ETHANOL UNFOLDS FIREFLY LUCIFERASE WHILE COMPETITIVE INHIBITORS ANTAGONIZE UNFOLDING - DSC AND FTIR ANALYSES

被引:18
作者
CHIOU, JS
UEDA, I
机构
[1] UNIV UTAH,SCH MED,SALT LAKE CITY,UT 84148
[2] VET ADM MED CTR,DEPT ANAESTHESIA,SALT LAKE CITY,UT 84148
关键词
ANESTHESIA THEORY; ALCOHOLS; PROTEIN FOLDING; ENZYME PROTEIN; NONCOMPETITIVE INHIBITOR; PROTEIN PHASE TRANSITION; CALORIMETRY;
D O I
10.1016/0731-7085(94)00045-X
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
Firefly luciferase has gained popularity as a protein model in elucidating anaesthesia mechanism because the bioluminescence of the purified enzyme system is extremely sensitive to volatile anaesthetics. This study analysed the thermal unfolding of firefly luciferase by differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR). DSC showed that the transition of firefly luciferase from the folded (N) to unfolded (D) state occurred at 41.7 degrees C with the excess heat flow of 1.6 cal g(-1) protein. Ethanol decreased the transition temperature dose dependently. In contrast, luciferin competitors, anilinonaphthalenesulphonate (ANS), toluidinonaphthalenesulphonate (TNS), and myristic acid increased the transition temperature. The competitive inhibitors antagonized unfolding and stabilized the N-state. Ethanol promoted unfolding and stabilized the D-state. Temperature scan by FTIR agreed with the DSC data. The intensities of amide-I' and amide-II' bands started to increase at 20-25 degrees C. This temperature coincides with the temperature where the bioluminescence of firefly luciferase is maximal. The unfolding effect of ethanol was evident even at 5 degrees C. ANS, TNS, and myristic acid completely protected the enzyme from the thermal unfolding. This is the first demonstration that the noncompetitive inhibitors induce the isothermal first-order phase transition in a functional protein, whereas competitive inhibitors protect the enzyme from thermal unfolding. The action mode of competitive inhibitors on firefly luciferase is completely different from that of noncompetitive inhibitors.
引用
收藏
页码:969 / 975
页数:7
相关论文
共 30 条
[1]   TO FOLD OR NOT TO FOLD [J].
AGARD, DA .
SCIENCE, 1993, 260 (5116) :1903-1904
[2]  
Ainsworth S., 1977, STEADY STATE ENZYME
[3]  
ALOIA RC, 1991, ADV MEMBRANE FLUIDIT, V5
[4]   BEHAVIOR OF FATTY-ACID DURING THE INCUBATION OF BOVINE SERUM-ALBUMIN - ENTITY OF THE SERUM-ALBUMIN RESISTANT TO HEAT [J].
AOKI, K ;
HAYAKAWA, N ;
NODA, K ;
TERADA, H ;
HIRAMATSU, K .
COLLOID AND POLYMER SCIENCE, 1983, 261 (04) :359-364
[5]  
BOYER PD, 1946, J BIOL CHEM, V162, P181
[6]  
BRANDT J, 1976, INT J PEPT PROT RES, V8, P33
[7]   ALCOHOLS DEHYDRATE LIPID-MEMBRANES - AN INFRARED STUDY ON HYDROGEN-BONDING [J].
CHIOU, JS ;
KRISHNA, PR ;
KAMAYA, H ;
UEDA, I .
BIOCHIMICA ET BIOPHYSICA ACTA, 1992, 1110 (02) :225-233
[8]   THE ALPHA-HELIX TO BETA-SHEET TRANSITION IN POLY(L-LYSINE) - EFFECTS OF ANESTHETICS AND HIGH-PRESSURE [J].
CHIOU, JS ;
TATARA, T ;
SAWAMURA, S ;
KAMINOH, Y ;
KAMAYA, H ;
SHIBATA, A ;
UEDA, I .
BIOCHIMICA ET BIOPHYSICA ACTA, 1992, 1119 (02) :211-217
[9]  
CLARK AH, 1981, INT J PEPT PROT RES, V17, P353
[10]   HYDROPHOBIC NATURE OF ACTIVE SITE OF FIREFLY LUCIFERASE [J].
DELUCA, M .
BIOCHEMISTRY, 1969, 8 (01) :160-&