Abstract
For nearly 50 years, succinyl-CoA synthetase in animals was thought to be specific for guanine nucleotides. Recently, we purified and characterized both an ADP-forming succinyl-CoA synthetase from pigeon breast muscle and the GDP-forming enzyme from liver (Johnson, J. D., Muhonen, W. W., and Lambeth, D. O. (1998) J. Biol. Chem. 273, 27573-27579). Using the sequences of the pigeon enzymes as queries in BLAST searches, we obtained genetic evidence that both enzymes are expressed in a wide range of animal species (Johnson, J. D., Mehus, J. G., Tews, K., Milavetz, B. I., and Lambeth, D. O. (1998) J. Biol. Chem. 273, 27580-27586). Here we extend those observations by presenting data from Western and Northern blots and enzymatic assays showing that both proteins are widely expressed in mammals with the relative amounts varying from tissue to tissue. We suggest that both succinyl-CoA synthetases catalyze the reverse reaction in the citric acid cycle in which the ADP-forming enzyme augments ATP production, whereas the GDP-forming enzyme supports GTP-dependent anabolic processes. Widely accepted shuttle mechanisms are invoked to explain how transport of P-enolpyruvate across mitochondrial membranes can transfer high energy phosphate between the cytosol and mitochondrial matrix.
Highlights
In earlier reports [1, 2], we reviewed data prior to 1990 suggesting that animals have an ADP-forming succinyl-CoA synthetase (A-SCS1; EC 6.2.1.5) in addition to the GDP-forming succinyl-CoA synthetase (G-SCS; EC 6.2.1.4) discovered in 1954 [3], almost 20 years after formulation of the citric acid cycle by Krebs and Johnson [4]
Following evidence obtained by RT-PCR and analysis of expressed sequence tags in databases that the gene for A-SCS is widely expressed in animal tissues [2], we have further studied the relative levels of A-SCS and G-SCS in selected tissues by using enzymatic assays and Western and Northern blots
We established by RT-PCR that messages encoding the  chains of A-SCS and G-SCS are well represented in a variety of tissues in mouse and human [2]
Summary
A-SCS, ADP-forming succinyl-CoA synthetase; G-SCS, GDP-forming succinyl-CoA synthetase; A-,  subunit of A-SCS; G-,  subunit of G-SCS; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DTNB, 5,5Ј-dithiobis(2-nitrobenzoic acid.; RT, reverse transcriptase. The group that aligned closely with the G- subunit aligned more distantly with A- and vice versa. This strongly indicated that the two  subunits arose by gene duplication and provided evidence that both types of  subunits are expressed in mammals. Results obtained by using Western blotting and a different enzyme assay show that A-SCS is prominently expressed at the protein level in a wide range of mammalian tissues. Knowledge that both ATP- and GDP-forming succinyl-CoA synthetases occur in mammalian tissues has prompted us to further consider the role of the GDP-forming enzyme. Under “Discussion,” we propose that currently accepted shuttles between mitochondrial matrix and cytosol would allow the phosphorylation potential of P-enolpyruvate to be transferred from mitochondrial matrix to cytosol and vice versa
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