[Aldehyde-dehydrogenase from human liver: Purification, partial characterization and influence of malondialdehyde]. / Aldehyddehydrogenase aus Humanleber: Isolierung, partielle Charakterisierung und Einfluss von Malondialdehyd.

A procedure to separate the isozymes E1 and E2 of aldehyde dehydrogenase (ALDH, aldehyde: NAD oxidoreductase, EC 1.2.1.3) from human liver, avoiding 5'-AMP-Sepharose, was worked out. It results in fairly purified isozymes. The isoelectric points could be re-estimated for E1 at pH 5.21 +/- 0.04 and for E2 at pH 4.90 +/- 0.05. The pH-optimum of both the isozymes is dependent on the buffer used, the best buffer being sodium pyrophosphate/HCI. In this buffer the enzyme activity is also dependent on ionic strength. Malondialdehyde (MDA), at concentrations which are found in patient serum, is converted by the ALDH. The Km-values of this reaction are 1.71 mmol/l for MDA and 0.19 mmol/l for NAD (E1) and 0.21 mmol/l for MDA and 0.24 mmol/l for NAD (E2) resp. The highest rates of conversion are reached at concentrations of 0.08 mmol/l MDA (E1) and 0.16 mmol/l MDA (E2). At higher concentrations of MDA substrate excess inhibition is observed (Kss = 0.17 mmol/l for E1 and 0.20 mmol/l for E2).

[A membrane-bound alanine aminopeptidase from Acinetobacter calcoaceticus. 1. Isolation and purification of the enzyme]. / Eine membrangebundene Alaninaminopeptidase aus Acinetobacter calcoaceticus. 1. Isolierung und Reinigung des Enzyms.

The alanine aminopeptidase of Acinetobacter calcoaceticus was found to be bound to the inner membranes only. The enzyme was solubilized by Triton X-100 and purified approximately 480-fold by gel filtration and affinity chromatography on alanine methyl ketone-AH-Sepharose 4B. The purified alanine aminopeptidase has a molecular mass of 212 kDa, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme selectively catalyses the hydrolysis of N-terminal alanine residues of peptides. The enzyme is inhibited by p-hydroxy-mercuribenzoate, 1,10-phenanthroline, and puromycin, but was activated by CO2(+)-ions.

[Kinetics of membrane-bound aldehyde dehydrogenase from Acinetobacter calcoaceticus]. / Zur Kinetik der membrangebundenen Aldehyd-Dehydrogenase aus Acinetobacter calcoaceticus.

In recent investigations we were able to demonstrate that the NADP-dependent aldehyde dehydrogenase of Acinetobacter calcoaceticus is an inducible enzyme localized in intracytoplasmic membranes limiting alkane inclusions. Long-chain aliphatic hydrocarbons and alkanols are inducers of the enzyme. It was purified by us and now kinetically characterized using the enzyme-micelle form, which contains bacterial phospholipids and a detergent (sodium cholate), too. The pH optimum of aldehyde dehydrogenase was determined to be at pH 10. The enzyme showed substrate inhibition (by aldehyde excess). The Ks and Km values of the leading substrate NADP+ were found to be 8.6 X 10(-5) and 10.3 X 10(-5)M independent of the chain-length of the aldehydes. The Km values of the aldehydes decreased depending on increasing chain-length (butanal: 1.6 X 10(-3), decanal: 1.5 X 10(-6)M). The Ki values (for inhibition by aldehyde excess) showed a similar behaviour (butanal: 7.5 X 10(-3), decanal: 3.5 X 10(-5)M) as well as the optimal aldehyde concentrations inducing the "maximal" reaction velocity (butanal: 5mM, decanal: 6 microM). The number of inhibiting aldehyde molecules per enzyme-substrate complex was determined to be n = 1. NADPH showed product inhibition kinetics (Ki(NADPH) = 2.2 X 10(-4)M), fatty acids did not. We were unable to measure a reverse reaction. The following ions and organic compounds were non-competitive inhibitors of the enzyme: Sn2+, Fe2+, Cu2+, BO3(3-), CN-, EDTA, o-phenanthroline, p-chloromercuri-benzoate, mercaptoethanol, phenylmethylsulfonyl fluoride, and diisopropylfluorophosphate; iodoacetate did not influence enzyme activity. Chloral hydrate was a competitive inhibitor of the aldehydes. Ethyl butyrate activates the enzyme, dependent on the chain-length of the aldehyde substrates.

[Proteases in different membrane fractions of Acinetobacter calcoaceticus]. / Proteasen in differenten Membranfraktionen von Acinetobacter calcoaceticus.

Distinct protease activities were found in membrane fractions from Acinetobacter calcoaceticus grown on acetate-NH4+ medium until early stationary phase. Mechanical or enzymatic cell disintegration followed by membrane fractionation through sucrose gradient revealed higher activities in the outer membrane than in the cytoplasmic membrane. Using azocasein and synthetic p-nitroanilides as substrates we found very low proteinase activities in intracytoplasmic membrane fractions. However, these fractions contained a significant aminopeptidase activity which was absent from cell envelope membranes. Peptidolytic activities in intracytoplasmic membranes of gram-negative bacteria have not been described before.

[Cell envelope-bound proteinase activities of Acinetobacter calcoaceticus]. / Zellhüllgebundene Proteinase-Aktivitäten in Acinetobacter calcoaceticus.

Acinetobacter calcoaceticus contains proteolytic activity after growth on various culture media. The enzyme activity could be found in the cytosolic fraction as well as in the cell envelopes (also containing the intracytoplasmic membranes). The highest proteolytic activity could be detected during the transition from the logarithmic growth phase to the stationary phase and in the early stationary phase, respectively. In the culture medium proteolytic activity was only evident in the later stationary phase. This activity was very instable and was liberated apparently by autolysis of the cells. The concentration of the nitrogen source (NH4+) in the medium (i.g. with acetate as carbon source) influences the proteinase activities of the cells. When nitrogen is limited, the proteolytic activity increases strongly in the stationary phase. The pH-profile of the azocaseinolytic activities of the cytosol was nearly the same as that of the cell envelopes (pH-optima are between 7 and 9). A partial inhibition of the proteolytic activities in the cytosol as well as in the cell envelopes could be attained by serine proteinase inhibitors. Inhibitors of thiol- and metalloproteinases showed no effects.

Ultracytochemical localization of aldehyde dehydrogenase in Acinetobacter calcoaceticus.

A membrane-bound aldehyde dehydrogenase is induced in Acinetobacter calcoaceticus grown on aliphatic hydrocarbons as sole carbon source. This enzyme is NADP-dependent and is able to oxidize medium- and long-chain aliphatic aldehydes to their corresponding fatty acids. Electron micrographs of sectioned alkane-adapted bacteria showed hydrocarbon inclusions in the cytoplasmic matrix. The cytochemical phenazine methosulphate-tetranitro tetrazolium blue capture reaction allowed to localize the activity of the aldehyde dehydrogenase near the surface of these inclusions. At the same location we also found a NADPH tetrazolium-reducing activity.

[Temperature dependence of membrane-bound aldehyde dehydrogenase from Acinetobacter: effect of solubilization and chain length of the substrates]. / Temperaturabhängigkeit der membrangebundenen Aldehyddehydrogenase aus Acinetobacter: Einfluss der Solubilisierung und der Kettenlänge der Substrate.

The NADP+-dependent aldehyde dehydrogenase of intracytoplasmic membranes of Acinetobacter calcoaceticus shows a characteristic behaviour of its reaction velocities in dependence on temperature. The calculated "real" activation energies depend on the increasing chain-length of the homologous aldehydes and grow appropriate to the membrane-bound and to the micellar form of the enzyme with 6.2 and 7.3 kJ/mole CH2-group, respectively. The values of the activation energy of the membrane-bound enzyme are - for each aldehyde of the homologous series - 12 kJ/mole larger than those of the solubilized enzyme (present as mixed micelles of enzyme, cholate and bacterial phospholipids).

[Optimization of culture conditions for Acinetobacter calcoaceticus grown on n-alkanes in a laboratory fermenter]. / Optimierung von Kulturbedingungen für Acinetobacter calcoaceticus beim Wachstum auf n-Alkanen in einem Laborfermentor.

The growth conditions for the cultivation of Acinetobacter calcoaceticus in a commercial laboratory fermenter have been optimized. This was done taking former results of shake cultures as a basis. At the end of the logarithmic phase up to 10 g/l dry weight were obtained. The following culture conditions have been used: The medium contained 10 ml/l n-alkane and 6 g/l NH4Cl. The pH was adjusted to 6.5 and pO2 employed to 70% saturation. A 12 times higher amount of dry weight was observed compared with shake culture technique.