In this paper we continue the study of a number of properties of protomitochondria--small young mitochondrial organelles in the animal cells. Protomitochondria were obtained by filtration of total suspension of mitochondria of rat liver through Millipore filters. Protomitochondria contain an active respiratory chain as evidenced by the high rate of oxygen consumption during succinate and NADH oxidation. A shunt succinate:tetrazolium-reductase activity of protomitochondria was lower and NADH-tetrazolium-reductase activity was higher than that in mitochondria. Electrophoresis and gel filtration found no qualitative differences between protomitochondria, 0.25-0.45 μm in diameter, and mitochondria in major protein composition, but some quantitative differences in several bands were found. Perhaps, these differences reflect the process of intracellular maturation of protomitochondria to mitochondria. The data obtained are important for understanding the mitochondriogenesis in the animal cells.

Passage of light through a suspension is accompanied by a competition of processes of absorption and scattering for each individual particle. As a result, a hypochromism phenomenon (a decrease in the extinction coefficient) takes place. The hypochromism value increases with the growth of a particle sizes or its refractive index. Since Tyndall’s light scattering in a suspension where the size of every particle is considerably larger than the wavelength weakly depends on the wavelength, the absorption (or excitation) spectrum is almost uniformly attenuated. A simple method for determination of the true extinction coefficients of the absorption and excitation spectra of diluted suspensions which have no multiple light scattering is proposed. Experimental data on the spectra of hemoglobin in erythrocytes, actinomycin in DNA, and flavines in mitochondria are discussed.

Изучено действие внеклеточных бактериолитических ферментов Lysobacter sp. на грамотрицательные бактерии. Показано, что бактериологические ферменты способны гидролизовать пептидогликан, выделенный из клеток этих бактерий. Липополисахарид клеточных стенок полностью ингибирует процесс гидролиза пептидогликана бактериолитическими ферментами. Лизис нативных клеток грамотрицательных микроорганизмов становится возможным после изменения проницаемости их внешней мембраны путем обработки клеток этих бактерий полимиксином В.

The effect of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-negative bacteria was studied. These enzymes were found to be able to hydrolyze the peptidoglycan that was isolated from the gram-negative bacteria, the hydrolysis being completely inhibited by the cell wall lipopolysaccharide of these bacteria. The native cells of the gram-negative bacteria became susceptible to the bacteriolytic enzymes after the permeability of the outer membrane of the cells was altered by treating them with polymyxin B.

The study of the extracellular bacteriolytic enzymes of Lysobacter sp. showed that they can efficiently hydrolyze the peptidoglycan of gram-positive bacteria provided that there is an electrostatic interaction of these enzymes with the cell wall anionic polymers, teichoic and teichuronic acids in particular. The hydrolytic action of bacteriolytic enzymes on the cell wall largely depends on the negative charge of the teichoic and teichuronic acids rather than on their chemical composition.

Specificity of Staphylococcus aureus 209P cell wall hydrolysis by the L1 and L2-bacteriolytic enzymes from lysoamidase lytic complex was studied. L1-peptidase was shown to display both glycyl-glycine endopeptidase and N-acetylmuramyl-L-alanine amidase enzymatic activities on the S. aureus peptidoglycan molecule, whereas L2-peptidase acts as N-acetylmuramyl-L-alanine amidase.

Lysoamidase, a bacteriolytic complex from the culture liquid of Xanthomonas sp., hydrolyzed the cells walls of Staphylococcus aureus, Streptomyces chrysomallus, and Streptomyces azureus, which contain ribitol teichoic acids in addition to peptidoglycan. The cell walls of Streptomyces roseoflavus, Glycomyces harhinensis, and Nocardiopsis dassonvillei, containing glycerol teichoic acids, were not hydrolyzed by lysoamidase. The extent of the hydrolysis of 20-h Str. chrysomallus cells and cell walls, containing 40% ribitol teichoic acids, was considerably higher than that of 40-h cells and cell walls, containing 15% teichoic acids. Homogeneous bacteriolytic enzymes of the lysoamidase complex (muramidase and two bacteriolytic peptidases) most efficiently hydrolyzed S. aureus and Str. chrysomallus cell walls, characterized by the highest content of ribitol teichoic acids, and did not hydrolyze purified peptidoglycan.

The bacteriolytic peptidase L1 has been isolated from the enzyme preparation of lysoamidase capable to lyze cell walls of gram-positive bacteria using ion-exchange chromatography and gel filtration. Some physico-chemical properties of the enzyme have been established. The molecular mass of L1 is 21 kDa, the pH optimum for Staphylococcus aureus cell lysis is 7-11. The optimal concentration of the buffer is 50 mM; the temperature optimum is 70 degrees C; the half-inactivation temperature is 55 degrees C.

It was shown that long-term cultivation of Xanthomonas sp. - a bacteriolytic enzyme-complex producer-on media providing for high bacteriolytic activity caused the development of cells with low activity that, due to their more rapid,growth on these media, outgrew the active cells. The bacteriolytic complex produced by the active variant of Xanthomonas sp. contained three enzymes: muramidase and two peptidases (L(1) and L(2)), with peptidase L(1) as the dominant protein and muramidase and peptidase L(2) as minor components. The bacteriolytic complex of the low-activity variant was composed of two enzymes: muramidase and peptidase L(2) Peptidase L(1) was not secreted by this strain. It was concluded that the total bacteriolytic activity of the active Xanthomonas sp. variant was mainly determined by the enzyme L(1), whereas muramidase and peptidase L(2) provided insignificant lyric activity.

Lysoamidase, a bacteriolytic complex from the culture liquid of Xanthomonas sp., hydrolyzed the cells walls of Staphylococcus aureus, Streptomyces chrysomallus, and Streptomyces azureus, which contain ribitol teichoic acids in addition to peptidoglycan. The cell walls of Streptomyces roseoflavus, Glycomyces harbinensis, and Nocardiopsis dassonvillei, containing glycerol teichoic acids, were not hydrolyzed by lysoamidase. The extent of the hydrolysis of 20-h Str. chrysomallus cells and cell walls, containing 40% ribitol teichoic acids, was considerably higher than that of 40-h cells and cell walls, containing 15% teichoic acids. Homogeneous bacteriolytic enzymes of the lysoamidase complex (muramidase and two bacteriolytic peptidases) most efficiently hydrolyzed S. aureus and Str chrysomallus cell walls, characterized by the highest content of ribitol teichoic acids, and did not hydrolyze purified peptidoglycan.

A bacteriolytic muramidase was isolated to highly purified state from culture filtrate of Xanthomonas sp. bacteria using ion-exchange chromatography and gel filtration. The enzyme has molecular weight 22.4 kD, pH optimum for Staphylococcus aureus cell lysis at 8.0, and optimum reaction temperature 60 degrees C. Incubation of the enzyme at 65 degrees C for 15 min resulted in 50% loss of activity. The activity of the enzyme increased with decreasing buffer concentration in the assay medium with maximal activity being observed in H2O.