Improving ready biodegradability testing of fatty amine derivatives.

This study assesses the biodegradation potential of a number of fatty amine derivatives in tests following the OECD guidelines for ready biodegradability. A number of methods are used to reduce toxicity and improve the bioavailability of the fatty amine derivatives in these tests. Alkyl-1,3-diaminopropanes and octadecyltrimethylammonium chloride are toxic to microorganisms at concentrations used in OECD ready biodegradability tests. The concentration of these fatty amine derivatives in the aqueous phase can be reduced by reacting humic, or lignosulphonic acids with the derivatives or through the addition of silica gel to the test bottles. Using these non-biodegradable substances, ready biodegradability test results were obtained with tallow-1,3-diaminopropane and octadecyltrimethylammonium chloride. Demonstration of the ready biodegradability of the water-insoluble dioctadecylamine under the prescribed standard conditions is almost impossible due to the limited bioavailability of this compound. However, ready biodegradability results were achieved by using very low initial test substance concentrations and by introducing an organic phase. The contents of the bottles used to assess the biodegradability of dioctadecylamine were always mixed. False negative biodegradability results obtained with the fatty amine derivatives studied are the result of toxic effects and/or limited bioavailability. The aids investigated therefore improve ready biodegradability testing.

Purification, characterisation, and gene cloning of transglutaminase from Streptoverticillium cinnamoneum CBS 683.68.

The transglutaminase (TGase; EC 2.3.2.13) from Streptoverticillium cinnamoneum CBS 683.68 has been purified, characterised and its gene cloned. The purified enzyme had a relative molecular mass of 37,660 determined by mass spectrometry and contained a single Cys residue that was essential for the catalytic activity. Contrary to eukaryotic TGases, this enzyme was calcium-independent. The fact that TGase was capable to incorporate a wide variety of aliphatic and aromatic non-polar compounds suggested that the amine fixation site could be an hydrophobic pocket. S cinnamoneum CBS 683.68 TGase was synthesised as a protein precursor of 411 amino acid residues corresponding to a signal peptide of 81 amino acid residues and a mature TGase of 330 amino acid residues. Amino acid sequence analysis revealed that the S cinnamoneum CBS 683.68 TGase had little sequence homology with eukaryotic TGases, but shared high identity with the sequence of Streptoverticillium strain S-8112. In accordance with kinetics data, hydropathy analysis showed that the active site of the enzyme was in an hydrophobic environment as for eukaryotic TGases.

Rat platelet phospholipase A2. Kinetic characterization using the monomolecular film technique.

We have determined some kinetic parameters of rat platelet phospholipase A2, such as surface pressure dependency and substrate specificity, using the monomolecular film technique. We found that rat platelet phospholipase A2 is very specific for phospholipids having a negatively charged headgroup, no activity was detected when using zwitterionic phospholipids such as phosphatidylcholine. Furthermore, the interfacial pressure window which permits enzyme activity is very narrow as compared to pancreatic phospholipase A2. Maximal enzyme activity is found at 22 mN/m when using 1,2-dilauroylphosphatidylglycerol as substrate. Studies of the competitive inhibition of mixed films containing 2-acylaminophosphatidylglycol show that platelet phospholipase A2 is less sensitive than pancreatic and intestinal phospholipase A2. These results imply that, despite the high degree of sequence similarity, one must be very cautious in extrapolating inhibition data from one phospholipase A2 to similar enzymes from other origins.

Competitive inhibition of lipolytic enzymes. V. A monolayer study using enantiomeric acylamino analogues of phospholipids as potent competitive inhibitors of porcine pancreatic phospholipase A2.

For the first time, we have shown that a stereospecific interaction occurs between porcine pancreatic phospholipase A2 and a monomolecular film of amidophospholipid used as inhibitor. Direct binding experiments, using radiolabelled phospholipase A2, showed that 13 times more enzyme was bound to phospholipid films of the L series by comparison with films of the D series. These results were confirmed by indirect binding studies using re-spreading experiments. Kinetic studies of the porcine pancreatic PLA2, using enantiomeric acyl-amino phospholipid analogues, have shown that: (1) inhibitors of the L series are more potent than inhibitors of the D series, (2) inhibitors having a negative charge are more potent than zwitterionic inhibitors, (3) inhibitory power values are greater when evaluated in micellar system than in a the monolayer system, (4) the inhibitory power increases continuously with surface pressure.

Competitive inhibition of lipolytic enzymes. I. A kinetic model applicable to water-insoluble competitive inhibitors.

It is now becoming clear from the abundant lipolytic enzyme literature that any meaningful interpretation of inhibition data has to take into account the kinetics of enzyme action at the lipid/water interface. We attempt in the present paper to provide a kinetic model applicable to water-insoluble competitive inhibitors, in order to quantitatively compare the results obtained at several laboratories. We derived kinetic equations applicable to the pre-steady state as well as steady state. By measuring the inhibitory power, as described in the present paper, it is possible to obtain a normalized estimation of the relative efficiency of various potential inhibitors. Furthermore, with the kinetic treatment developed here, it is possible to make quantitative comparisons with the same inhibitor placed under various physico-chemical situations, i.e., micellar or monolayer states.