(full paper is archived in the Miller Library)
Title: Macromolecular crowding: effects on MDH stability and LDH kinetics
Student Author(s): Wahlstrand, Benjamin D.
Faculty Advisor(s): Somero, George
Location: Final Papers Biology 175H
Date: June 1998
Abstract: Biochemistry is usually carried out under in vitro conditions that do not closely approximate those found in vivo. Molecular crowding, resulting from high intracellular protein concentrations, has been shown to affect macromolecular function (Garner & Burg, 1994). To study this effect, the thermal stabilities of mitochondrial and cytoplasmic isoforms of malate dehydrogenase (MDH), an enzyme involved in the Krebs cycle, were studied under simulated in vivo conditions of high protein concentration. The mitochondrion contains much higher protein concentrations than those found in the cytoplasm; protein crystals have been used to model the mitochondrial matrix (Srere, 1981). The mitochondrial isoform of MDH (mMDH) showed substantial stability enhancement in media crowded with bovine serum albumin, a model protein. The cytoplasmic isoform (cMDH), which is more thermally stable than the mitochondrial isoform, exhibited no significant enhancement of stability in crowded media. This difference can be seen as an adaptation to local protein concentration. mMDH must be more flexible to function well in a more crowded medium, rendering it less thermally stable and more responsive to the stabilizing effects of a crowding agent. In order to study enzyme function, which is closely related to stability, apparent Michaelis-Menten constants (Km's) and Vmax's were determined in crowded and viscous conditions for the LDH of three species. The LDH of Parachaenichthys charcoti , an Antarctic fish, appears less susceptible to the effects of agents that slow enzyme function than two homologous enzymes from organisms that maintain a warmer body temperature. LDH from colder temperature organisms appears to be more flexible than LDH from organisms adapted to warm temperatures (Fields and Somero, in prep). This flexibility may explain the results obtained.