George L. Mendz

NMR studies of myelin basic protein: X. Conformation of a determinant encephalitogenic in the rabbit

Residues 67 to 75 in myelin basic protein from several species comprise the sequence Thr-His-Tyr-Gly-Ser-Leu-Pro-Gln-Lys that acts as an encephalitogenic determinant in the rabbit. Proton magnetic resonance spectra of human, bovine and porcine proteins display nuclear Overhauser effects between the delta-CH of Tyr-69 and the delta-CH3 of Leu-72, which indicate reverse-turn conformations about the Gly-Ser residues. This effect occurs also in physiological saline solution at pH 6.0 but in dimethylsulfoxide solution the nuclear Overhauser effect disappears. Circular dichroism indicates that the protein when bound to ganglioside micelles acquires 30-40% alpha-helical conformation, but the reverse turn still persists in the sequence of the rabbit encephalitogen. These results suggest that the encephalitogenic region of the protein remains at the aqueous interface of the micelles.

Stimulation of human erythrocyte 2,3-bisphosphoglycerate phosphatase by vanadate

The rates of vanadate-stimulated hydrolysis of 2,3-bisphosphoglycerate in metabolically competent erythrocytes and in hemolysates were determined from data on time courses up to 35 min employing 31P nuclear magnetic resonance spectroscopy. The enhanced rate of hydrolysis of the bisphosphate was attributed principally to the activation of the phosphatase activity of 2,3-bisphosphoglycerate synthase both in cell suspensions and in hemolysates. Information on the concentrations of vanadate and vanadyl present in the preparations was obtained employing 51V nuclear magnetic resonance spectroscopy and electron paramagnetic resonance spectroscopy. Redox reactions involving vanadium ions appeared to be important in establishing the final equilibrium concentrations of the oxy- and oxo-ions (vanadate and vanadyl, respectively), but the data suggested that the activation of the enzyme resulted from direct action of the vanadium ions on the enzyme and not as a consequence of the alteration in the equilibrium of intracellular oxidants and reductants.

NMR studies of myelin basic protein. IX. Complete assignments of the tyrosine residues by proton NMR of proteins from six species.

All the proton resonances from the tyrosine residues are assigned in 400 MHz NMR spectra in aqueous solution of myelin basic proteins from human, cow, pig, rabbit, rat (small protein) and chicken. Assignments are based on species comparisons, spectra of enzymatic cleavage products of the basic protein, pH titrations, broadening effects of Gd(III), and nuclear Overhauser effects. The mobile extended polypeptide chain structure of the protein facilitates the detection of interactions between nearest neighbors. Evidence is found for reverse turns in the structure in regions of encephalitogenic determinants.

NMR studies of myelin basic protein. XIII. Assignment of histidine residues in rabbit, bovine and porcine proteins

Myelin basic protein from three species (rabbit, cow and pig) and peptides from enzymatic digests or cleavage of the proteins have been examined in aqueous solutions by proton nuclear magnetic resonance (NMR) at 400 MHz. The epsilon 1-CH and delta 2-CH resonances of all the histidine residues in the three proteins have been assigned and the pK values have been measured. The heterogeneity of chemical shifts among these resonances can be variously ascribed to persistent localized secondary structures and to effects arising from charged side-chains, particularly those of aspartic acid residues, and from side-chains of aromatic moieties.

Conformation of two antigenic regions in myelin basic protein

Four different regions of myelin basic protein from various species have been reported to be the antigenic sites (epitopes) for seven monoclonal antibodies evoked in rats or mice by guinea pig or monkey basic protein. The structures of the epitopes located in the amino-terminal region and in the eight-residue sequence including S-133, were examined by proton n. m. r at 400 MHz in aqueous solutions of peptides obtained by enzymatic cleavage of the rabbit protein. The data suggest conformational similarities between the two regions.

Interactions of Na+ with haemoglobin-organic phosphate complexes

Longitudinal and transverse relaxation times were measured in aqueous solutions containing haemoglobin and 2,3-bisphosphoglycerate and in dilute lysates of human erythrocytes. Analysis of the data in terms of calculated excess relaxation rates shows that Na+ interacts with the protein-organic phosphate complex. The comparable magnitude of the effect in the model system and in dilute lysate suggests that intracellular Na+ binds to the haemoglobin-bisphosphate complex. Results obtained with adenosine triphosphate and D-glucose indicate that there is also interaction between Na+ and haemoglobin complexes of these molecules.

31P Magnetization transfer in the phosphoglyceromutase-enolase coupled enzyme system

The rate of exchange of phosphoryl groups between 2-phosphoglycerate, 3-phosphoglycerate and phosphoenolpyruvate by the coupled phosphoglyceromutase-enolase enzyme system using one- and two-dimensional 31P NMR spectroscopy was measured. Magnetization exchange in one-dimensional experiments was achieved by saturation transfer with selective irradiation at both one and two sites in this three-site exchange system using the DANTE pulse sequence. The two-dimensional magnetization exchange experiment avoids the need to selectively saturate at one or more frequencies which may be difficult in complex exchange systems. Analysis of the two-dimensional exchange experiment by the back transformation method yielded exchange rate constants in good agreement with the saturation transfer method.

Magnetic potential and field gradients of a model cell

The magnetic potential within and outside a nucleated cell placed in a uniform magnetic field is described for a model consisting of two concentric diamagnetic spheres. The analytical description of the magnetic potential in and around a system consisting of a finite number of concentric diamagnetic spheres in a uniform magnetic field was derived. The solution was employed to calculate the field gradients outside and inside a model chicken red blood cell. The form and magnitude of the gradients provide a theoretical basis on which to discuss experimental results relating to the attenuation of signals obtained using proton nuclear magnetic resonance spectroscopy with chicken erythrocyte suspensions. The form of the magnetic field, field difference and field gradients in the external medium of a cell suspension was simulated for a distribution of spheres in a uniform magnetic field, such as might occur in an idealised dilute cell suspension.