Hise M K, Mehta P S
Department of Internal Medicine, University of Maryland Medical School, Baltimore 21201.
Endocrinology. 1988 Sep;123(3):1553-8. doi: 10.1210/endo-123-3-1553.
The substrate specificities of calcium/phospholipid-dependent kinase-C (PKC) were examined in rat kidney cortex, and localization of the protein was studied after the induction of diabetes. The cytosolic kinase was eluted from an anion exchange resin using a linear gradient of 0-0.15 M NaCl. A sharp peak of activity was demonstrated at approximately 80 mM using histone as a substrate. The kinase demonstrated a broad pH optimum of 6.5-8.0. ATP was the preferred phosphorus donor. The Ka for ATP averaged 2.6 +/- 0.1 microM (n = 4) and was not different in diabetic animals. Lysine-rich histones, but not arginine-rich or mixed histones, were the most suitable phosphorus acceptors. Phosphatidylserine stimulated kinase activity with Ka of 4.5 +/- 0.7 microM in the presence of 20 microM diolein (n = 3). Twenty micromolar diolein in the presence of 25 microM phosphatidylserine lowered the apparent Ka for calcium from 17.2 +/- 1.4 to 3.3 +/- 1.5 microM (n = 3; P less than 0.01). Similar data were evident in diabetic animals. Diabetic renal growth was induced by the injection of streptozotocin (35 mg/kg, iv). At the end of 4 weeks, blood glucose averaged 119.6 +/- 7.4 mg/dl in vehicle-injected controls and 548.7 +/- 21.6 mg/dl in diabetic animals (n = 5; P less than 0.001). Despite reduced weight gains in diabetic animals, renal protein content was increased in this group compared to the control value. Neither cytosolic nor proximal tubule basolateral membrane PKC activity changed after the induction of diabetes; however, luminal brush border PKC activity increased from 83.8 +/- 4.6 pmol/mg.min in control animals to 107.3 +/- 55 pmol/mg.min in diabetic animals (n = 5; P less than 0.02). The increased activity in the brush border membrane may have important consequences for the growth response of the kidney in diabetes.
在大鼠肾皮质中检测了钙/磷脂依赖性激酶-C（PKC）的底物特异性，并在诱导糖尿病后研究了该蛋白的定位。使用0-0.15M NaCl的线性梯度从阴离子交换树脂上洗脱胞质激酶。以组蛋白为底物时，在约80mM处显示出一个尖锐的活性峰。该激酶的最适pH范围较宽，为6.5-8.0。ATP是首选的磷供体。ATP的Ka平均值为2.6±0.1μM（n = 4），在糖尿病动物中无差异。富含赖氨酸的组蛋白，而非富含精氨酸或混合组蛋白，是最合适的磷受体。在存在20μM二油精的情况下，磷脂酰丝氨酸刺激激酶活性，Ka为4.5±0.7μM（n = 3）。在存在25μM磷脂酰丝氨酸的情况下，20μM二油精将钙的表观Ka从17.2±1.4降低至3.3±1.5μM（n = 3；P＜0.01）。在糖尿病动物中也有类似的数据。通过注射链脲佐菌素（35mg/kg，静脉注射）诱导糖尿病性肾生长。4周结束时，注射载体的对照组血糖平均为119.6±7.4mg/dl，糖尿病动物血糖平均为548.7±21.6mg/dl（n = 5；P＜0.001）。尽管糖尿病动物体重增加减少，但该组肾蛋白含量相对于对照值有所增加。糖尿病诱导后，胞质或近端小管基底外侧膜PKC活性均未改变；然而，管腔刷状缘PKC活性从对照动物的83.8±4.6pmol/mg·min增加至糖尿病动物的107.3±55pmol/mg·min（n = 5；P＜0.02）。刷状缘膜中活性增加可能对糖尿病时肾脏的生长反应具有重要影响。
