Flavocytochrome c from the Gram-negative, food-spoiling bacterium Shewanella putrefaciens is a soluble, periplasmic fumarate reductase. We have isolated the gene encoding flavocytochrome c and determined the complete DNA sequence. The predicted amino acid sequence indicates that flavocytochrome c is synthesized with an N-terminal secretory signal sequence of 25 amino acid residues. The mature protein contains 571 amino acid residues and consists of an N-terminal cytochrome domain, of about 117 residues, with four heme attachment sites typical of c-type cytochromes and a C-terminal flavoprotein domain of about 454 residues that is clearly related to the flavoprotein subunits of fumarate reductases and succinate dehydrogenases from bacterial and other sources. A second reading frame that may be cotranscribed with the flavocytochrome c gene exhibits some similarity with the 13-kDa membrane anchor subunit of Escherichia coli fumarate reductase. The sequence of the flavoprotein domain demonstrates an even closer relationship with the product of the yeast OSM1 gene, mutations in which result in sensitivity to high osmolarity. These findings are discussed in relation to the function of flavocytochrome c.

The major soluble cytochrome isolated from microaerobically grown cells of Shewanella putrefaciens has been shown to be a novel type of flavocytochrome with fumarate reductase activity. This flavocytochrome, located in the periplasmic fraction of cell extracts, has been purified to homogeneity and shown to contain 4 mol of haem c and 1 mol of non-covalently bound FAD per mol of protein. An M(r) value of 63,800 is estimated from sequence analysis assuming 4 mol of haem/mol of protein. In the presence of the artificial electron donor, reduced methyl viologen, the flavocytochrome catalysed the reduction of fumarate but not that of nitrite, dimethylsulphoxide, trimethylamine-N-oxide or sulphite. The pH optimum was 7.4 with calculated pKa values of 6.8 and 8.0 for contributing catalytic groups. The Km and kcat. values for fumarate reduction were 21 microM and 250 s-1 respectively, whereas the corresponding values for succinate oxidation with 2,6-dichlorophenol-indophenol as electron carriers were 200 microM and 0.07 s-1 respectively. Mesaconic acid was a competitive inhibitor of fumarate reduction with a Ki of 2 microM. Zymogram staining of polyacrylamide gels with purified protein showed a band of fumarate reductase activity. Polyclonal antibodies, raised to the purified flavocytochrome, were shown to titrate out fumarate reductase activity. We conclude that the physiological role of this enzyme is as a fumarate reductase. Optical absorption spectra of the flavocytochrome indicated that all the haems were of the c-type and gave alpha, beta and gamma peaks at 552.3, 523 and 418 nm in the reduced spectrum with epsilon values of 30.2, 15.9 and 188.2 mM-1.cm-1 respectively. Oxidized spectra showed no 695 nm band that would be indicative of His-Met coordination. Two redox potentials were resolved at -220 mV and -320 mV. The cytochrome was reduced by formate in the presence of particulate cell fractions. The relationship of this cytochrome to other low-potential flavocytochromes c is discussed.

We report the isolation, nucleotide sequence determination, and bacterial expression of a partial cDNA for the immunoglobulin ic chain from the Epstein-Barr virustransformed human lymphoid cell line GM131. The cDNA, cloned in pBR322 by use ofollgo(dG)-oligo(dC) tails, yields two Pst I fragments of 250 and 600 base pairs (bp). Various restriction enzyme fragments of the cDNA were subdloned in the vectors M13 mplO and M13 mpll for sequence analysis. As a result of instability of the 250-bp M13 subclones, the base sequence of the 250-bp Pst I fagment could not be determined. The 600-bp Pst I fragment contains coding sequences for part of the variable (V) region (residues 78-95) and all of thejoining (J) (residues 96-108) and constant (C) regions (residues 109-212) and extends 148 bp into the 3' fnking region.Although the C-and 3'-flanking-region sequences are identical to germ-line sequences, the J-region sequence does not correspond to any of the five human germ-line J regions. The sequence is most similar to that of J4, with three base changes resulting in one silent mutation and two amino acid substitutions, at residues 103 (Lys --Tyr) and 106 (Ile --Met). The silent mutation appears to be the result of RNA splicing between the J and the C regions. The V-region sequence differs from published V-region germ-line sequences at several codons and from the more common amino acid sequences at two positions, residues 91 and 93. At these positions, histdine residues are found in place of the more common tyrosine and serine, respectively. None of the four amino acd substitutions observed for the GM131 sc-chain are unique, suggesting that the changes, which most likely contribute to antigenic specificity, are compatible with antibody structure and function.The 600-bp Pst I fragment was subeloned in two prokaryotic expression vectors, pATHil and pUC8. In both instances, a oc-chain fusion protein detectable by immunoblotting was produced.Immunoglobulins represent an excellent model system for studies on protein-protein and protein-ligand interactions.Not only must a finite number of amino acids in two individual chains (H and L, heavy and light) associate in such a manner as to form a unique antigen-binding pocket, but that binding pocket must retain sufficient variability, presumably by alterations in the composite amino acids, to recognize and distinguish between as many as 109 different antigens (1). However, in spite of or in addition to this variability, these same two polypeptide chains must conform to a relatively constant structure to allow for chain-chain interactions as well as effector functions associated with antibodies.X-ray crystallographic studies have shown that the six hypervariable loops, three contributed by the variable (V) region of each chain, form the antigen-binding site (2, 3). The V domains are formed from two layers of (sheet (4-8) with each of the six hypervariable loops forming a connection between two antiparallel strands of /-sheet. The VH-VL interaction forms a barrel structure that ...

The reactivity of 23 mouse monoclonal Ig with a rabbit polyclonal antiserum to VH of anti-alpha(1----6)dextran 19.22.1 and with a monoclonal anti-VH of anti-DNP MOPC315, when correlated with amino acid sequence, identified several residues in the first and third framework regions as being of potential importance in forming the epitope. Inhibition studies using synthetic peptides corresponding to residues 1-15 of the monoclonal Ig used to produce the poly- and monoclonal reagents provide evidence that the epitopes are predominantly, if not exclusively, specific for the N-terminal strand of the domain. Examination of known x-ray structures of mouse VH suggests that the primary difference between the two epitopes in the N-terminal strands is determined by the peptide chain structure due to Pro at position 9. Pro 9 appears essential for the epitope reactive with anti-VH MOPC315.

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