体内失活大鼠心脏丙酮酸脱氢酶磷酸化位点的占据情况。

Occupancy of sites of phosphorylation in inactive rat heart pyruvate dehydrogenase phosphate in vivo.

作者信息

Sale G J, Randle P J

出版信息

Biochem J. 1981 Mar 1;193(3):935-46. doi: 10.1042/bj1930935.

DOI:10.1042/bj1930935

PMID:7305968

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1162688/

Abstract

Inactive pyruvate dehydrogenase phosphate complexes were partially purified from hearts of fed, starved or alloxan-diabetic rats by using conditions that prevent phosphorylation or dephosphorylation. 2. Unoccupied sites of phosphorylation were assayed by incorporation of 32P from [gamma-32P]ATP into the complexes. Total sites of phosphorylation were assayed by the same method after complete reactivation, and thus dephosphorylation, of complexes by incubation with pyruvate dehydrogenase phosphate phosphatase. Occupancy is assumed from the difference (total sites--unoccupied sites). Percentage incorporation into individual sites was measured by high-voltage electrophoresis after tryptic digestion. 3. Values (means +/- S.E.M., in nmol of phosphate/unit of inactive complex) for total sites, occupied sites and percentage occupancies, with numbers of observations in parentheses were: fed, 2.1 +/- 0.04, 1.15 +/- 0.04, 54.8 +/- 1.6% (39); starved, 2.05 +/- 0.03, 1.85 +/- 0.03, 90.2 +/- 1.4% (28); alloxan-diabetic, 1.99 +/- 0.03, 1.72 +/- 0.03, 86.4 +/- 1.4% (68%). 4. Values (means +/- S.E.M. for percentage occupancy) for individual sites of phosphorylation in pyruvate dehydrogenase phosphate given in the order sites 1, 2 and 3 were : fed, 100 +/- 2.7, 27.8 +/- 1.6, 33.9 +/- .9; starved, 100 +/- 1.4, 76.2 +/- 2.0, 92.4 +/- 1.5; alloxan-diabetic, 100 +/- 1.2, 64.0 +/- 1.7, 94.6 +/- 1.4. 5. It is concluded that starvation or alloxan-diabetes leads to a 2--3-fold increase in the occupancy of phosphorylation sites 2 and 3 in pyruvate dehydrogenase phosphate in rat heart in vivo.

摘要

通过采用防止磷酸化或去磷酸化的条件，从喂食、饥饿或四氧嘧啶糖尿病大鼠的心脏中部分纯化出无活性的磷酸丙酮酸脱氢酶复合物。2. 通过将[γ-32P]ATP中的32P掺入复合物中来测定未占据的磷酸化位点。在用丙酮酸脱氢酶磷酸磷酸酶孵育使复合物完全重新激活并因此去磷酸化后，用相同方法测定总的磷酸化位点。占据率由差值（总位点 - 未占据位点）得出。胰蛋白酶消化后，通过高压电泳测量掺入各个位点的百分比。3. 总位点、占据位点和占据百分比的值（以每单位无活性复合物中磷酸盐的纳摩尔数表示，均值±标准误，括号内为观察次数）分别为：喂食组，2.1±0.04、1.15±0.04、54.8±1.6%（39）；饥饿组，2.05±0.03、1.85±0.03、90.2±1.4%（28）；四氧嘧啶糖尿病组，1.99±0.03、1.72±0.03、86.4±1.4%（68）。4. 磷酸丙酮酸脱氢酶中磷酸化各个位点的占据百分比值（均值±标准误），按照位点1、2和3的顺序分别为：喂食组，100±2.7、27.8±1.6、33.9±0.9；饥饿组，100±1.4、76.2±2.0、92.4±1.5；四氧嘧啶糖尿病组，100±1.2、64.0±1.7、94.6±1.4。5. 得出结论：饥饿或四氧嘧啶糖尿病导致大鼠心脏中体内磷酸丙酮酸脱氢酶的磷酸化位点2和3的占据率增加2至3倍。