One-step molybdate method for rapid determination of inorganic phosphate in the presence of protein

Piper, Jeanna; Lovell, Stephen J.

doi:10.1016/0003-2697(81)90693-x

Cited by 36 publications

(19 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Thus the acid-quenched pre-steady state dTTPase kinetics indicates that dTTP hydrolysis on the helicase active site is faster than the subsequent steps of product release and the burst amplitude being close to one indicates that only one dTTP is hydrolyzed during the first turnover. 18 (21), has been used to analyze the reversibility of the hydrolysis step of many ATPases. Briefly, high concentrations of dTDP (6 mM) and [ 18 O]P i (20 mM) were mixed with gp4AЈ, and incorporation of unlabeled oxygen from water into P i due to any reversible dTTPase reaction was monitored with time.…”

Section: Resultsmentioning

confidence: 99%

“…Briefly, high concentrations of dTDP (6 mM) and [ 18 O]P i (20 mM) were mixed with gp4AЈ, and incorporation of unlabeled oxygen from water into P i due to any reversible dTTPase reaction was monitored with time. The loss of 18 O label in P i was monitored by 31 P NMR experiments. It is already known that 18 O bound to the phosphorous atom shifts the 31 P NMR by about 0.02 ppm up-field compared that of the 16 O species (19).…”

Section: Resultsmentioning

confidence: 99%

“…The loss of 18 O label in P i was monitored by 31 P NMR experiments. It is already known that 18 O bound to the phosphorous atom shifts the 31 P NMR by about 0.02 ppm up-field compared that of the 16 O species (19). A control experiment was conducted without gp4AЈ to obtain the distribution of […”

Section: Resultsmentioning

confidence: 99%

“…The fractional contribution of each [ 18 O]P i species to the spectrum was evaluated directly from the peak area. The observed 18 O distribution in the phosphate were compared with those obtained from the control experiment, which was performed without gp4AЈ.…”

Section: Phosphate-water Oxygen Exchange Experiment-[mentioning

confidence: 99%

See 3 more Smart Citations

Kinetic Pathway of dTTP Hydrolysis by Hexameric T7 Helicase-Primase in the Absence of DNA

Jeong

Kim

Patel

2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

Bacteriophage T7 gp4A protein is a hexameric helicase-primase protein that separates the strands of a duplex DNA in a reaction coupled to dTTP hydrolysis. Here we reexamine in more detail the kinetic mechanism of dTTP hydrolysis by a preassembled T7 helicase hexamer in the absence of DNA. Pre-steady state dTTP hydrolysis kinetics showed a distinct burst whose amplitude indicated that a preformed hexamer of T7 helicase hydrolyzes on an average one dTTP per hexamer. The presteady state chase-time experiments provided evidence for sequential hydrolysis of two dTTPs. The medium [ 18 O]P i exchange experiments failed to detect dTTP synthesis, indicating that the less than six-site hydrolysis observed is not due to reversible dTTP hydrolysis on the helicase active site. The P i -release rate was measured directly using a stopped-flow fluorescence assay, and it was found that the rate of dTTP hydrolysis on the helicase active site is eight times faster than the P i -release rate, which in turn is three times faster than the dTDP release rate. Thus, the rate-limiting step in the pathway of helicase-catalyzed deoxythymidine triphosphatase Helicases are motor proteins that translocate along nucleic acids using the energy of NTP 1 hydrolysis. This activity facilitates many nucleic acid metabolic processes including those that require the separation of double-stranded DNA into their component single strands (1-3). A class of helicases assembles into ring-shaped hexamers, and helicases belonging to this class have been found to function in genome replication and recombination that require processive action of the helicase (1). The assembly of helicase subunits into hexamers is generally induced by NTP binding, and the hexamer is also stabilized by DNA (1, 4 -6). Bacteriophage T7 gp4 protein is one of the better-studied ring helicases whose function is to facilitate T7 genome replication and recombination. T7 gp4 proteins gp4AЈ and gp4B assemble into rings in the presence of nucleotide triphosphate, preferably dTTP or its non-hydrolyzable analog dTMP-PCP. The hexamer has a high affinity for ssDNA that binds within the central channel of the ring. It has been proposed that the helicase passes only one strand of the doublestranded DNA through the central channel and excludes the complementary strand from the central channel as it translocates and separates the strands of the double-stranded DNA (7-10).The translocation and strand separation activity of T7 helicase is fueled by dTTP hydrolysis. The mechanism by which dTTP hydrolysis is coupled to translocation and strand separation is not known. Our previous studies indicate that the dTTPase mechanism of gp4AЈ shows striking similarity to the binding change mechanism of the F 1 -ATPase protein (11) despite there being no amino acid sequence homology between them. These studies showed that two-three dTTPs bound tightly but did not get hydrolyzed at the steady state rate, and these were referred to as the noncatalytic sites. Two additional dTTPs, referred to as catalytic sites, we...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Phosphate-water Oxygen Exchange Experiment-[mentioning

confidence: 99%

See 2 more Smart Citations

Kinetic Pathway of dTTP Hydrolysis by Hexameric T7 Helicase-Primase in the Absence of DNA

Jeong

Kim

Patel

2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Mant-ATP Hydrolysis-The kinetics of mant-ATP hydrolysis in the presence of RNA was measured by quantifying the product phosphate using the molybdate method (22). Mant-ATP hydrolysis was not observed in the absence of RNA.…”

Section: Methodsmentioning

confidence: 99%

Nucleotide Binding Induces Conformational Changes in Escherichia coli Transcription Termination Factor Rho

Jeong

Kim

Patel

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

The Escherichia coli Rho protein uses the energy of ATP binding and hydrolysis to translocate along RNA and cause transcription termination. Using fluorescence stopped-flow kinetic studies, we have discerned the conformational changes in the Rho protein that occur upon nucleotide and nucleic acid binding. We show that the 2,(3)-O-[N-methylanthraniloyl] derivative of ATP (mant-ATP) is a good fluorescent substrate of Rho and is hydrolyzed with a K m comparable with that for ATP but a k cat five to six times slower than that for ATP. The kinetics of ATP and mant-ATP binding indicates that, in the absence of RNA, the Rho protein is structurally distinct from the Rho hexamer found when bound to RNA or DNA. In the absence of RNA, the nucleotidebinding rates are 50-to 70-fold slower, and the dissociation rates are 40-to 120-fold slower than the corresponding rates in the presence of RNA. We conclude that RNA or DNA binding to the primary nucleic acid binding sites causes conformational changes in the Rho hexamer that result in the opening of the subunit interfaces. Furthermore, the kinetic studies revealed a unique protein conformational change in the Rho⅐RNA complex upon ATP binding that is a result of RNA contacting the secondary nucleic acid binding sites in the central channel of the Rho ring. This conformational change seems to render the Rho ring competent in ATP hydrolysis and translocation.The Escherichia coli transcription termination factor Rho is a hexameric protein that has the ability to unwind RNA/DNA heteroduplexes (1-5). Although the exact mechanism by which Rho causes transcription termination is not known, Rho protein binds nascent mRNA at specific loading sites, and it is believed that Rho translocates along RNA until it reaches the transcription complex, where it disrupts the transcription ternary complex (3, 6). Translocation along nucleic acid is, therefore, a basic activity of Rho similar to helicases (7, 8), and understanding it requires the knowledge of how nucleotide binding and hydrolysis events at the multiple nucleotide-binding sites on the Rho hexamer are coordinated to the mechanics of protein translocation. In this study, we measured the kinetics of nucleotide binding to Rho complexed to RNA or DNA that reveal conformational changes in the Rho protein that we believe are important for RNA binding and ATP hydrolysis.E. coli Rho protein contains two types of nucleic acid binding sites, referred to as the primary and the secondary sites (9). The primary nucleic acid binding sites reside in the N-terminal domains that form a crown over the Rho hexamer (10), and these sites bind pyrimidine-rich DNA or RNA with a high affinity (11). In addition to wrapping around the primary sites, the RNA but not the DNA interacts with the secondary sites in the central channel of the hexamer (12). The kinetics of RNA binding to the Rho hexamer showed that the initial binding of RNA occurs at a diffusion-limited rate constant to the primary sites (13). The primary sites act as loading sites that facilitate th...

show abstract

Isolation and characterization of Brush border fragments from mosquito mesenterons

Houk

Arcus

Hardy

1986

Arch Insect Biochem Physiol

View full text Add to dashboard Cite

Brush border fragments were isolated from homogenates of mesenterons from the mosquito, Culex tarsalis, by a combination of Ca2+ precipitation and differential centrifugation. These preparations were routinely enriched seven-to eightfold for the brush border marker enzyme, leucine aminopeptidase. Alkaline phosphatase, a putative brush border marker for both vertebrate and invertebrate brush borders, was found to be unsuitable for Cx. tarsalis. lsoelectric focusing electrophoresis coupled with histochemical enzyme detection was used to enumerate isozymic species of nonspecific esterases [3], leucine aminopeptidase [I], and alkaline phosphatase [ I ] in isolated brush border fragments. Leucine aminopeptidase activity was solubilized by papain digestion, suggesting an extrinsic active site for this membrane-bound enzyme. The predominant nonspecific esterase isozyme remained membrane-bound. Conventional staining (ie, Coomassie Blue and silver) of proteins separated by isoelectric focusing, sodium dodecylsulfate, and two-dimensional electrophoresis indicated a simple pattern for brush border fragments, with two proteins predominating among the 11-14 routinely detected.

show abstract

One-step molybdate method for rapid determination of inorganic phosphate in the presence of protein

Cited by 36 publications

References 8 publications

Kinetic Pathway of dTTP Hydrolysis by Hexameric T7 Helicase-Primase in the Absence of DNA

Kinetic Pathway of dTTP Hydrolysis by Hexameric T7 Helicase-Primase in the Absence of DNA

Nucleotide Binding Induces Conformational Changes in Escherichia coli Transcription Termination Factor Rho

Isolation and characterization of Brush border fragments from mosquito mesenterons

Contact Info

Product

Resources

About