Regulation of acetyl-CoA carboxylase in rat mammary gland. Effects of incubation with Ca2+, Mg2+ and ATP on enzyme activity in tissue extracts

E M McNeillie; R A Clegg; V A Zammit

摘要

p1. The effect of preincubation of extracts of lactating rat mammary gland with ATP, Mg2+ and micromolar concentrations of Ca2+ on the activity of acetyl-CoA carboxylase was studied. 2. Both Mg2+ and Ca2+ activated the enzyme. Activation with Mg2+ (5 mM) was larger than that with Ca2+ (calculated free Ca2+ concentration = 20-50 microM), but the activity decreased after reaching a peak. The activation obtained with Ca2+ was stable for up to 180 min. 3. Incubation with Ca2+ and Mg2+ together resulted in an activation that was slightly higher than that with Mg2+ only and was stable (compare the results for Ca2+ alone). 4. Preincubation in the absence of Mg2+, but not in the absence of Ca2+, resulted in the impairment of subsequent activation with either Mg2+ (when preincubation was with Ca2+ alone) or Mg2+ plus Ca2+. 5. KF (50 mM) prevented the activation of acetyl-CoA carboxylase by Ca2+ and Mg2+. 6. MgATP2- reversed (Mg2+ + Ca2+)-mediated activation and decreased the activity of acetyl-CoA carboxylase to about 10% of initial activity. Inhibition by ATP was unaffected by addition of cyclic AMP or cyclic AMP-dependent protein kinase inhibitor. 7. 32P was incorporated into acetyl-CoA carboxylase when incubations were carried out in the presence of [gamma-32P]ATP. Subsequent removal of ATP from the incubation medium resulted in rapid loss of 32P from acetyl-CoA carboxylase. 8. It is suggested that extracts of rat mammary gland contain endogenous protein kinase and phosphatase activities that modulate acetyl-CoA carboxylase activity through reversible phosphorylation and dephosphorylation. The phosphatase activity is sensitive to both Mg2+ and micromolar concentrations of Ca2+, whereas the kinase does not appear to be cyclic AMP-dependent./p

机译：> 1。研究了用ATP，Mg2 +和微摩尔浓度的Ca2 +预孵育哺乳期大鼠乳腺提取物对乙酰辅酶A羧化酶活性的影响。 2. Mg2 +和Ca2 +均激活了该酶。 Mg2 +（5 mM）的活化作用大于Ca2 +的活化作用（计算的游离Ca2 +浓度= 20-50 microM），但活性达到峰值后降低。用Ca2 +所获得的活化作用稳定长达180分钟。 3.与Ca2 +和Mg2 +一起温育会导致活化程度略高于仅与Mg2 +一起，并且是稳定的（比较仅Ca2 +的结果）。 4.在不存在Mg2 +的情况下进行预孵育，但在不存在Ca2 +的情况下进行预孵育，会导致随后的Mg2 +激活（当仅与Ca2 +一起进行预孵育）或Mg2 +加Ca2 +的激活受到损害。 5. KF（50 mM）阻止了Ca2 +和Mg2 +激活乙酰辅酶A羧化酶。 6. MgATP2-逆转了（Mg2 + + Ca2 +）介导的活化，并将乙酰辅酶A羧化酶的活性降低到初始活性的10％。 ATP的抑制作用不受添加环状AMP或环状AMP依赖性蛋白激酶抑制剂的影响。 7.当在[γ-32P] ATP存在下进行孵育时，将32P掺入乙酰辅酶A羧化酶中。随后从温育培养基中除去ATP，导致乙酰辅酶A羧化酶迅速失去32P。 8.建议大鼠乳腺提取物含有内源性蛋白激酶和磷酸酶活性，它们通过可逆的磷酸化和去磷酸化来调节乙酰辅酶A羧化酶活性。磷酸酶活性对Mg2 +和微摩尔浓度的Ca2 +均敏感，而该激酶似乎不是环AMP依赖性的。

Regulation of acetyl-CoA carboxylase in rat mammary gland. Effects of incubation with Ca2+, Mg2+ and ATP on enzyme activity in tissue extracts

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