Molecular mobilities of soluble components in the aqueous phase of chromaffin granules

• Published in: Biochimica et biophysica acta Vol. 497; no. 1; pp. 260 - 271
• Main Authors: Sharp, Robert R., Richards, Edward P.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 29.03.1977
• Subjects: Adenosine Triphosphate; Adrenal Medulla - cytology; Animals; Cattle; Chemical Phenomena; Chemistry; Epinephrine; Magnetic Resonance Spectroscopy; Protons
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(77)90159-3

NMR relaxation times have been used to characterize molecular motion and intermolecular complexes in the aqueous phase of bovine chromaffin granules. Partially relaxed 13C and proton spectra have been obtained at 3 and 25°C. T 1 measurements of five protonated carbons on epinephrine (C 2, C 5, C 6 CHOH and NCH 3) give a correlation time of 0.15 (10 −9) s at 25°C for the catechol ring and methine carbon, while the effective correlation time for the NCH 3 group is somewhat shorter due to its internal degree of rotational freedom. Resonances of protonated carbons on the soluble protein chromogranin give very similar corerlation times: 0.20 (10 −9) s for the peptide α-carbon and 0.2 (10 −9) s for the methylene sidechain carbons of glutamic acid. The correlation time ( τ R) of ATP was not measured direrctly using 13C T 1 data due to the weakness of its spectrum, but its reorinetation appears to be substantially slower than that of epinephrine or chromogranin. This conclusion is based on three observations: (1) the qualitative temperature dependence of T 1 for H 2 and H 8 on the adenine ring places τ R for ATP to the right of the T 1 minimum, or τ R ⩾ 1.0 (10 −9) s; (2) 13C resonances of ATP have anomalously low amplitudes compared with epinphrine resonances, a fact that is readily explained only if ATP undergoes substantially slower reorientation; and (3) a comparision of the T 1 data on H 8 on chromaffin granules and in a dilute aqueous solution, where ρ R for ATP cam be measured directly, indicates that τ R ∼ 1.0 (10 −9 s at 25°C in the granules. The relaxation data are consistent with the concept of a storage complex based on electrostatic interaction between a polyion (chromogranin) and its counterious (ATP and epinephrine), in which ATP cross-links cationic sidechains of the protein.