AJP - Regulatory, Integrative and Comparative Physiology, Vol 258, Issue 5 1132-R1139, Copyright © 1990 by American Physiological Society
Direct observation of the phosphate acceptor and phosphagen pool sizes in vivo
A. Van Waarde, G. van den Thillart, M. Verhagen, C. Erkelens, A. Addink and J. Lugtenburg
Department of Biology, University of Leiden, The Netherlands.
Earthworms were subjected to environmental anoxia (200 min) and electrical
stimulation (5 min, 6 V, 50 Hz). The levels of high-energy phosphate
compounds and the intracellular pH were monitored during anoxia, muscular
contraction, and recovery by in vivo phosphorus-31 nuclear magnetic
resonance (31P-NMR). Several key metabolites were determined by enzymatic
analysis and reversed-phase high-performance liquid chromatography of
perchlorate extracts. Because lombricine, the phosphagen phosphate acceptor
of earthworms, is a phosphodiester, 31P-NMR permitted direct analysis of
the extent of phosphorylation of the lombricine pool in vivo. Total
lombricine, lombricine phosphate, ATP, and H+ were measured by NMR, and
free ADP was calculated from the lombricine kinase equilibrium constant.
The ADP concentration was not significantly changed by anoxia, but it rose
threefold after stimulation. L-Lactate accumulated on stimulation, whereas
multiple end products (L-lactate, alanine, succinate, and glutamine) were
formed during environmental anaerobiosis.
