The Purification, Properties and Subunit Structure of Glycerol Dehydrogenase

<p>The inducible, NAD-linked glycerol dehydrogenase (E.C. 1.1.1.6) of a guanine requiring mutant of A. aerogenes has been purified to homogeneity. The molecular weight of the pure enzyme was found to be 3.4 x 10⁵ daltons. The sedimentation coefficient of the enzyme was 10.7 x 10^-13 sec.^-1. The diffusion constant was 3.07 x 10^-7 cm²/sec.</p> <p>The partial specific volume calculated from the amino acid composition was 0.72 ml/g. The following kinetic parameters were determined; K_m glycerol, 2.4 x 10^-3 m, K_m NAD, 2.8 x 10^-4 M, K_m dihydroxyacetone, 5.1 x 10^-4 M, K_mNADH, 2.3 x 10^-5 M.</p> <p>During the course of the purification of the enzyme it was found that multiple peaks of enzyme activity were eluted from ion-exchange chromatography experiments. Ultracentrifugation experiments with the enzyme treated with urea and dithiothreitol, and with dithiothreitol and p- hydroxymercuribenzoate in the presence of guanidinium chloride indicated that the isolated enzyme is composed of subunits. The untreated enzyme, with a molecular weight of 3.4 x 10⁵ daltons, is a hexamer of a species of molecular weight 5.6 x 10⁴ daltons. This species in turn is a dimer of a species of molecular weight 2.8 x 10⁴ daltons. Disulfide bonds between the smallest species are responsible for the stability of the dimer, while the untreated hexamer of the dimers is stabilized by ionic and hydrophobic forces Thirteen major peaks and one minor peak of enzyme activity were consistently eluted during ion-exchange experiments with the native enzyme. Polyacrylamide gel electrophoresis of the enzyme treated with dithiothreitol and urea indicated the presence of two species of protein. The amino acid composition of these two proteins and of the thirteen major peaks are consistent with the hypothesis that these two proteins represent the proteins of molecular weight 2.8 x 10⁴ daltons observed in the ultracentrifuge and that the stiochiometry of the thirteen major peaks may be represented by the formula (A (12-n) B_n), n = 0, 1,..., 12, where A and B represent the two proteins separated by polyacrylamide gel electrophoresis.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Year dc:date.available

1969

Author dc:creator

• Barrett, Michael James

Contributors dc:contributor

• Charles Walter

• Analytical, Diagnostic and Therapeutic Techniques and Equipment
• Chemicals and Drugs
• Enzymes and Coenzymes
• Investigative Techniques
• Medical Biochemistry
• Medical Sciences
• Medicine and Health Sciences

Repository record dc:identifier

https://dc.uthsc.edu/dissertations/578

OAI identifier oai:identifier

oai:dc.uthsc.edu:dissertations-1578