Effects of growth hormone on ATPase and fluorescence of isolated liver membranes utilizing the fluorescent substrate, 1, N6-etheno-adenosine triphosphate

Abstract

The influence of bovine growth hormone on Mg 2+-ATPase (EC 3.6.1.4) in isolated liver plasma membranes of hypophysectomized rats has been investigated in vitro by means of spectrofluorescence measurements in parallel with Mg 2+-ATPase assays, using 1, N 6-etheno-ATP as substrate and fluorescence probe. Bovine growth hormone, at concentrations of 10 −14 m and above, enhanced significantly Mg 2+-ATPase activity in the presence of GTP at concentrations from 10 −6 m to 10 −10 m. Moreover, bovine growth hormone decreased fluorescence intensity of membrane protein at the peak at 330 nm and of 1, N 6-etheno-ATP at its peak at 395 nm as well. The greatest decrease in fluorescence intensity of 1, N 6-etheno-ATP was observed in the presence of 5 m m MgCl 2 and 10 −8 m GTP, consistent with the stimulating effect of bovine growth hormone on Mg 2+-ATPase activity. In addition, bovine growth hormone caused a small decrease in fluorescence intensity of 1, N 6-etheno-ADP, but not the corresponding fluorescent analogs of AMP, cyclic AMP, and adenosine. The decrease in fluorescence intensity of 1, N 6-etheno-ATP by bovine growth hormone was completely eliminated by addition of ATP, ADP, and cyclic AMP at concentrations five times that of 1, N 6-etheno-ATP. Neither AMP nor adenosine exerted any effects. Bovine growth hormone also increased the fluorescence polarization of 1, N 6-etheno-ATP from 0.177 ± 0.006 to 0.212 ± 0.010 at 300 nm under the same conditions employed for the Mg 2+-ATPase assay. These observations suggest that bovine growth hormone produced changes in tertiary structure of membrane proteins in general and probably Mg 2+-ATPase in particular with consequent enhanced enzyme activity.