J.H. Harrison

Selective chemical modification of arginine residues in mitochondrial malate dehydrogenase

Mitochondrial malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) from porcine heart exhibits a time dependent loss in enzymatic activity in the presence of the reagent butanedione. The inhibition occurs concomitant with the modification of 2.4 residues of arginine per molecular weight of 70,000. The presence of the reduced coenzyme, NADH, protects the enzyme from inhibition by butanedione and from modification of arginine residues, suggesting that the residues modified are located near the coenzyme binding site and hence at or near the enzymatic active center of this enzyme.

The reaction of 4,4′-bis-dimethylaminodiphenylcarbinol with the sulfhydryl group: A new reagent for sulfhydryl analysis

Abstract 4,4′-bis-Dimethylaminodiphenylcarbinol (BDC-OH) dissociates in aqueous buffers at pH values below neutrality to form a resonance-stabilized carbonium-immonium ion (BDC+) which exhibits an absorbance maximum at 606 nm. In the presence of 4.0 M guanidine hydrochloride, BDC+ has an apparent molar absorption coefficient of 70,800 M−1cm−1 and an absorbance maximum of 612 nm. Sulfhydryl groups react with the cation to form S-(4,4′-bis-dimethylaminodiphenylmethyl-) derivatives with a concomitant quantitative loss of the 612-nm absorbance. This quantitative interaction has been exploited in the development of a new and convenient technique for the quantitative determination of sulfhydryl groups in proteins. Results of sulfhydryl determinations on simple thiols and five proteins are presented, along with comparison data obtained via other sulfhydryl techniques.

The differential effect of tetranitromethane on the proteinase and esterase activity of bovine thrombin

Abstract Some aspects of the reaction of bovine thrombin with tetranitromethane have been studied. Treatment of highly purified bovine thrombin with this reagent inhibits the proteinase (fibrinogen-clotting) activity of this enzyme but the esterase activity remains unchanged. Amino acid analysis indicated that 4–5 tyrosyl residues are modified under these reaction conditions. The inhibition of proteinase activity is not prevented by the presence of benzamidine, a competitive inhibitor of thrombin. It is concluded that the reaction of bovine thrombin with tetranitromethane results in a change in the conformation of the enzyme molecule with subsequent distortion of the substrate binding site which leads to an alteration in the specific proteinase activity but not in the less specific esterase activity.

The relation of the pH and concentration-dependent dissociation of porcine heart mitochondrial malate dehydrogenase

Abstract The relationship of the pH-dependent and concentration-dependent dissociation of porcine heart mitochondrial malate dehydrogenase (L-malate: NAD + oxidoreductase, EC 1.1.1.37) was investigated by means of gel filtration chromatography utilizing a standardized Sephacryl S-200 column. The results obtained indicate that the dimeric form of this enzyme dissociates to yield monomers at conditions of low protein concentration or at pH values below neutrality. In addition it is apparent that as the pH is lowered, the minimum concentration of protein required to maintain the enzyme in the dimeric form is increased.

4,4′-Bis dimethylaminodiphenylcarbinol: A new reagent for selective chemical modification. Interaction with porcine malate dehydrogenase

Abstract 4,4- bis Dimethylaminodiphenylcarbinol (BDC-OH) has recently been reported to be a highly sensitive reagent for the quantitative determination of sulfhydryl residues in biological materials (1). In this communication the effectiveness of BDC-OH as a reagent for selective chemical modification of “active center” cysteine residues was investigated. The supernatant and mitochondrial forms of malate dehydrogenase were chosen for investigation by this reagent. Supernatant malate dehydrogenase which has never been found to contain an “active center” cysteine is unaffected by this reagent. Mitochondrial malate dehydrogenase (L malate: NAD + oxidoreductase, EC 1.1.1.37) from porcine heart can be irreversibly inactivated by a 20 fold M excess of the reagent. Chemical modification of two essential sulfhydryl residues is prevented by the presence of the coenzyme, NAD + , suggesting that the site of interaction is located at or near the coenzyme binding site and hence at or near the enzymatic center of this enzyme.

Regulation of mitochondrial malate dehydrogenase: kinetic modulation independent of subunit interaction

Porcine heart mitochondrial malate dehydrogenase (EC 1.1.1.37), a dimeric enzyme of Mr = 70,000, is both allosterically activated and inhibited by citrate. Using an affinity elution procedure based upon citrate binding to malate dehydrogenase, the isolation of pure heterodimer (a dimeric species with one active subunit and one iodoacetamide-inactivated subunit) has been achieved. Investigations utilizing this heterodimer in conjunction with resin-bound monomers of malate dehydrogenase have allowed the formulation of a definite conclusion concerning the role of subunit interactions in catalysis and regulation of this enzyme. The citrate kinetic effects, oxaloacetate inhibition, malate activation, and the effects of 2-thenoyl-trifluoroacetone (TTFA) are shown to be independent of interaction between catalytically active subunits. Previous kinetic data thought to support a reciprocating catalytic mechanism for this enzyme may be reinterpreted upon closer analysis in relation to an allosteric, conformationally specific binding model for malate dehydrogenase.

Interaction of pyridoxal 5' phosphate and malate dehydrogenase.

Abstract Mitochondrial malate dehydrogenase (L-malate-NAD + oxidoreductase, E.C.1.1.1.37) of porcine heart is totally and irreversibly inhibited by pyridoxal-5′-phosphate. The course of inactivation appears to be biphasic in nature. The coenzyme, NADH, was observed to fully protect the enzyme from inactivation, suggesting interaction at or near the enzymatic active center.

Structural studies of porcine malate dehydrogenase selective chemical modification

Abstract Evidence is presented indicating that residues at or near the active center of mitochondrial malate dehydrogenase (L-malate-NAD+ oxidoreductase, E.C.1.1.1.37) of porcine heart can be selectively carboxamidomethylated with the use of iodoacetamide. Protection against inactivation by addition of the coenzyme NADH and quantitation of incorporation of modifying reagent are discussed.

Concomitant purification of three porcine heart mitochondrial enzymes: Citrate synthase, aspartate aminotransferase, and malate dehydrogenase☆

The mitochondrial enzymes citrate synthase, malate dehydrogenase, and aspartate aminotransferase were purified to homogeneity from porcine hearts by use of Bio-Rex 70, carboxymethylcellulose CM32, and Affi-Gel blue chromatography. This procedure provides relatively rapid, large-scale preparation of the three enzymes based on their differential binding to commercially available cation-exchange resins followed by a final affinity chromatography step.

Kinetics of formation of the 4,4′-bis-dimethylaminodiphenyl carbonium ion (BDC+) and its reaction with sulfhydryl residues

Abstract The use of 4,4′-bis-dimethylaminodiphenylcarbinol (BDC-OH) as an analytical reagent for sulfhydryl residues and as a specific chemical modification reagent for proteins is dependent upon the unique properties of the BDC + cation present in aqueous buffers below a pH of 6.5. In the presence of aqueous buffers, pH 5.1, BDC + exhibits a λ max of 606 nm with an apparent molar absorption coefficient of 10,000 m −1 cm −1 . Upon the addition of 4 m guanidine hydrochloride this apparent coefficient is enhanced to 70,800 m −1 cm −1 . The true molar extinction coefficient for BDC + was determined to be 128,000 m −1 cm −1 . The reaction of BDC + with sulfhydryl residues of proteins or simple thiols is rapid and leads to a complex devoid of visible color. In the pH range 3.0–7.0, a complex equilibrium is established among the three species BDC-OH, BDC + , BDCH ++ . The formation of this equilibrium is proton mediated, and is discussed in terms of the equilibrium, rate, and acid dissociation constants.