Sanghera J S, Vance D E
Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Canada.
J Biol Chem. 1989 Jan 15;264(2):1215-23.
The role of phosphorylation/dephosphorylation in the regulation of CTP:phosphocholine cytidylyltransferase activity was investigated. Incubation of post mitochondrial supernatant with cAMP-dependent protein kinase (50 units) led to an increased (28%) recovery of the cytidylyltransferase in the cytosolic fraction, while incubation with an intestinal alkaline phosphatase (20 units) led to an increased (61%) recovery in the microsomal fraction. When pure cytidylyltransferase was incubated with washed microsomes in the presence of cAMP-dependent protein kinase (133 units), the enzyme associated with the supernatant fraction increased (3.12 +/- 0.02 to 3.77 +/- 0.03 nmol/min/ml) while that of the microsomal fraction decreased (1.36 +/- 0.01 to 0.56 +/- 0.05 nmol/min/ml) by 2.5-fold. The increase in the cytidylyltransferase activity in the supernatant corresponded to an increase in 32P incorporation into the cytidylyltransferase. Treatment with alkaline phosphatase (40 units) decreased the cytidylyltransferase activity in the supernatant (3.61 +/- 0.08 to 2.88 +/- 0.07 nmol/min/ml) while the activity in the microsomal fraction increased (0.56 +/- 0.08 to 1.16 +/- 0.06 nmol/min/ml) by 2-fold. The decrease in the cytidylyltransferase activity in the supernatant corresponded to a decrease in 32P incorporation into the cytidylyltransferase. Incubation of cytidylyltransferase with phosphatidylcholine vesicles in the presence of cAMP-dependent protein kinase (110 units) decreased the cytidylyltransferase activity by 30%. The decrease in cytidylyltransferase activity corresponded to an increase in 32P incorporation into the cytidylyltransferase. Treatment with alkaline phosphatase (20 units) resulted in a 41% increase in the cytidylyltransferase activity. The increase in cytidylyltransferase activity corresponded to a decrease in 32P incorporation into the cytidylyltransferase. Incubation of the cytidylyltransferase with [gamma-32P] ATP and cAMP-dependent protein kinase led to incorporation of 32P into the serine residues of cytidylyltransferase. If the cytidylyltransferase were preincubated with alkaline phosphatase prior to incubation with cAMP-dependent protein kinase, 2-fold more 32P (0.2 mol P/mol cytidylyltransferase) was incorporated into the cytidylyltransferase. Collectively, this data is in agreement with a role for reversible phosphorylation in the regulation of cytidylyltransferase.
研究了磷酸化/去磷酸化在CTP：磷酸胆碱胞苷转移酶活性调节中的作用。线粒体后上清液与cAMP依赖性蛋白激酶（50单位）一起孵育导致胞苷转移酶在胞质部分的回收率增加（28%），而与肠碱性磷酸酶（20单位）一起孵育导致其在微粒体部分的回收率增加（61%）。当将纯胞苷转移酶与洗涤后的微粒体在cAMP依赖性蛋白激酶（133单位）存在下孵育时，与上清液部分相关的酶增加（从3.12±0.02至3.77±0.03 nmol/min/ml），而微粒体部分的酶减少（从1.36±0.01至0.56±0.05 nmol/min/ml）达2.5倍。上清液中胞苷转移酶活性的增加与胞苷转移酶中32P掺入量的增加相对应。用碱性磷酸酶（40单位）处理使上清液中胞苷转移酶活性降低（从3.61±0.08至2.88±0.07 nmol/min/ml），而微粒体部分的活性增加（从0.56±0.08至1.16±0.06 nmol/min/ml）达2倍。上清液中胞苷转移酶活性的降低与胞苷转移酶中32P掺入量的减少相对应。在cAMP依赖性蛋白激酶（110单位）存在下，将胞苷转移酶与磷脂酰胆碱囊泡一起孵育使胞苷转移酶活性降低30%。胞苷转移酶活性的降低与胞苷转移酶中32P掺入量的增加相对应。用碱性磷酸酶（20单位）处理导致胞苷转移酶活性增加41%。胞苷转移酶活性的增加与胞苷转移酶中32P掺入量的减少相对应。将胞苷转移酶与[γ-32P]ATP和cAMP依赖性蛋白激酶一起孵育导致32P掺入胞苷转移酶的丝氨酸残基中。如果在与cAMP依赖性蛋白激酶孵育之前，先将胞苷转移酶与碱性磷酸酶预孵育，则有2倍多的32P（0.2 mol P/mol胞苷转移酶）掺入胞苷转移酶中。总体而言，这些数据与可逆磷酸化在胞苷转移酶调节中的作用一致。
