Investigation of the complex between rabbit muscle glyceraldehyde-3-phosphate dehydrogenase and aldolase using fluorescence laser spectroscopy

Aydin Orstan; Ari Gafni

doi:10.1117/12.17752

1 May 1990 Investigation of the complex between rabbit muscle glyceraldehyde-3-phosphate dehydrogenase and aldolase using fluorescence laser spectroscopy

Aydin Orstan, Ari Gafni

Author Affiliations +

Proceedings Volume 1204, Time-Resolved Laser Spectroscopy in Biochemistry II; (1990) https://doi.org/10.1117/12.17752
Event: OE/LASE '90, 1990, Los Angeles, CA, United States

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.

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Aydin Orstan and Ari Gafni "Investigation of the complex between rabbit muscle glyceraldehyde-3-phosphate dehydrogenase and aldolase using fluorescence laser spectroscopy", Proc. SPIE 1204, Time-Resolved Laser Spectroscopy in Biochemistry II, (1 May 1990); https://doi.org/10.1117/12.17752

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KEYWORDS

Luminescence

Polarization

Laser spectroscopy

Biochemistry

Anisotropy

Mass attenuation coefficient

Absorbance

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