Phosphorylation state of acetyl-coenzyme A carboxylase. II. Variation with nutritional condition.
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| Abstract |
Acetyl-CoA carboxylase from liver exhibits a linear inverse relationship between the ratio of enzymic activities at 0 and 2 mM citrate and the extent of phosphorylation by its kinase, and this citrate activity ratio method was used to examine the effect of nutritional conditions on the phosphorylation state of the enzyme. This method showed that the calculated phosphorylation state, being the extent of phosphorylation at sites accessible to carboxylase kinase, was highest in the livers of starved rats, lower in those fed normally, and lower still in starved rats which had been refed for 48 h on a fat-free diet. The actual values were 0.44, 0.26, and 0 mol of P/subunit, respectively, provided that liver samples were frozen rapidly to liquid nitrogen temperatures and extracted with stopping buffers at temperatures well below freezing. Normal homogenization with stopping buffers (containing inhibitors for protein kinases and phosphatases) resulted in much higher calculated phosphorylation states. The effect of nutritional conditions on the phosphorylation state as estimated reported above was confirmed by purifying the carboxylase from livers of rats, measuring the amount of phosphate which could be incorporated by carboxylase kinase, and comparing this with the phosphorylation state calculated from the citrate activity ratio method or the specific activity. Furthermore, treatment with protein phosphatase of carboxylase from starved rats resulted in the largest increase in specific activity, that from the starved/refed rats in the least. Finally, the effects of hyperglycemia on carboxylase and phosphorylase characteristics in the livers of intact rats were ascertained by taking liver samples and preparing crude extracts by the rapid freezing method described above. Hyperglycemia caused a rapid increase in the activity of the carboxylase and a rapid decrease in its putative phosphorylation state as measured by the citrate activity ratio method. Phosphorylase was also dephosphorylated, as indicated by a decrease in phosphorylase a activity. We conclude that the citrate activity ratio method is a valid test for the phosphorylation state of acetyl-CoA carboxylase in crude extracts of tissue.
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| Authors | H Jamil, N B Madsen |
| Journal | The Journal of biological chemistry (J Biol Chem) Vol. 262 Issue 2 Pg. 638-42 (Jan 15 1987) ISSN: 0021-9258 [Print] United States |
| PMID | 2879834 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't) |
| Chemical References |
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