John M. Brewer

Artifact Produced in Disc Electrophoresis by Ammonium Persulfate

Ammonium persulfate, a common polymerizing agent for acrylamide gels, can inactivate yeast enolase and produce increased electrophoretic heterogeneity during disc electrophoresis in gels containing 8M urea. The use of riboflavin and light for polymerization or thioglycolate for removal of the persulfate are feasible alternatives.

The inactivation of yeast enolase by 2,3-butanedione

Abstract Yeast enolase has been reacted with 2,3-butanedione in borate buffer and the number of arginine residues modified has been measured as a function of enzymatic activity. Activity losses are linear with respect to arginine modification, with complete inactivation being correlated with the alteration of one arginine per subunit. The tryptic peptide whose arginine is modified has been isolated using O-(triethylaminoethyl)-cellulose chromatography and paper electrophoresis and is a dipeptide, leucyl-arginine. Protection against loss of activity is afforded by the addition of substrate, 2-phosphoglyceric acid, or a competitive inhibitor, 3-aminoenolpyruvate-2-phosphate (AEP), and Mg; elimination of the Mg, a prerequisite for substrate or inhibitor binding, eliminates the protection afforded by substrate. It is suggested that a single arginine residue per subunit of enolase is necessary for enzymatic activity and is located at or near the substrate binding site (active site). The binding of a chromophoric competitive inhibitor, AEP, to native and butanedione-reacted enolase was examined by spectrophotometric titrations. Compared to the native enzyme, the butanedione-inactivated enolase binds AEP, but with an extinction coefficient for the inhibitor that is about two-thirds of the native value; the binding affinities are comparable. The strong binding of the modified enzyme for AEP suggests that the arginine, although critical for enzymatic activity, is not significantly involved in the overall binding of the inhibitor.

A conserved region in the σ54-dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation

Rhizobium melioti DctD activates transcription from the dctA promoter by catalysing the isomerization of closed complexes between σ54‐RNA polymerase holoenzyme and the promoter to open complexes. DctD must make productive contact with σ54‐holoenzyme and hydrolyse ATP to catalyse this isomerization. To define further the activation process, we sought to isolate mutants of DctD that had reduced affinities for σ54‐holoenzyme. Mutagenesis was confined to the well‐conserved C3 region of the protein, which is required for coupling ATP hydrolysis to open complex formation in σ54‐dependent activators. Mutant forms of DctD that failed to activate transcription and had substitutions in the C‐terminal half of the C3 region were efficiently cross‐linked to σ54 and the β‐subunit of RNA polymerase, suggesting that they bound normally to σ54‐holoenzyme. In contrast, some mutant forms of DctD with amino acid substitutions in the N‐terminal half of the C3 region had reduced affinities for σ54 and the β‐subunit in the cross‐linking assay. These data suggest that the N‐terminal half of the C3 region of DctD contains a site that may contact σ54‐holoenzyme during open complex formation.

A spectrophotometric method for quantitation of carboxyl group modification of proteins using woodward's reagent K

Reaction of proteins with Woodwards Reagent K in 0.05 ionic strength Tris-HCl, pH 7.8, followed by removal of excess reagent by chromatography on Sephadex G-25 in the same buffer, results in covalently attached chromophores with an absorption maximum at 340 nm and an extinction coefficient of 7000 M-1 cm-1. This absorbance can be used to quantitate the reaction of Woodwards Reagent K with carboxyl groups in proteins, provided sulfhydryl groups do not react. The chromophore also enables specific detection and identification of carboxyl-modified peptides upon separation by chromatography or electrophoresis.

Characterization of the interaction of yeast enolase with polynucleotides.

Yeast enolase is inhibited under certain conditions by DNA. The enzyme binds to single-stranded DNA-cellulose. Inhibition was used for routine characterization of the interaction. The presence of the substrate 2-phospho-D-glycerate reduces inhibition and binding. Both yeast enolase isozymes behave similarly. Impure yeast enolase was purified by adsorption onto a single-stranded DNA-cellulose column followed by elution with substrate. Interaction with RNA, double-stranded DNA, or degraded DNA results in less inhibition, suggesting that yeast enolase preferentially binds single-stranded DNA. However, yeast enolase is not a DNA-unwinding protein. The enzyme is inhibited by the short synthetic oligodeoxynucleotides G6, G8 and G10 but not T8 or T6, suggesting some base specificity in the interaction. The interaction is stronger at more acid pH values, with an apparent pK of 5.6. The interaction is prevented by 0.3 M KCl, suggesting that electrostatic factors are important. Histidine or lysine reverse the inhibition at lower concentrations, while phosphate is still more effective. Binding of single-stranded DNA to enolase reduces the reaction of protein histidyl residues with diethylpyrocarbonate. The inhibition of yeast enolase by single-stranded DNA is not total, and suggests the active site is not directly involved in the interaction. Binding of substrate may induce a conformational change in the enzyme that interferes with DNA binding and vice versa.

Avian thymic hormone (ATH) is a parvalbumin

Amino acid sequence analysis of a protein from chicken thymus tissue which promotes immunological maturity in chicken bone marrow cells in culture has established sequences of a 45-residue fragment, a 24-residue fragment and a 9-residue and an 8-residue peptide. Independent comparison of the 45- and 24-residue fragments with known amino acid sequences by computer search has unequivocally identified avian thymic hormone as a parvalbumin. This is the first demonstration that a protein previously identified by a biological function is a parvalbumin.

Specificity and mechanism of action of metal ions in yeast enolase

Enolase Specificity Yeast

Hydrolysis of p-nitrophenylphosphorylcholine by alkaline phosphatase and phospholipase C from rabbit sperm-acrosome.

Abstract p-Nitrophenylphosphorylcholine, used as an artificial substrate for phospholipase C, is readily hydrolyzed by alkaline phosphatases also, despite the reported requirement of alkaline phosphatases for a terminal phosphate for activity. p-Nitrophenylphosphorylcholine hydrolyzing activity in rabbit semen is concentrated in Hyamine-Triton extracts of sperm acrosomes or cytoplasmic droplets rather than seminal plasma. Disc electrophoresis of these extracts shows well separated zones of phosphatase and phospholipase C-like activity. Only phosphatase activity is observed in seminal plasma. It is suggested that phospholipase C is located in the sperm acrosome.

Binding of inhibitory metals to yeast enolase.

Certain divalent cations can inhibit yeast enolase by binding at sites that are distinct from those metal binding sites normally associated with catalytic activity, i.e., the conformational and catalytic binding sites. By using a buffer that does not compete with metal ions (tetrapropylammonium borate) Zn, Co, Mn, Cu, Cd, and Ni are found to exhibit similar inhibitory characteristics. Inhibition by those metals is alleviated by the addition of imidazole or tris buffer and, for zinc, by a metal chelating agent (Calcein). Inhibition by zinc was examined in detail through binding studies and enzymatic activity measurement. In tetrapropylammonium buffers at pH 8.0, enolase binds up to four moles of zinc per mole of enzyme (two moles per subunit). An imidazole concentration of 0.05 M reduces the binding: in the absence of substrate, just two moles of zine per enzyme are bound. The enzyme will bind two additional moles of zinc upon the addition of substrate in either buffer, but the enzyme in tetrapropylammonium buffer is nearly inactive. Inhibition is, therefore, correlated with the binding of two moles of zinc per mole of enzyme. Some additional metal ions, Ca, Tb, Hg, and Ag also caused inhibition of yeast enolase but not by binding to the inhibitory site described.

The increase in yeast enolase fluorescence produced by substrates and competitive inhibitors in the presence of excess Mg2

Abstract 1. 1. Substrates or competitive inhibitors produced an increase in the fluorescence of yeast enolase (2-phospho- d -glycerate hydro-lyase, EC 4.2.1.11) in the presence of a high concentration (0.01 M) of Mg2+ or other activating metal ions. 2. 2. The increase in fluorescence is ascribed to a decrease in quenching of one or more tryptophans in the enzyme upon binding metal ion and substrate. 3. 3. Fluorometric titrations of the enzyme with substrate in the presence of excess Mg2+ have given substrate binding constants in agreement with substrate Michaelis constants. 4. 4. Titrations with Mg2+ in the presence of excess substrate show two binding sites. The site with the smallest dissociation constant is identical with the site associated with structural changes in the enzyme when the metal ion is bound. The second site appears only in the presence of substrate and is not connected with enzymic activity, but perhaps with actual inhibition.