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A cDNA coding for human breast cancer cell cytosolic NADP(+)-dependent malic enzyme was obtained. This cDNA is composed of a length of 2084 base pairs, with 1698 base pairs coding for 565 amino acid residues and a length of 386 base pairs representing a 3'-noncoding region. Comparing this nucleotide sequence with that from the normal human tissue [Loeber, G., Dworkin, M. B., Infante, A., and Ahorn, H. (1994), FEBS Lett. 344, 181-186] reveals that three nucleotides in the open reading frame and the length of 3'-noncoding region of the cDNA are different. One of the changes results in a substitution of serine at position 438 for proline, which, however, may not cause significant changes in the predicted secondary structure. A partial cDNA lacking the first 84 nucleotides in the open reading frame was successfully cloned and expressed functionally in Escherichia coli cells. Its Km value for L-malate (1.21 +/- 0.11 mM) is four times higher than that for the natural human breast cancer cell malic enzyme (0.29 +/- 0.04 mM) but similar to that for the full-length recombinant enzyme (1.06 +/- 0.07 mM). The Km values for Mn2+ and NADP+ (0.26 +/- 0.03 and 0.97 +/- 0.4 microM, respectively) are similar to those for the natural enzyme (0.12 +/- 0.02 and 1.9 +/- 0.3 microM, respectively) or the recombinant wild-type enzyme (0.56 +/- 0.04 and 0.44 +/- 0.02 microM, respectively). A recombinant pigeon liver malic enzyme without the first 13 amino acid residues was used for comparison. The Km values for L-malate and Mn2+ of the truncated enzyme (11.2 +/- 0.9 mM and 61.2 +/- 4.6 microM, respectively) are over 40 times larger than those for the natural pigeon liver malic enzyme (0.21 +/- 0.02 mM and 1.06 +/- 0.08 microM, respectively) or the recombinant wild-type enzyme (0.25 +/- 0.01 mM and 1.48 +/- 0.05 microM, respectively). We suggest that the N-terminus of malic enzyme may be required for the substrate binding during the catalytic cycle.

The active site of pigeon liver malic enzyme was labeled with a fluorescent affinity label, the periodate-oxidized aminopyridine adenine dinucleotide phosphate. The modified enzyme was subjected to proteolytic digestion with trypsin. The resulted peptides were then separated with reversed-phase high-performance liquid chromatography on Waters muBondapak C18 column. Two pure fluorescent peptides were obtained after three runs of the chromatography. The peptides were then subjected to automatic Edman degradation on a Beckman peptide sequencer and subsequently separated and identified with phenylthiohydantoin C18 column. No sequence was obtained. The possible reasons for the failure in sequencing the periodate-oxidized nucleotides labeled active site peptide and some possible pitfalls in using these reagents were discussed.

Kinetic comparison of epsilon-crystallins isolated from the avian and reptilian species and the authentic lactate dehydrogenases (LDHs) was undertaken in order to clarify the identities of these structural lens proteins in relation to their enzymatic activity. Caiman epsilon-crystallin similar to the previously characterized duck epsilon-crystallin appeared to possess a genuine and stable LDH activity as detected by nitro blue tetrazolium staining on polyacrylamide gels and conventional kinetic assays. Kinetic parameters for pyruvate, L-lactate, NAD+, and three structural analogues of the coenzyme in this epsilon-crystallin catalyzed reaction were also determined and compared. Despite the structural similarities between epsilon-crystallins and chicken heart LDH, differences in charge and kinetic properties have been revealed by native isozyme electrophoresis and kinetic analysis as examined by initial velocity and substrate inhibition studies. It is found that the kinetic data analyzed for caiman epsilon-crystallin were more fitted with a compulsory ordered Bi-Bi sequential mechanism similar to those for the authentic LDHs and duck epsilon-crystallin. Caiman epsilon-crystallin has for the first time been established as a heart-type LDH based on the kinetic analysis and comparison with the authentic heart- and muscle-type LDHs from pig and chicken.

Rabbit antibodies against pigeon liver malic enzyme (EC 1.1.1.40) were prepared. The antiserum gave single precipitation line with crude pigeon liver extract. Cross reaction was observed with partially purified malic enzyme or crude extract from chicken liver. Positive cross reaction was also observed with the concentrated cytosolic fraction of two human carcinoma cell lines which were demonstrated to contain high malic enzyme activity. All other proteins examined did not react with the antibodies. When purified pigeon liver malic enzyme was mixed with the antiserum in vitro, a time-dependent inactivation of the enzyme activity was observed. Protection of the enzyme activity against antiserum inactivation was afforded by NADP+ or L-malate. Metal Mn2+ gave little protection.