Detection of thymosin beta 4 in situ in a guinea pig cerebral cortex preparation using 1H NMR spectroscopy.
In the present work we have investigated the macromolecules that contribute to the brain 1H NMR spectrum. The cerebral cortex showed distinct resonances at the uncrowded methyl- and methylene chemical shift scale of the spin-echo 1H NMR spectrum. The peaks at 1.22 and 1.40 ppm (relative to the methyl protons of N-acetyl aspartate at 2.02 ppm) arise from cerebral macromolecules without evidence for co-resonances from low molecular weight metabolites as shown by the spin-spin relaxation decays of these resonances. In addition to these NMR signals, peaks at 0.9 and 1.7 ppm from macromolecules were detected. These resonances are from proteins, and we have identified the polypeptides that contributed to the 1H NMR peaks. Two proteins that were present at concentrations of 250 and 350 micrograms/g of dryed tissue showed 1H NMR spectra that resembled the macromolecular pattern in the cerebral 1H NMR spectrum. They were identified as thymosin beta 4 and histone H1, respectively. Thymosin beta 4 was present in soluble high speed cytoplasmic fraction and in P2 pellet, whereas histone H1 was detected in nuclear enriched fraction. A chemical shift-correlated two-dimensional 1H NMR spectrum of thymosin beta 4 in vitro revealed a coupling pattern that matched the macromolecule in the cerebral cortex which we have previously noted (Kauppinen R. A., Kokko, H., and Williams, S. R. (1992) J. Neurochem. 58, 967-974). On the basis of both one- and two-dimensional NMR evidence, subcellular distribution and high concentration, we assign the 1H NMR signals at 0.9, 1.22, 1.40, and 1.7 ppm in the cerebral cortex to thymosin beta 4.
Kauppinen
R A
RA
NRM Research Group, University of Kuopio, Finland.
Nissinen
T
T
Kärkkäinen
A M
AM
Pirttilä
T R
TR
Palvimo
J
J
Kokko
H
H
Williams
S R
SR
D000595:Amino Acid Sequence; D000818:Animals; D002540:Cerebral Cortex / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006168:Guinea Pigs; D006859:Hydrogen; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D011506:Proteins / Q000032:analysis; D013947:Thymosin / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D000099201:Thymosin beta(4)
D006859:Hydrogen; D011506:Proteins; D013947:Thymosin; D000099201:Thymosin beta(4)