Analysis of positional isotope exchange in ATP by cleavage of the. beta. P-O. gamma. P bond. Demonstration of negligible positional isotope exchange by myosin

Journal Article · Tue Dec 15 04:00:00 EST 1987 · Biochemistry; (United States)

OSTI ID:5446218

Dale, M P; Hackney, D D

A method for analysis of positional isotope exchange (PIX) during ATP in equilibrium HOH oxygen exchange is presented that uses a two-step degradation of ATP resulting in cleavage of the ..beta..P-O..gamma..P bond. This cleavage yields P/sub i/ derived from the ..gamma..-phosphoryl of ATP that contains all four of the ..gamma.. oxygens. Both PIX between the ..beta.., ..gamma..-bridge and ..beta..-nonbridge positions and washout of the ..gamma..-nonbridge oxygens can be simultaneously followed by using ATP labeled with /sup 17/O at the ..beta..-nonbridge positions and /sup 18/O at the ..beta..,..gamma..-bridge and ..gamma..-nonbridge positions. Application of this method to ATP in equilibrium HOH exchange during single turnovers of myosin indicates that the bulk of the ATP undergoes rapid washout of ..gamma..-nonbridge oxygens in the virtual absence of PIX. At 25/sup 0/C with subfragment 1 the scrambling rate is at the limit of detectability of approximately 0.001 s/sup -1/, which is 50-fold slower than the steady-state rate. This corresponds to a probability of scrambling for the ..beta..-oxygens of bound ADP of 1 in 10,000 for each cycle of reversible hydrolysis of bound ATP. A fraction of the ATP, however, does not undergo rapid washout. With myosin and stoichiometric ATP at 0/sup 0/C, this fraction correspond to 10% of the ATP remaining at 36 s, or 2% of the initial ATP, and an equivalent level of ATP is found that does not bind irreversibly to myosin in a cold chase experiment. A significant level of apparent PIX is observed with subfragment 1 in the fraction that resists washout, and this apparent PIX is shown to be due to contaminant adenylate kinase activity. This apparent PIX due to adenylate kinase provides a possible explanation for the PIX observed by Geeves et al. with subfragment 1.

• Research Organization: Carnegie Mellon Univ., Pittsburgh, PA
• OSTI ID: 5446218
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:25; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
ATP
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMISTRY
CHEMICAL BONDS
CHEMICAL REACTIONS
CHEMISTRY
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
DEUTERIUM COMPOUNDS
ENZYMATIC HYDROLYSIS
ENZYMES
EVEN-EVEN NUCLEI
EVEN-ODD NUCLEI
GLOBULINS
HYDROGEN COMPOUNDS
HYDROLYSIS
ISOTOPES
ISOTOPIC EXCHANGE
LABELLED COMPOUNDS
LIGHT NUCLEI
LYSIS
MAMMALS
MUSCLES
MYOSIN
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
OXYGEN 17
OXYGEN 18
OXYGEN COMPOUNDS
OXYGEN ISOTOPES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PROTEINS
RABBITS
RADIOISOTOPES
SOLVOLYSIS
STABLE ISOTOPES
STOICHIOMETRY
TRANSFERASES
VERTEBRATES
WATER