Chemical modification of Lactobacillus casei thymidylate synthetase with 1-fluoro-2,4-dinitrobenzene and 1,5-difluoro-2,4-dinitrobenzene

Previous studies of the pH dependence of sulfhydryl group modification in thymidylate synthetase (W. A. Munroe, C. A. Lewis and R. B. Dunlap, 1978, Biochem. Biophys. Res. Commun.80, 355–360) suggested that a neighboring general base residue enhanced the nucleophilicity of the catalytic cysteinyl side chain. In an effort to identify the latter residue by active site crosslinking, chemical modification of the enzyme by 1,5-difluoro-2,4-dinitrobenzene was investigated and compared with results of modification by 1-fluoro-2,4-dinitrobenzene. Incubation of enzyme with 1-fluoro-2,4-dinitrobenzene led to rapid inactivation and loss of ability to form ternary complexes. Paper chromatography of the acid hydrolysate of enzyme modified with 1-fluoro-2,4-dinitrobenzene yielded two yellow spots, identified as dinitrophenylenecysteine and dinitrophenylenelysine. Specific active site labeling was indicated by substrate protection with dUMP, by the release of 1.65 of fluoride ion per enzyme dimer during inactivation, and by the fact that 70% of the activity was recovered after incubation of the inactivated enzyme with 2-mercaptoethanol, The results of a similar series of studies with 1,5-difluoro-2,4-dinitrobenzene indicated quite specific active site modification. The equivalents of fluoride ion released during modification, 3.5 per enzyme dimer, and the fact that thiolysis of the totally inactivated enzyme led to a recovery of only 18% of the original activity provided evidence for active site crosslinking with the catalytic cysteine as one of the modification sites. Characterization of the modified enzyme, its yellow acid hydrolysate fragments, and a variety of dinitrophenylene crosslinked models suggested that 1,5-difluoro-2,4-dinitrobenzene had modified the enzyme by crosslinking cysteine and serine residues.     

Mutagenicity of 1-fluoro-2,4-dinitrobenzene is affected by bacterial glutathione

Recently we have shown that Salmonella typhimurium tester strains have high levels of the tripeptide glutathione (GSH) and activity of GSH S-transferases (Summer et al., 1979). In continuation of the GSH-dependent suppression of mutagenicity of 1-chloro-2,4-dinitrobenzene in presence of S9 fraction (Summer et al., 1979), this paper is focused on the GSH-dependent detoxifying capacity of the bacterial tester strains. 1-Fluoro-2,4-dinitrobenzene (FDNB), an electrophilic agent, which is used to identify terminal amino acids in proteins (Sanger reagent), readily reacts with GSH leading to a dose-dependent depletion of bacterial GSH. Additionally, FDNB is a strong mutagen for Salmonella typhimurium TA100, TA1538 and TA98 without metabolic activation.Presumably owing to conjugation with bacterial GSH, FDNB in concentrations which were lower or equal to those of bacterial GSH were found to be not mutagenic. Accordingly, increasing amounts of bacteria in the test system require increasing amounts of FDNB for expression of mutagenicity.     

Crystallization and partial characterization of glutamate dehydrogenase from ox liver nuclei.

Glutamate dehydrogenase have been obtained in crystalline form from purified ox liver nuclear fractions. The enzyme appeared homogeneous, as judged by several electrophoretic techniques at two pH values. A comparative study with the widely known ox liver mitochondrial glutamate dehydrogenase revealed several common features, such as the allosteric effect of the nucleotides ADP and GTP, the activation at high concentrations of the cofactor NAD+, and the existence of a concentration-dependent reversible monomer-polymer(s) equilibrium. However, the two enzymes differed in many other respects. Inorganic phosphate activated nuclear glutamate dehydrogenase to a much greater extent than the mitochondrial enzyme; the substrate NH4+ showed cooperative homotropic interactions only with nuclear glutamate dehydrogenase; kinetic differences were detected with most of the reaction substrates, as well as different rates of oxidative deamination of other L-amino acids, the nuclear enzyme had a higher anodic mobility and a different chromatographic behavior on anionic exchangers. The latter evidence indicates that the glutamate dehydrogenase activity in liver is associated with two proteins which are structurally different, thus confirming the results of a separate immunological study. Preliminary evidence suggests that the enzyme in nuclei is attached to the nuclear envelope, probably the inner membrane, from which it can be solubilized by the addition of salts.     

Regulation of ox liver glutamate dehydrogenase activity by coenzymes

The effects of coenzymes NAD(P) and NAD(P)H on the kinetics of the ox liver glutamate dehydrogenase reaction have been studied. The oxidized coenzymes were shown to activate alpha-ketoglutarate amination at inhibiting concentrations of NADH and NADPH. The reduced coenzymes, NADH and NADPH, inhibit glutamate deamination with both NAD and NADP as coenzymes. The data obtained are discussed in terms of literature data on the mechanisms of the coenzyme effects on the glutamate dehydrogenase activity and are inconsistent with the theory of direct ligand--ligand interactions. It was shown that the peculiarities of the glutamate dehydrogenase kinetics can easily be interpreted in the light of the two state models.     

Spin-labelling of phosphorylase b using a paramagnetic 1-fluoro-2,4-dinitrobenzene derivative

Phosphorylase b (1,4-alpha-D-glucan:1,6-alpha-D-glucan 6-alpha-glucosyltransferase, EC 2.4.1.1) can be specifically spin-labelled at a site essential for the catalytic action of the enzyme. A paramagnetic analogue of 1-fluoro-2,4-dinitrobenzene was synthesized and used as a dinitrophenylating agent. Reaction of phosphorylase b with the paramagnetic probe combined with the thiolysis method, leads to spin-labelling of a single -NH2 group (0.75 groups per subunit) with concomitant loss of 50% of the catalytic activity. Dinitrophenylation does not change the sedimentation profile of the enzyme. The ESR spectrum of modified phosphorylase b indicates that the attached label has rather limited segmental mobility and its environment is slightly hydrophobic. Small but subtle conformational changes induced by ligands in this critical site of the macromolecule can be directly detected by the spin-label. Also, sulfhydryl group modification of the spin-labelled enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) has a pronounced effect on the resonance spectrum.     

A peptide containing a reactive lysyl group from ox liver glutamate dehydrogenase

1. Inhibition of ox liver glutamate dehydrogenase with N-(N'-acetyl-4[(35)S]-sulphamoylphenyl)maleimide (ASPM) is more specific at pH7.3 than at pH6.9. At pH7.3 inhibition accompanies the incorporation at 1 mole of ASPM residues into about 53000g. of protein. 2. Digestion of the modified protein with chymotrypsin and trypsin yields a unique radioactive peptide. 3. Acid hydrolysis of 1 mole of this peptide yields 1 mole of N(in)-succin-2-yl-lysine. The in-amino group of a lysyl residue is thus the site of modification of the protein. 4. The sequence containing the modified lysyl residue is: [Formula: see text] where Asx respresents either aspartic acid or asparagine.     

Reaction of tRNAPhe from yeast with 1-fluoro-2,4-dinitrobenzene. Attachment sites of the potential antigenic-determining 2,4-dinitrophenyl residues.

The reaction of 1-fluoro-2,4-dinitrobenzene with tRNAPhe from yeast, for the introduction of antigenic-determining 2,4-dinitrophenyl residues into tRNA, took place only at adenosine residues in tRNAPhe. After reaction at pH 8.0 and 50 degrees C two kinds of products were detected: one was ribose-modified adenosine which was derived from the 3' terminus of tRNA, and the other was base-modified adenosine. The sites and extent of the modification of each particular adenosine residue of tRNAPhe were determined as follows: 5 (6% modified), 31 (2%), 35 (36%), 67 (5%), and 76 (51%). Thus mainly the terminal adenosine and one adenosine in the anticodon loop bear the 2,4-dinitrophenyl residue.     

Reaction of 1-fluoro-2,4-dinitrobenzene and 2,4,6-trinitrobenzenesulphonic acid with membranes of sarcoplasmic reticulum.

Membranes of sarcoplasmic reticulum were labelled with 1-fluoro-2,4-dinitro[3H]benzene at pH 6.5 and with 2,4,6-trinitrobenzenesulphonate at pH 9.2. Conditions were chosen to restrict reaction to amino groups, and the effect of blockings of these groups by methyl acetimidate was determined. All proteins were labelled to some extent by both reagents, but, whereas the trinitrophenylation of both lipid and protein amino groups was almost completely blocked by methyl acetimidate, the dinitrophenylation of the ATPase at pH 6.5 was much less affected. The seven amino groups on the ATPase that were labelled under these conditions did not react with methyl acetimidate. This reagent can therefore be used to enhance the specificity of fluorodinitrobenzene for amino groups in a hydrophobic environment. The amino groups on the minor proteins and on the phospholipids that reacted with fluorodinitrobenzene at pH 6.5 were probably in an aqueous environment, since the reaction was blocked by methyl acetimidate.     

L-lactate-2-monooxygenase. Sequence of peptides containing residues modified by 1-fluoro-2,4-dinitrobenzene

L-Lactate-2-monooxygenase (EC 1.13.12.4) inactivated by 1-fluoro-2,4-dinitrobenzene essentially as described previously (Choong, Y. S., Shepherd, M. G., and Sullivan, P. A. (1978) Biochem. J. 173, 255-262) incorporated 2.8 mol of the dinitrophenyl (DNP) moiety per mole of flavin. The inhibitors 2-methyl lactate or sulfite decreased the incorporation to 0.9 mol of DNP per mole of flavin. Peptide mapping by high performance liquid chromatography of radioactively labeled protein digested with trypsin showed three peaks of radioactivity. DNP-amino acid analysis and peptide sequencing showed that 2 distinct cysteine residues and a histidine residue had been modified. Both cysteine peptides were protected from modification by either of the inhibitors, whereas the histidine was only partially protected. The sum of the 2 cysteine peptides accounted for nearly 1 mole of label per mole of monomer. Since both of the cysteines are protected by inhibitors, they both must be in or near the substrate-binding site of the enzyme and appear to be modified in a mutually exclusive fashion. The histidine, on the other hand, does not lie directly in the substrate-binding site. It is possible that this histidine is the positively charged residue that is postulated to be near the N-1 position of the flavin.     

Reversible association of ox liver glutamate dehydrogenase with the inner mitochondrial membrane

A comparative study on the catalytic and allosteric properties of particulate and soluble forms of ox liver glutamate dehydrogenase has been carried out. The response of the bound enzyme to release by various effectors was investigated. The particulate enzyme was found to have catalytic activities similar to the free enzyme in contrast to its behaviour when bound to pure anionic phospholipids. Possible reasons for such outstanding differences are discussed.     

Essential residues in angiotensin converting enzyme: modification with 1-fluoro-2,4-dinitrobenzene

The peptidase and esterase activities of rabbit pulmonary angiotensin converting enzyme (ACE) are rapidly abolished on reaction with 1-fluoro-2,4-dinitrobenzene (Dnp-F). Inactivation follows first-order kinetics with respect to the reagent and is accompanied by stoichiometric incorporation of 3,5-[3H]Dnp, indicating that the effect is due to a specific modification of the enzyme. Thin-layer chromatography of an acid hydrolysate of the modified enzyme indicates that most of the radioactive label is present as O-Dnp-tyrosine (65 to greater than 95%) and the rest as N epsilon-Dnp-lysine. The pH dependence of the reaction is consistent with modification of either tyrosine or lysine. The presence of a competitive inhibitor effectively protects the enzyme against inactivation by Dnp-F. Acetylation of ACE with N-acetylimidazole also protects the enzyme against modification with Dnp-F. The results indicate the presence of catalytically essential tyrosine and lysine residues at the active site of ACE.