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The syntrophin family of dystrophin-associated proteins consists of three isoforms, ␣1, ␤1, and ␤2, each encoded by a distinct gene. We have cloned and characterized the mouse ␣1-and ␤2-syntrophin genes. The mouse ␣1-syntrophin gene (>24 kilobases) is comprised of eight exons. The mouse ␤2-syntrophin gene (>33 kilobases) contains seven exons, all of which have homologues at the corresponding position in the ␣1-syntrophin gene. Primer extension analysis reveals two transcription initiation sites in the ␣1-syntrophin gene and a single site in the ␤2-syntrophin gene. The sequence immediately 5 of the transcription start sites of both genes lacks a TATA box but is GC-rich and has multiple putative SP1 binding sites. The ␣1-syntrophin gene is located on human chromosome 20 and mouse chromosome 2, while the ␤2-syntrophin gene is on human chromosome 16 and mouse chromosome 8. Analysis of the amino acid sequence of the syntrophins reveals the presence of four conserved domains. The carboxylterminal 56 amino acids are highly conserved and constitute a syntrophin unique domain. Two pleckstrin homology domains are located at the amino-terminal end of the protein. The first pleckstrin homology domain is interrupted by a domain homologous to repeated sequences originally found in the Drosophila discs-large protein.Syntrophin is a peripheral membrane protein of M r ϳ58,000 that was first identified in the postsynaptic membrane of Torpedo electric organ and subsequently shown to be present in many mammalian tissues (1). Interest in syntrophin came first from its location at the neuromuscular junction and more recently from the demonstration that it is directly associated with dystrophin, the product of the Duchenne/Becker muscular dystrophy gene. Although the precise function of syntrophin is unknown, a potential role for the dystrophin-associated proteins in agrin-stimulated nicotinic acetylcholine receptor clustering has implicated syntrophin in the process of synaptogenesis (2).Three different but highly conserved syntrophin isoforms encoded by distinct genes have been identified and cloned. Each syntrophin has approximately 50% amino acid identity with the other two (3). The three syntrophins can be separated into two classes based on isoelectric point (4). The acidic isoform, ␣1-syntrophin, (pI ϳ6.7) has been cloned from Torpedo, mouse, rabbit, and human (5-7). There are two basic forms, ␤1-and ␤2-syntrophin (pI ϳ 9.0). A full-length human cDNA encoding ␤1-syntrophin has been cloned, and the gene has been localized to human chromosome 8q23-24 (8). Partial clones encoding mouse and human ␤2-syntrophin have been reported previously (5,8).The function of syntrophin is likely to be related to its association with dystrophin and other members of the dystrophin protein family (9 -13). Proteins of the dystrophin family are derived from a combination of three genes, the use of alternative promoters within these genes, and alternative splicing. Dystrophin, the major product in skeletal muscle, is a 427-kDa protein with an actin-bi...

Glutaryl-CoA dehydrogenase catalyzes the oxidation of glutaryl-CoA to crotonyl-CoA and CO(2) in the mitochondrial degradation of lysine, hydroxylysine, and tryptophan. We have characterized the human enzyme that was expressed in Escherichia coli. Anaerobic reduction of the enzyme with sodium dithionite or substrate yields no detectable semiquinone; however, like other acyl-CoA dehydrogenases, the human enzyme stabilizes an anionic semiquinone upon reduction of the complex between the enzyme and 2,3-enoyl-CoA product. The flavin potential of the free enzyme determined by the xanthine-xanthine oxidase method is -0.132 V at pH 7.0, slightly more negative than that of related flavoprotein dehydrogenases. A single equivalent of substrate reduces 26% of the dehydrogenase flavin, suggesting that the redox equilibrium on the enzyme between substrate and product and oxidized and reduced flavin is not as favorable as that observed with other acyl-CoA dehydrogenases. This equilibrium is, however, similar to that observed in isovaleryl-CoA dehydrogenase. Comparison of steady-state kinetic constants of glutaryl-CoA dehydrogenase with glutaryl-CoA and the alternative substrates, pentanoyl-CoA and hexanoyl-CoA, suggests that the gamma-carboxyl group of glutaryl-CoA stabilizes the enzyme-substrate complex by at least 5.7 kJ/mol, perhaps by interaction with Arg94 or Ser98. Glu370 is positioned to function as the catalytic base, and previous studies indicate that the conjugate acid of Glu370 also protonates the transient crotonyl-CoA anion following decarboxylation [Gomes, B., Fendrich, G. , and Abeles, R. H. (1981) Biochemistry 20, 3154-3160]. Glu370Asp and Glu370Gln mutants of glutaryl-CoA dehydrogenase exhibit 7% and 0. 04% residual activity, respectively, with human electron-transfer flavoprotein; these mutations do not grossly affect the flavin redox potentials of the mutant enzymes. The reduced catalytic activities of these mutants can be attributed to reduced extent and rate of substrate deprotonation based on experiments with the nonoxidizable substrate analogue, 3-thiaglutaryl-CoA, and kinetic experiments. Determination of these fundamental properties of the human enzyme will serve as the basis for future studies of the decarboxylation reaction which is unique among the acyl-CoA dehydrogenases.

Syntrophin, a 58-kDa membrane-associated protein, is one component of a protein complex associated with dystrophin and other members of the dystrophin family, including the 87-kDa homologue (87K protein). To characterize interactions between syntrophin and 87K protein, we used an in vitro overlay binding assay. We demonstrate that purified Torpedo syntrophin binds directly to dystrophin and 871(. By expressing overlapping regions of the 87K protein as bacterial fusion proteins for binding targets, we show that a 52-amino acid region of 87K (residues 375-426) is sufficient for binding syntrophin.Key words: Syntrophin; Dystrophin; Binding site; Torpedo into two groups based on isoelectric point: acidic (~, pI = 6.7) or basic (ill and f12, pI = 9 +) [19]. In skeletal muscle, c~-syntrophin is found on the sarcolemm a, in a distribution like that of dystrophin and the 87K protein, while fl2-syntrophin is localized to the neuromuscular junction [20].Here, we characterize the binding of Torpedo syntrophin to dystrophin and 87K. Using an in vitro blot overlay assay, we show that Torpedo syntrophin (an ~-syntrophin) binds directly to dystrophin and 87K. Using fusion proteins encoding portions of 87K, we map a binding site of syntrophin to residues 375-426.