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Biomedical subjects

O Jardetzky

Publications and source records attributed to O Jardetzky.

At least 73 records · Page 4Linked to original sources
PubMed1984

Measurement of mitochondrial ATPase activity in maize root tips by saturation transfer p nuclear magnetic resonance.

We show that saturation transfer (31)P nuclear magnetic resonance can be used to measure the activity of the mitochondrial ATPase of maize (Zea mays L. hybrid WW x Br38) root tips in vivo. Unidirectional rates of ATP synthesis were determined in the steady state (i.e. ATP and cytoplasmic orthophosphate constant) under various conditions. These measurements, and determinations of oxygen consumption, give a P/O ratio (measured in the living tissue) close to 3. In succinate-fed root tips the P/O ratio is approximately 2. Cyanide inhibits the rate of ATP synthesis by two-thirds (P/O ratio approximately 1), with an effective inhibitor constant of approximately 35 micromolars. We show that the alternative electron pathway cannot make ATP, and does not normally operate in this tissue. This method of studying plant mitochondrial metabolism avoids potential artifacts encountered in studies using isolated and purified mitochondria. The method also allows, for the first time, direct and simultaneous examination of the relationship between the rate of ATP synthesis and levels of metabolites such as ATP, and derived parameters such as phosphorylation potential.

Journal Article↗
PubMed1984

Salt stimulation of phosphate uptake in maize root tips studied by p nuclear magnetic resonance.

The effects of external salt and inorganic phosphate (Pi) on the concentrations of vacuolar Pi, and cytoplasmic Pi, ATP, glucose-6-phosphate and UDP-glucose in maize root tips were examined using (31)P nuclear magnetic resonance spectroscopy. We observed a more than two-fold stimulation of Pi uptake from 10 millimolar KH(2)PO(4) solutions when root tips were exposed to 100 millimolar NaCl + CaCl(2). This stimulation of Pi uptake was associated with an increase in the concentration of cytoplasmic Pi in root tip cells. Thus, the molar ratio of cytoplasmic Pi to Pi + ATP + glucose-6-phosphate + UDP-glucose increased greatly in root tips exposed to salt and Pi. We speculate that it is this disturbance in relative concentrations of cytoplasmic phosphates (which we show are normally tightly regulated) that is responsible for both the greater rate of uptake of Pi by vacuoles of excised maize root tips, and the previously documented stimulation of Pi translocation from root to shoot in whole maize plants exposed to salt and Pi.

Journal Article↗
PubMed1983

Actin-induced changes in the dynamics of myosin subfragment-1 detected by nuclear magnetic resonance.

Analysis of high resolution 1H NMR spectra for myosin and myosin subfragment-1 (S-1) indicates that S-1 has an unusual structure, about 20% of which is mobile. The rest of the myosin molecule and F-actin are rigid by comparison. A wide variety of perturbations do not affect the S-1 internal mobility and suggest that the mobile structure is located in the interior of S-1. Actin binding uniquely quenches the internal motions entirely. The F and G forms have a similar effect. Nucleotide binding restores the internal motions under conditions known to cause dissociation of the acto-S-1 complex. A model of force generation by the actomyosin-nucleotide system, which incorporates this striking actin-induced change in S-1 structural dynamics, is proposed and discussed.

Actins↗
PubMed1982

Continuous-flow NMR culture system for mammalian cells.

A continuous-flow NMR culture system for mammalian cells has been developed on which 31P-NMR experiments under complete and strictly physiologic conditions have been performed. Observations on the response of the cellular metabolism to stresses such as starvation, low temperature and changes in environmental pH monitored by 31P-NMR are reported. The response of the intracellular pH relative to the external pH of the growth medium is studied. We find that under the experimental conditions used there exists a delta pH varying between less than 0.2 and more than 0.6 pH units. These results are compatible with those obtained using other techniques.

Animals↗
PubMed1982

Bending motions and internal motions in myosin rod.

Depolarized light scattering and high-resolution 1H NMR measurements were made on solutions of light meromyosin (LMM) and myosin rod in 0.6 M KCl-0.010 M pyrophosphate, pH 9.5, at 20 degrees C. The light scattering data indicated LMM is a rigid molecule. Myosin rod is best described as a once-broken rod with domains that can freely diffuse in conical volumes. The maximum angle that the cone surface makes with the myosin rod axis is 128 degrees, indicating the bending motion of the domains is largely unrestrained. NMR data indicated the fraction of the structure that could be in a random-coil configuration was equal and less than 0.04 for both hydrodynamically rigid LMM and bending myosin rod. Thus, the flexible bending of myosin rod appears to not be due to a random-coil structure.

Animals↗
PubMed1982

Regulation of Cytoplasmic and Vacuolar pH in Maize Root Tips under Different Experimental Conditions.

(31)P-Nuclear magnetic resonance spectra of perfused maize (Zea mays L., hybrid WW x Br 38) root tips, obtained at 10-minute intervals over 12 hours or longer, indicate that no cytoplasmic or vacuolar pH changes occur in these cells in the presence of 25 millimolar K(2)SO(4), which induces extrusion of 4 to 5 microequivalents H(+) per gram per hour. In contrast, hypoxia causes cytoplasmic acidification (0.3-0.6 pH unit) without a detectable change in vacuolar pH. The cytoplasm quickly returns to its original pH on reoxygenation. Dilute NH(4)OH increases the vacuolar pH more than it does the cytoplasmic pH; after NH(4)OH is removed, the vacuole recovers its original pH more slowly than does the cytoplasm. The results indicate that regulation of cytoplasmic pH and that of vacuolar pH in plant cells are separate processes.

Journal Article↗
PubMed1981

Intracellular pH measurements by 31P nuclear magnetic resonance. Influence of factors other than pH on 31P chemical shifts.

Titration curves plotting chemical shift vs. pH for inorganic phosphate and glucose 6-phosphate in solutions of various composition are presented. Physiological concentrations of K+ (0.1 M) and Mg2+ (5 mM) are shown to significantly shift the titration curve. The Mg2+ effect can be partly or completely reversed by addition of sufficient quantities of adenosine triphosphate or organic acids. The acidic protein bovine serum albumin and soluble maize root tip protein have no noticeable effect on the titration curves, whereas the basic protein protamine exerts a profound effect. The results clearly indicate that knowledge of intracellular ionic strength and free Mg2+ concentrations in the sample are required if the determination of intracellular pH by 31P NMR is to be considered accurate within +/- 0.05-01 pH unit.

Cells↗
PubMed1981

pH dependence of the high-resolution proton nuclear magnetic resonance spectrum of the lac repressor headpiece.

The pH dependence of the N-terminal 51 amino acid headpiece (HP) of the lac repressor has been followed by using 1H NMR spectroscopy to monitor the chemical shifts of resolved aromatic and methyl resonances. The NMR evidence reveals the folded HP domain to be stable from pH 1 to 10 at 23 degrees C. All observed resonances shift toward their expected random-coil positions above pH 10, which suggests that a general unfolding occurs. The four tyrosine rings reflect a combination of unfolding and titration in the order 7 greater than 17, 12 greater than 47. This pH-induced unfolding is completely reversible. In addition, strikingly similar pH behavior for selected tyrosine and methyl resonances at acid pH values suggests that clusters of various tyrosine, methyl, and carboxyl side chains exist in the native structure.

Amino Acids↗
PubMed1981

High-resolution proton nuclear magnetic resonance studies of the exchangeable resonances of the lac repressor headpiece.

The exchangeable hydrogens of the N-terminal 51 amino acid headpiece (HP) of the lac repressor have been examined with high-resolution 1H NMR. Resonances from 35 +/- 3 protons are observable in 2H2O solutions at p2H 4.2. The exchange rates for some of these have been measured over the p2H range 1.5-5. In H2O, resonances from 70 +/- 6 protons are observable at pH 3.2 and 35 +/- 5 protons at pH 7.1. The temperature dependence of the spectrum at neat neutral pH shows the existence of a group of approximately 22 protons particularly slow to exchange. The pH dependence of the spectrum in H2O solution over the range 1-11 is discussed. Results are compared with NMR studies of the nonexchanging protons and CD and IR results.

Deuterium↗
PubMed1981

Internal motions in myosin. 2.

The high-resolution 1H NMR detected internal motions in myosin and myosin subfragment 1 (S1) [Highsmith, S., Akasaka, K., Konrad, M., Goody, R., Holmes, K., Wade-Jardetzky, N., & Jardetzky, O. (1979) Biochemistry 18, 4238--4244] were unperturbed by induced changes in the rate of protein tumbling, and the mobile regions proved inaccessible to added surface-directed paramagnetic probes. The rate of tumbling was changed by changing the solvent viscosity for S1 or by aggregation to thick filaments for myosin. Neither manipulation caused a measurable broadening of the narrow lines in the spectrum. Sulfhydryl-directed covalently attached nitroxide spin-labels, soluble nitroxide spin-labels, and MnCl2 were used to probe the surface. Unique labeling at the fastest reacting thiol of S1 had no effect on the NMR spectrum. Multiple labeling of thiols caused a small but detectable broadening of the narrow peaks. Soluble spin-labels and MnCl2 had a very small effect on the narrow bands even in great excess. The results substantiate the notion that myosin has internal motions that are independent of the overall rate of rotation and suggest that the mobile structure is mainly in the interior of the S1 moiety. This supports a model in which actin quenches the internal motions of myosin by changing the structure of myosin upon binding.

Animals↗
PubMed1981

High-resolution nuclear magnetic resonance studies of the Lac repressor. 1. Assignments of tyrosine resonances in the N-terminal headpiece.

The DNA binding site of the lac repressor protein has been implicated to lie within the N-terminal 51 amino acid fragment termed headpiece (HP-51 or LR-51). High-resolution NMR suggests that isolated HP-51 retains most of the secondary and tertiary structure which it has in the whole repressor. Four of the eight tyrosines of repressor are in HP-51. 1H NMR spectra (360 MHz) over the aromatic region of native HP-51 show that the four tyrosines are nonequivalent with an unusual distribution of chemical shifts. Denaturation leads to loss of these chemical shift differences. Homonuclear decoupling and a two-dimensional autocorrelated spectrum allow unequivocal pairing of resonances from Tyr A at 6.99 and 6.79 ppm, Tyr B at 6.98 and 6.39 ppm, Tyr C at 6.70 and 6.54 ppm, and Tyr D at 6.39 and 6.33 ppm. The 2,6 protons are low field of the 3,5 protons for each Tyr residue. Selective chemical modification with nitration reagents allows assignments of Tyr A to Tyr-47, Tyr B to Tyr-7, Tyr C to Tyr-12, and Tyr D to Tyr-17 in HP-51. All four tyrosines are essential for maintaining the structure of the isolated headpiece, and Tyr-7, -12, and -17 appear to be stacked.

Escherichia coli↗
PubMed1981

High-resolution nuclear magnetic resonance studies of the Lac repressor. 2. Partial analysis of the aliphatic region of the Lac repressor headpiece spectrum.

The 360-MHz 1H NMR spectrum of native lac repressor headpiece (HP-51 or HP-59) contains a large number (greater than 30%) of aliphatic side-chain methyl and backbone alpha-CH resonances and three of four aromatic tyrosine multiplet resonances shifted to high-field chemical shift positions, indicating the presence of extensive folded structure. Denaturation leads to loss of the NMR chemical shift differences. Resonance identifications of the 27 methyl-possessing amino acids in HP-59 have been made by using resolution enhancement, double-resonance, and difference spectra. There are three firmly assigned methyl resonances and 21 pairwise identifications of methyl resonances in HP-51. Comparison of HP-51 and HP-59 allows identification of four additional methyl groups in amino acid residues 52--59. The sequence HP-50--59 is not essential to maintain the structure of HP-59, but it is of interest itself as the flexible hinge portion connecting HP to the tetrameric core of whole repressor.

Escherichia coli↗