PHOSPHORYLATION STATES OF ACTIN FILAMENT ADENINE-NUCLEOTIDES IN DETERGENT-EXTRACTED NEURONAL CYTOSKELETAL FRACTIONS

High performance liquid chromatography of nucleotides from Triton X-100 cytoskeletal extracts has permitted analysis of the ATP and ADP content of actin filaments isolated intact from PC12 pheochromocytoma cells. We observed that the adenine nucleotide content matched the actin content of these cytoskeletal extracts, a finding consistent with the unit stoichiometry of nucleotide binding. Efficient assembly-linked ATP hydrolysis occurs in vivo, and based on a boundary hydrolysis model for nucleotide-promoted assembly, the observed ADP/ATP ratio indicates that the average microfilament in nonmuscle cells has 24 ATP-actin molecules at its growing end. Studies with NB41A3 neuroblastoma cells indicate that the ATP content of assembled actin filaments is about 3-4 times lower. (C) 1994 Academic Press, Inc.