Analysis of Ribonucleotides by Reverse-Phase HPLC Using Ion Pairing on Radially Compressed Or Stainless Steel Columns

Darwish, A.; Prichard, Roger K.

doi:10.1080/01483918108064826

Cited by 31 publications

(6 citation statements)

References 13 publications

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“…Both proteins were >95% pure as judged by SDS-PAGE. All preparations were analyzed for their nucleotide content by RP-HPLC (Darwish & Prichard, 1981;John et al, 1990). Purified proteins were stored at -70 °C in buffer A (50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 0.5 mM DTE, 1 mM NaN3, 0.1 mM PMSF) containing 0.1 mM GDP.…”

Section: Methodsmentioning

confidence: 99%

An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21

et al. 1991

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We have measured the Raman spectrum of GDP bound to the elongation factor protein, EF-Tu, and the c-Harvey-ras protein, p21, two proteins of the guanine nucleotide binding family. In order to separate the Raman spectrum of the nucleotide from the much more intense protein spectrum, we investigate the feasibility of "tagging" the normal modes of the nucleotide by isotopic substitution, here by incoporating deuterium-labeled guanine at the C8 position into the active site. A difference spectrum between the labeled and unlabeled protein-nucleotide complex shows the changes in the Raman spectrum of the bound nucleotide that arise from the isotopic exchange. We find that surprisingly good Raman spectra of bound ligands can be obtained with this method and that the method can be easily generalized to other systems. The data show that the guanine amino group of the nucleotide interacts differently with both EF-Tu and p21 than it does with water, showing a change in hydrogen-bonding properties upon binding. On the other hand, no change in hydrogen bonding is observed at guanine's N7. The data strongly suggest that the conformation of the nucleotide when bound to EF-Tu and that p21 is the C2' endo pucker of the ribose ring and anti about the glycosidic bond. These results are compared to previous structural and chemical studies.

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Section: Methodsmentioning

confidence: 99%

An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21

et al. 1991

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“…Cdc42Hs does not bind to the Q-Sepharose column under these conditions, and the protein-containing flow-through was pooled and concentrated to ϳ20 mg/ml using an ultrafiltration cell (Amicon). In the case of the constitutively active Cdc42Hs(Q61L) protein, reverse phase high performance liquid chromatographic analysis confirmed that more than 90% of the purified, E. coli expressed protein was bound to GTP (20). The concentrated protein was stored at Ϫ20°C after dialysis into HMA plus 40% glycerol.…”

Section: Proteinsmentioning

confidence: 99%

Fluoride Activation of the Rho Family GTP-binding Protein Cdc42Hs

Hoffman¹,

Nassar²,

Oswald³

et al. 1998

Journal of Biological Chemistry

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“…The rest ofthe solution was analysed for nucleotide composition by ion-pair reverse phase h.p.l.c. (Darwish & Prichard, 1981). For this purpose an analytical C-18 h.p.l.c.…”

Section: Protocolmentioning

confidence: 99%

The kinetics of magnesium adenosine triphosphate cleavage in skinned muscle fibres of the rabbit.

Ferenczi¹,

Homsher²,

Trentham³

1984

The Journal of Physiology

136

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SUMMARY1. The time course of magnesium adenosine triphosphate (Mg ATP) cleavage in chemically skinned muscle fibres of the rabbit was measured by a method in which Mg ATP cleavage was initiated by photolytic release of ATP from P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP) and terminated by rapid freezing 50 ms to 8 s later. Up to 5 mM-ATP was released following a single 50 ns laser pulse at 347 nm. Mg ATP cleavage was measured at 19 0C in the presence and absence of calcium ions, for fibres near rest length and stretched beyond overlap of the myofilaments.2. At full overlap and in the absence of calcium (< 10-8 M) and nucleotide, the fibres developed rigor.tension. Following the laser pulse the tension decreased to that of a relaxed fibre in two distinct phases. The first phase lasted about 40 ms and was followed by a second phase during which tension decreased to zero with an approximately exponential time course with a rate constant of 11 s51. In the presence of 2 x 10-5 M-free calcium ions, the initial phase following the laser flash lasted approximately 13 ms, and was followed by an exponential rise of tension with a rate constant of 28 s-1. The active tension reached by the muscle fibres was 54 kN/m2. For fibres stretched beyond overlap, no change in tension was observed following the release of Mg ATP.3. Under all conditions the time course of Mg ATP cleavage was biphasic, and consisted of a rapid initial burst of ADP formation, complete within 50 ms, followed by a slower steady-state rate of Mg ATP cleavage. The number of molecules of Mg ATP cleaved during the burst was approximately equal to the number of myosin subfragment 1 heads for fibres at full myofilament overlap, and equal to 0-7 molecules per myosin subfragment 1 head for fibres stretched beyond overlap. At full overlap in the presence of calcium ions, the steady-state rate equalled 1'8 mol Mg ATP cleaved per mole myosin subfragment 1 head per second. In all other cases the steady-state rate of Mg ATP cleavage was at least 10-fold less.4. When fibres at full overlap were pre-incubated with 2 mM-ADP, the initial phase M. A. FERENCZI, E. HOMSHER AND D. R. TRENTHAMof the tension response was somewhat prolonged, but the burst of ADP formation was also complete within 50 ms. However, in the case of fibres stretched beyond overlap, the presence of ADP reduced the rate of the initial burst of ADP formation by at least a factor of 3. 5. A low steady-state rate of Mg ATP cleavage for fibres stretched beyond overlap in the presence of calcium ions showed that other ATPases such as the calcium ion-dependent ATPase of the sarcoplasmic reticulum did not cleave Mg ATP significantly. This conclusion was supported by control experiments in which fibres had been treated with Triton X-100.6. The results show that during an isometric contraction most of the myosin heads are in the form of product intermediate states and that this steady state is reached rapidly (<50 ms). These properties are similar to those of the Mg-dependent actomyosin subfra...

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Analysis of Ribonucleotides by Reverse-Phase HPLC Using Ion Pairing on Radially Compressed Or Stainless Steel Columns

Cited by 31 publications

References 13 publications

An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21

An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21

Fluoride Activation of the Rho Family GTP-binding Protein Cdc42Hs

The kinetics of magnesium adenosine triphosphate cleavage in skinned muscle fibres of the rabbit.

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