Corbin J D, Sugden P H, Lincoln T M, Keely S L
J Biol Chem. 1977 Jun 10;252(11):3854-61.
In rabbit heart homogenates about 50% of the cAMP-dependent protein kinase activity was associated with the low speed particulate fraction. In homogenates of rat or beef heart this fraction represented approximately 30% of the activity. The percentage of the enzyme in the particulate fraction was not appreciably affected either by preparing more dilute homogenates or by aging homogenates for up to 2 h before centrifugation. The particulate enzyme was not solubilized at physiological ionic strength or by the presence of exogenous proteins during homogenization. However, the holoenzyme or regulatory subunit could be solubilized either by Triton X-100, high pH, or trypsin treatment. In hearts of all species studied, the particulate-bound protein kinase was mainly or entirely the type II isozyme, suggesting isozyme compartmentalization. In rabbit hearts perfused in the absence of hormones and homogenized in the presence of 0.25 M NaCl, at least 50% of the cAMP in homogenates was associated with the particulate fraction. Omitting NaCl reduced the amount of particulate-bound cAMP. Most of the particulate-bound cAMP was probably associated with the regulatory subunit in this fraction since approximately 70% of the bound nucleotide was solubilized by addition of homogeneous catalytic subunit to the particulate fraction. The amount of cAMP in the particulate fraction (0.16 nmol/g of tissue) was approximately one-half the amount of the regulatory subunit monomer (0.31 nmol/g of tissue) in this fraction. The calculated amount of catalytic subunit in the particulate fraction was 0.18 nmol/g of tissue. Either epinephrine alone or epinephrine plus 1-methyl-3-isobutylxanthine increased the cAMP content of the particulate and supernatant fractions. The cAMP level was increased more in the supernatant fraction, possibly because the cAMP level became saturating for the regulatory subunit in the particulate fraction. The increase in cAMP was associated with translocation of a large percentage of the catalytic subunit activity from the particulate to the supernatant fraction. The distribution of the regulatory subunit of the enzyme was not significantly affected by this treatment. The catalytic subunit translocation could be mimicked by addition of cAMP to homogenates before centrifugation. The data suggest that the regulatory subunit of the protein kinase, at least that of isozyme II, is bound to particulate material, and theactive catalytic subunit is released by formation of the regulatory subunit-cAMP complex when the tissue cAMP concentration is elevated. A model for compartmentalized hormonal control is presented.
在兔心脏匀浆中，约50%的环磷酸腺苷（cAMP）依赖性蛋白激酶活性与低速颗粒部分相关。在大鼠或牛心脏匀浆中，该部分的活性约占30%。无论是制备更稀的匀浆，还是在离心前将匀浆放置长达2小时，颗粒部分中该酶的百分比均未受到明显影响。在生理离子强度下或匀浆过程中存在外源蛋白时，颗粒酶都不会溶解。然而，全酶或调节亚基可通过Triton X - 100、高pH值或胰蛋白酶处理而溶解。在所研究的所有物种的心脏中，颗粒结合的蛋白激酶主要或完全是II型同工酶，提示同工酶的区室化。在无激素灌注并在0.25 M NaCl存在下匀浆的兔心脏中，匀浆中至少50%的cAMP与颗粒部分相关。省略NaCl会减少颗粒结合的cAMP量。该部分中大部分颗粒结合的cAMP可能与调节亚基相关，因为向颗粒部分添加均一的催化亚基可使约70%结合的核苷酸溶解。颗粒部分中cAMP的量（0.16 nmol/g组织）约为该部分调节亚基单体量（0.31 nmol/g组织）的一半。颗粒部分中催化亚基的计算量为0.18 nmol/g组织。单独肾上腺素或肾上腺素加1 - 甲基 - 3 - 异丁基黄嘌呤均可增加颗粒部分和上清部分的cAMP含量。上清部分的cAMP水平升高更多，可能是因为颗粒部分中调节亚基的cAMP水平达到饱和。cAMP的增加与大部分催化亚基活性从颗粒部分转位至上清部分相关。该处理对酶调节亚基的分布没有显著影响。在离心前向匀浆中添加cAMP可模拟催化亚基的转位。数据表明，蛋白激酶的调节亚基，至少II型同工酶的调节亚基，与颗粒物质结合，当组织cAMP浓度升高时，活性催化亚基通过调节亚基 - cAMP复合物的形成而释放。本文提出了一种区室化激素控制模型。
