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Acinetobacter lwoffii K24 is a known aniline-degrading bacterium. In previous studies, two catechol branches of the β-ketoadipate pathway were reported to be induced for aniline degradation, and related enzymes (CatA 1 and CatA 2 ) were identified from the aniline-induced proteome of A. lwoffii K24 [S.I. Kim, S.H. Leem, J.S. Choi, Y.H. Chung, S. Kim, Y.M. Park, Y.K. Park, Y.N. Lee, K.S. Ha, Cloning and characterization of two catA genes in Acinetobacter lwoffii K24, J. Bacteriol. 179 (1997) 5226–5231; and E.A. Kim, J.Y. Kim, S.J. Kim, K.R. Park, H.J. Chung, S.H. Leem, S.I. Kim, Proteomic analysis of Acinetobacter lwoffii K24 by 2-D gel electrophoresis and electrospray ionization quadrupole-time of flight mass spectrometry, J. Microbiol. Methods 57 (2004) 337-349]. A. lwoffii K24 has also been found to utilize other aromatic compounds such as p -hydroxybenzoate, salicylate, and benzoate. In this study, we performed a comparative 2-DE/MS analysis of a benzoate-induced proteome and found that a new catechol 1,2-dioxygenase (CatA 3 ) and benzoate 1,2-dioxygenase were up-regulated as the primary dioxygenases responsible for benzoate degradation in A. lwoffii K24. However, CatA 1 and CatA 2 were not detected on the same 2D gel as CatA 3 . Transcription analysis of three catA genes from A. lwoffii K24 showed that these cat genes were specifically expressed under certain growth conditions using different aromatic compounds as the carbon source. While catA 1 and catA 2 were expressed under the aniline culture condition, catA 3 was expressed under the benzoate culture condition. A new cat gene cluster ( catB 3 C 3 A 3 F 3 ) was cloned and found to share sequence homology and a similar gene structure with the cat genes of Acinetobacter radioresistens . This result suggests that the third catechol branch ( cat 3 ) of the β-ketoadipate pathway was selectively induced for the degradation of benzoate in A. lwoffii K24. It also provides evidence of multiple catechol branches in the β-ketoadipate pathway and the independent regulation of monocyclic aromatic compound degradation in A. lwoffii K24.

Residue Phe375 of cephalosporin acylase has been identified as one of the residues that is involved in substrate specificity. A complete mutational analysis was performed by substituting Phe375 with the 19 other amino acids and characterising all purified mutant enzymes. Several mutations cause a substrate specificity shift from the preferred substrate of the enzyme, glutaryl-7-ACA, towards the desired substrate, adipyl-7-ADCA. The catalytic efficiency ( [Formula: see text] (cat)/ [Formula: see text] (m)) of mutant SY-77(F375C) towards adipyl-7-ADCA was increased 6-fold with respect to the wild-type enzyme, due to a strong decrease of [Formula: see text] (m). The [Formula: see text] (cat) of mutant SY-77(F375H) towards adipyl-7-ADCA was increased 2.4-fold. The mutational effects point at two possible mechanisms by which residue 375 accommodates the long side chain of adipyl-7-ADCA, either by a widening of a hydrophobic ring-like structure that positions the aliphatic part of the side chain of the substrate, or by hydrogen bonding to the carboxylate head of the side chain.

Acinetobacter lwoffii K24 known as an aniline degrading bacterium has also been found to utilize p-hydroxybenzoate as a sole carbon source. In this study, 2-DE using Q-Sepharose column separation was attempted for fast screening of protocatechuate 3,4-dioxygenase for catabolism of p-hydroxybenzoate in A. lwoffii K24. Two protocatechuate 3,4-dioxygenase subunits, pcaG and pcaH were detected and identified with N-terminal and internal sequencing, suggesting proteomics using a column separation may be helpful for the identification of specific protein spots and maximizing the detectable protein spots on the 2-DE gel. The PCR process using degenerate primers for protocatechuate 3,4-dioxygenase and sequence analyses of the PCR products revealed the existence of pcaH and pcaG in A. lwoffii K24. These two subunits were found to be closely located and share extensive homology with pcaH and pcaG of Pseudomonas marginata or Pseudomonas cepacia, providing the evidence that A. lwoffi K24 has the protocatechuate branches as well as catechol branches of beta-ketoadipate pathway.

Native chromatin was reacted with bifunctional protein cross-linking reagents of varying molecular sizes. Short cross-linkers do not produce significant yields of polymeric histones whereas intermediate-sized materials such as dimethyladipimidate produce extensive interaction between histones. Dimethylsuberimidate reacts with all histone fractions but only generates polymers of F1 and of F2b and F2a2.

The six identical polypeptide chains of the smallest enzymatically active unit of beef liver glutamate dehydrogenase are shown to be arranged in two trimers. Cross-linking with bifunctional reagents of varying chain length and subsequent SDS polyacrylamide gel electrophoresis of the protein shows main bands corresponding to molecular weights of 168,000 and 336,000 daltons which is three times and six times, respectively, the molecular weight of the polypeptide chain (56,000 daltons). This finding supports a model for the quaternary structure of the glutamate dehydrogenase proposed by Reisler and Eisenberg (Biopolymers 9 , 877 (1970)).

Studies in vivo and in vitro were made of the metabolism of 5-hydroxy-4-keto-valeric acid HKV , which is formed by decarboxylation of 2-hydroxy-3-keto-adipate, the primary condensation product of the 2-ketoglutarate:glyoxylate carboligase reaction. Evidence is presented for the formation of 14C-aspartate, 14C-glutamate and 14C-delta-aminolevulinic acid when 14C-HKV is incubated with sonicated rat liver mitochondria. Formation of these products suggests that HKV may be converted to 2-keto-glutamate and delta-aminolevulinic acid via 4,5-dioxo-valerate. In vivo oxidation of 14C-HKV to 14CO2 is also shown in the rat.

Citrate synthase (EC 4.1.3.7.) from roots of bean seedlings and mitochondria of cauliflower buds was purified 400-fold and had specific activity of 6 to 10. Such enzyme was stimulated by the plant hormone indoleacetic acid (IAA). Consistent with the accepted molecular size of the enzyme, it was eluted as a single peak off Sephadex G-200. Treatment with IAA increased the molecular size of the enzyme. It is demonstrated that while increasing its activity and modifying its size, the hormone binds with the enzyme.

Catechol is degraded by meta - cleavage in a naphthalene-grown pseudomonad to yield 2-hydroxymuconic semialdehyde. It is shown that there exist two enzymes in naphthalene-grown cells which degrade 2-hydroxymuconic semialdehyde, one NAD+-dependent and one non NAD+-dependent. Both are induced by growth upon naphthalene. It is suggested that the more important of these enzymic reactions in vivo is the NAD+-dependent dehydrogenation to γ-oxalocrotonate.

Human erythrocyte ghosts were treated with a bifunctional cross-linking reagent, dimethyl adipimidate dihydrochloride. On SDS-polyacrylamide electrophoresis of the cross-linked membrane proteins after solubilization, sialoglycoproteins and the proteins disappeared from the original band positions and appeared in a new band of aggregates.

Purified chick skin collagen constituent chains were examined for the content of the aldol condensation product of two residues of α-aminoadipic δ-semialdehyde after reduction with NaBT 4 and alkali hydrolysis. The single chains, α1 and α2, contained none whereas their cross-linked dimer, β 12 , contained one equivalent. These findings were confirmed by the similar studies performed on the peptides derived from the cross-link region of collagen by CNBr cleavage. Data presented here strongly indicate that the aldol condensation product is the intramolecular cross-link.

Evidence is presented to support the view that the conversion of α-aminoadipic acid to α-aminoadipic-δ-semialdehyde involves three different steps. Cell-free preparations of two of the lysine auxotrophs, ly 2 and ly 5 , fail to accomplish this conversion. These two mutants possess the activity for activation (step 1) and reduction (step 2), but lack the activity for hydrolysis (step 3). These mutants complement each other in the diploid and are mapped on two different chromosomes. However, they fail to complement in , vitro , indicating control of one biosynthetic step by two unlinked genes.

Purified chick skin collagen was aggregated into native type fibrils in vitro and the intermolecular cross-links were stabilized by reduction with NaBT 4 . Ion exchange chromatography and scintillation spectrometry of the resulting insoluble collagen after acid or alkaline hydrolysis indicated the presence of ϵ-hydroxylysinonorleucine, lysinonorleucine and a third radioactive component of unknown structure eluting near histidine. The intramolecular cross-link, the aldol condensation product of two residues of α-aminoadipic δ-semialdehyde was no longer detectable in these reconstituted fibrils. Data presented here suggest that the intermolecular cross-link formation closely involves the intramolecular cross-link.

Dimethyl adipimidate (DMA), a bifunctional cross-linking agent, is known to be a powerful anti-sickling agent in vitro . The hemoglobin isolated from DMA-treated erythrocytes is heterogeneous with respect to charge and size as shown by starch gel electrophoresis and sedimentation velocity studies; some hemoglobin tetramers being cross-linked together resulting in high molecular weight species. Measurement of transverse water proton relaxation times of DMA-treated sickle erythrocytes shows that while some aggregation may take place within deoxygenated erythrocytes, it is not of sufficient extent to cause the cells to change shape. Therefore, it is suggested that the anti-sickling effect of DMA is due to its ability to react with hemoglobin S inside sickle erythrocytes and inhibit the aggregation process.

Summary The following observations strongly suggest that L-kynurenine aminotransferase (KAT) and L-α-aminoadipate aminotransferase (AAT) activities are associated with the same protein: 1. a similar distribution in the supernatant and mitochondrial fractions of kidney and liver of male rats, 2. a similar distribution in these fractions of kidney and liver of female rats, 3. a similar sex difference in the supernatant and mitochondrial fractions of kidney with no sex difference in liver fractions, 4. a similar pattern of inhibition with a homologous series of dicarboxylic acids with adipate being most effective, 5. competitive inhibition of KAT with d , l -α-aminoadipate (αAA), 6. substrate inhibition of KAT with α-ketoadipate (αKA), and 7. AAT activity in a highly purified preparation of KAT. A scheme for the interaction of the two activities in lysine and tryptophan metabolism is presented.

Summary Studies using subcellular fractions of rat liver have revealed that α-ketoadipate dehydrogenase is principally localized in the mitochondrial fraction and that α-ketoadipate reductase is principally localized in the cell supernatant fraction. Both enzymes have the highest activity in heart muscle. The ratio of the dehydrogenase activity to the reductase activity is highest in kidney followed by heart, brain, liver, leg muscle, and small intestine. This pattern is consistent with the relative mitochondrial density of the various tissues tested, and suggests that the reductase has a dominant role in α-ketoadipate metabolism in tissues that have a low mitochondrial density.

The α-ketoglutarate dehydrogenase complex (KGDC) of baker's yeast was isolated and shown to have a sedimentation velocity (S 20, W ) of 20.9 S. The enzyme complex catalyzes the oxidation of 7.7 μmoles of KG per min per mg protein. The activity of the purified KGDC has an absolute dependence on Co A and NAD and a partially dependence on TPP and Mg ++ . The enzymic reaction has a pH optimum of 7.2. The Km value for KG was determined to be 0.15 mM. The complex is specific for the oxidation of KG. Its visible spectrum has the characteristics of a flavoprotein.

Soluble collagen extracted by 0.2 M cysteamine from rat skin contains 2.5 to 2.7 residues of peptide bound α-aminoadipic semi-aldehyde per α-chain. When reconstituted into fibers and maintained at 37° C these aldehydes become involved in the formation of stable non-reducible crosslinks. Using collagen biosynthetically labeled at the lysine and hydroxylysine positions, these stable crosslinks have been partially identified. Lysinonorleucine and its hydroxyderivative account for 50% of the initial aldehyde residues present on the collagen molecule while two unidentified peaks (post-lysine and post-histidine) account for the other 50%.

It was shown previously that lysine was degraded in the mitochondria in the presence of α-ketoglutarate and, under these conditions, saccharopine accumulated (1). The obligatory requirement of α-ketoglutarate for lysine breakdown as well as for saccharopine formation suggest that lysine is metabolized, in mammalian liver, by a reversal of reaction sequence described for its biosynthesis in yeast (2–6). The present report describes that saccharopine is further metabolized to α-aminoadipic acid in the mitochondria and that NAD rather than NADP is required in this reaction.

In collagenous tissues, borohydride-reduced crosslinks and their reduced aldehyde precursors are present in varying proportions, depending upon the nature of the tissue, the animal species, and the age of the animal. In the case of bovine Achilles tendon, a predominant reduced aldehyde is δ, e-dihydroxynorleucine. Furthermore, one of the most abundant crosslinks is a previously undescribed compound, hydroxylysinohydroxynorleucine. This compound is not present in solutions of tendon collagen but appears in reconstituted fibrils and probably is an intermolecular crosslink. It probably arises by reduction of the Schiff base formed between the α-amino group of hydroxylysine and the carbonyl moiety of α-amino-δ-hydroxy adipic acid-δ-semialdehyde. We have isolated and also synthesized the reduction product of the latter compound and located it in the chromatograms.