Preparation and some physico-chemical properties of L-canaline

A new and simple method for the preparation of L-canaline for use in biological studies is described. This material, an analogue of ornithine, was prepared by enzymatic hydrolysis of L-canavanine followed by fractionation under physiological conditions by gel filtration on Sephadex G10. The behaviour in thin-layer chromatography of the separated material is identical with that of canaline prepared by two different routes of organic synthesis. Further physico-chemical characterisation gave data compatible with the molecular structure proposed for canaline (2-amino-4-aminoxybutyric acid). Addition of L-canaline results in a change in the absorption spectrum of pyridoxal phosphate, maximum effect being observed in the presence of equimolar amounts. Under these conditions, a single addition product is detected in thin-layer chromatography. Each of the canaline preparations tested is identical in this respect. The biological significance of this reaction is discussed.     

Anthracycline gels. II. Spectroscopic study on doxorubicin-lecithin association products.

Thixotropic and thermally reversible gels have been prepared from doxorubicin-lecithin association products (DL12) by addition of salts to their aqueous solutions. The gel formation and the melting profiles have been followed by several spectroscopic techniques (1H NMR, UV-Vis, Circular Dichroism). The transition temperatures increase as the concentration of both the salt and the DL12 is increased, suggesting a progressive closer approach of the gel forming species. The process of the gel formation is cooperative and causes immobilization of the doxorubicin molecules of DL12.     

Antimicrobial activity and physico-chemical properties of some alkyldimethylbenzylammonium chlorides

A series of alkyldimethylbenzylammonium chlorides have been synthesized with n-alkyl chain lengths of C1 leads to C18. Octanol/water partition coefficients were determined and the antimicrobial activity assessed as the minimum growth inhibitory concentrations towards twelve strains of micro-organisms, representative of Gram-negative and Gram-positive bacteria, yeasts and fungi. The data were subjected to a numerical analysis. Antimicrobial activity of the compounds was found to be a parabolic function of their lipophilicity and maximized with n-alkyl chain lengths of between C12 and C16. The data fit to quadratic functions estimated for low (C1-C7) and high (C8-C16) alkyl chain length compounds was better than for a single quadratic describing the activity of the complete series (C1-C18). These maximized at log P values corresponding to alkyl-chain lengths of approximately C7 and C14 respectively, and were suggestive of low and high affinity binding sites upon the cell surface. The data analysis allowed the chain lengths of compounds with optimal activity towards the various groups of organisms to be determined. Generally yeasts and fungi were most sensitive towards C12, Gram-positive bacteria towards C14, and the Gram-negative bacteria towards C16. Gram-negative cells were the most resistant towards all the compounds and Gram-positive cells the least.     

Purification and some physico-chemical properties of myocardial adenylate deaminase

A procedure for isolation of adenylate deaminase from duck heart muscle has been developed. The method includes extraction of enzyme, chromatography on cellulose phosphate, fractionation by ammonium sulfate, chromatography on Sephadex G-25 and ion-exchange chromatography on DEAE-cellulose. The enzyme was purified approximately 4000-fold with a yield of 25%. Electrophoresis in polyacrylamide gel revealed that the enzyme contains no proteins other than adenylate deaminase. The enzyme has a UV absorption spectrum typical for proteins which contain no nucleic acid impurities. Using sievorptive chromatography, it was shown that the myocardial extract contains two adenylate deaminase forms, which are tetramers with mol. weights of 190 000 and 240 000. The molecular weights of the subunits are 47 000 and 63 000, respectively. In the oligomeric form the enzyme is only detected at high enzyme concentrations and in the presence of large amounts of substrate.     

Effects of sterilization treatments on some properties of alginate solutions and gels.

Aqueous sodium alginate solutions were subjected to various heat sterilization treatments. Sodium alginate powder was also treated by both gamma-irradiation and ethylene oxide sterilization. The effects of these treatments on the viscosities of sodium alginate solutions and both the diameter and strength of the beads formed in 0.1 M CaCl2 solutions were determined quantitatively. The viscosity of sodium alginate solutions and the gel strength of the calcium alginate beads decreased with increasing sterilization temperature while the bead diameters were found to increase. All these effects can be attributable to a reduction in the degree of polymerization of the alginate molecules as a result of the heat treatments. Ethylene oxide and gamma-irradiation treatments caused similar effects. Standard conditions for sterilization are necessary for comparative studies with alginate beads.     

Purification and some physico-chemical and enzymatic properties of tissue kallikrein from human urine

A procedure for obtaining tissue kallikrein (EC 3.4.21.35) from large specimens of human urea (100 l) has been developed. The isolation procedure included primary extraction of the protein with chitosan (a crustacean chitin deacylated by alkaline treatment), desorption from chitosan with 1 M NH3, affinity chromatography on contrical-Sepharose, ion-exchange chromatography on DEAE-Sepharose and gel filtration on Sephadex G-100. This method permits to obtain tissue kallikrein preparations purified 1080-fold (with respect to AcPheArg-OEt esterase) and 1360-fold (with respect to kininogenase) with 33 and 40% yields, respectively. Tissue kallikrein preparations were homogeneous as could be judged from the results of electrophoresis performed in 12% PAAG in the presence of 0.1% SDS as well as from the presence of one N-terminal amino acid identified as isoleucine. Purified tissue kallikrein had specific activities of 133 mumol/min/mg protein (with respect to AcPheArg-OEt hydrolysis) and 8.8 mumol/min/mg protein (with respect to D-Val-Leu-Arg-pNa hydrolysis) and liberated 462 micrograms equiv. of bradykinin/min/mg protein from heated human blood plasma used as a kininogen source. The protein exhibited the highest stability at pH 8.0-9.0; the pH optimum is at pH 8.0 with AcPheArg-OMe as substrate. The enzyme revealed a high thermostability and was fully inactivated only after 1-hour heating in a boiling water bath. The identity of the urine enzyme to tissue kallikrein could be confirmed by the resistance of the enzyme activity to SIT, high sensitivity to the inhibiting effect of aprotinin (Ki = 0.94 x 10(-10) M) and by an exceedingly low value of the second order inhibition constant for DPP (4.6 M-1 min-1). The fact that this value differs drastically from that for human blood plasma kallikrein (EC 3.4.21.34) which is equal to 360 M-1 min-1 points to marked differences in the structure of the active centers of the both kallikreins as well as to the uniqueness of the tissue kallikrein active center.     

Effect of gamma-irradiation on some biological and physico-chemical properties of polyene antibiotics]

Deep effect of gamma-rays on polyenic antibiotics was studied. It was shown that gamma-radiation induced radiation-chemical oxidation of the substances. The chromatographic analysis showed that the levorin degradation products were identical to the polyenic products of the antibiotic oxidative destruction. As for mycoheptin and amphotericin B, destruction of their molecules to non-polyenic products was observed. It was found that toxicity of the levorin aromatic heptaen did not practically change after gamma-irradiation in high doses. The toxicity of mycoheptin and amphotericin B, non-aromatic heptaens increased after exposure to high doses of gamma-rays.     

Ammonification in soil aggregates: Effect of some physical,physico-chemical and chemical properties

Aggregate diameter affected significantly the intensity of ammonification in chernozemic rendzina but not in lessivē soil. In the latter the process was influenced significantly by the number of microorganisms able to grow on asparagine agar. A high correlation, though not significant at the level of 0.05, was found between the ammonification intensity and the content of pores of radius: 3–1.5, 7.5–5.0, 0.5–0.25 and 0.01–0.005 μm in chernozemic rendzina and those measuring 1.5–0.5, 0.025–0.01, 0.01–0.005 and >7.5 μm in lessivē soil aggregates as well as the percentage of soil particles of 100–50 μm in chernozemic rendzina aggregates and the internal surface area and organic C in aggregates of lessivē soil.