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Subunit composition and ATP site labeling of the coated vesicle proton-translocating adenosinetriphosphatase

Journal Article · · Biochemistry; (United States)
OSTI ID:5403173
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The partially purified proton-translocating adenosinetriphosphatase ((H/sup +/)-ATPase) from clathrin-coated vesicles has been reported to contain eight polypeptides of molecular weights 15,000-116,000. To determine whether these polypeptides form a single macromolecular complex, the authors have isolated three monoclonal antibodies which recognize the reconstitutively active (H/sup +/)-ATPase in the native, detergent-solubilized state. All three monoclonal antibodies precipitate the same set of polypeptides from either the partially purified enzyme or the detergent-solubilized coated vesicle membrane proteins. The immunoprecipitated polypeptides have molecular weights of 100,000, 73,000, 58,000, 40,000, 38,000, 34,000, 33,000, 19,000, and 17,000. These results thus indicate that this set of polypeptides forms a single macromolecular complex and suggest that they correspond to subunits of the coated vesicle (H/sup +/)-ATPase. To identify the ATP-hydrolytic subunit of the coated vesicle (H/sup +/)-ATPase, the purified enzyme was reacted with N-ethylmaleimide (NEM) and 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-C1), both of which inhibit activity in an ATP-protectable manner. Labeling was carried out by using (/sup 3/H)NEM or (/sup 14/C)NBD-C1, and the specificity of the reaction was increased by prelabeling of the protein with the nonradioactive reagents in the presence of ATP and by taking advantage of the nucleotide specificity of protection. The principal polypeptide labeled by both (/sup 3/H)NEM and (/sup 14/C)NBD-C1 had a molecular weight of 73,000. In addition, this protein was the only polypeptide whose labeling was significantly reduced in the presence of ATP. These results suggest that the 73,000-dalton polypeptide participates in ATP hydrolysis by the coated vesicle (H/sup +/)-ATPase.

Research Organization:
Tufts Univ. School of Medicine, Boston, MA
OSTI ID:
5403173
Journal Information:
Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:21; ISSN BICHA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ANIMALS
ANTIBODIES
ANTIMITOTIC DRUGS
ATP
ATP-ASE
BARYONS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMISTRY
BODY
BRAIN
CALVES
CARBON 14 COMPOUNDS
CATTLE
CENTRAL NERVOUS SYSTEM
CHEMICAL REACTIONS
CHEMISTRY
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
DOMESTIC ANIMALS
DRUGS
ELEMENTARY PARTICLES
ENZYMATIC HYDROLYSIS
ENZYME IMMUNOASSAY
ENZYME INHIBITORS
ENZYMES
FERMIONS
HADRONS
HYDROLASES
HYDROLYSIS
IMIDES
IMMUNOASSAY
ISOTOPES
LABELLED COMPOUNDS
LABELLING
LIGHT NUCLEI
LYSIS
MAMMALS
MEMBRANE TRANSPORT
MOLECULAR WEIGHT
MONOCLONAL ANTIBODIES
NEM
NERVOUS SYSTEM
NUCLEI
NUCLEONS
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PEPTIDES
PHOSPHOHYDROLASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
POLYPEPTIDES
PROTEINS
PROTONS
RADIOISOTOPES
RADIOSENSITIZERS
RUMINANTS
SOLVOLYSIS
TRITIUM COMPOUNDS
VERTEBRATES