Bovine and porcine lactate dehydrogenases, in solutions of 0.1-10 microM at neutral pH, dissociate into monomers upon application of hydrostatic pressures of up to 2 kbar. The dissociation was determined from observations of the polarization of fluorescence under pressure in seeming equilibrium conditions and by occasional hybridization experiments of the H4 and M4 isozymes. Decompression is followed by the rapid association of the monomers into tetramers and by slow, and sometimes incomplete, return of the enzymic activity. The dissociation curves obtained on compression and decompression differ, indicating that association results in partial loss of subunit affinity. These phenomena are attributed to a slow conformational drift that follows the loss of contact of the monomers with each other and to an even slower reversal of the drift that takes place upon reassociation.

Solutions of porcine lactate dehydrogenase of micromolar concentration kept at 4 degrees C for several days lose the greater part of their enzymic activity but recover it when returned to room temperature. The rate of spoiling decreases and the rate of recovery increases with the concentration of the solutions. The decrease in tetramer stability in the cold is shown by experiments of pressure dissociation at various temperatures and confirmed because isozyme hybridization occurs in parallel with the inactivation at low temperature but is absent at room temperature. Cold-inactivated solutions contain tetramers that dissociate much more readily than those of the fully active solutions. It is postulated that cryoinactivation, like pressure inactivation, takes place through a cycle of dissociation, conformational drift [King, L., & Weber, G. (1986) Biochemistry (second paper of three in this issue)] and reassociation into inactive tetramers.

Rabbit skeletal myosin rod, which is the coiled-coil alpha-helical portion of myosin, contains two tryptophan residues located in the light meromyosin (LMM) portion whose fluorescence contributes 27% to the fluorescence of the entire myosin molecule. The temperature dependence of several fluorescence parameters (quantum yield, spectral position, polarization) of the rod and its LMM portion was compared to the thermal unfolding of the helix measured with circular dichroism. Rod unfolds with three major helix unfolding transitions: at 43, 47, and 53 degrees C, with the 43 and 53 degrees C transitions mainly located in the LMM region and the 47 degrees C transition mainly located in the subfragment 2 region. The fluorescence study showed that the 43 degrees C transition does not involve the tryptophan-containing region and that the 47 degrees C transition produces an intermediate with different fluorescence properties from both the completely helical and fully unfolded states. That is, although the fluorescence of the 47 degrees C intermediate is markedly quenched, the tryptophyl residues do not become appreciably exposed to solvent until the 53 degrees C transition. It is suggested that although the intermediate that is formed in the 47 degrees C transition contains an extensive region which is devoid of alpha-helix, the unfolded region is not appreciably solvated or flexible. It appears to have the properties of a collapsed nonhelical state rather than a classical random coil.

No abstract

Gizzard smooth muscle myosin rod, an alpha-helical coiled coil, exhibits two cooperative thermal or denaturant-induced helix unfolding transitions in solutions containing 0.6 M NaCl at neutral pH, when monitored by circular dichroism at 222 nm. The first smaller transition unfolds part of the subfragment 2 (S2) domain, and the main transition unfolds the remaining helix including the light meromyosin (LMM) domain. These unfolding domains were identified by monitoring the fluorescence of acrylodan, an environmentally sensitive fluorescence probe, and the ESR signal of a maleimide spin-label, sensitive to motion, both specifically attached to Cys 43 in the S2 region of the rod sequence. The identities of the domains were verified by studying the unfolding of the S2 and LMM coiled-coil peptides obtained by proteolytic cleavage of spin-labeled and unlabeled rod. The fluorescence of acrylodan-labeled rod indicated that although the S2 intermediate is unfolded, it is not in a random-coil conformation. The unfolded S2 region stabilized the LMM domain against unfolding, possibly by a direct interaction with the LMM region. Such an interaction may be involved in the salt- and phosphorylation-dependent 6S to 10S shift in configuration of the myosin molecule.