cpm) at 37" for 10 min in a total volume of 2 ml. The incubated mixture which showed 75% inhibition of enzyme activity was then applied to the Sepharose 4B column (3 x 40 cm), equilibrated, and eluted at O-4" with buffer containing 50 m&i glycylglycine, pH 7.4, 5 mrvr EDTA, 5 rnM dithiothreitol, and 15% glycerol.
Fractions of 2.5 ml were collected from the start to determine the glycogen peak which was eluted in the void volume. Glycogen peak was determined by the absorption reading at 480 nm. The radioactivity associated with glycogen and the enzyme subunit were determined as the percentage of radioactivity recovered from the column in an 0.5-ml aliquot. B, Sepharose 4B elution profile of palmityl-CoA-treated glycogen synthase D. Enzyme (1 mg of protein) was incubated with [l-14Clpalmityl-CoA (0.1 mM, 100,000 cpm) in a total volume of 2 ml at 37" for 10 min. The incubated mixture was then applied to a Sepharose 4B column (3 x 40 cm), equilibrated, and eluted at O-4" with buffer containing 50 rnM glycylglycine, pH 7.4, 5 rnM EDTA, 5 rnM dithiothreitol, and 15% glycerol.
Fractions of 2.5 ml were collected for the determination of enzyme activity and radioactivity. The native enzyme peak was determined as percentage of total activities recovered from the column in an 0.4-ml aliquot.
The enzyme subunit peak with associated [l-"'Clpalmityl-CoA was determined as percentage of total radioactivity recovered from the column in an aliquot of 0.5 ml. and found to be 74,000. This experiment shows that in the presence of palmityl-CoA the enzyme dissociates into monomers and that palmityl-CoA binds to the monomers. Fig. 7A