The homologous cytosolic and mitochondrial isozymes of aspartate aminotransferase (c- and mAspAT, respectively) seem to follow very different folding pathways after synthesis in rabbit reticulocyte lysate, suggesting that the nascent proteins interact differently with molecular chaperones (Mattingly, J. R., Jr., Iriarte, A., and Martinez-Carrion, M. (1993) J. Biol. Chem. 268, 26320-26327). In an attempt to discern the structural basis for this phenomenon, we have begun to study the effect of temperature on the refolding of the guanidine hydrochloride-denatured, purified proteins and their interaction with the groEL/groES molecular chaperone system from Escherichia coli. In the absence of chaperones, temperature has a critical effect on the refolding of the two isozymes, with mAspAT being more susceptible than cAspAT to diminishing refolding yields at increasing temperatures. No refolding is observed for mAspAT at physiological temperatures. The molecular chaperones groEL and groES can extend the temperature range over which the AspAT isozymes successfully refold; however, cAspAT can still refold at higher temperatures than mAspAT. In the absence of groES and MgATP, the two isozymes interact differently with groEL, groEL arrests the refolding of mAspAT throughout the temperature range of 0-45 degrees C. Adding only MgATP releases very little mAspAT from groEL; both groES and MgATP are required for significant refolding of mAspAT in the presence of groEL. On the other hand, the extent to which groEL inhibits the refolding of cAspAT depends upon the temperature of the refolding reaction, only slowing the reaction at 0 degrees C but arresting it completely at 30 degrees C. MgATP alone is sufficient to effect the release of cAspAT from groEL at any temperature examined; inclusion of groES along with MgATP has no effect on the refolding yield but does increase the refolding rate at temperatures greater than 15 degrees C. These results demonstrate that groEL can have significantly different affinities for proteins with highly homologous final tertiary and quarternary structures and suggest that dissimilarities in the primary sequence of the protein substrates may control the structure of the folding intermediates captured by groEL and/or the composition of the surfaces through which the folding proteins interact with groEL.

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A Fourier-transform infrared spectroscopic study of hen egg phosvitin and ovalbumin has been carried out. Bands arising from monoanionic and dianionic phosphate monoester [Shimanouchi, T., Tsuboi, M., & Kyogoku, Y. (1964) Adv. Chem. Phys. 8, 435-498] can be identified easily in the 1300-930 cm-1 region in spectra of solutions of O-phosphoserine and phosvitin, a highly phosphorylated protein. On the other hand, spectra of ovalbumin show a relatively strong absorption above 1000 cm-1 arising from the protein moiety. Below 1000 cm-1, a single band at 979 cm-1 is observed; this band is not present in spectra of dephosphorylated ovalbumin, and therefore, it has been assigned to the symmetric stretching of the phosphorylated Ser-68 and Ser-344 in the dianionic ionization state. In addition, bands arising from symmetric and antisymmetric stretchings of the monoanionic ionization state, and from the antisymmetric stretching of the dianionic state, can be detected above 1000 cm-1 in difference spectra of ovalbumin minus dephosphorylated ovalbumin. The effect of pH on the infrared spectra of O-phosphoserine, phosvitin, and ovalbumin is consistent with the phosphoserine residues undergoing ionization with pK values about 6. This study demonstrates that Fourier-transform infrared spectroscopy can be a useful technique to assess the ionization state of phosphoserine residues in proteins in solution.

Rat liver mitochondrial aspartate aminotransferase (a homodimer) was shown to catalyse a β-lyase reaction with three nephrotoxic halogenated cysteine S-conjugates [S-(1,1,2,2-tetrafluoroethyl)-l-cysteine, S-(1,2-dichlorovinyl)-l-cysteine and S-(2-chloro-1,1,2-trifluoroethyl)-l-cysteine], and less effectively so with a non-toxic cysteine S-conjugate [benzothiazolyl-l-cysteine]. Transamination competes with the β-lyase reaction, but is not favourable. The ratio of β elimination to transamination in the presence of S-(1,1,2,2-tetrafluoroethyl)-l-cysteine and 2-oxoglutarate is >100. Syncatalytic inactivation by the halogenated cysteine S-conjugates is also observed. The enzyme turns over approx. 2700 molecules of halogenated cysteine S-conjugate on average for every monomer inactivated. Kidney mitochondria are known to be especially sensitive to toxic halogenated cysteine S-conjugates. Evidence is presented that 15—20% of the cysteine S-conjugate β-lyase activity towards S-(1,1,2,2-tetrafluoroethyl)-l-cysteine in crude kidney mitochondrial homogenates is due to mitochondrial aspartate aminotransferase. The possible involvement of mitochondrial aspartate aminotransferase in the toxicity of halogenated cysteine S-conjugates is also discussed.