MODULATION OF SOLUBLE OVARIAN ADENOSINE 3',5'-MONOPHOSPHATE-DEPENDENT PROTEIN-KINASE ACTIVITY DURING PREPUBERTAL DEVELOPMENT OF THE RAT
MODULATION OF SOLUBLE OVARIAN ADENOSINE 3',5'-MONOPHOSPHATE-DEPENDENT PROTEIN-KINASE ACTIVITY DURING PREPUBERTAL DEVELOPMENT OF THE RAT
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DOI：
10.1210/endo-115-1-302
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发表时间：
1984-01-01
期刊：
影响因子：
4.8
通讯作者：
WEST, DE
中科院分区：
文献类型：
--
作者：
HUNZICKERDUNN, M;JUNGMANN, RA;WEST, DE
进行研究以评价在整个青春期前卵巢成熟（第5-37天）期间获得的大鼠卵巢可溶性部分中cAMP依赖性蛋白激酶的个体发生。cAMP刺激的蛋白激酶活性和抑制的热稳定的蛋白激酶抑制剂，指定的cAMP依赖性蛋白激酶活性，已经存在于卵巢5日龄大鼠。该激酶活性随后在第16天和第18天之间增加至最大值，然后在第21天和第23天之间暴跌4倍至最低点。cAMP依赖性蛋白激酶活性在第24天再次开始升高，增加5倍，在第27天达到最大值。进行实验以评估21-23日龄大鼠卵巢可溶性提取物中cAMP依赖性蛋白激酶活性降低80%的基础。为了确定激酶活性的降低是否伴随着cAMP结合活性的降低，评价了卵巢胞质溶胶结合[3 H]cAMP的能力。在cAMP依赖性蛋白激酶活性降低时，总可溶性cAMP结合活性未显著降低。此外，通过8-N3-[32 P]cAMP光亲和标记检测到的调节亚基RI和RII（cAMP依赖性蛋白激酶的I型和II型同工酶形式的调节亚基）的cAMP结合活性没有改变。为了确定激酶活性的下降是否是由于催化亚基从21-23日龄大鼠卵巢的可溶性卵巢提取物中实际消失所致，使用针对牛心脏催化亚基的抗血清通过酶联免疫吸附测定来定量催化亚基的相对量。蛋白激酶催化活性的降低不是由于催化亚基数量的减少。进行实验以确定蛋白激酶催化活性的降低是否不是由于催化亚基的量的减少。进行实验以确定在阴离子交换层析后未分级胞质溶胶中蛋白激酶催化活性的降低是否被表达。21-23日龄大鼠可溶性卵巢提取物经DEAE-纤维素层析后测得的cAMP刺激蛋白激酶活性估计值不再降低。由于在21-23日龄大鼠卵巢提取物中催化激酶活性降低80%，在未分级胞质溶胶中在DEAE-纤维素层析之前而不是之后可检测到，因此对胞质溶胶组分中催化激酶活性的内源性抑制剂的存在进行了试验。测定21-23日龄或任何其他青春期前年龄大鼠中热稳定蛋白激酶抑制剂的水平。然而，当测试21-24日龄大鼠的胞质溶胶调节外源性催化活性的能力时，观察到外源性催化活性的明显浓度依赖性抑制。这种抑制被取消的热处理的胞质溶胶和不存在的卵巢老年大鼠。在21-23日龄大鼠可溶性卵巢提取物中检测到cAMP依赖性蛋白激酶催化活性在DEAE-纤维素层析之前而非之后降低80%，这是一种尚未鉴定的热不稳定抑制剂的结果，该抑制剂在阴离子交换层析中释放。这种抑制剂的身份和生理意义目前尚不清楚。
Studies were conducted to evaluate the ontogeny of cAMP-dependent protein kinases in the soluble fraction of rat ovaries obtained throughout prepubertal ovarian maturation (days 5-37). Protein kinase activity stimulated by cAMP and inhibited by the heat-stable protein kinase inhibitor, designated cAMP-dependent protein kinase activity, was already present in ovaries of 5 day old rats. This kinase activity subsequently increased to reach maximal values between days 16 and 18, then plunged 4-fold to a nadir between days 21 and 23. cAMP-dependent protein kinase activity began to rise again by day 24 increasing 5-fold to a maximum on day 27. Experiments were conducted to evaluate the basis for the 80% reduction of cAMP-dependent protein kinase activity in the soluble extract of ovaries of 21-23 day old rats. To determine whether the decrease in kinase activity was accompanied by a concomitant reduction of cAMP-binding activity, the ability of ovarian cytosol to bind [3H]cAMP was evaluated. At the time of decreased cAMP-dependent protein kinase activity, total soluble cAMP-binding activity was not significantly reduced. Additionally, the cAMP-binding activity of the regulatory subunits RI and RII(regulatory subunits of the type I and II isoenzyme forms of cAMP-dependent protein kinase), as detected by photoaffinity labeling with 8-N3-[32P]cAMP, was not changed. To determine whether the decline in kinase activity was due to an actual disappearance of the catalytic subunit from the soluble ovarian extracts of ovaries of 21-23 day old rats, the relative amounts of catalytic subunit were quantified by an enzyme-linked immunosorbent assay using an antiserum directed against bovine heart catalytic subunit. The decrease in protein kinase catalytic activity was not due to a reduction in the amount of catalytic subunits. Experiments were conducted to determine whether the reduction of protein kinase catalytic activity was not due to a reduction in the amount of catalytic subunits. Experiments were conducted to determine whether the reduction of protein kinase catalytic activity in unfractionated cytosol was expressed after anion exchange chromatography. The estimated total cAMP-stimulated protein kinase activity measured after DEAE-cellulose chromatography of the soluble ovarian extracts of 21-23 day old rats was no longer depressed. Since the 80% reduction of catalytic kinase activity in ovarian extracts of 21-23 day old rats was detectable in unfractionated cytosol before but not after DEAE-cellulose chromatography, tests were made for the presence of an endogenous inhibitor of catalytic kinase activity in the cytosol fraction. Determinations of levels of the heat-stable protein kinase inhibitor in 21-23 day old rats or at any other prepubertal age. However, when cytosol of 21-24 day old rats was tested for its ability to modulate the activity of exogenous catalytic activity, an apparent concentration-dependent inhibition of exogenous catalytic activity was observed. This inhibition was abolished on heat treatment of the cytosol and was absent in ovaries of older rats. The 80% reduction of cAMP-dependent protein kinase catalytic activity detected in soluble ovarian extracts of 21-23 day old rats before but not after DEAE-cellulose chromatography is the result of an as yet unidentified heat-labile inhibitor which is freed on anion exchange chromatography. Neither the identity nor the physiological significance of this inhibitor is currently known.