1H NMR spectroscopy: an approach to evaluation of diseased skin in vivo.

1H nuclear magnetic resonance (NMR) spectroscopy was tested for its applicability in evaluating diseased skin. In order to explore potential spectral markers characteristic of diseased tissue, perchloric acid (PCA) extracts of psoriasis and malignant melanoma tissues were compared with normal skin, and changes in melanoma after heat treatment were monitored. In psoriatic plaque extract, the spectral peak intensity ratios of Glu: Ser, creatine: Gly, and taurine: Ala were approximately three-fold compared with symptom-free or normal skin, whereas Val: Leu/Ile was one-half the normal skin ratio. In melanoma extracts, the phosphorylcholine (PC)/glycerophosphorylcholine (GPC): Ala, Glu: Ser, and lactate: Ala ratios were five-, three-, and two-fold higher, respectively, than normal skin and the Val: Leu/Ile ratio was two-thirds of normal skin. With heat treatment, PC/GPC: Ala and Glu: Ser ratios decreased, whereas lactate: Ala and Val: Leu/Ile ratios increased three-fold and one-third, respectively. Results indicate that 1H NMR spectroscopy is a sufficiently sensitive technique to distinguish normal from diseased skin. The main attraction of this technique lies in the possibility of non-invasive study of various skin diseases, malignant transformation of benign tumors, and responses to treatment. Several methodologic problems remain to be resolved before a meaningful interpretation of in vivo observations becomes feasible. Correlation of in vivo and in vitro findings is an essential step toward this goal.

Comparison of intracellular pH measurements by 31P NMR and weak acid partitioning in Chinese hamster ovary fibroblasts.

Intracellular pH measurements obtained by 31P NMR and DMO partitioning are compared. A continuous-flow culture system was used to measure the intracellular pH of Chinese hamster ovary fibroblasts in response to changes in the extracellular pH. These measurements were repeated with the CHO cells in suspension and under similar experimental conditions using the weak acid partitioning technique employing 14C-DMO. It is shown that the pHi values are identical, within experimental error, for both techniques in the 6.80-7.70 pH range, with the DMO technique giving slightly more acidic intracellular pH in the 6.00-6.80 range. It is concluded that both techniques give similar values for intracellular pH in the physiological range, with the DMO partitioning giving more acidic values at pH's less than 6.80. The range of validity for pHi measurements using 31P NMR lies between 6.70 and 7.50.

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.

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.

High-resolution nuclear magnetic resonance studies of the Lac repressor. 3. Unfolding of the Lac repressor headpiece.

At temperatures below 20 degrees C, the lac repressor headpiece (N-terminal amino acids 1--51) has a well-defined structure which is independent of ionic strength. Its unfolding with increasing temperature proceeds gradually with a characteristic transition temperature which depends on ionic strength. Unfolding has been studied by using NMR and CD. Shifts of several methyl and all of the tyrosyl resonances can be followed, allowing a detailed analysis of the temperature denaturation. At high ionic strength (1 M), the unfolding is complete at 85 degrees C, while at low ionic strength (0.01 M), it is complete by 65 degrees C. Native and partially unfolded structures are in rapid exchange during the unfolding, and the process appears completely reversible at all ionic strengths.

Small-angle X-ray studies of the quaternary structure of the lac repressor from Escherichia coli.

The quarternary structures of the lac repressor molecule from Escherichia coli and its tetrametic core, which can be derived from it by proteolytic cleavage, were studied in dilute solutions by small-angle X-ray scattering. The dimensions and general shape of the lac repressor and of the tetrameric core are reported. The core itself appears to be an elongated structure, and in the intact repressor the headpieces are located at its ends. The results ar derived from model calculations and from the following molecular parameters determined from the scattering curve and the pair distance distribution function: for lac repressor, radius of gyration 5.30 +/- 0.02 nm, radius of gyration of the cross section 2.20 +/- 0.03 nm, maximum diameter 18.0 +/- 0.5 nm, hydrated volume 329 +/- 20 nm3, relative molecular mass 149 000 +/- 15 000, for tetrameric core, radius of gyration 4.92 +/- 0.02 nm, radius of gyration of the cross section 2.24 +/- 0.03 nm, maximum diameter 16.0 +/- 0.5 nm, hydrated volume 278 +/- 15 nm3, relative molecular mass 120 000 +/- 10 000.