TRANSPORT ADENOSINE TRIPHOSPHATASE CYTOCHEMISTRY I. BIOCHEMICAL CHARACTERIZATION OF A CYTOCHEMICAL MEDIUM FOR THE ULTRASTRUCTURAL LOCALIZATION OF OUABAIN-SENSITIVE, POTASSIUM-DEPENDENT PHOSPHATASE ACTIVITY IN THE AVIAN SALT GLAND
TRANSPORT ADENOSINE TRIPHOSPHATASE CYTOCHEMISTRY I. BIOCHEMICAL CHARACTERIZATION OF A CYTOCHEMICAL MEDIUM FOR THE ULTRASTRUCTURAL LOCALIZATION OF OUABAIN-SENSITIVE, POTASSIUM-DEPENDENT PHOSPHATASE ACTIVITY IN THE AVIAN SALT GLAND
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转运腺苷三磷酸酶细胞化学 I. 鸟类盐腺中哇巴因敏感、钾依赖性磷酸酶活性超微结构定位的细胞化学介质的生物化学表征
DOI：
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发表时间：
1972
影响因子：
3.2
通讯作者：
S. A. Ernst
中科院分区：
文献类型：
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作者：
S. A. Ernst
K-依赖，哇巴因敏感的水解p-硝基苯磷酸钾-硝基苯磷酸酶的最佳动力学参数，在接近那些采用细胞化学的条件下，确定在禽盐腺的酶的超微结构定位的必要先决条件。在50 µ低温恒温器切片中对多聚甲醛固定的组织进行表征，室温下在含有5 mM硝基苯磷酸盐、10 mM MgCl 2、20 mM SrCl 2和100 mM Tris-HCl缓冲液（pH 9.0）（含或不含10 mM KCl）的培养基中孵育。为了进行比较，在不存在Sr的情况下进行平行测定，Sr是用于沉淀磷酸盐用于细胞化学的重金属盐。酶活性是通过测定水解的硝基苯酚的量来确定的。在这个系统中，Sr作为一个纯粹的非竞争性抑制剂的酶，导致50%的抑制在3 mM和87%在20 mM。的Km为酶是4.5 mM。Sr（20 mM）导致8倍减少的表观亲和力的酶镁，但对K亲和力的影响不大。哇巴因的酶的敏感性降低50倍，在20 mM Sr的存在下。这种酶的活性的关系，钠-K-激活的腺苷三磷酸酶和应用这种确定的培养基运输腺苷三磷酸酶细胞化学进行了讨论。
The optimal kinetic parameters for the K-dependent, ouabain-sensitive hydrolysis of p-nitrophenyl phosphate by K-nitrophenyl phosphatase, under conditions closely approximating those employed for cytochemistry, were determined in the avian salt gland as a necessary prerequisite for the ultrastructural localization of the enzyme. The enzyme was characterized in 50-µ cryostat sections of paraformaldehyde-fixed tissue, incubated at room temperature in a medium containing 5 mM nitrophenyl phosphate, 10 mM MgCl2, 20 mM SrCl2 and 100 mM Tris-HCl buffer (pH 9.0), either with or without 10 mM KCl. For comparison, parallel assays were conducted in the absence of Sr, the heavy metal salt used to precipitate phosphate for cytochemistry. Enzymatic activity was determined by measuring spectrophotometrically the amount of nitrophenol hydrolyzed. In this system, Sr acts as a pure noncompetitive inhibitor of the enzyme, causing 50% inhibition at 3 mM and 87% at 20 mM. The Km for the enzyme is 4.5 mM. Sr (20 mM) causes an 8-fold reduction in the apparent affinity of the enzyme for Mg but has little effect on K affinity. The sensitivity of the enzyme to ouabain is decreased 50-fold in the presence of 20 mM Sr. The relationship of this enzymatic activity to Na-K-activated adenosine triphosphatase and the application of this defined medium to transport adenosine triphosphatase cytochemistry are discussed.