Active subunits in hybrid-modified malate dehydrogenase.

S.R Jurgensen,J.H Harrison

doi:10.1016/s0021-9258(19)68401-2

Abstract

Inactivation of porcine heart mitochondrial malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) by selective modification of an active center histidine residue with the reagent iodoacetamide has been further investigated to examine the existence of and the enzymatic activity of a hybrid (half)-modified dimer. The loss of enzymatic activity during iodo(1-14C) acetamide modification is linear with 14C incorporation. Enzyme was modified to various extents and the reaction was quenched. Microzonal electrophoresis was performed to separate native dimeric enzyme, hybrid-modified enzyme, and doubly modified enzyme. The distribution of each species was quantitated by scanning densitometry. The distribution generated throughout the time course of inactivation indicates that both subunits are modified independently and at the same rate. It is apparent that the hybrid-modified dimer contributes one-half of the enzymatic activity of a native dimer in the standard assay. Kinetic studies were performed and the results indicate that there is no apparent change in kinetic parameters between a subunit of the native dimer and the active subunit in the hybrid-modified dimer. Dissociation and reassociation of a mixture of native enzyme and doubly-iodoacetamide-modified enzyme indicates that there is no preferential association of a modified subunit with another modified subunit, or of a native subunit with another native subunit, but rather, association is random with respect to native and iodoacetamide-modified subunits.

Highlights

1.1.1.37) by selective modification of an active center investigation of the enzyme which has been immobilized on histidine residue with the reagent iodoacetamide has Sepharose beads has indicated that the mitochondrial malate been further investigated to examine the existence of dehydrogenase monomer can exhibit significant cataand the enzymatic activity of a hybrid-modified dimer
Dissociation and reassocia- Materials-Porcineheart mitochondrial malate dehydrogenase tion of a mixture of native enzyme and doubly-iodo- was purified from acetone powders of fresh pig hearts asdescribed by acetamide-modified enzyme indicates that there is no preferential association of a modified subunit with another modified subunit, or of a native subunit with another native subunit, but rather, association is random with respect to native and iodoacetamide-modified subunits
Incorporation of [‘4C]Zodoacetamide into Mitochondrial Malate malonate have led to the proposal of a reciprocating mecha- Dehydrogenase-The incorporation of i~do(l-’~C)acetamidineto nism in which the subunits do not carry out catalysis independently but rather function in concert using a “flip flop” catalytic sequence [1,2]

Summary

Protein concentration was determined spectrophotometrically using

Aliquots (400 or 500 pl) were placed half-of-the-siteseffects have not been apparent [4,5,6,7,8,9,10] It has been observed tha t t he dimeric enzyme, mitochondrial in 10 ml of aquasol scintillation mixture (New England Nuclear) and monitored for radioactivity. The costs of publication of this article were defrayed in part by the branes were equilibrated in a running buffer o1f.m4 M sodium payment of page charges. Steady State Kinetics-The apparent K,,, and V,,, values for malateand NAD’ with both native and partially IAM-modified enzyme,were determined with the assay described previously, except that sodium pyrophosphate buffer at pH 9.0, rather than 10.6, was used due to decreased stability of the hybrid-modified enzyme a t pH. An expanded absorbance scale of 00.2 full scale was used, and assays at each substrate concentration were performed in triplicate and theresults averaged

AND DISCUSSION

Isozymdeoublme od ax

SAMPLE native enzyme