Physical and Chemical Properties of Reversibly Inactivated Lactate Dehydrogenases

Alan S Levi,Nathan O Kaplan

doi:10.1016/s0021-9258(19)34132-8

Alan S Levi, Nathan O Kaplan

Open Access

https://doi.org/10.1016/s0021-9258(19)34132-8

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Abstract

Abstract The effect of reversible inactivation on the physical and chemical properties of dogfish M4 and chicken heart H4 lactate dehydrogenases has been studied. Reduced diphosphopyridine nucleotide was found to have a significant effect on the rate of recovery of the enzymatic activity, but not on the over-all recovery. Optical rotatory dispersion, circular dichroism, fluorescence, and immunological studies have shown structural differences between the native and reassociated enzyme. Further evidence for the difference in conformational structure was obtained from the pattern of reactivity of sulfhydryl groups of the reactivated enzyme. The native enzyme was also found to be more stable to heat and urea denaturation than the reassociated form. Pyruvate inhibition, and the catalytic activity with coenzyme analogues, was found to be different for the native and the reassociated enzyme. These results indicate the possibility that a conformational difference exists between the native and the reassociated enzyme for both dogfish M4 and chicken H4 lactate dehydrogenase. Studies on the lactate dehydrogenase reassociated in the presence of reduced DPN showed structural characteristics which are closer to the native dehydrogenase than the enzyme which was reassociated in the absence of the reduced coenzyme. Incubation of the enzyme with DPNH after reassociation does not convert the reassociated enzyme to a structure similar to that of the native enzyme. These studies suggest a possible role of the reduced coenzyme in the folding of the peptide chain or in the subunit interaction and in the attainment of the native conformation of the enzyme.

Highlights

Kaplan show that the ~20,~ value for the reassociated protein is not significantly different from that of the native enzyme
These findings indicate that following the refolding, the peptide chains have the capa.city to form the tetramer
Rates of Protein Fluorescence and Catalytic Activity Recoveries upon Reassociation of DogJish Md Lactate Dehydrogenases-Chilson et al [3] have demonstrated that after the reversible denaturation of the chicken Hd lactate dehydrogenase with 8 M urea, the recovery of fluorescence paralleled the recovery of enzymatic activity

Summary

Methods

Measurement of Enzyme Activity-Enzyme activity was measured by the method of Pesce et al [13], with a Zeiss Spectrophotometer model PhlQ II. Protein determinations were carried out according to the method of Lowry et al [14]. Enzyme Preparation-Dogfish hlr and chicken Hq lactate dehydrogenase were prepared and crystallized in accordance with the method described by Pesce et al [15]. In the case of the dogfish M4 lactate dehydrogenase a minor contaminant was removed by Sephadex G-100 column chromatography. The enzyme was eluted from the column with a 0.05 M Tris-HCl buffer, pH 7.5.

Results

Discussion

Conclusion