Transport of C4-dicarboxylic acids in salmonella typhimurium.

C₄-Dicarboxylic acids are transported into Salmonella typhimurium by stereospecific systems of both high and low affinity. Succinate and l-malate are accumulated in a tricarboxylic acid cycle mutant as was d(+)-malate in induced wild-type cells. Accumulated dicarboxylates are exchangeable with exogenous dicarboxylates. The trichloroacetic acid cycle dicarboxylates are the best inducers of their own transport. Specific mutants devoid of dicarboxylate transport activity (dct) were isolated and differed from tricarboxylate transport mutants (tct) with respect to growth and transport. A mutant devoid of α-ketoglutarate dehydrogenase was unable to transport dicarboxylic acids but citrate transport remained unaffected. Tricarboxylic acid cycle mutants were markedly dependent on an exogenous energy source for the transport of succinate, proline, or leucine. Dicarboxylate transport was largely inhibited by various metabolic inhibitors but could only be inhibited by N,N'-dicyclohexylcarbodiimide anaerobically. ATPase mutants were unimpaired in their ability to transport succinate or proline aerobically.     

Reaction of aspartate aminotransferase with C5-dicarboxylic acids: comparison with the reaction with C4-dicarboxylic acids

The reaction of Escherichia coli aspartate aminotransferase (AspAT) with glutamate and other C5-dicarboxylates was analyzed in order to compare its mechanism of action toward C5 substrates with that toward C4 substrates, which had been extensively characterized. The association of the amino-group protonated and unprotonated forms of glutamate (SH(+) and S, respectively) with the Schiff-base protonated and unprotonated forms of the enzyme (E(L)H(+) and E(L), respectively) yields at least three forms of the Michaelis complex, whereas in the case of aspartate, only two species of this complex exist, E(L).SH(+) and E(L)H(+).S. The reaction of AspAT with 2-methylglutamate can be explained only when we consider all the protonation states of the Michaelis complex. Based on the previous crystallographic studies [Miyahara et al. (1994) J. Biochem. 116, 1001-1012], we consider that glutamate binds to the open form of AspAT and takes an extended conformation in the Michaelis complex, with the alpha-amino group of glutamate oriented in the opposite direction to the Schiff base. This is in contrast to the Michaelis complex of aspartate, in which a strong interaction of the alpha-amino group of aspartate and the Schiff base excludes the presence of the species E(L)H(+).SH(+). It is concluded that AspAT recognizes the two types of dicarboxylates with different chain lengths by changing the gross conformation of the enzyme protein.     

Genetics of complement C4. Two homoduplication haplotypes C4S C4S and C4F C4F in a family

Summary A family in which two homoduplicated C4 haplotypes (or supergenes) segregate is described. One haplotype C4F ^* 3 C4F ^*2.2 is composed of two C4F alleles and the other C4S ^* 5.1 C4S ^*1 of two C4S alleles. The C4F duplication haplotype is a partial inhibitor of the Rodgers antigen, and judged from our family and population material, it seems to be rather frequent and associated with HLAB ^*35, Bf ^* F, and HLAD/DR ^*1. The C4S duplication haplotype is Rg(a-) and is not identified in individuals without another S, Ch(a+) variant.This work was supported by grant No 12-1727 from the Danish Medical Research Council     

The N terminus of human myelin basic protein consists of C2, C4, C6, and C8 alkyl carboxylic acids.

Peptide 1-21, generated by cyanogen bromide cleavage of each of two highly purified components of human myelin basic protein, components 1 and 8, gave a series of peaks in the fast atom bombardment mass spectra with m/z 2299, 2327, 2355, 2383, and 2411, indicating additions of 42, 70, 98, 126, and 154 atomic mass units respectively with m/z 2327 and 2355 as the dominant species. The pentafluorobenzyl esters prepared from an acid hydrolysate analyzed by negative ion chemical ionization gas chromatography mass spectrometry confirmed that C6, C8, and C10 fatty acids were present. These data demonstrated (i) that the N terminus of a myelin basic protein is not simply acetylated but contains C2, C4, C6, C8, and C10 fatty acids with C4 and C6 as the dominant species, (ii) the two components studied (C-1 and C-8) showed different relative amounts of C2 and C8 in particular, and (iii) human myelin basic protein is the first protein to be reported with a complex N terminus consisting of several alkyl carboxylic acid species.     

A cluster of positively charged amino acids in the C4BP alpha-chain is crucial for C4b binding and factor I cofactor function.

C4b-binding protein (C4BP) is a regulator of the classical complement pathway, acting as a cofactor to factor I in the degradation of C4b. Computer modeling and structural analysis predicted a cluster of positively charged amino acids at the interface between complement control protein modules 1 and 2 of the C4BP alpha-chain to be involved in C4b binding. Three C4BP mutants, R39Q, R64Q/R66Q, and R39Q/R64Q/R66Q, were expressed and assayed for their ability to bind C4b and to function as factor I cofactors. The apparent affinities of R39Q, R64Q/R66Q, and R39Q/R64Q/R66Q for immobilized C4b were 15-, 50-, and 140-fold lower, respectively, than that of recombinant wild type C4BP. The C4b binding site demonstrated herein was also found to be a specific heparin binding site. In C4b degradation, the mutants demonstrated decreased ability to serve as factor I cofactors. In particular, the R39Q/R64Q/R66Q mutant was inefficient as cofactor for cleavage of the Arg937-Thr938 peptide bond in C4b. In contrast, the factor I mediated cleavage of Arg1317-Asn1318 bond was less affected by the C4BP mutations. In conclusion, we identify a cluster of amino acids that is part of a C4b binding site involved in the regulation of the complement system.     

Structural basis for the C4d.1/C4d.2 serologic allotypes of murine complement component C4

The C4d.1 antigenic specificity was first defined serologically in 1959 as an H-2-associated cellular alloantigen first designated "G," later H-2.7. It was subsequently shown to be an allotype of component C4 of the C system, with the antigenic determinant carried on the C4d proteolytic fragment of the alpha-chain, thus the designation C4d.1. Alloantisera defining an antithetical Ag, C4d.2, were also prepared. Previous studies in our laboratory showed that the structural difference between the two specificities resides in a single tryptic peptide of C4d. As an efficient approach to definition of the amino acid difference(s) involved, genomic clones covering the C4d regions from two H-2 haplotypes of the C4d.1 type have been prepared and sequenced, and compared with two sequences already available for C4d.2-type molecules. The results indicate that the rather striking serologic difference between C4d.1 and C4d.2 is attributable to the single amino acid substitution of arginine in C4d.2 for glutamine in C4d.1. The substituted residue is in a highly hydrophilic region of the C4 molecule, at a position homologous to one that contributes to the Chido/Rodgers serologic difference of human C4 molecules. This substitution also determines a new Pst I site in C4d.1 strains. A HindIII restriction fragment length polymorphism between C4d.1 and C4d.2 has also been observed.     

Surface binding of amino acids by L1210 cells at 4 degrees C

Surface binding of amino acids by L1210 cells at 4 °C was studied. 1. Alanine binds very poorly. Phenylalanine binds promptly and hydroxylation of the phenyl moiety (tyrosine) reduces the binding. 2. Leucine was taken up much more rapidly than isoleucine and threonine more rapidly than valine, suggesting that the β-methyl group hinders surface binding. 3. Histidine binds slowly at pH 5.5, but increases with the increase of pH to a peak at pH 7.4, then decreases at pH 8. The presence of net charge on histidine apparently inhibits surface binding.