Damuni Z, Merryfield M L, Humphreys J S, Reed L J
Proc Natl Acad Sci U S A. 1984 Jul;81(14):4335-8. doi: 10.1073/pnas.81.14.4335.
Branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase was purified about 8000-fold from extracts of bovine kidney mitochondria. The highly purified phosphatase exhibited a molecular weight of approximately 460,000, as estimated by gel-permeation chromatography. Another form of the phosphatase, with an apparent molecular weight of approximately 230,000, was also detected under conditions of high dilution. In contrast to pyruvate dehydrogenase phosphatase, BCKDH phosphatase was active in the absence of divalent cations. BCKDH phosphatase was inactive toward 32P-labeled phosphorylase a, but exhibited approximately 10% maximal activity with 32P-labeled pyruvate dehydrogenase complex. BCKDH phosphatase activity was inhibited by GTP, GDP, ATP, ADP, UTP, UDP, CTP, and CDP. Half-maximal inhibition occurred at about 60, 200, 200, 400, 100, 250, 250, and 400 microM, respectively. These inhibitions were reversed completely by 2 mM Mg2+. GTP was replaceable by guanosine 5'-(beta, gamma-imido)triphosphate. GMP, AMP, UMP, CMP, NAD, and NADH showed little effect, if any, on BCKDH phosphatase activity at concentrations up to 1 mM. Heparin showed half-maximal inhibition at 2 micrograms/ml. This inhibition was only partially (30%) reversed by 2 mM Mg2+. CoA and various acyl-CoA compounds exhibited half-maximal inhibition at 150-300 microM. These inhibitions were not reversed by 2 mM Mg2+. BCKDH phosphatase activity was stimulated 1.5- to 3-fold by protamine, poly(L-lysine), and poly(L-arginine) at 3.6 micrograms/ml.
支链α-酮酸脱氢酶（BCKDH）磷酸酶从牛肾线粒体提取物中纯化了约8000倍。通过凝胶渗透色谱法估计，高度纯化的磷酸酶分子量约为460,000。在高稀释条件下还检测到另一种形式的磷酸酶，其表观分子量约为230,000。与丙酮酸脱氢酶磷酸酶不同，BCKDH磷酸酶在没有二价阳离子的情况下具有活性。BCKDH磷酸酶对32P标记的磷酸化酶a无活性，但对32P标记的丙酮酸脱氢酶复合物表现出约10%的最大活性。BCKDH磷酸酶活性受到GTP、GDP、ATP、ADP、UTP、UDP、CTP和CDP的抑制。半数最大抑制分别发生在约60、200、200、400、100、250、250和400微摩尔处。这些抑制作用可被2 mM Mg2+完全逆转。GTP可被鸟苷5'-（β，γ-亚氨基）三磷酸替代。在浓度高达1 mM时，GMP、AMP、UMP、CMP、NAD和NADH对BCKDH磷酸酶活性几乎没有影响（如果有影响的话）。肝素在2微克/毫升时表现出半数最大抑制。这种抑制作用仅被2 mM Mg2+部分（30%）逆转。辅酶A和各种酰基辅酶A化合物在150 - 300微摩尔时表现出半数最大抑制。这些抑制作用不能被2 mM Mg2+逆转。在3.6微克/毫升时，鱼精蛋白、聚（L-赖氨酸）和聚（L-精氨酸）可将BCKDH磷酸酶活性刺激1.5至3倍。
