Isolation and characterization of mitochondrial F(1)-ATPase from crayfish (Orconectes virilis) gills.
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 15-fold purification was estimated. The properties for both soluble and membrane-bound enzyme were studied. Both enzyme forms were stable at 4 to -70 degrees C 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(2)=8.55, optimum pH 7.49). The anion-stimulated activities were in the order HCO(3)(-)>SO(4)(2-)>Cl(-). The apparent K(a) values for soluble enzyme were 11.4, 11.2, and 10.9 mM, respectively, but the K(a) of HCO(3)(-) 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 microM, respectively, but were ineffective in inhibiting soluble enzyme. Both enzyme forms shared identical sensitivity to DIDS (I(50)=12.5 microM) and vanadate (I(50)=9.0 mM). Soluble ATPase was significantly more sensitive to pCMB (I(50)=0.15 microM) and NO(3)(-) (I(50)=28.6 mM) than membrane-bound enzyme (I(50)=1.04 microM pCMB and 81.5 mM NO(3)(-)). In addition, soluble F(1)-ATPase was slightly more sensitive to azide (I(50)=91.8 microM) and NBD-Cl (I(50)=9.18 microM) than membrane-bound enzyme (I(50)=111.6 microM azide and 12.88 microM 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(1). In addition, an unmodified F(0) stabilizes F(1) and decreases F(1) sensitivities to inhibitors and modulators.
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Mount Sinai School of Medicine, Department of Human Genetics, 1425 Madison Ave., New York, NY 10029, USA.
Neufeld
G J
GJ
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000818:Animals; D000838:Anions; D003400:Astacoidea; D001386:Azides / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005880:Gills / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D009840:Oligomycins / Q000494:pharmacology; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification*; D013010:Sonication; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014638:Vanadates / Q000494:pharmacology; D020245:p-Chloromercuribenzoic Acid / Q000494:pharmacology
D000838:Anions; D001386:Azides; D004791:Enzyme Inhibitors; D009840:Oligomycins; D013439:Sulfhydryl Reagents; D014638:Vanadates; D020245:p-Chloromercuribenzoic Acid; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid