Date of Award

7-22-1999

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

David O. Lambeth

Abstract

Two isoforms of succinyl-CoA synthetase (SCS) have been purified from breast muscle and liver in pigeon, one highly specific for adenine nucleotides (A-SCS), the other for guanine nucleotides (G-SCS), respectively. Nucleotide specificity studies demonstrated that A-SCS could not utilize guanine nucleotides and that G-SCS could not use adenine nucleotides. Enzymatic assays of tissue extracts detected both isoforms in kidney, heart, and brain, while only A-SCS was in breast muscle, and G-SCS in liver. Highly purified A-SCS and G-SCS each possessed the α and β subunits that are common to all previously purified SCSs. Characterization studies of the two isoforms by SDS-PAGE electrophoresis, isoelectric focusing, reversed-phased HPLC, and tryptic peptide mapping, demonstrated that the α-subunits were virtually identical, but the β-subunits were clearly different. This work provides evidence that the β-subunit must contain the residues that are critical in determining nucleotide specificity of SCS in pigeon. Total cDNA pools, prepared from purified mRNAs of breast muscle and liver from pigeon, were used in PCR to amplify the sequences that encode the mature α-subunits and β-subunits of A-PCS and G-SCS. Cloning and sequencing of the α-subunits of A-SCS and G-SCS in pigeon showed their deduced amino acid sequences to be identical, whereas the β-subunits were only 53% identical. The amino acid sequence of the α-subunit from pigeon shows 90% identity to that from pig and 88% identity to that from rat. The nucleotide sequences for the β-subunits of pigeon were used as queries to identify numerous homologous ESTs for human and mouse in GenBank. The mature β-subunits of G-SCS in mouse and human, and A-SCS in mouse, human, and pig, were then cloned and sequenced. The β-subunits of A-SCS and G-SCS from human, pig, mouse, and pigeon show pairwise homology within a specific isoform (A-SCS or G-SCS) exceeding 89%. However, within any of the four species, the homology between A-SCS and G-SCS is in the range of 51–54%. This work demonstrates that A-SCS, as well as G-SCS, is expressed in vertebrates, and that the respective β-subunits are products of separate genes and not the result of alternative splicing of a single gene.

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