L-pipecolic acid oxidation in the rabbit and cynomolgus monkey. Evidence for differing organellar locations and cofactor requirements in each species

L-Pipecolic acid oxidation was studied in the rabbit and cynomolgus monkey. Tissue homogenates from both species incubated with L-[2,3,4,5,6-3H]pipecolic acid produced a single radioactive product identified as alpha-aminoadipic acid. In the rabbit, L-pipecolic acid oxidation was greatest in kidney cortex with progressively lesser specific activities in liver, heart, and brain. When rabbit kidney cortex was fractionated by differential centrifugation or on Percoll gradients, activity paralleled that of the mitochondrial marker, glutamate dehydrogenase. In sonicated mitochondria, 92% of the activity was in the soluble fraction. Activity was inhibited by both rotenone and antimycin A and was maximal when FAD, phenazine ethosulfate, and glycerol were included in the assay; Km,app was 0.74 +/- 0.16 mM. Nipecotic acid, piperidine, and cis-2,4-piperidine dicarboxylic acid did not inhibit L-pipecolic acid oxidation, while L-proline had a Ki greater than or equal to 10 mM. D-Alanine and kojic acid, substrate and inhibitor of D-amino acid oxidase, respectively, were also not inhibitory. When monkey kidney cortex was fractionated on Percoll gradients, L-pipecolic acid oxidation activity paralleled that of the peroxisomal marker, catalase. After organellar subfractionation, the activity was membrane-associated and maximal at pH 8.5; Km,app was 4.22 +/- 0.30 mM. L-Pipecolic acid oxidation produced hydrogen peroxide, suggesting involvement of an oxidase in alpha-aminoadipic acid formation. Antimycin A did not inhibit the reaction. No specific cofactor requirements were identified and phenazine ethosulfate inhibited the reaction. D-Pipecolic acid, L-proline, and the other compounds cited above did not significantly inhibit the activity.     

Conformational and amino acid residue requirements for the saposin C neuritogenic effect.

Prosaposin is the precursor of four activator proteins, termed saposins A, B, C, and D, that are required for much of glycosphingolipid hydrolysis. The intact precursor also has neurite outgrowth activity ex vivo and in vivo that is localized to amino acid residues 22-31 of saposin C. Across species, this saposin C region has a high degree of identity and similarity with amino acids in the analogous region of saposin A. Wild-type and mutant saposins C and A from human and mouse were expressed in E. coli. Pure proteins, synthetic peptide analogues, conformation-specific antibodies, and CD spectroscopy were used to evaluate the basis of the ex vivo neuritogenic effect. Wild-type saposin A had no neuritogenic activity whereas reduced and alkylated saposin A did. Introduction of the conserved saposin A Tyr 30 (Y30) into saposin C at the analogous position 31, a conserved Ala(A)/Gly(G)31, diminished neuritogenic activity by 50-60%. Nondenatured saposin A with an introduced A30 acquired substantial neuritogenic activity. Polyclonal antibodies directed against the NH2-terminus of saposin C cross-reacted well with reduced and alkylated saposins C and A, wild-type saposin C, and saposin A [Y30A], poorly with saposin C [A31Y], and not at all with wild-type saposin A. CD spectra of wild-type and mutant saposins C and A, the corresponding neuritogenic region of saposin C, and the analogous region of saposin A showed that more "saposin C-like" molecules had neuritogenic properties. Those with more "saposin A-like" spectra did not. These studies show that the neuritogenic activity of saposin C requires specific placement of amino acids, and that Y30 of saposin A significantly alters local conformation in this critical region and suppresses neuritogenic activity.     

Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase.

We have determined the sequence requirements for a protein hinge in triosephosphate isomerase. The codons encoding the hinge at the C-terminus of the active-site lid of triosephosphate isomerase were replaced with a genetic library of all possible 8,000 amino acid combinations. The most active of these 8,000 mutants were selected using in vivo complementation of a triosephosphate isomerase deficient strain of E. coli, DF502. Approximately 3% of the mutants complement DF502 with an activity that is above 70% of wild-type activity. The sequences of these hinge mutants reveal that the solutions to the hinge flexibility problem are varied. Moreover, these preferences are sequence dependent; that is, certain pairs occur frequently. They fall into six families of similar sequences. In addition to the hinge sequences expected on the basis of phylogenetic analysis, we selected three new families of 3-amino-acid hinges: X(A/S)(L/K/M), X(aromatic/beta-branched)(L/K), and XP(S/N). The absence of these hinge families in the more than 60 known species of triosephosphate isomerase suggests that during evolution, not all of sequence space is sampled, perhaps because there is no neutral mutation pathway to access the other families.     

Analysis of amino acid requirements ofClostridium thermocellum

Summary Clostridium thermocellum is a species able to convert cellulose to ethanol on complex medium. A systematic study of changes in the concentration of amino acids in the medium during growth of the bacterium has permitted us to obtain a chemically defined medium more effective than that hitherto used and to obtain some information about the use of amino acids by this bacterium.     

Differential amino acid requirements for sporulation in Bacillus subtilis

The amino acid requirements for sporulation were studied by use of auxotrophic mutants of Bacillus subtilis 168. Cells were grown to T(0) in medium containing the test amino acid and were then transferred to a minimal medium lacking that amino acid. Omission of leucine caused no reduction in sporulation. Omission of methionine, lysine, and phenylalanine appeared to cause reduced levels of sporulation, and sporulation was completely inhibited when isoleucine, tryptophan, and threonine were omitted. The amino acids in this third class showed a sequence of requirements, with tryptophan required earlier than isoleucine, which in turn was required earlier in the sporulation process than threonine. Isoleucine omission did not affect the early sporulation functions of extracellular protease formation or septum formation, but prevented the increased levels of protein synthesis and oxygen consumption that normally accompany early sporulation stages. Isoleucine did not appear to be metabolized to other compounds in significant amounts during sporulation. The role of isoleucine in the sporulation process remains unclear.