Sonia R. Anderson

The reversible acid dissociation and hybridization of lactic dehydrogenase

Abstract Polarization spectra of the intrinsic fluorescence of lactic dehydrogenase (LDH) demonstrate a reversible structural transition in acidic solutions. The pH region of this transition varies for different LDHs. The transition is marked by loss of coenzyme binding and catalytic activity, enhanced ability to bind 1-anilinonaphtha-lene-8-sulfonate, and a decrease in rotational relaxation time from 188 nseconds at pH 7.1 to 77 nseconds at pH 2.5. Renaturation by dilution into neutral solution leads to a recovery of more than 90% LDH with catalytic and physical properties identical to those of the untreated enzyme. Hybrids of beef heart LDH X beef muscle LDH and of beef heart LDH X chicken heart LDH have been prepared by neutralization of solutions of LDH incubated in acid. Our results indicate that a reversible dissociation, either partial or complete, is the basis of hybridization. Measurements of fluorescence polarization, coenzyme binding, and ease of hybridization demonstrate that the hybrids of beef LDH are more readily dissociated by acid than either of the parent types, beef M 4 or beef H 4 . This indicates that interaction among subunits is maximal when all are of the same type.

FLEXIBILITY INVOLVING THE INTERMOLECULAR DITYROSYL CROSS-LINKS OF ENZYMATICALLY POLYMERIZED CALMODULIN

The role of dityrosine as a fluorescent crossbridge between adjacent calmodulin molecules within the high molecular mass polymers that are generated by Arthromyces peroxidase-catalyzed cross-linking [Malencik, D. A., and Anderson, S. R. (1996) Biochemistry 35, 4375] has been examined in frequency domain fluorescence anisotropy studies. Measurements on a polymer fraction possessing a range of molecular masses > 96 000 in NaDodSO4 polyacrylamide gel electrophoresis demonstrate predominating fast local rotations involving the dityrosyl moieties. Normal distribution analyses of the results show peak rotational correlation times of 0.6 ns (zero Ca2+) and 1.2 ns (+Ca2+), values that are smaller than the principal correlation times determined for the global rotation of the free calmodulin monomer in either the presence or absence of Ca2+. The intermolecularly cross-linked segments of the polymers retain a degree of the mobility that is characteristic of the tyrosine-containing sequences of native calmodulin. The half-widths of the normal distribution curves range from 13 ns (zero Ca2+) to approximately 90 ns (5 mM Ca2+), thus encompassing varying rates of segmental motion within the polymers. When Ca2+ is present, possible contributions from the global rotations of polymer molecules are detected near the operating limits of the method. Experiments with the intramolecularly cross-linked calmodulin monomer give global rotational correlation times of 7.9 ns (zero Ca2+) and 11.4 ns (+Ca2+), which compare to values of 7.2 ns and 9.9 ns found previously in time domain measurements [Small, E. W., and Anderson, S. R. (1988) Biochemistry 27, 419]. Rotations of apparent phi2 = 0.2 to 0.3 ns also are detected, accounting for 31% (-Ca2+) to 23% (+Ca2+) of the anisotropy.