Activation of
acetyl-CoA carboxylase during incubation of
crude extracts of lactating rat mammary gland with Mg2+ and
citrate can be blocked by NaF, suggesting that it represents a dephosphorylation of the
enzyme. The greater extent of activation in extracts from 24 h-starved rats (200%) compared with fed controls (70%) implies that the decrease in
acetyl-CoA carboxylase activity in response to 24 h
starvation may involve increased phosphorylation of the
enzyme.
Acetyl-CoA carboxylase was purified from the mammary glands of lactating rats in the presence of
protein phosphatase inhibitors by
avidin-
Sepharose chromatography.
Starvation of the rats for 24 h increased the concentration of
citrate giving half-maximal activation by 75%, and decreased the Vmax. of the purified
enzyme by 73%. This was associated with an increase in the
alkali-labile
phosphate content from 3.3 +/- 0.2 to 4.5 +/- 0.4 mol/mol of
enzyme subunit.
Starvation of lactating rats for 6 h, or short-term
insulin deficiency induced by
streptozotocin injection, did not effect the kinetic parameters or the
phosphate content of
acetyl-CoA carboxylase purified from mammary glands. The effects of 24 h
starvation on the kinetic parameters and
phosphate content of the purified
enzyme were completely reversed by re-feeding for only 2.5 h. This effect was blocked if the animals were injected with
streptozotocin before re-feeding, suggesting that the increase in plasma
insulin that occurs on re-feeding was responsible for the activation of the
enzyme. The effects of re-feeding 24 h-starved rats on the kinetic parameters and
phosphate content of
acetyl-CoA carboxylase could be mimicked by treating
enzyme purified from 24 h-starved rats with
protein phosphatase-2A in vitro. Our results suggest that, in mammary glands of 24 h-starved lactating rats,
insulin brings about a dephosphorylation of
acetyl-CoA carboxylase in vivo, which may be at least partly responsible for the reactivation of mammary lipogenesis in response to re-feeding.