A soluble F-1-ATPase was isolated from the mitochondria of crayfish (Orconectes virilis) gill tissue. The maximal mitochondrial disruption rate (95%) was obtained by sonicating for 4 min at pH 8.6. A Ii-fold purification was estimated. The properties for both soluble and membrane-bound enzyme were studied. Both enzyme forms were stable at 4 to - 70 degreesC when kept in 20% glycerol. Soluble F-1-ATPase was more stable at room temperature than membrane-bound enzyme. It displayed a narrower pH profile (pK(1) = 6.58. pK(2) = 7.68) and more acid pH optimum (7.13) than membrane-bound enzyme (pK(1) = 6.42, pK, = 8.55, optimum pH 7.49). The anion-stimulated activities were in the order HCO3- SO42->Cl-. The apparent K-a values for soluble enzyme were 11.4, 11.2. and 10.9 mM, respectively, but the K-a of HCO3- for membrane-bound enzyme (14.9 mM) was higher than for soluble enzyme. Oligomycin and DCCD inhibited membrane-bound F-1-ATPase with I-50 of 18.6 ng/ml and 2.2 muM respectively, but were ineffective in inhibiting soluble enzyme. Both enzyme forms shared identical sensitivity to DIDS (I-50 = 12.5 muM) and vanadate I-50 = 9.0 mM). Soluble ATPase was significantly more sensitive to pCMB (I-50 = 0.15 muM) and NO; (I,,, = 28.6 mM) than membrane-bound enzyme (I-50 = 1.04 muM pCMB and 81.5 mM NO3-). In addition, soluble F-1-ATPase was slightly more sensitive to azide (I-50 = 91.8 muM) and NBD-Cl (I-50 = 91.8 muM) than membrane-bound enzyme (I-50 = 111.6 muM azide and 12.88 muM NBD-Cl). These data suggest a conformational change transmission between F-0 and F-1 sectors and slight conformational differences between soluble F-1 and membrane-bound F,. In addition, an unmodified F-0 stabilizes F-1 and decreases F-1 sensitivities to inhibitors and modulators. (C) 2001 Elsevier Science Inc. All rights reserved.
