Diethyl Pyrocarbonate Inactivation Research Articles

Monoacylglycerol acyltransferase. Evidence that the activities from rat intestine and suckling liver are tissue-specific isoenzymes.

The monoglycerol acyltransferase (EC 2.3.1.22) (recommended name acylglycerol palmitoltransferase) activities from rat intestinal mucosa and suckling liver microsomes were compared in order to determine why substrate specificities differed in the two tissues. Suckling liver monoacylglycerol acyltransferase activity was highly specific for sn-2-mono-C18:1 glycerol and acylated rac-1-mono-C18:1 glycerol and 1- and 2-mono-C18:1 glycerol ethers poorly. In contrast, the substrate specificity of intestinal monoacylglycerol acyltransferase activity was broad. 1-Acyl- and 1- and 2-alkylglycerols were acylated at rates that were 45-78% of the rate observed with the preferred substrate sn-2-mono-C18:1 glycerol. Partial heat inactivation did not alter these relative specific activities, making it unlikely that intestinal microsomes contained a second acyltransferase capable of acylating the alternate substrates. The hypothesis that intestine and liver contain non-identical monoacylglycerol acyltransferase activities was further tested. Intestinal mucosa monoacylglycerol acyltransferase was much more thermolabile than the liver activity. Incubation with 50 microM diethylpyrocarbonate inactivated liver monoacylglycerol acyltransferase activity 84% but had little effect on the intestinal activity. Hydroxylamine completely reversed diethylpyrocarbonate inactivation, suggesting that critical histidine residues were more accessible in liver monoacylglycerol acyltransferase. 2,4,6-Trinitrobenzene sulfonic acid inactivated hepatic monoacylglycerol acyltransferase more than the intestinal activity, suggesting that critical lysine residues were more accessible. The intestinal and liver activities were also differently affected by acetone, detergents, MgCl2, phospholipids, and bovine serum albumin. Taken as a whole, the data strongly suggest that rat intestinal mucosa and suckling liver contain tissue-specific monoacylglycerol acyltransferase isoenzymes.

Diethylpyrocarbonate inactivation of NAD-malic enzyme from Ascaris suum

Treatment with diethylpyrocarbonate results in a first-order loss of the malate oxidative decarboxylase activity of NAD-malic enzyme. First-order plots are biphasic, with about 40–50% activity loss in the first phase. The inactivation process is not saturable, and the second-order rate constant is 4.7 spm −1s −1 Malate (250 m m) provides complete protection against inactivation (as measured by a decrease in the inactivation rate), and less malate is required with Mg 2+ present. Partial protection (50%) is afforded by either NAD + (1 m m) or Mg 2+ (50 m m). Treatment of modified (inactive) enzyme with hydroxylamine restores activity to 100% of the control when corrected for the effect of hydroxylamine on unmodified enzyme. A total of 10–13 histidine residues/subunit are acylated concomitant with loss of activity while 1–2 tyrosines are modified prior to any activity loss. The presence of Mg 2+ and malate at saturating concentrations protect 1–2 histidine residues/subunit. The intrinsic fluorescence of the enzyme decreases with time after addition of diethylpyrocarbonate, but the rate constant for this process is at least 10-fold too low to account for the biphasicity observed in the first order plots. The histidine modified which is responsible for loss of activity has a p K of 8.3 as determined from the pH dependence of the rate of inactivation. The histidine titrated is still modified under conditions where the residue is completely protonated but at a rate 1 100 the rate of the unprotonated histidine. The results suggest that 1–2 histidines are in or near the malate binding site and are required for malate oxidative decarboxylation.

Essential histidyl residues of ferredoxin-NADP+ oxidoreductase revealed by diethyl pyrocarbonate inactivation.

Diethyl pyrocarbonate inhibited diaphorase activity of ferredoxin-NADP+ oxidoreductase with a second-order rate constant of 2 mM-1 X min-1 at pH 7.0 and 20 degrees C, showing a concomitant increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives. Activity could be restored by hydroxylamine, and the pH curve of inactivation indicated the involvement of a residue having a pKa of 6.8. Derivatization of tyrosyl residues was also evident, although with no effect on the diaphorase activity. Both NADP+ and NADPH protected the enzyme against inactivation, suggesting that the modification occurred at or near the nucleotide binding domain. The reductase lost all of its diaphorase activity after about two histidine residues had been blocked by the reagent. In differential-labeling experiments with NADP+ as protective agent, it was shown that diaphorase inactivation resulted from blocking of only one histidyl residue per mole of enzyme. Modified reductase did not bind pyridine nucleotides. Modification of the flavoprotein in the presence of NADP+, i.e., with full preservation of diaphorase activity, resulted in a significant impairment of cytochrome c reductase activity, with a second-order rate constant for inactivation of about 0.5 mM-1 X min-1. Reversal by hydroxylamine and spectroscopic data indicated that this second residue was also a histidine. Ferredoxin afforded only slight protection against this inhibition. Conversely, carbethoxylation of the enzyme did not affect complex formation with the ferrosulfoprotein. Redox titration of the modified reductase with NADPH and with reduced ferredoxin suggested that the second histidine might be located in the electron pathway between FAD and ferredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of glycerate-2,3-P 2—V. Histidine-specific reagents inactivate the phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase activities of rabbit muscle phosphoglycerate mutase

1. 1. Both treatment with diethylpyrocarbonate and photo-oxidation with rose bengal produces the loss of the three enzymatic activities of rabbit muscle phosphoglycerate mutase: phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase. 2. 2. The synthase and the phosphatase activities are less affected than the mutase activity. 3. 3. Glycerate-2,3-P 2 and glycerate-3-P protect against diethylpyrocarbonate inactivation, but do not protect against inactivation produced by photo-oxidation. 4. 4. Hydroxylamine reactivates the diethylpyrocarbonate-treated enzyme. 5. 5. Chemical modification of phosphoglycerate mutase markedly reduces its ability to form the functionally active phosphoenzyme. 6. 6. These results provide evidence of the intrinsic character of the three enzymatic activities of phosphoglycerate mutase. In addition, they support the existence of two separate binding sites for monophosphoglycerates and for bisphosphoglycerates.

Effect of diethylpyrocarbonate on lactose/proton symport in Escherichia coli membrane vesicles.

Exposure of Escherichia coli ML 308-225 membrane vesicles to the histidine-specific reagent diethylpyrocarbonate (DEPC) led to concentration- and time-dependent inactivation of active lactose transport, and the sensitivity of the system to inactivation was enhanced when an electrochemical proton gradient (delta- muH+, interior negative and alkaline) was generated across the vesicle membrane. Although beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside blocked DEPC inactivation, binding of p-nitrophenyl alpha-D-galactopyranoside was not significantly altered, indicating that DEPC does not react at the binding sites of the lac carrier protein. Strikingly, vesicles treated with DEPC exhibited an increased apparent Km for delta- muH+-driven lactose transport and counterflow but no change in the Vmax of these reactions and no change in the apparent Km or Vmax of facilitated diffusion. Moreover, DEPC treatment increased the apparent Km observed for delta- muH+-driven proline and D-lactate transport with no change in Vmax. Finally, the lactose counterflow activity of DEPC-treated vesicles was regenerated by subsequent exposure to hydroxylamine. It is suggested that a histidyl residue(s) in the lac carrier or another protein in the translocation complex is involved either in the binding and translocation of protons or in a conformational change that may occur upon protonation of the lac carrier protein.