Colley A M, Law M L
Department of Ophthalmology, Emory University School of Medicine, Emory Eye Center, Atlanta, GA 30322.
Metab Pediatr Syst Ophthalmol (1985). 1987;10(1):24-31.
Cyclic nucleotide-dependent protein kinase activity and cyclic nucleotide binding were assayed in cell homogenates and subcellular fractions of cultured rabbit corneal epithelium, and effects of carbamylcholine (1 mM) on activity in each fraction were evaluated. In cell homogenates and in nuclei, carbamylcholine significantly elevated cGMP-dependent kinase activity and [3H]cGMP binding, and reduced cAMP-dependent kinase activity and [3H]cAMP binding. In the cytosol, the drug significantly reduced cAMP kinase and cAMP binding but did not alter cGMP binding or kinase activity. In both mitochondria/lysosomes and microsomes, cGMP binding was significantly enhanced and cAMP binding reduced, but differences in protein kinase activity were not significant. The drug did not alter cyclic nucleotide-independent protein kinase activity. All observed effects were blocked by atropine. Kinase activity in the purified nuclear fraction also was assayed over a range of carbamylcholine, substrate, and cyclic nucleotide concentrations. Vmax for cGMP kinase in control nuclei was 295 +/- 18 pmol/mg protein/min (KM = 367 +/- 70 micrograms/ml histone) vs. Vmax = 846 +/- 6 pmol/mg/min (KM = 131 +/- 13 mu/ml histone) in carbamylcholine-treated nuclei. Vmax for cAMP kinase in control nuclei was 282 +/- 12 pmol/mg/min (KM = 172 +/- 7 micrograms/ml histone); 1 mM carbamylcholine abolished nuclear cAMP-dependent kinase activity. Cyclic nucleotide concentrations at half-maximal binding were 5.4 +/- 0.9 nM cGMP and 9.3 +/- 0.4 nM cAMP, as compared to 15.4 +/- 5.0 nM cGMP and 36.2 +/- 6.2 nM cAMP for half-maximal protein kinase activity. Regression analysis of Hill plots for variation of nuclear cyclic nucleotide binding and kinase activity as a function of carbamylcholine concentration indicated half-maximal drug effects on activity of both enzymes at approximately 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
在培养的兔角膜上皮细胞匀浆和亚细胞组分中测定了环核苷酸依赖性蛋白激酶活性和环核苷酸结合情况，并评估了氨甲酰胆碱（1 mM）对各组分中活性的影响。在细胞匀浆和细胞核中，氨甲酰胆碱显著提高了cGMP依赖性激酶活性和[3H]cGMP结合，并降低了cAMP依赖性激酶活性和[3H]cAMP结合。在胞质溶胶中，该药物显著降低了cAMP激酶和cAMP结合，但未改变cGMP结合或激酶活性。在线粒体/溶酶体和微粒体中，cGMP结合均显著增强，cAMP结合减少，但蛋白激酶活性差异不显著。该药物未改变非环核苷酸依赖性蛋白激酶活性。所有观察到的效应均被阿托品阻断。还在一系列氨甲酰胆碱、底物和环核苷酸浓度范围内测定了纯化细胞核组分中的激酶活性。对照细胞核中cGMP激酶的Vmax为295±18 pmol/mg蛋白/分钟（KM = 367±70微克/毫升组蛋白），而氨甲酰胆碱处理的细胞核中Vmax为846±6 pmol/mg/分钟（KM = 131±13微升/毫升组蛋白）。对照细胞核中cAMP激酶的Vmax为282±12 pmol/mg/分钟（KM = 172±7微克/毫升组蛋白）；1 mM氨甲酰胆碱使细胞核中cAMP依赖性激酶活性丧失。与半最大蛋白激酶活性时的15.4±5.0 nM cGMP和36.2±6.2 nM cAMP相比，半最大结合时的环核苷酸浓度分别为5.4±0.9 nM cGMP和9.3±0.4 nM cAMP。对细胞核中环核苷酸结合和激酶活性随氨甲酰胆碱浓度变化的希尔图进行回归分析表明，在约1 microM时药物对两种酶活性的半最大效应。（摘要截断于250字）
