1. Academic Validation
  2. Nonidentity of the cDNA sequence of human breast cancer cell malic enzyme to that from the normal human cell

Nonidentity of the cDNA sequence of human breast cancer cell malic enzyme to that from the normal human cell

  • J Protein Chem. 1996 Apr;15(3):273-9. doi: 10.1007/BF01887116.
W Y Chou 1 S M Huang G G Chang
Affiliations

Affiliation

  • 1 Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Abstract

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.

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