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Proceedings Paper

Investigation of the complex between rabbit muscle glyceraldehyde-3-phosphate dehydrogenase and aldolase using fluorescence laser spectroscopy
Author(s): Aydin Orstan; Ari Gafni
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Paper Abstract

The interaction of rabbit muscle aldolase with glyceraldehyde-3-phosphate dehydrogenase (GPDH) labeled with fluorescein-5-isothiocyanate (FITC) has been investigated at 25°C in Tris buffer, pH 7. 5. The addition of 5 to1 0 fold excess of aldolase to 0.1-1 micron GPDH labeled with FITC (GPDHFITC) causes a large increase in both the fluorescence and polarization of FITC over a period of several hours, reflecting the formation ofa complex between the enzymes. When GPDH-FITC is incubated with either 1 mM NAD or ADP, the fluorescence of FITC increases while the polarization decreases, indicating that these nucleotides may increase the degree of dissociation of tetrameric GPDH. The rate of approach to equilibrium during the formation of the complex between the two enzymes increases in the presence of either NAD or ADP but decreases with increasing concentrations ofGPDH. Therefore, the interaction of the enzymes may involve the dissociation of tetrameric GPDH into smaller units. Aldolase causes no changes in the fluorescence properties of probes that are attacted to the active site cysteine residues of GPDH, such as fluorescein acrylamide, indicating that when the active site of GPDH is blocked the formation of the complex between the two enzymes is prevented. When GPDH-FITC is incubated with either excess NAD or aldolase, the average fluorescence lifetime of FITC slowly increases and approaches that of free FITC. Since fluorescein is quenched by tryptophan, it is likely that the increases observed both in the fluorescence intensities and lifetimes of GPDH-FITC during its interaction with aldolase arise from the removal of tryptophan residues from the vicinity of the FITC groups as a result of changes either in the conformation or in the degree of dissociation of tetrameric GPDH.

Paper Details

Date Published: 1 May 1990
PDF: 9 pages
Proc. SPIE 1204, Time-Resolved Laser Spectroscopy in Biochemistry II, (1 May 1990); doi: 10.1117/12.17752
Show Author Affiliations
Aydin Orstan, Univ. of Michigan (United States)
Ari Gafni, Univ. of Michigan (United States)

Published in SPIE Proceedings Vol. 1204:
Time-Resolved Laser Spectroscopy in Biochemistry II
Joseph R. Lakowicz, Editor(s)

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