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Isocitrate dehydrogenase from bovine heart: primary structure of subunit 3/4
1Department of Biochemistry, State University of New York Health Science Center at Brooklyn 11203, USA.
Insights
Bovine NAD(+)-dependent isocitrate dehydrogenase exhibits subunit heterogeneity, confirmed by N-terminal sequencing. This study reveals conserved catalytic residues between bovine and E. coli enzymes, suggesting functional importance.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Bovine NAD(+)-dependent isocitrate dehydrogenase (IDH) previously showed four distinct subunits (approx. 40 kDa) with varied peptide maps.
- Previous findings suggested subunit heterogeneity, but potential allelic variation remained.
- Updated purification methods and single-animal enzyme sources were employed to confirm this heterogeneity.
Purpose of the Study:
- To confirm and characterize the subunit heterogeneity of bovine NAD(+)-dependent isocitrate dehydrogenase.
- To elucidate the N-terminal amino acid sequences of the bovine enzyme's subunits.
- To compare the bovine enzyme's subunits with homologous subunits of the pig heart enzyme and related NADP(+)-dependent enzymes.
Main Methods:
- Enzyme purification using updated techniques.
- N-terminal amino acid sequencing of enzyme subunits.
- cDNA cloning and sequencing.
- Peptide mapping and electrophoretic analysis.
- Sequence comparison with homologous and related enzymes.
Main Results:
- Heterogeneity of bovine NAD(+)-dependent IDH subunits confirmed, ruling out allelic variability.
- Subunits 1 and 2 differentiated by N-terminal sequencing; subunits 3 and 4 showed identical sequences.
- N-terminal sequences of bovine subunits 1 and 2 showed homology to pig heart beta- and gamma-subunits, respectively.
- Bovine subunits 3/4 shared sequence identity with the N-terminus of the pig heart alpha-subunit.
- Full-length sequence of a bovine protein (339 amino acids, 36,685 Da) deduced from cDNA, with a processed N-terminus matching subunits 3/4.
- High sequence identity (92 residues) between bovine subunit 3/4 and pig heart alpha-subunit peptides suggests structural importance.
- Electrophoretic heterogeneity observed in pig heart alpha-subunit, mirroring bovine subunits 3/4.
- Sequence comparison with E. coli NADP(+)-dependent IDH revealed conserved residues critical for binding isocitrate, Mg2+, and NAD+.
Conclusions:
- Bovine NAD(+)-dependent isocitrate dehydrogenase exhibits intrinsic subunit heterogeneity, not due to allelic variation.
- The alpha-subunit's three-dimensional structure is likely crucial for enzyme function, as indicated by conserved sequences with the pig heart enzyme.
- Conserved catalytic residues between bovine and E. coli IDH suggest a conserved catalytic mechanism across different enzyme forms.
Abstract:
Bovine NAD(+)-dependent isocitrate dehydrogenase was shown previously to contain four subunits of approx. 40 kDa (subunits 1-4) possessing different peptide maps and electrophoretic properties [Rushbrook and Harvey (1978) Biochemistry 17, 5339-5346]. In this study the heterogeneity is confirmed using enzyme purified by updated methods and from single animals, ruling out allelic variability. Subunits 1 and 2 were differentiated from each other and from subunits 3 and 4 by N-terminal amino acid sequencing. Subunits 3 and 4 (subunits 3/4) were identical in sequence over 30 residues. The N-terminal residues of subunits 1 and 2 were homologous but not identical with the beta- and gamma-subunits respectively of the comparable pig heart enzyme. Subunits 3/4 were identical over 30 residues with the N-terminus of the pig heart alpha-subunit. Full-length sequence, including that for mitochondrial import, is presented for a protein with the processed N-terminus of subunits 3/4, deduced from cloned cDNA obtained utilizing the N-terminal sequence information. The derived amino acid sequence for the mature protein contains 339 amino acids and has a molecular mass of 36,685 Da. Complete identity with N-terminal and Cys-containing peptides totalling 92 residues from the alpha-subunit of the pig heart enzyme [Huang and Colman (1990) Biochemistry 29, 8266-8273] suggests that maintenance of a particular three-dimensional structure in this subunit is crucial to the function of the enzyme. An electrophoretic heterogeneity within the pig heart alpha-subunit, similar to that shown by bovine subunits 3/4, was demonstrated. One reordering of the Cys-containing peptides of the pig heart alpha-subunit is indicated. Sequence comparison with the distantly related NADP(+)-dependent enzyme from Escherichia coli, for which the three-dimensional structure is known [Stoddard, Dean and Koshland (1993) Biochemistry 32, 9310-9316] shows strong conservation of residues binding isocitrate, Mg2+ and the NAD+ moiety of NADP+, consistent with a catalytic function.