Cooper R H, Sul H S, McCullough T E, Walsh D A
J Biol Chem. 1980 Dec 25;255(24):11794-801.
This study reports the purification of bovine cardiac phosphorylase kinase to near homogeneity (approximately 1240-fold), as based upon SDS gel electrophoresis and sucrose density gradient ultracentrifugation (co-migration of enzyme activity and protein with constant specific activity throughout the peak). The molecular weight of the enzyme following purification is identical with that in the soluble extract from beef hearts, and similar to that of the rabbit skeletal muscle isoenzyme (i.e. approximately 1.3 X 10(6)) based on co-migration in sucrose density gradient ultracentrifugation. The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 134,000; beta, 125,000; gamma, 48,000. The subunit stoichiometry is determined to be alpha 1 beta 1.01 gamma 1.35 with a faint band co-migrating with purified bovine brain calmodulin. The enzyme displays little, if any, activity below pH 6.0, but activity increases markedly in the pH range of 6.8 to 8.2. After phosphorylation with pure cAMP-dependent protein kinase catalytic subunit and [gamma-32P]ATP (0.14 mM ATP, 4 mM magnesium acetate) in which the beta subunit was maximally phosphorylated (stoichiometry, 0.25 mol/mol; ratio of 32P in the alpha' and beta subunits, 1.90:1), enzyme activity was increased approximately 2-fold at pH 6.8, while at higher pH values, the effect of phosphorylation was less marked (20 to 50% increase at pH 8.2). Ca2+ is required for enzyme activity; ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (0.5 mM) completely inhibited the activity of th nonphosphorylated and phosphorylated enzyme, and activity was restored by the addition of Ca2+ (Ka for Ca2+, 1.94 and 1.35 microM for the nonphosphorylated and phosphorylated enzyme, respectively.
本研究报告了牛心肌磷酸化酶激酶的纯化，其纯度接近均一（约1240倍），这是基于SDS凝胶电泳和蔗糖密度梯度超速离心法（在整个峰中酶活性和蛋白质共迁移，比活性恒定）得出的。纯化后该酶的分子量与牛肉心脏可溶性提取物中的分子量相同，并且基于在蔗糖密度梯度超速离心法中的共迁移，与兔骨骼肌同工酶的分子量相似（即约1.3×10⁶）。通过SDS凝胶电泳测定的亚基分子量为：α′，134,000；β，125,000；γ，48,000。亚基化学计量比确定为α₁β₁.₀₁γ₁.₃₅，有一条 faint带与纯化的牛脑钙调蛋白共迁移。该酶在pH 6.0以下几乎没有活性，但在6.8至8.2的pH范围内活性显著增加。用纯的cAMP依赖性蛋白激酶催化亚基和[γ-³²P]ATP（0.14 mM ATP，4 mM乙酸镁）进行磷酸化后，其中β亚基被最大程度地磷酸化（化学计量比，0.25 mol/mol；α′和β亚基中³²P的比率，1.90:1），在pH 6.8时酶活性增加约2倍，而在较高pH值下，磷酸化的影响不太明显（在pH 8.2时增加20%至50%）。酶活性需要Ca²⁺；乙二醇双（β-氨基乙基醚）-N,N,N′,N′-四乙酸（EGTA）（0.5 mM）完全抑制未磷酸化和磷酸化酶的活性，通过添加Ca²⁺可恢复活性（未磷酸化和磷酸化酶的Ca²⁺的Ka分别为1.94和1.35 μM）。
