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Abstract
Fragments of porcine lactate dehydrogenase M4 and H4 were isolated by a combination of gel chromatography and ion exchange chromatography. For sequence determination the peptides were degraded by the manual Edman procedure using a Sequemat solid phase sequenator or using a Beckman Model 890 c Sequencer. Amino acids were identified by gas liquid chromatography and TLC. Porcine lactate dehydrogenase H chain has 333 residues whereas the M chain is 2 residues shorter due to deletions in positions 16 (16) and 332 (330 B); 250 residues, i.e. 75%, are identical in both peptide chains. Amino acid exchanges are not homogeneously spread over the peptide chains. In the NAD binding domain including the loop region, and in the internal part of the cat lytic domain, only 15.5% of the residues are exchanged. The external part of the catalytic domain and the aminoterminal arm show a drastically increased exchange rate of 47% and 60%, respectively. The major parts of these 2 regions are on the surface of the tetrameric molecule. The high amino acid exchange rate on the protein surface could explain the lack of immunological cross reaction between the isoenzymes. Several amino acids are known to influence enzymatic activity after chemical modification: histidine, arginine, cysteine, tyrosine and lysine. The important amino acids are located in the positions: cysteine 163 (165), histidine 193 (195) and tyrosine 239 (237). The functional roles of 1 histidine, 2 arginine and 1 cysteine residue are well understood. The tyrosine was noted to be far from the active center on the basis of the tentative lactate dehydrogenase sequence. With the primary structure presented here tyrosine 239 (237) is now located near the substrate binding site. The special function of the former lysine (250) now isoleucine 252 (250) and of glutamic acid (140) now asparagine 138 (140) needs new interpretation.