Abstract
The thiol groups of mitochondrial malate dehydrogenases (EC 1.1.1.37) from pig, chicken, and tuna hearts were alkylated with 1-[14C]iodoacetate in 8 M urea, and the alkylated enzymes digested with trypsin and with mixed trypsinchymotrypsin. Peptide mapping of the digests revealed a carboxymethylcysteine-containing peptide which appeared to be the same in all three malate dehydrogenases in its position on the peptide maps, its migration during electrophoretic purification at pH 3.5, and its amino acid composition. This peptide common to the three malate dehydrogenases contained one residue each of lysine, [14C]carboxymethylcysteine, threonine, serine, glutamic acid (or glutamine), proline, and isoleucine when isolated from tryptic peptide maps. When isolated from tryptic-chymotryptic maps it contained all the same residues except for the absence of threonine. A second and larger peptide containing carboxymethylcysteine also appeared to be common to the tryptic peptide maps of all three enzymes and contained the same residues as the first peptide plus one additional residue each of threonine or serine, glycine, alanine, isoleucine, leucine, and possibly proline. The first peptide may be a chymotryptic fragment of the second, and both appear to represent a thiol-containing region in mitochondrial malate dehydrogenases which is conserved during evolution. The effect of iodoacetate on native chicken heart mitochondrial malate dehydrogenase in the presence and absence of substrates and coenzymes has been studied. A tryptic digest of the enzyme after total thiol alkylation in 8 M urea has been fractionated on a cation-exchange column, and the carboxymethylcysteine-containing peptides purified by paper electrophoresis and anion-exchange column chromatography. The peptides so isolated have been compared to those obtained from the peptide maps. The use of comparative peptide mapping in the identification of functionally essential regions in proteins is discussed.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1001-1010 |
| Number of pages | 10 |
| Journal | Biochemistry |
| Volume | 9 |
| Issue number | 4 |
| State | Published - 1970 |
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- Biochemistry
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A comparison of thiol peptides of heart mitochondrial malate dehydrogenases from pig, chicken, and tuna. / Fondy, Thomas P; Barrie Kitto, G.; Driscoll, Geraldine A.
In: Biochemistry, Vol. 9, No. 4, 1970, p. 1001-1010.Research output: Contribution to journal › Article
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TY - JOUR
T1 - A comparison of thiol peptides of heart mitochondrial malate dehydrogenases from pig, chicken, and tuna
AU - Fondy, Thomas P
AU - Barrie Kitto, G.
AU - Driscoll, Geraldine A.
PY - 1970
Y1 - 1970
N2 - The thiol groups of mitochondrial malate dehydrogenases (EC 1.1.1.37) from pig, chicken, and tuna hearts were alkylated with 1-[14C]iodoacetate in 8 M urea, and the alkylated enzymes digested with trypsin and with mixed trypsinchymotrypsin. Peptide mapping of the digests revealed a carboxymethylcysteine-containing peptide which appeared to be the same in all three malate dehydrogenases in its position on the peptide maps, its migration during electrophoretic purification at pH 3.5, and its amino acid composition. This peptide common to the three malate dehydrogenases contained one residue each of lysine, [14C]carboxymethylcysteine, threonine, serine, glutamic acid (or glutamine), proline, and isoleucine when isolated from tryptic peptide maps. When isolated from tryptic-chymotryptic maps it contained all the same residues except for the absence of threonine. A second and larger peptide containing carboxymethylcysteine also appeared to be common to the tryptic peptide maps of all three enzymes and contained the same residues as the first peptide plus one additional residue each of threonine or serine, glycine, alanine, isoleucine, leucine, and possibly proline. The first peptide may be a chymotryptic fragment of the second, and both appear to represent a thiol-containing region in mitochondrial malate dehydrogenases which is conserved during evolution. The effect of iodoacetate on native chicken heart mitochondrial malate dehydrogenase in the presence and absence of substrates and coenzymes has been studied. A tryptic digest of the enzyme after total thiol alkylation in 8 M urea has been fractionated on a cation-exchange column, and the carboxymethylcysteine-containing peptides purified by paper electrophoresis and anion-exchange column chromatography. The peptides so isolated have been compared to those obtained from the peptide maps. The use of comparative peptide mapping in the identification of functionally essential regions in proteins is discussed.
AB - The thiol groups of mitochondrial malate dehydrogenases (EC 1.1.1.37) from pig, chicken, and tuna hearts were alkylated with 1-[14C]iodoacetate in 8 M urea, and the alkylated enzymes digested with trypsin and with mixed trypsinchymotrypsin. Peptide mapping of the digests revealed a carboxymethylcysteine-containing peptide which appeared to be the same in all three malate dehydrogenases in its position on the peptide maps, its migration during electrophoretic purification at pH 3.5, and its amino acid composition. This peptide common to the three malate dehydrogenases contained one residue each of lysine, [14C]carboxymethylcysteine, threonine, serine, glutamic acid (or glutamine), proline, and isoleucine when isolated from tryptic peptide maps. When isolated from tryptic-chymotryptic maps it contained all the same residues except for the absence of threonine. A second and larger peptide containing carboxymethylcysteine also appeared to be common to the tryptic peptide maps of all three enzymes and contained the same residues as the first peptide plus one additional residue each of threonine or serine, glycine, alanine, isoleucine, leucine, and possibly proline. The first peptide may be a chymotryptic fragment of the second, and both appear to represent a thiol-containing region in mitochondrial malate dehydrogenases which is conserved during evolution. The effect of iodoacetate on native chicken heart mitochondrial malate dehydrogenase in the presence and absence of substrates and coenzymes has been studied. A tryptic digest of the enzyme after total thiol alkylation in 8 M urea has been fractionated on a cation-exchange column, and the carboxymethylcysteine-containing peptides purified by paper electrophoresis and anion-exchange column chromatography. The peptides so isolated have been compared to those obtained from the peptide maps. The use of comparative peptide mapping in the identification of functionally essential regions in proteins is discussed.
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M3 - Article
VL - 9
SP - 1001
EP - 1010
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 4
ER -