Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase.

A probe has been developed that can rapidly measure micromolar concentrations of inorganic phosphate (Pi), in particular to follow the release of Pi in real time from enzymes such as phosphatases. Its application is described to investigate the mechanism of actomyosin subfragment 1 ATPase. The probe uses the A197C mutant of Escherichia coli phosphate binding protein (PBP), generated by oligonucleotide-directed mutagenesis. A new fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), was attached to the single cysteine to produce the reporter molecule that was purified free of unlabeled protein and unattached MDCC. The labeled protein has an excitation maximum at 425 nm and emission maximum at 474 nm in the absence of Pi, shifting to 464 nm with a 5.2-fold increase in fluorescence (lambda max/lambda max) when complexed with Pi at pH 7.0, low ionic strength, 22 degrees C. The fluorescence increase is not much altered by change to pH 8 or by increase in ionic strength to 1 M. Pi binds tightly (Kd approximately 0.1 microM) and rapidly (1.36 x 10(8) M-1 s-1) and the dissociation rate constant is 21 s-1, at pH 7.0, low ionic strength, 22 degrees C. A variety of phosphate esters were tested to investigate the specificity of the MDCC-PBP and none gave a significant fluorescence increase at 100 microM or higher concentration. ATP weakly inhibited the Pi-induced fluorescence change, indicating that it binds at least 3000-fold weaker than Pi. Because Pi is a widespread contaminant, the probe is used in conjunction with a "Pi mop", consisting of 7-methylguanosine and purine nucleoside phosphorylase, to remove free Pi from solutions by its conversion to ribose 1-phosphate. Because the equilibrium constant of this reaction is > 100, free Pi can be reduced below 0.1 microM. The probe was used to measure the rate of Pi release during a single turnover of ATP hydrolysis with actomyosin subfragment 1 from rabbit skeletal muscle, to determine to what extent Pi release contributes to the rate limitation of this ATPase. Using a stopped-flow apparatus, a small lag prior to rapid Pi release was detected at pH 7.0, low ionic strength, between 5 and 22 degrees C at both high and low [ATP]. For measurements of a single turnover at low [ATP], the observed rate increased with [actin], showing saturation with a Km with respect to actin of 26 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

[1]  J. Eccleston,et al.  Elementary processes of the magnesium ion-dependent adenosine triphosphatase activity of heavy meromyosin. A transient kinetic approach to the study of kinases and adenosine triphosphatases and a colorimetric inorganic phosphate assay in situ. , 1972, The Biochemical journal.

[2]  S. Lehrer,et al.  Intrinsic fluorescence of actin. , 1972, Biochemistry.

[3]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[4]  F. Quiocho,et al.  Crystallization and x-ray diffraction studies of a phosphate-binding protein involved in active transport in Escherichia coli. , 1986, Journal of Biological Chemistry.

[5]  C. Wu,et al.  N-(1-pyrene)maleimide: a fluorescent cross-linking reagent. , 1976, Biochemistry.

[6]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[7]  D. Jans,et al.  Structural gene for the phosphate-repressible phosphate-binding protein of Escherichia coli has its own promoter: complete nucleotide sequence of the phoS gene , 1984, Journal of bacteriology.

[8]  J A McCammon,et al.  Hinge-bending in L-arabinose-binding protein. The "Venus's-flytrap" model. , 1982, The Journal of biological chemistry.

[9]  S. Roy,et al.  A continuous fluorimetric assay for ATPase activity. , 1990, The Biochemical journal.

[10]  Clive R. Bagshaw,et al.  The reversibility of adenosine triphosphate cleavage by myosin. , 1973, The Biochemical journal.

[11]  K. Shukla,et al.  Mechanism of oxygen exchange in actin-activated hydrolysis of adenosine triphosphate by myosin subfragment 1. , 1977, Biochemistry.

[12]  M. Webb.  The role of nucleoside triphosphate hydrolysis in transducing systems: p21ras and muscle. , 1992, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[13]  A. G. WEEDS,et al.  Separation of subfragment-1 isoenzymes from rabbit skeletal muscle myosin , 1975, Nature.

[14]  A. Bzowska,et al.  Properties of two unusual, and fluorescent, substrates of purine-nucleoside phosphorylase: 7-methylguanosine and 7-methylinosine. , 1986, Biochimica et biophysica acta.

[15]  E. Eisenberg,et al.  Intermediate oxygen exchange catalyzed by the actin-activated skeletal myosin adenosinetriphosphatase. , 1989, Biochemistry.

[16]  F. Eckstein,et al.  Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis. , 1986, Nucleic acids research.

[17]  K. Kitagishi,et al.  Cryoenzymic studies on actomyosin ATPase: kinetic evidence for communication between the actin and ATP sites on myosin. , 1991, Biochemistry.

[18]  J. Biosca,et al.  Transient kinetics of the binding of ATP to actomyosin subfragment 1: evidence that the dissociation of actomyosin subfragment 1 by ATP leads to a new conformation of subfragment 1. , 1984, Biochemistry.

[19]  H. Shinagawa,et al.  Nucleotide sequence of the phoS gene, the structural gene for the phosphate-binding protein of Escherichia coli , 1984, Journal of bacteriology.

[20]  M. Malamy,et al.  Control of the synthesis of alkaline phosphatase and the phosphate-binding protein in Escherichia coli , 1976, Journal of bacteriology.

[21]  M. Webb.  A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[22]  J. Corrie.  A convenient synthesis of N-(7-dimethylamino-4-methylcoumarin-3-yl)-maleimide incorporating a novel variant of the Pechmann reaction , 1990 .

[23]  J. Sleep,et al.  Exchange between inorganic phosphate and adenosine 5'-triphosphate in the medium by actomyosin subfragment 1. , 1980, Biochemistry.

[24]  F. Travers,et al.  Is a four‐state model sufficient to describe actomyosin ATPase? , 1990, FEBS letters.

[25]  D. Trentham,et al.  The mechanism of ATP hydrolysis catalyzed by myosin and actomyosin, using rapid reaction techniques to study oxygen exchange. , 1981, The Journal of biological chemistry.

[26]  S. Rosenfeld,et al.  The ATPase mechanism of skeletal and smooth muscle acto-subfragment 1. , 1984, The Journal of biological chemistry.

[27]  F. Quiocho,et al.  High specificity of a phosphate transport protein determined by hydrogen bonds , 1990, Nature.

[28]  E. Eisenberg,et al.  The rate-limiting step in the actomyosin adenosinetriphosphatase cycle. , 1984, Biochemistry.

[29]  Y. Goldman,et al.  Kinetics of the actomyosin ATPase in muscle fibers. , 1987, Annual review of physiology.

[30]  D. Trentham,et al.  Relationships between chemical and mechanical events during muscular contraction. , 1986, Annual review of biophysics and biophysical chemistry.

[31]  P. Boyer,et al.  Effect of actin concentration on the intermediate oxygen exchange of myosin; relation to the refractory state and the mechanism of exchange. , 1978, Biochemistry.

[32]  F. Eckstein,et al.  The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. , 1985, Nucleic acids research.

[33]  F. Quiocho,et al.  Crystallographic evidence of a large ligand-induced hinge-twist motion between the two domains of the maltodextrin binding protein involved in active transport and chemotaxis. , 1992, Biochemistry.

[34]  H. Rosenberg,et al.  The phosphate-binding protein of Escherichia coli , 1970 .

[35]  P. V. von Hippel,et al.  Calculation of protein extinction coefficients from amino acid sequence data. , 1989, Analytical biochemistry.

[36]  K. Makino,et al.  Hyperproduction of phosphate-binding protein, phoS, and pre-phoS proteins in Escherichia coli carrying a cloned phoS gene. , 1983, European journal of biochemistry.

[37]  T. Noll,et al.  Enzymic determination of inorganic phosphates, organic phosphates and phosphate-liberating enzymes by use of nucleoside phosphorylase-xanthine oxidase (dehydrogenase)-coupled reactions. , 1985, The Biochemical journal.