Adenosine cyclic 3',5'-monophosphate dependent protein kinase: fluorescent affinity labeling of the catalytic subunit from bovine skeletal muscle with o-phthalaldehyde

Abstract

The catalytic subunit of adenosine cyclic 3',5'-monophosphate dependent protein kinase from bovine skeletal muscle was rapidly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The reaction followed pseudo-first-order kinetics, and the second-order rate constant was 1.1 X 10(2) M-1 s-1. Absorbance and fluorescence spectroscopic data were consistent with the formation of an isoindole derivative (1 mol/mol of enzyme). The reaction between the catalytic subunit and o-phthalaldehyde was not reversed by the addition of reagents containing free primary amino and sulfhydryl functions following inactivation. The reaction, however, could be arrested at any stage during its progress by the addition of an excess of cysteine or less efficiently by homocysteine or glutathione. The catalytic subunit was protected from inactivation by the presence of the substrates magnesium adenosine triphosphate and an acceptor serine peptide substrate. The decrease in fluorescence emission intensity of incubation mixtures containing iodoacetamide- or 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified catalytic subunit and o-phthalaldehyde paralleled the loss of phosphotransferase activity. Catalytic subunit denatured with urea failed to react with o-phthalaldehyde. Inactivation of the catalytic subunit by o-phthalaldehyde is probably due to the concomitant modification of lysine-72 and cysteine-199. The proximal distance between the epsilon-amino function of the lysine and the sulfhydryl group of the cysteine residues involved in isoindole formation in the native enzyme is estimated to be approximately 3 A. The molar transition energy of the catalytic subunit-o-phthalaldehyde adduct was 121 kJ/mol and compares favorably with a value of 127 kJ/mol for the 1-[(beta-hydroxyethyl)thio]-2-(beta-hydroxyethyl)isoindole in hexane, indicating that the active site lysine and cysteine residues involved in formation of the isoindole derivative of the catalytic subunit are located in a hydrophobic environment. o-Phthalaldehyde probably acts as an active site specific reagent for the catalytic subunit.

Inactivation of an NADPH‐dependent succinic semialdehyde reductase by o‐phthalaldehyde

“…The molar transition energies can be calculated from the fluorescence emission maximum of an isoindole by the empirical relationship [20,21]:…”

“…The calculated ET for the succinic semialdehyde reductase derivative (232 kJ/mol) is greater than those reported for most isoindole-modified proteins (116-187 kJ/mol [12][13][14]19]), indicating that the microenvironment of the site modified by o-phthalaldehyde in succinic semialdehyde reductase is less hydrophobic than in most other enzymes (see refs. [20,21] for details). Native and completely modified succinic semialdehyde reductase showed identical mobilities when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (data not shown).…”

Update and Evaluation of the Effectiveness of Different Thiols and Micellar Media in Roth's Fluorimetric Method for the Determination of Primary Amino Compounds

“…The fact that the emission wavelength shifts to shorter values with a decrease in dielectric constant of the bulk solvent is thought to be due to diminished dipole-dipole interaction between the fluorescent isoindole and bulk solvent molecules (139). A linear correlation has been reported between the fluorescence emission wave- length maximum of 1-(b-hydroxyethylthio)-2-b-hydroxyethylisoindole (formed by reaction of OPA/2-ME with ethanolamine) and some measure of the pure bulk solvent's micropolarity (such as Dimroth's E T scale) (140,141), with the l em markedly shifting toward shorter wavelength as the solvent polarity is decreased. The decrease in l em of isoindoles upon their transfer from a bulk water to a micellar-containing solution suggests that the isoindole is binding to the micellar aggregate (Fig.…”

“…(5,7,11,12,25,37,41,71,79,98,101,105,(136)(137)(138)(139). The emission wavelength is dependent upon the solvent polarity and decreases as the solvent polarity decreases (140,141).…”

Flow cytometric analysis of protein thiol groups in relation to the cell cycle and the intracellular content of glutathione in rat hepatocytes

“…The three cell lines differ in their growth characteristics (33) and in their tumorigenic potential (28). Measurement of protein thiols was made by using the probe o-phthalaldehyde (OPT), the reaction of which with protein thiols and neighbouring amino groups results in the formation of highly fluorescent isoindole adducts (7,21,35,40,48). In ethanol-fixed cells, the thiol groups are those associated with structural proteins as ethanol-soluble thiols are lost during fixation.…”