Characterization of the residues modified when the bovine mitochondrial F/sub 1/-ATPase is inactivated with 7-chloro-4-nitrobenzofurazan
When bovine heart mitochondrial F/sub 1/-ATPase (MF/sub 1/), taken as ..cap alpha../sub 3/b/sub 3/..gamma..deltaepsilon with a molecular weight of 375,000, was inactivated by greater than 90% with a 4-fold molar excess of 7-chloro-4-nitro(/sup 14/C)benzofurazan ((/sup 14/C)Nbf-Cl) at pH 7.4, a total of 1.42 mol (/sup 14/C)Nbf was incorporated per mol of enzyme. Treatment of the inactivated enzyme with dithiothreitol led to restoration of the lost activity and removed 0.99 mol (/sup 14/C)Nbf per mol of MF/sub 1/, indicating that this much was present as (/sup 14/C)Nbf-O-Tyr derivatives. The major radioactive tryptic peptide derived from the ..beta.. subunit was purified by gel filtration on Sephadex G-75 followed by reverse phase HPLC on a C-18 column and sequence analysis of it demonstrated that the /sup 14/C was released at the position corresponding to ..beta..-Tyr-311. In another experiment, when MF/sub 1/ was inactivated with (/sup 14/C)Nbf-Cl as described above, the enzyme incorporated a total of 1.15 mol (/sup 14/C)Nbf per mol of enzyme, 0.82 mol/mol of which was present as (/sup 14/C)Nbf-O-Tyr derivatives and 0.33 mol/mol of which was present as (/sup 14/C)Nbf-N/sup epsilon/-Lys derivatives. Incubation of the modified enzyme at pH 9.0 for 18 hr at room temperature led to an increase of 0.64 mol of (/sup 14/C)Nbf-N/sup epsilon/-Lys per mol of F/sub 1/ that was due to an O ..-->.. N migration of the (/sup 14/C)Nbf group. A single major radioactive tryptic peptide resulted as a consequence of the O ..-->.. N migration, the sequence analysis of which demonstrated that ..beta..-Lys-162 was the residue specifically labelled.
- Research Organization:
- California Univ., San Diego (USA)
- OSTI ID:
- 6640398
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ATP-ASE
MOLECULAR STRUCTURE
CARBON 14 COMPOUNDS
CATTLE
GEL PERMEATION CHROMATOGRAPHY
HEART
LIQUID COLUMN CHROMATOGRAPHY
LYSINE
MITOCHONDRIA
NITRO COMPOUNDS
TRACER TECHNIQUES
TYROSINE
ACID ANHYDRASES
AMINO ACIDS
ANIMALS
BODY
CARBOXYLIC ACIDS
CARDIOVASCULAR SYSTEM
CELL CONSTITUENTS
CHROMATOGRAPHY
DOMESTIC ANIMALS
ENZYMES
HYDROLASES
HYDROXY ACIDS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MAMMALS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANOIDS
ORGANS
PHOSPHOHYDROLASES
RUMINANTS
SEPARATION PROCESSES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques