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
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), Vol. 26:25
- Country of Publication:
- United States
- Language:
- English
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ATP
ENZYMATIC HYDROLYSIS
ISOTOPIC EXCHANGE
MYOSIN
BIOCHEMISTRY
CHEMICAL BONDS
DEUTERIUM COMPOUNDS
LABELLED COMPOUNDS
MUSCLES
OXYGEN 17
OXYGEN 18
PHOSPHORUS 32
PHOSPHOTRANSFERASES
RABBITS
STOICHIOMETRY
WATER
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHEMICAL REACTIONS
CHEMISTRY
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
ENZYMES
EVEN-EVEN NUCLEI
EVEN-ODD NUCLEI
GLOBULINS
HYDROGEN COMPOUNDS
HYDROLYSIS
ISOTOPES
LIGHT NUCLEI
LYSIS
MAMMALS
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
OXYGEN ISOTOPES
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PROTEINS
RADIOISOTOPES
SOLVOLYSIS
STABLE ISOTOPES
TRANSFERASES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques