A Calcium-Dependent Protease Exhibiting Circadian Rhythm in Synechococus RF-1

Abstract

自聚球藻RF-1品系中分離出一種分子量約為180 kDa之蛋白酶（P-180），此蛋白酶位於細胞膜，具有概日韻律之特性。當藻株以12 h光／12 h暗設定後，藻細胞之P-180活性出現於光週期，在暗週期消失，此24小時之週期變化在細胞轉移到連續照光後仍然可以維持。由於P-180與 pepstatin具有專一之親和性，故應屬於天門冬胺酸類之蛋白酶。添加EGTA於溶液中可抑制P-180之活性，故此蛋白酶之活性需要鈣離子。經由光／暗週期設定之藻細胞，如於進入光週期前添加氯黴素則測不到P-180之活性，故P-180韻律之調控與新蛋白質之合成有關。此文同時對聚球藻細胞膜上之另一蛋白酶（P-55）加以分析比較，P-55不具韻律現象，故在探討P-180之韻律時，通常以P-55做為內在之對照指標。

A protease with an experimental molecular weight of 180 kDa (P-180) was isolated from Synechococcus RF-1 which exhibited a circadian rhythm under free running conditions after L/D entrainment. When Synechococcus RF-1 cultures were entrained at a 12 h L/12 h D regimen, the activity of P-180 appeared at light phase and disappeared during dark phase. P-180 bound specifically on the affinity gel column containing immobilized pepstatin, so it presumptively belongs to the aspartic type proteases. Calcium was shown to be required for its activity because its activity was inhibited in the presence of EGTA. When chloramphenicol was added before the occurrence of P-180 activity, its activity could not be found. Thus, the rhythmic activity of P-180 was regulated by rhythmic protein synthesis. In addition to P-180, another protease isolated from Synechococcus RF-1 with an experimental molecular weight of 55 kDa (P-55) was also studied. The activity of P-55 remained constant even when the cultures were grown under a L/D regimen, so it was used as an internal standard during the examination of the rhythmic activity of P-180.