ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 215, No. 2, May, pp. 498-507, 1982 The Reactions of Pyridoxal S-Phosphate with the M, and H4 lsoenzymes of Pig Lactate Dehydrogenase’ KEITH G. GOULD2 AND PAUL C. ENGEL Department of Biochemistry, Th.e University of Sbf$e.!d, Sheffield SlO .2TN, United Kingdom Received October 1, 1981, and in revised form December 9, 1981 The H4 and M4 isoenzymes of pig lactate dehydrogenase are both inactivated by reaction with pyridoxal 5’-phosphate. In the early stages, inactivation is largely re- versible by the addition of lysine in excess, but may be made irreversible by reduction with borohydride. This indicates that modification of lysine residues probably causes the initial inactivation. Both isoenzymes also undergo a slower process of irreversible inactivation which becomes more evident with increasing concentrations of pyridoxal 5’-phosphate and higher temperature. Although coenzymes give only partial protection of enzyme activity, they nevertheless completely prevent irreversible inactivation. Nei- ther pyruvate nor lactate alone gives any protection. With the M4 isoenzyme, complete protection against inactivation by pyridoxal 5’-phosphate may be achieved in ternary complexes, but no conditions have been found for complete protection of the H4 iso- enzyme. In the course of irreversible inactivation of H4 lactate dehydrogenase, complete loss of activity can be correlated with the loss of approximately two free thiol groups per subunit. Present findings with regard to the importance of temperature and reagent concentration in determining the outcome of the chemical modification appear to re- solve earlier controversy. Three distinct types of subunits are found in the tetrameric isoenzymes of lac- tate dehydrogenase (EC 1.1.1.2’7) (LDH)4 in higher animals. The M and H (A and B) subunits are widely distributed in var- ious combinations, and the C subunit is found in the C4 or X isoenzyme of sperm. Although the homotetrameric isoenzymes M4, H4, and C4 are all inactivated (1,2) by pyridoxal 5’-phosphate (PLP), a reagent 1 This work was supported by the Science Research Council through the award of a Research Grant to P.C.E. and a Scholarship for Training in Research Methods to K.G.G. 2 Present address: Sir William Dunn School of Pa- thology, University of Oxford, South Parks Road, Oxford, United Kingdom. 3 To whom reprint requests should be directed. 4 Abbreviations used: LDH, lactate dehydrogenase (EC 1.1.1.27); PLP, pyridoxal B-phosphate, DTNB, 5,5’-dithiobis(2-nitrobenzoic acid). commonly used for modifying lysine res- idues, it remains a matter of controversy whether any or all of these enzymes pos- sess essential lysine residues. Inactivation of the M4 (pig and dogfish muscle) and C4 (mouse) isoenzymes by PLP has been at- tributed to modification of lysine residues (l-3). In a series of papers, however, Pflei- derer and his co-workers argue (e.g., Ref. (4), p. 338) that no essential lysine is pres- ent in lactate dehydrogenase. Their ar- guments are based on experiments with the H4 isoenzyme under conditions very different from ours, and although the iso- enzymes are homologous, the attempt to extrapolate and generalize seems risky in the absence of direct supporting evidence. Indeed different responses to chemical modifications for the M4 and H4 isoen- zymes were reported by Rajewsky (5) who found that M4 was much more susceptible 0003-9861/82/060498-10$02.00/O 498 Copyright Q 1982 by Academic Press, Inc. All righta of reproduction in any form reserved.