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.     

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.     

Corbular sarcoplasmic reticulum of rabbit cardiac muscle

The structure of corbular sarcoplasmic reticulum as part of the sarcoplasmic reticulum (SR) in perfusion-fixed rabbit cardiac muscle was studied by thin sections and freeze fracture. In thin sections, processes on the surface of corbular SR have all the anatomical features of junctional processes of junctional SR. By freeze fracture, the E face of corbular SR was particle poor and showed deep pits; the P face was particle rich. The demonstrated structural homology of corbular SR to all forms of junctional SR justifies its inclusion in that group.     

Two-step modification of aspartate aminotransferase with 1,5-difluoro-2,4-dinitrobenzene. Cross-link localization

Rabbit muscle pyruvate kinase has been shown to catalyze the decarboxylation of oxalacetate (Creighton, D.J. and Rose, I.A. (1976) J. Biol. Chem. 251, 61). Noncovalent and covalent modifiers of the enzyme have been used to assess whether the decarboxylase and kinase reactions take place at a common site. Phosphoenol-alpha-ketobutyrate, an analog of the substrate phosphoenol-pyruvate, inhibits decarboxylase and kinase competitively and with nearly identical Ki values (5.7 micron and 4.8 micron, respectively). Oxalate, an analog of enol-pyruvate, inhibits each competitively and with similar Ki values (11 micron and 4.7 micron, respectively). Both activities are lost in parallel upon reaction with dithionitrobenzoate, which is active-site specific. These results indicate that the two activities share a common site on the enzyme. But, effects of the following modifiers suggest that different amino acid residues at that site participate in the two reactions: phenylalanine inhibition and fructose 1,6-bisphosphate activation are more effective with the decarboxylase; iodoacetamide preferentially inactivates decarboxylase while trinitrobenzenesulfonate preferentially inactivates kinase.     

Identification of intramolecular crosslinks in bovine growth hormone after two-step modification with 1,5-difluoro-2,4-dinitrobenzene

Derivatives of bovine growth hormone, containing monoaminotyrosyl residues in positions 35, 42 and 174, were treated at pH 3.6 with a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Under these conditions aminotyrosyl groups reacted. On changing the pH to 9.3, the second fluorine atom of the reagent was substituted with the sterically adjacent side groups of lysine, since the excess of reagent had been previously removed. The modified protein underwent cyanogen bromide treatment. Peptides containing the crosslinks were purified from tryptic digests of the cyanogen bromide fragments by HPLC. Results show that aminoTyr 174 was able to form dinitrophenylene bridges with Lys 111, Lys 29 and Lys 170. AminoTry 35 was found crosslinked to Lys 29. Taking into account the size of the reagent, it may be inferred that Lys 29, 111 and 170 are located at approximately 5 A from Tyr 174 in the bovine growth hormone molecule.     

Activated states of phosphorylase kinase as detected by the chemical cross-linker 1,5-difluoro-2,4-dinitrobenzene

When phosphorylase kinase from rabbit skeletal muscle was activated by phosphorylation and then cross-linked with 1,5-difluoro-2,4-dinitrobenzene at pH 6.8, dimers of beta subunits were formed that were not observed during cross-linking of nonphosphorylated enzyme under the same conditions. The ability to form these dimers was due to phosphorylation of the beta subunit because when enzyme phosphorylated in the alpha and beta subunits was incubated with a protein phosphatase relatively specific for the beta subunit (Ganapathi, M.K., Silberman, S.R., Paris, H., and Lee, E.Y.C. (1981) J. Biol. Chem. 256, 3213-3217), the ability to form the cross-linked beta dimers was lost. Significant amounts of two complexes also judged to be dimers of beta subunits were observed when nonphosphorylated phosphorylase kinase was cross-linked after preincubation with Ca2+ plus Mg2+ ions, after proteolysis by chymotrypsin, or when it was cross-linked at pH 8.2, three conditions known to stimulate the activity of the nonphosphorylated enzyme. From these results, we conclude that 1,5-difluoro-2,4-dinitrobenzene can serve as a structural probe for activated states of phosphorylase kinase. The activation is associated with a conformational change in which two beta subunits either move closer together or have a reactive group on one, or both, of them unmasked. Our results suggest that the diverse mechanisms listed above for stimulating phosphorylase kinase activity cause a common conformational change to occur.     

Calcium transport ATPase of canine cardiac sarcoplasmic reticulum. A comparison with that of rabbit fast skeletal muscle sarcoplasmic reticulum.

To define the mechanism responsible for the slow rate of calcium transport by cardiac sarcoplasmic reticulum, the kinetic properties of the Ca2+-dependent ATPase of canine cardiac microsomes were characterized and compared with those of a comparable preparation from rabbit fast skeletal muscle. A phosphoprotein intermediate (E approximately P), which has the stability characteristics of an acyl phosphate, is formed during ATP hydrolysis by cardiac microsomes. Ca2+ is required for the E approximately P formation, and Mg2+ accelerates its decomposition. The Ca2+ concentration required for half-maximal activation of the ATPase is 4.7 +/- 0.2 muM for cardiac microsomes and 1.3 +/- 0.1 muM for skeletal microsomes at pH 6.8 and 0 degrees. The ATPase activities at saturating concentrations of ionized Ca2+ and pH 6.8, expressed as ATP hydrolysis per mg of protein, are 3 to 6 times lower for cardiac microsomes than for skeletal microsomes under a variety of conditions tested. The apparent Km value for MgATP at high concentrations in the presence of saturating concentrations of ionized Ca2+ is 0.18 +/- 0.03 ms at pH 6.8 and 25 degrees. The maximum velocity of ATPase activity under these conditions is 0.45 +/- 0.05 mumol per mg per min for cardiac microsomes and 1.60 +/- 0.05 mumol per mg per min for skeletal microsomes. The maximum steady state level of E approximately P for cardiac microsomes, 1.3 +/- 0.1 nmol per mg, is significantly less than the value of 4.9 +/- 0.2 nmol per mg for skeletal microsomes, so that the turnover number of the Ca2+-dependent ATPase of cardiac microsomes, calculated as the ratio of ATPase activity to the E approximately P level is similar to that of the skeletal ATPase. These findings indicate that the relatively slow rate of calcium transport by cardiac microsomes, whem compared to that of skeletal microsomes, reflects a lower density of calcium pumping sites and lower Ca2+ affinity for these sites, rather than a lower turnover rate.     

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.     

Isolation of two proteolipids from rabbit skeletal muscle sarcoplasmic reticulum

We have isolated two proteolipids from rabbit skeletal muscle sarcoplasmic reticulum by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. One, PL-II, is identical to the proteolipid previously obtained by others using organic solvent extraction. The other, PL-I, has an amino acid composition very similar to those of proteolipids we previously isolated from canine cardiac SR and lamb kidney (Na,K)-ATPase.     

Actin modifying system of the sarcoplasmic reticulum from rabbit muscle

By interaction with sarcoplasmic reticulum from rabbit muscle G-actin loses the capability to polymerize. The modification of actin is due to the concomitant action of the Ca²⁺ ATPase and of an ADPase embedded in the sarcoplasmic reticulum vesicles. Methyl isobutyl xanthine increases the rate of modification of actin by the sarcoplasmic reticulum vesicles by increasing the rate of the exchange of the actin-bound nucleotide.     

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.     

The location of spermine in bacterial ribosomes as indicated by 1,5-difluoro-2,4-dinitrobenzene and by ethidium bromide

1. When the binding of ethidium bromide to rRNA is measured both in the presence and in the absence of spermine, by spectrophotometric titrations, by gel filtration, or by the changes in fluorescence intensity, spermine competes with ethidium bromide for sites on the rRNA; under the conditions used in these experiments ethidium bromide is bound to the double-stranded regions of rRNA. 2. When an excess of ethidium bromide is added to ribosomes from Bacillus stearothermophilus approx. 80% of the endogenous spermine is displaced from the ribosomes. 3. [(14)C]Spermine is fixed to ribosomes by either formaldehyde or 1,5-difluoro-2,4-dinitrobenzene. Most of the [(14)C]spermine, fixed to ribosomes by 1,5-difluoro-2,4-dinitrobenzene, attaches to the ribosomal protein. 4. It is concluded that most of the endogenous spermine is bound to the double-stranded RNA in ribosomes, and that some of these double-stranded regions to which spermine is attached also have ribosomal proteins bound to them.