In vivo observation of lactate methyl proton magnetization transfer in rat C6 glioma.

Magnetic resonance spectroscopy (MRS) measurements of the lactate methyl proton in rat brain C6 glioma tissue acquired in the presence of an off-resonance irradiation field, analyzed using coupled Bloch equation formalism assuming two spin pools, demonstrated the occurrence of magnetization transfer. Quantitative analysis revealed that a very small fraction of lactate (f = 0.0012) is rotationally immobilized despite a large magnetization transfer effect. Off-resonance rotating frame spin-lattice relaxation studies demonstrated that deuterated lactate binds to bovine serum albumin and the proteins present in human plasma, thereby providing a possible physical basis for the observed magnetization transfer effect. These results demonstrate that partial or complete saturation of the motionally restricted lactate pool (as well as other metabolites) by the application of an off-resonance irradiation field, such as that used for water presaturation, can lead to a substantial decrease in resonance intensity by way of magnetization transfer effects, resulting in quantitation errors.

Rotational correlation times of internuclear vectors in a DNA duplex with G-A mismatch determined in aqueous solution by complete relaxation matrix analysis of off-resonance ROESY (O-ROESY) spectra.

Complete relaxation matrix analysis of the off-resonance ROESY (O-ROESY) spectra is presented and demonstrated for a synthetic DNA duplex with two G-A mismatches, d(GCTGTC-GAAAGC)2, in solution. The internuclear distance and the rotational correlation time of the internuclear vector could be garnered simultaneously using complete relaxation matrix analysis of O-ROESY, by which spin diffusion effects could be accommodated. Correlation times in the terminal and the mismatched regions were significantly reduced compared to those in other regions, indicating the conformational flexibility of the mismatched pair. The average structure obtained by restrained molecular dynamics simulation with inclusion of variations of the rotational correlation times also indicated a general tendency of the mismatched and contiguous bases to flip to the outside of the double strand. Off-resonance ROESY combined with the complete relaxation matrix analysis method may offer an alternative way to investigate the structures and dynamics of biological macromolecules.

Oxidative-stress induced protein glutathione mixed-disulfide formation in the ocular lens.

The biochemistry of protein-glutathione mixed disulfide formation in the ocular lens was examined by 13C-NMR spectroscopic measurements of glutathione oxidative metabolism in intact rabbit lenses maintained in organ culture. Lenticular amino acid uptake and glutathione biosynthetic mechanisms were employed to facilitate the incorporation of L-[3-13C]cysteine from the incubation medium into the cysteinyl residue of glutathione. Subsequent exposure to increasing levels of oxidative stress induced by tert-butylhydroperoxide resulted in decreased levels of ([3-13C]cysteinyl)-glutathione and a loss of 13C NMR resonance intensity, a reflection of protein-glutathione mixed disulfide formation. The rate of ([3-13C]cysteinyl)-glutathione loss depended on the concentration of tert-butylhydroperoxide; 13C-labeled oxidized glutathione was observed only at the highest concentration (2 mM) of oxidant tested. Removal of the oxidative stress led to a partial recovery of ([3-13C]cysteinyl)-glutathione levels and 13C resonance intensity. Recovery was significantly enhanced by the addition of 2-mercaptoethanol. The mechanism of protein-glutathione adduct formation was further characterized by the in vitro monitoring of the reaction of oxidized glutathione with bovine lens gamma-II crystallin protein using proton NMR spectroscopy. These experiments provided insight into the role of the cellular glutathione redox-couple, [GSH]/[GSSG], in maintaining reduced protein thiol groups, and suggested that protein-glutathione adduct formation may function as a mechanism for modulating the glutathione redox buffer under conditions of oxidative stress in ocular tissue. In addition, the results demonstrate the feasibility of direct chemical reduction of protein-glutathione disulfide bonds in vivo which may reflect a mechanism for the inhibition of disulfide-linked light scattering protein aggregate formation.

Pharmacokinetic properties of several novel oligonucleotide analogs in mice.

Biophysical and pharmacokinetic properties of five analogs of ISIS 3082, a 20-mer phosphorothioate oligodeoxynucleotide that inhibits the expression of mouse intercellular adhesion molecule 1, were evaluated. Compared to the parent compound, ISIS 3082, the 2'-propoxy modified phosphodiester, ISIS 9044 and the 2'-propoxy phosphorothioate, ISIS 9045, had greater affinity for complementary RNA and were more lipophilic. A chimeric oligonucleotide comprised of 2'-propoxy diester wings and a phosphorothioate deoxy center (ISIS 9046) had equal affinity. It was also more lipophilic than ISIS 3082, but less so than the other 2'-propoxy modified analogs. The two analogs with 5'-lipophilic conjugates, ISIS 9047 (5'-octadecylamine) and ISIS 8005 (5'-(2'-O-hexylamino-carbonyl-oxycholesterol) were more lipophilic than ISIS 3082 (3- and 7-fold, respectively) but had similar affinity for complementary RNA. Binding of ISIS 3082 to bovine serum albumin was salt-dependent and, at physiological concentration (320 mOsmol), the dissociation constant (Kd) was 140 microM. Similarly, the 2'-propoxy phosphodiester, ISIS 9044, displayed salt-dependent bovine serum albumin binding, but not binding was measurable at physiological salt conditions. In contrast, the more lipophilic phosphorothioate analogs displayed much higher affinity to bovine serum albumin at 320 mOsmol than ISIS 3082. After bolus injection to mice, the initial volumes of distribution of the more lipophilic phosphorothioate analogs, ISIS 9045, ISIS 9047 and ISIS 8005, were less and the initial clearance from plasma was slower than ISIS 3082. The pharmacokinetics of the other analogs was similar to ISIS 3082. Distribution of ISIS 3082 into peripheral tissues was similar to that reported for other phosphorothioates with liver and kidney accumulating the highest fraction of the dose. The only modification to markedly influence distribution was the very lipophilic cholesterol conjugate (ISIS 8005), which increased substantially the fraction of the dose accumulated by the liver. Little intact drug was found in urine or feces for any analog, and the patterns of metabolites suggested that for all analogs the principal metabolic pathway was due to 3'-exonuclease activity. The metabolism of ISIS 3082 was similar to that reported for other phosphorothioates. After 2 hr, most of the radioactivity in plasma represented metabolites but, in tissues, intact ISIS 3082 was present for much longer periods of time and metabolites accumulated more slowly. The 24-hr exposure to ISIS 3082 of liver and kidney was 20.7 and 67.9 microM/hr, respectively. The rates of metabolism in plasma, liver and kidney of the two 5'-conjugates, ISIS 9047 and ISIS 8005, were similar to ISIS 3082, as was the pattern of metabolism. The rate of metabolism of ISIS 9044 (2'-propoxy phosphodiester oligonucleotide) was much more rapid in liver and plasma, but surprisingly much slower in the kidney. ISIS 9045 (full 2-propoxy phosphorothioate) was much more stable than ISIS in all tissues, the enhanced stability of ISIS 9045 resulted in increased exposure of liver and kidney to the drug, whereas the exposure of the liver to the two more lipophilic analogs, ISIS 9047 and ISIS 8005, was greater because a higher fraction of the dose was distributed to the liver. The exposure of the kidney to ISIS 9044 was also greater than that to ISIS 3082 due to the surprising stability of the drug in the kidney.

Deuterium off-resonance rotating frame spin-lattice relaxation of macromolecular bound ligands.

Deuterated 3-trimethylsilylpropionic acid binding to bovine serum albumin was used as a model system to examine the feasibility and limitations of using the deuterium off-resonance rotating frame spin-lattice relaxation experiment for the study of equilibrium ligand-binding behavior to proteins. The results of this study demonstrate that the rotational-diffusion behavior of the bound species can be monitored directly, i.e., the observed correlation time of the ligand in the presence of a protein is approximately equal to the correlation time of the ligand in the bound state, provided that the fraction of bound ligand is at least 0.20. The presence of local ligand motion and/or chemical exchange contributions to relaxation in the bound state was inferred from the observation that the correlation time of the bound ligand was somewhat smaller than the correlation time characterizing the overall tumbling of the protein. An approximate value for the fraction of bound ligand was obtained from off-resonance relaxation experiments when supplemental spin-lattice or transverse relaxation times were employed in the analysis. Incorporation of local motion effects for the bound species into the theoretical relaxation formalism enabled the evaluation of an order parameter and an effective correlation time, which in conjunction with a wobbling in a cone model, provided additional information about ligand motion in the bound state.

13C-NMR spectroscopic studies of 2-mercaptoethanol-stimulated glutathione synthesis in the intact ocular lens.

13C-NMR spectroscopy was employed non-invasively for the real-time measurement of the rates of glutathione synthesis in intact rabbit lenses supported in organ culture containing L-[3-13C]cysteine. Supplementation of the organ culture medium with 2-mercaptoethanol resulted in a dose- dependent enhancement of lenticular glutathione synthesis rates (dose for 50% maximal effect = 125 microM). At the most effective concentration (400 microM) 2-mercaptoethanol increased the rate of glutathione synthesis 163% relative to the rate observed under control conditions. The mechanism of action for this effect was investigated in intact lenses using antagonists of specific amino acid uptake systems. These experiments demonstrated that the enhanced rates of glutathione synthesis observed in the presence of 2-mercaptoethanol were due to the affinity of the mixed-disulfide formed between cysteine and 2-mercaptoethanol for L system amino acid uptake, thereby providing a mechanism for increasing intracellular cysteine levels by circumventing normal cellular cysteine uptake pathways in the lens. Because of the role of cysteine as the rate limiting substrate in lenticular glutathione biosynthesis, these results suggest a potential strategy to prevent tissue opacification associated with depleted glutathione levels.

13C-NMR off-resonance rotating frame spin-lattice relaxation studies of bovine lens gamma-crystallin self association: effect of 'macromolecular crowding'.

The NMR technique of 13C off-resonance rotating frame spin-lattice relaxation, which provides an accurate assessment of the effective rotational correlation time (tau 0, eff) for macromolecular rotational diffusion, was applied to the study of gamma-crystallin association as a function of protein concentration and temperature. Values of the effective rotational correlation time for gamma-crystallin rotational diffusion were obtained at moderate to high protein concentrations (80-350 mg/ml) and at temperatures above, and below, the cold cataract phase transition temperature. With increasing concentration gamma-crystallin was observed to increasingly associate as reflected by larger values of tau 0, eff Decreasing temperature in the range of 35 to 22 degrees C was found to result in no change in the temperature corrected value of tau 0, eff at a gamma-crystallin concentration of 80 mg/ml, whereas at temperatures of 18 degrees C or below, this parameter was approx. twofold larger, suggesting the occurrence of a well defined phase transition, which correlated well with the cold cataract phase transition temperature. At higher protein concentrations, by contrast, tau 0, eff (temperature corrected) was found to increase by approx. 1.6- to 2-times in the temperature interval 35 degrees C to 22 degrees C, a result consistent with the dependence of the cold cataract phase transition temperature on gamma-crystallin concentration. Analysis of intensity ratio dispersion curves, using an assumed model of isodesmic association, permitted the estimation of the association constant characterizing the aggregation under particular conditions of concentration and temperature. The significant increase in the value of the association constant with moderate increases in protein concentration was rationalized by invoking the effect of 'macromolecular crowding'. The results obtained in this study suggest that in the intact lens, where high protein concentrations prevail, gamma-crystallin is unlikely to be found in the monomeric state, but more likely, as a significantly aggregated species, representing a broad molecular weight distribution.

Modified Jeener solid-echo pulse sequences for the measurement of the proton dipolar spin-lattice relaxation time (T1D) of tissue solid-like macromolecular components.

Modified Jeener solid-echo pulse sequences are proposed for the measurement of the proton dipolar spin-lattice relaxation time, T1D, of motionally restricted (solid-like) components in the presence of mobile molecular species, such as encountered in biological tissue. A phase-cycled composite-pulse sequence was used for detection of the dipolar signal and cancellation of the Zeeman signal. A homospoil gradient pulse was added to the Jeener echo pulse sequence to enhance dephasing of the transverse magnetization components of mobile species, thereby aiding in elimination of the Zeeman signal during dipolar signal acquisition. A modified Jeener echo sequence incorporating water suppression is also proposed as a means to further depress the Zeeman signal arising from mobile components. The modified Jeener echo sequences were successfully used for the measurement of proton T1D values of solid 2,6-dimethylphenol and Sephadex gels of differing degrees of cross linking and hydration.

Off-resonance rotating-frame spin-lattice relaxation of quadrupolar (spin-1) nuclei.

The validity of the formalism for the off-resonance rotating-frame spin-lattice relaxation experiment applicable to spin-1 quadrupolar nuclei was experimentally examined by considering two model systems, deuterated glycerol and deuterated benzene in castor oil, at different temperatures. When appropriately implemented, the deuterium off-resonance rotating-frame spin-lattice relaxation experiment provides spectral-intensity-ratio-dispersion data which agree remarkably well with those predicted by the theoretical formalism. The assumption of quadrupolar relaxation as the dominant relaxation mechanism, and rigid-rotor isotropic tumbling, permits the assessment of rotational diffusion behavior, i.e., the determination of a rotational correlation time, of a variety of molecular systems. With the inclusion of an additional relaxation measurement, T1 or T2, the 2H off-resonance rotating-frame spin-lattice relaxation experiment becomes a convenient method for the estimation of the 2H quadrupolar coupling constant, provided that a realistic reorientational motional model is assumed in the theoretical relaxation expressions used in the analysis. Because the motional window of the 2H off-resonance rotating-frame spin-lattice relaxation experiment includes intermediate molecular motion with correlation times as short as 1 or 2 ns, this experiment is appropriate for the investigation of the rotational diffusion behavior of deuterated molecules varying in size from moderately small to macromolecular. The 2H off-resonance rotating-frame spin-lattice relaxation experiment is applicable to in vitro and in vivo experimental situations.

T1 discrimination contributions to proton magnetization transfer in heterogeneous biological systems.

Water proton spin-lattice relaxation times are commonly used as a guide in establishing the off-resonance irradiation time as well as the repetition time of the magnetization transfer experiment. T1 discrimination effects occur if the motionally restricted spin bath longitudinal magnetization does not reach thermal equilibrium. In this study we developed the formalism necessary for the evaluation of T1 discrimination contributions to proton magnetization transfer arising from the use of a short repetition time relative to the spin-lattice relaxation time of the motionally restricted spin bath. The results of computer simulation indicate that T1 discrimination contributions occur when the repetition time is small relative to the spin-lattice relaxation time of the motionally restricted spin bath, and when the off-resonance irradiation is weak and far off-resonance. For somewhat longer repetition times, T1 discrimination contributions become important only when the cross relaxation rate is small, and the fractional amount of motionally restricted component large. The occurrence of T1 discrimination effects results in distortion of water proton intensity ratio dispersion curves thereby resulting in the estimation of erroneous magnetization transfer parameters, whereas in magnetization transfer contrast enhanced imaging, such contributions are manifested by a decrease in image contrast.

Relaxation-matrix formalism for rotating-frame spin-lattice proton NMR relaxation and magnetization transfer in the presence of an off-resonance irradiation field.

The derivation of a generalized relaxation matrix equation for the off-resonance rotating-frame spin-lattice experiment, representing N macromolecular components, is presented. The applicability of the derived formalism was demonstrated using water proton off-resonance rotating-frame spin-lattice relaxation data obtained for calf lens cortical and nuclear homogenates, a tissue system characterized by the presence of both mobile and solid-like protein domains, whose relative amounts vary in a protein-concentration-dependent manner. Protein concentration and temperature were utilized as variables in the magnetization-transfer experiments. Curve fitting to obtain the relevant magnetization-transfer parameters was accomplished by simulated annealing and the method of steepest descents. In all cases, the best fit, as reflected by the smallest root-mean-square deviation, was obtained by assuming the presence of three components representing bulk-water and mobile and solid-like macromolecular components, characterized by Lorentzian (water and mobile protein) and Gaussian proton resonance lineshapes (solid-like protein). A two-component relaxation model gave unsatisfactory fits. Dilution of nuclear homogenate resulted in physically realistic changes in the derived magnetization-transfer parameters, which included a decrease in the fraction of solid-like protein, in agreement with previously published 13C NMR studies. Changes in magnetization-transfer parameters occurred as a result of the cold cataract phase transition in nuclear homogenate. The utility and limitations of the derived formalism are discussed.

Determination of proton magnetization transfer rate constants in heterogeneous biological systems.

Two procedures are currently in use for the determination of proton magnetization transfer rate constants between macromolecular tissue components and water. The first method assumes that there are only two spin baths (macromolecular plus solvent) and that during off-resonance irradiation complete saturation of the "immobile" proton spin bath occurs (S. H. Koenig, R. D. Brown, III, R. Ugolini, Magn. Reson. Med. 29, 311 (1993)). This approach neglects the possibility of incomplete saturation and polydispersity, and yields an apparent magnetization transfer rate constant, Kapp. The second approach utilizes a formalism which can account for polydispersity and incomplete saturation of the immobile spin bath (K. Kuwata, D. Brooks, H. Yang, T. Schleich, J. Magn. Reson., in press). In this work magnetization transfer rate constants derived by the use of both methods for two systems, ocular lens tissue and cross-linked bovine serum albumin (BSA) were compared. For both samples Kapp was dependent on B2 off-resonance irradiation frequency and power when the first method was used. The second method provided values of the magnetization transfer rate constant that were similar to the values obtained by the first method, as the limit of complete saturation was approached.

Cytokeratin 19 fragment CYFRA 21-1 compared with carcinoembryonic antigen, squamous cell carcinoma antigen and neuron-specific enolase in lung cancer. Results of an international multicentre study.

The diagnostic value of the water-soluble cytokeratin 19 fragment CYFRA 21-1 in lung cancer was assessed in comparison with carcinoembryonic antigen, squamous cell carcinoma antigen, and neuron-specific enolase. The cut-off value, defined as 95% specificity versus a group of 526 patients suffering from benign chest diseases, was set at 3.3 micrograms/l for cytokeratin 19 fragment CYFRA 21-1 (carcinoembryonic antigen: 7.8 micrograms/l, squamous cell carcinoma antigen: 1.9 micrograms/l, neuron-specific enolase: 13.7 micrograms/l). Elevated pretreatment cytokeratin 19 fragment CYFRA 21-1 concentrations were recorded: in 112 of 244 (46%) patients with all histological types of lung cancer (carcinoembryonic antigen: 32%, squamous cell carcinoma antigen: 25%, neuron-specific enolase: 28%), in 89 of 177 (50%) patients with non-small cell lung cancer (carcinoembryonic antigen: 33%, squamous cell carcinoma antigen: 24%, neuron-specific enolase: 12%), in 47 of 81 (58%) patients with squamous cell carcinoma (carcinoembryonic antigen: 23%, squamous cell carcinoma antigen: 32%, neuron-specific enolase: 14%), in 27 of 63 (42%) patients with adenocarcinoma (carcinoembryonic antigen: 44%, squamous cell carcinoma antigen: 14%, neuron-specific enolase: 9%), in 15 of 33 (45%) patients with other non-small cell lung cancer (carcinoembryonic antigen: 36%, squamous cell carcinoma antigen: 24%, neuron-specific enolase: 14%), and in 20 of 55 (36%) patients with small cell lung cancer (carcinoembryonic antigen: 32%, neuron-specific enolase: 77%). Three of 12 patients with undefined histological type showed cytokeratin 19 fragment CYFRA 21-1 elevations. The best performance in terms of sensitivity and diagnostic accuracy was attained with the cytokeratin 19 fragment CYFRA 21-1 test in squamous cell carcinoma. In small cell lung cancer neuron-specific enolase was confirmed to be superior to the other markers. Cytokeratin 19 fragment CYFRA 21-1 concentrations increased with the extent of the malignant disease in non-small cell lung cancer. The positivity rate of cytokeratin 19 fragment CYFRA 21-1 in tumour stage TNM I was only 23% (carcinoembryonic antigen: 23%, squamous cell carcinoma antigen: 14%), i.e. the markers under study cannot be used for the diagnosis of early stage disease. Cytokeratin 19 fragment CYFRA 21-1 differentiated significantly between squamous cell carcinoma and the other histological types (p < 0.01). In addition, cytokeratin 19 fragment CYFRA 21-1 distinguished significantly the operable group TNM I-IIIa from inoperable TNM IIIb-IV (p < 0.05), but not TNM IIIa from IIIb. Out of 177 patients with non-small cell lung cancer, 90 individuals were monitored after surgery.(ABSTRACT TRUNCATED AT 400 WORDS)

Assessment of protein rotational diffusion by 13C off-resonance rotating frame spin-lattice relaxation: effect of backbone and side-chain internal motion.

The 13C off-resonance rotating frame spin-lattice relaxation technique is applicable to the study of protein rotational diffusion behavior in a variety of experimental situations. The original formalism of James and co-workers (1978) (J. Am. Chem. Soc. Vol. 100, pp. 3590-3594) incorporated random isotropic reorientational motion of a rigid spherical rotor with no provision for backbone or side-chain carbonyl group internal motion. Here we demonstrate that the failure to include such internal motion may lead to erroneous rotational correlation time determinations for overall reorientational motion. The effect becomes severe for protein molecular masses in excess of 100 kD. Inclusion of both backbone and side-chain carbonyl carbon internal motion, using reasonable parameters derived from the literature [R. Levy and M. Karplus (1979), Chemical Physics Letters, Vol. 65, pp. 4-11; G. Careri, P. Fasella, and E. Gratton (1975), Critical Reviews in Biochemistry, Vol. 3, pp. 141-164; G. Lipari, A. Szabo, and R. LEvy (1982), Nature, Vol. 300, pp. 197-198], plus corrections for anisotropic tumbling [C. F. Morgan, T. Schleich, G. H. Caines, and D. Michael (1990), Biopolymers, Vol. 29, pp. 469-480] and microscopic viscosity [S. H. Koenig (1980), ACS Symposium Series, Vol. 127, pp. 157-176], leads to reliable values for the correlation time describing overall protein reorientation up to molecular masses of approximately 1000 kD.

13C NMR studies of protein motional dynamics in bovine, human, rat, and chicken ocular lenses.

The motional dynamics of lens proteins were studied by two 13C nuclear magnetic resonance (NMR) techniques sensitive to molecular motion to define the effect of lens water content on the presence of solid-like protein domains in ocular lenses from bovine (juvenile and adult), human, rat, and chicken eyes. The solid state 13C NMR technique of proton dipolar decoupling was used to study slow (solid-like) motions (correlation time, tau o > or = 10 microseconds), whereas for intermediate (mobile) protein, rotational reorientational motion (tau o range of 1-500 nsec) the 13C off-resonance rotating frame spin-lattice relaxation technique was employed. 13C NMR studies of calf lens cortical and nuclear homogenates indicated a reversible loss of lens protein motional freedom with decreasing water content. Values of 6% and 63% solid-like protein contents were obtained for native cortical and nuclear calf lens homogenates, respectively. At equivalent total protein concentrations cortical and nuclear calf lens homogenates exhibited essentially the same solid-like (motionally restricted) protein content. Lens protein rotational correlation times determined by off-resonance rotating frame spin-lattice relaxation measurements were consistent with lens protein aggregation. The solid-like protein content of the bovine nuclear lens region was observed to increase with age, whereas no significant change was detected for the cortex. Across lens species an inverse correlation between the percentage of solid-like protein content and water content was observed. Very broad 13C NMR resonances, even in the presence of proton dipolar decoupling, were observed for the lens proteins present in the cataractous human lens, indicating the presence of highly aggregated protein species. The occurrence of solid-like protein domains in lens tissue has implications for the interpretation of proton nuclear magnetic resonance dispersion (NMRD) measurements of lens homogenates and for proton magnetization transfer contrast enhanced magnetic resonance imaging of lens. Solid-like protein domains may play a protective role in the maintenance of lens transparency by minimizing enhanced refractive index fluctuations created by protein packing defects resulting from post-translational modification.

13C NMR spectroscopic measurement of glutathione synthesis and antioxidant metabolism in the intact ocular lens.

A 13C NMR spectroscopic method for non-invasive, time-resolved measurements of glutathione function in the intact ocular lens maintained in organ culture is described. L-[beta-13C]cysteine (1 mM) included in the incubation medium is incorporated, by way of lenticular amino acid uptake and glutathione biosynthetic mechanisms, into the cysteinyl residue of intralenticular glutathione. 13C-NMR chemical shift measurements facilitate analysis of glutathione synthesis and anti-oxidant reactions in the intact tissue. The results of this preliminary study demonstrate the viability of a rapid non-invasive method for monitoring the multiple aspects of glutathione biosynthesis, metabolism, and function in intact tissue.

Sodium-23 and potassium-39 nuclear magnetic resonance relaxation in eye lens. Examples of quadrupole ion magnetic relaxation in a crowded protein environment.

Single and multiple quantum nuclear magnetic resonance (NMR) spectroscopic techniques were used to investigate the motional dynamics of sodium and potassium ions in concentrated protein solution, represented in this study by cortical and nuclear bovine lens tissue homogenates. Both ions displayed homogeneous biexponential magnetic relaxation behavior. Furthermore, the NMR relaxation behavior of these ions in lens homogenates was consistent either with a model that assumed the occurrence of two predominant ionic populations, "free" and "bound," in fast exchange with each other or with a model that assumed an asymmetric Gaussian distribution of correlation times. Regardless of the model employed, both ions were found to occur in a predominantly "free" or "unbound" rapidly reorienting state. The fraction of "bound" 23Na+, assuming a discrete two-site model, was approximately 0.006 and 0.017 for cortical and nuclear homogenates, respectively. Corresponding values for 39K+ were 0.003 and 0.007, respectively. Estimated values for the fraction of "bound" 23Na+ or 39K+ obtained from the distribution model (tau C greater than omega L-1) were less than or equal to 0.05 for all cases examined. The correlation times of the "bound" ions, derived using either a two-site or distribution model, yielded values that were at least one order of magnitude smaller than the reorientational motion of the constituent lens proteins. This observation implies that the apparent correlation time for ion binding is dominated by processes other than protein reorientational motion, most likely fast exchange between "free" and "bound" environments. The results of NMR visibility studies were consistent with the above findings, in agreement with other studies performed by non-NMR methods. These studies, in combination with those presented in the literature, suggest that the most likely role for sodium and potassium ions in the lens appears to be the regulation of cell volume by affecting the intralenticular water chemical potential.

Effect of differential saturation on the spatial localization performance of depth pulses.

Computer simulations of Depth pulse B (theta; (2 theta [+/- x, +/- y])2; acquire) and other Depth pulses, verified by experimental surface coil NMR studies utilizing phantom samples, reveal that the spatial localization performance of Depth pulses degrades when the repetition time is short relative to T1 because of differential saturation, i.e. T1 discrimination effects. Simulations of Depth pulse A (theta; 2 theta [+/- x, +/- y]; acquire) and Depth pulse B indicate that there is no phase-cycled pulse sequence delivery order which negates the untoward effect of T1 discrimination on spatial localization performance. The results of this study demonstrate the importance of consistent magnetization preparation prior to the delivery of each phase-cycled multiple pulse sequence in a Depth pulse cycle for obtaining optimal spatial localization performance. The untoward effects of inconsistent magnetization preparation, resulting from T1 discrimination, may be ameliorated by the application of many Depth pulse cycles.

L-alpha-glycerophosphate binding to bovine gamma-crystallin: a potential link between metabolism and supramolecular order.

The highly selective nature of protein-ligand interactions provides a sensitive mechanism for the modulation of cellular activity by proteins. In the eye lens the supramolecular order of the lens crystallins, which is expected to be susceptible to protein electrostatic charge, in part defines transparency. The binding of charged ligands to proteins is one way of achieving an alteration in protein electrostatic charge. Evidence is presented that L-alpha-glycerophosphate, a major phosphorus metabolite of eye lens metabolism, binds to the globular protein, gamma-crystallin with moderately high affinity and in a positive cooperative manner. The following binding parameters were obtained from equilibrium measurements: minimum number of binding sites, n = 2; Kassoc = 6.2 +/- 0.5 x 10(3) M-1; cooperativity parameter, alpha H = 1.9 +/- 0.1. Interactive computer graphics display techniques were used to locate putative ligand binding sites, and in turn, to identify the possible molecular interactions responsible for the binding of ligand to protein at one of the sites. One putative binding site was located in the cleft between the two domains of gamma II-crystallin. Arginyl residues 79 and 147 are involved in ligand binding as are the peptide carbonyl oxygens of residues Tyrosyl-50 and Aspartyl-156. Five hydrogen bonds between the ligand and the protein structure are predicted for the binding of L-alpha-glycerophosphate, whereas only 3 occur for the binding of the "unnatural" D-enantiomorph. Modulation of both lens protein supramolecular organization and lens metabolism is predicted to be a consequence of L-alpha-glycerophosphate binding to gamma-crystallin in the lens.

Off-resonance rotating frame spin-lattice NMR relaxation studies of phosphorus metabolite rotational diffusion in bovine lens homogenates.

The rotational diffusion behavior of phosphorus metabolites present in calf lens cortical and nuclear homogenates was investigated by the NMR technique of 31P off-resonance rotating frame spin-lattice relaxation as a means of assessing the occurrence and extent of phosphorus metabolite-lens protein interactions. 31P NMR spectra of calf lens homogenates were obtained at 10 and 18 degrees C (below and above the cold cataract phase transition temperature, respectively) at 7.05 T. Effective rotational correlation times (tau 0,eff) for the major phosphorus metabolites present in cortical and nuclear bovine calf lens homogenates were derived from nonlinear least-squares analysis of R vs omega e (spectral intensity ratio vs precessional frequency about the effective field) data with the assumption of isotropic reorientational motion. Intramolecular dipole-dipole (1H-31P, 31P-31P), chemical shift anisotropy (CSA), and solvent (water) translational intermolecular dipole-dipole (1H-31P) relaxation contributions were assumed in the analyses. In those cases where the limiting value of the spectral intensity ratio failed to reach unity at large offset frequency, a modified formalism incorporating chemical exchange mediated saturation transfer between two sites was used. Values of tau 0,eff for phosphorus metabolites present in the cortex varied from a low of ca. 2 ns [L-alpha-glycero-phosphocholine (GPC)] to a high of 12 ns (alpha-ATP) at 10 degrees C, whereas at 18 degrees C the range was from ca. 1 to 9 ns. For the nucleus the tau 0,eff values ranged from ca. 3 ns (GPC) to 41 ns (Pi) at 10 degrees C; at 18 degrees C the corresponding values ranged from 4 to 39 ns. For PME (phosphomonoester; in lens the predominant metabolite is L-alpha-glycerol phosphate) at 18 degrees C evidence was obtained for binding and subsequent exchange with solid like protein domains. The diversity in tau 0,eff values for lenticular phosphorus metabolites is suggestive of differential binding to more slowly tumbling macromolecular species, most likely lens crystallin proteins. Corresponding measurement of tau 0,eff values for the mobile protein fraction present in calf lens cortical and nuclear homogenates at 10 and 18 degrees C, by 13C off-resonance rotating frame spin-lattice relaxation, provided average macromolecular correlation times that were assumed to represent the bound metabolite state. A fast-exchange model (on the T1 time scale), between free and bound forms, was employed in the analysis of the metabolite R vs omega e curves to yield the