Abstract
Acetyl-CoA carboxylase of animal tissues is known to be dependent on citrate for its activity. The observation that dephosphorylation abolishes its citrate dependence (Thampy, K. G., and Wakil, S. J. (1985) J. Biol. Chem. 260, 6318-6323) suggested that the citrate-independent form might exist in vivo. We have purified such a form from rapidly freeze-clamped livers of rats. Sodium dodecyl sulfate gel electrophoresis of the enzyme gave one protein band (Mr 250,000). The preparation has high specific activity (3.5 units/mg in the absence of citrate) and low phosphate content (5.0 mol of Pi/mol of subunit). The enzyme isolated from unfrozen liver or liver kept in ice-cold sucrose solution for 10 min and then freeze-clamped has low activity (0.3 unit/mg) and high phosphate content (7-8 mol of Pi/mol of subunit). Citrate activated such preparations with half-maximal activation at greater than 1.6 mM, well above physiological range. The low activity may be due to its high phosphate content because dephosphorylation by [acetyl-CoA carboxylase]-phosphatase 2 activates the enzyme and reduces its dependence on citrate. Since freeze-clamping the liver yields enzyme with lower phosphate content and higher activity, it is suggested that the carboxylase undergoes rapid phosphorylation and consequent inactivation after the excision of the liver. The carboxylase is made up of two polymeric forms of Mr greater than or equal to 10 million and 2 million based on gel filtration on Superose 6. The former, which predominates in preparations from freeze-clamped liver, has higher activity and lower phosphate content (5.3 units/mg and 4.0 mol of Pi/mol of subunit, respectively) than the latter (2.0 units/mg and 6.0 mol of Pi/mol of subunit, respectively). The latter, which predominates in preparations from unfrozen liver, is converted to the active polymer (Mr greater than or equal to 10 million) by dephosphorylation. Thus, the two polymeric forms are interconvertible by phosphorylation/dephosphorylation and may be important in the physiological regulation of acetyl-CoA carboxylase.
Highlights
Acetyl-coA carboxylase of animal tissues is known nisms of short-term control involving allosteric modification to be dependent on citrate for itsactivity
The enzyme isolated from unfrozen liver or liver kept in ice-cold sucrose solution for 10 min and freeze-clamped has low activity (0.3 unit/mg) and high phosphate content
Ylase is observed at 2-7 mM citrate [12, 15, 16], severalfold higher than intracellular citrate(0.17-0.3 mM in freezeclamped liver and 0.45 mM in hepatocytes) [17].in light of conflicting data regarding intracellular citrate and rate of fatty acid synthesis [18], the role of citrate in the regulation of acetyl-coA carboxylase is not clear
Summary
Acetyl-coA carboxylase of animal tissues is known nisms of short-term control involving allosteric modification to be dependent on citrate for itsactivity. The preparation has high specific activity (3.5units/ mg in theabsence of citrate) andlow phosphate content (5.0molof PJmol of subunit). Citrate activated such containing 6-15 mol of Pi/mol of subunit [12, 19, 20] It is a preparations with half-maximal activataito>n1.6mM, good substrate for additional phosphorylations. The low activity may phorylation by CAMP-dependent and -independent protein be due to its high phosphate content because dephos- kinases [21,22] converts the inactive enzyme (citrate-dependphorylation by [acetyl-coA carboxylase]-phosphatase ent) tother inactive forms that are less sensitive to activation. Sincefreeze-clamping the liver yields enzyme kinases I and I1 results in the incorporation of up to 2 and with lower phosphate contenatnd higher activity, it is 0.6 mol of Pi/mol of subunit, respectively, with no resultant suggested that the carboxylase undergoes rapid phos- changes in carboxylase activity [23]. Dephosphorylation of phorylation and consequent inactivation after the ex- acetyl-coA carboxylase by protein phosphatases resultisn the cision of the liver
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