Selman-Reimer S, Merchant S, Selman B R
Biochemistry. 1981 Sep 15;20(19):5476-82. doi: 10.1021/bi00522a020.
Chloroplast thylakoid particles were prepared from wild-type Chlamydomonas reinhardi by gentle sonication. These particles catalyzed phenazine methosulfate dependent photophosphorylation with rates ranging from 300 to 700 mumol of adenosine 5'-triphosphate (ATP) formed (mg of chlorophyll)-1h-1. Photophosphorylation was not sensitive to tentoxin but was sensitive to an anticoupling factor 1 (CF1) antiserum preparation made against spinach CF1. The C. reinhardi chloroplast CF1 was isolated from thylakoid particles by either chloroform or ethylenediaminetraacetic acid extraction. The former enzyme appeared to be missing the gamma subunit and did not reconstitute with partially resolved thylakoid particles. The latter enzyme reconstituted with partially resolved particles and had a specific activity at 37 degrees C of 2-5 umol of ATP hydrolyzed (mg of protein)-1 min-1. The enzyme utilized both MnATP and MgATP. CaATP was a poor substrate, and SrATP was not hydrolyzed. The enzyme was not activated by heat or proteolysis but was stimulated approximately 2-fold by 50 mM dithiothreitol. Alcohols reversibly stimulated the ATPase activity of the enzyme 5-25-fold. Ethanol, 20%, dramatically lowered the temperature optimum from approximately 75 to approximately 45 degrees C and slightly lowered the pH optimum from 8.5 to 8.2. Ethanol had no effect on the activation energy of the ATPase reaction (17 +/- 1.7 kcal/mol). The kinetics of the ATPase reaction catalyzed by the C. reinhardi enzyme are complex. Both free divalent cations and divalent cation ATP inhibited the activity of the enzyme. The apparent Km for MgTAP (55 uM free Mg2+) was approximately 0.2 mM.
通过温和超声处理从野生型莱茵衣藻中制备叶绿体类囊体颗粒。这些颗粒催化了依赖硫酸甲酯吩嗪的光磷酸化反应，形成三磷酸腺苷（ATP）的速率范围为300至700 μmol（毫克叶绿素）⁻¹小时⁻¹。光磷酸化对抗霉素A不敏感，但对抗菠菜CF1制备的抗偶联因子1（CF1）抗血清敏感。莱茵衣藻叶绿体CF1通过氯仿或乙二胺四乙酸提取从类囊体颗粒中分离出来。前一种酶似乎缺少γ亚基，并且不能与部分解离的类囊体颗粒重组。后一种酶与部分解离的颗粒重组，在37℃时的比活性为2 - 5 μmol ATP水解（毫克蛋白质）⁻¹分钟⁻¹。该酶利用MnATP和MgATP。CaATP是一种较差的底物，而SrATP不被水解。该酶不被加热或蛋白水解激活，但被50 mM二硫苏糖醇刺激约2倍。醇类可逆地刺激该酶的ATP酶活性5 - 25倍。20%的乙醇显著降低了最适温度，从约75℃降至约45℃，并略微降低了最适pH，从8.5降至8.2。乙醇对ATP酶反应的活化能没有影响（17±1.7千卡/摩尔）。莱茵衣藻酶催化的ATP酶反应动力学很复杂。游离二价阳离子和二价阳离子ATP都抑制该酶的活性。MgTAP（游离Mg²⁺为55 μM）的表观Km约为0.2 mM。
