Hiroshi Souzu

The mechanism of indoleacetic acid oxidase reaction catalyzed by turnip peroxidase

Abstract In the iron-reducing activity of peroxidase in the presence of indoleacetic acid as electron donor, the kinetic and stoichiometric evidence supports a free-radical mechanism in which peroxidase catalyzes the formation of 2 moles indoleacetic acid radicals at the expense of 2 moles indoleacetic acid and 1 mole hydrogen peroxide. Inability of peroxidase to catalyze peroxidation of indoleacetic acid under anaerobic conditions is attributed to inactivating action of free radicals upon the enzyme, which is prevented by the addition of some oxidants. The effects of carbon monoxide and Mn 2+ were also examined.

Physical-chemical basis of the protection of slowly frozen human erythrocytes by glycerol.

One theory of freezing damage suggests that slowly cooled cells are killed by being exposed to increasing concentrations of electrolytes as the suspending medium freezes. A corollary to this view is that protective additives such as glycerol protect cells by acting colligatively to reduce the electrolyte concentration at any subzero temperature. Recently published phase-diagram data for the ternary system glycerol-NaCl-water by M. L. Shepard et al. (Cryobiology,13:9-23, 1976), in combination with the data on human red cell survival vs. subzero temperature presented here and in the companion study of Souzu and Mazur (Biophys. J.,23:89-100), permit a precise test of this theory. Appropriate liquidus phase-diagram information for the solutions used in the red cell freezing experiments was obtained by interpolation of the liquidus data of Shepard and his co-workers. The results of phase-diagram analysis of red cell survival indicate that the correlation between the temperature that yields 50% hemolysis (LT(50)) and the electrolyte concentration attained at that temperature in various concentrations of glycerol is poor. With increasing concentrations of glycerol, the cells were killed at progressively lower concentrations of NaCl. For example, the LT(50) for cells frozen in the absence of glycerol corresponds to a NaCl concentration of 12 weight percent (2.4 molal), while for cells frozen in 1.75 M glycerol in buffered saline the LT(50) corresponds to 3.0 weight percent NaCl (1.3 molal). The data, in combination with other findings, lead to two conclusions: (a) The protection from glycerol is due to its colligative ability to reduce the concentration of sodium chloride in the external medium, but (b) the protection is less than that expected from colligative effects; apparently glycerol itself can also be a source of damage, probably because it renders the red cells susceptible to osmotic shock during thawing.

Studies on the damage to Escherichia coli cell membrane caused by different rates of freeze-thawing

Freeze-thawing of Escherichia coli cells caused a release of cell membrane components such as protein, phospholipids and lipopolysaccharides. A greater amount of release and a lesser extent of cell survival were seen in slow freeze-thawing than in rapid freeze-thawing. Several dehydrogenases in the cells were also freed. The mode of release was also dependent on the rate of freeze-thawing. The materials released by slow freeze-thawing were found to be mostly composed of outer membrane components, whereas the materials released by rapid freeze-thawing contained cytoplasmic as well as outer membrane components. The chemical composition of these fragments differed significantly from that of the original membranes. The relative content of cytoplasmic membrane-bound enzymes in these fragments also differed from that of the cytoplasmic membrane. The fragmentation was assumed to have resulted mainly from the crystallization of external water. In slow fraeeze-thawing, it was considered that the phase separation of the membrane phospholipid bilayer increased the possibility of outer membrane fragmentation. Rapid freeze-thawing caused cytoplasmic membrane damage to the cells as well as to the outer membrane. In rapid freeze-thawing, the effect of phase separation appeared to be small because of rapid passage through the transition temperatures. The presence of 10% glycerol completely inhibited the release of cellular materials and enzymes. Cell survival was maintained at a high level in the glycerol-treated samples whether freeze-thawed slowly or rapidly.

Location of polyphosphate and polyphosphatase in yeast cells and damage to the protoplasmic membrane of the cell by freeze-thawing.

Abstract In an investigation with intact or frozen-thawed cells and protoplasts it was found that the polyphosphatase which can decompose polyphosphate between pH 4 and 8 was located inside the cell membrane, and that acid phosphatase and polyphosphate were in the cell wall. The freeze-thawing treatment resulted in damage to the protoplasmic membrane in the yeast cells; lactose and polyphosphate, which normally can penetrate only the cell wall, became capable of penetrating the protoplasmic membrane into the cell. From the results obtained, it was assumed that damage to the protoplasmic membrane caused by freeze-thawing may bring about contact of polyphosphatase and polyphosphate, which are located, respectively, inside and outside the membrane.

Fluorescence polarization studies on Escherichia coli membrane stability and its relation to the resistance of the cell to freeze-thawing. II. Stabilization of the membranes by polyamines

The effects of polyamines, spermine, spermidine and putrescine on the stabilization of the membrane organization of Escherichia coli cells were studied using measurements of fluorescence polarization change of extrinsic fluorescence probes in membrane specimens as a function of temperature. The effects of the polyamines on the restoration of the cell viability after freeze-thawing were also investigated. In logarithmic-phase membrane specimens, polyamines depressed the polarization ratio increase below the transition temperatures in a dose-dependent manner. The physiologically relevant concentration of polyamines repressed the ratios to the same levels as are obtained with the stationary-phase specimens. In the stationary-phase specimens, no effect of polyamines on repression of the polarization increase was observed. A preliminary exposure of logarithmic-phase cells to polyamines protected the cells from the reduction of viability in freeze-thawing. However, a considerably high concentration and a certain length of preincubation time were required in order to an effect to be exerted. These results indicate that the intracellular polyamines could stabilize the membrane organization of logarithmic-phase cells to the same extent as in the stationary-phase cell membranes. It is conjectured that the membrane stability which is mediated by the polyamines results in cellular resistance to freeze-thawing, as it is attained by increasing the growth phase of the cells.

Fluorescence polarization studies on Escherichia coli membrane stability and its relation to the resistance of the cell to freeze-thawing. I: Membrane stability in cells of differing growth phase

Physical properties of Escherichia coli membrane lipids in logarithmic- and stationary-phase cells were studied by measuring the fluorescence polarization change of cis- and trans-parinaric acid as a function of temperature. In aqueous dispersions of phospholipids extracted from cytoplasmic and outer membranes of cells of differing growth phase, a similar polarization increase was observed over the range from physiological temperature to below 0 degrees C, and nearly the same transition ratios were obtained in all samples. The cytoplasmic membrane of both of the growth-phase cells showed a higher polarization ratio above the transition temperatures, compared to that in the aqueous dispersion of phospholipids. The polarization ratios below the transition temperatures of these specimens were lower than the value obtained with the lipids, especially in the stationary-phase specimens. The outer membrane specimens showed a similar polarization change but the transition temperature ranges were considerably higher both in the logarithmic- and the stationary-phase specimens, compared to those in the cytoplasmic membrane specimens. Freeze-thawing of logarithmic-phase cells showed the emergence of activity of certain enzymes which are known to be located in the membranes. The stationary-phase cells did not suffer from any such deleterious effect and maintained a high level of cell viability in a similar treatment. These results indicate that in the stationary-phase cell membranes lipids are in a highly ordered state, and the lipid state causes a membrane stability which results in the high resistance of the cell to freeze-thawing.

Decomposition of polyphosphate in yeast cell by freeze-thawing

Abstract Following the freezing and thawing of yeast cells, it was shown that a polyphosphate in the cells was decomposed to orthophosphate and released from the cells. The release of phosphates was restricted to polyphosphate and orthophosphate native to the cells. Decomposition and liberation of polyphosphate in frozen-thawed cells was dependent upon the pH and was inhibited by an addition of sodium fluoride or heating of the cells. Polyphosphatase, also present in the cells, did not lose its activity by the freezing and thawing. Its behavior on decomposing polyphosphate coincided closely with that of polyphosphate decomposition in frozen-thawed cells, in the pH-activity curve, NaF sensitivity, and heat stability.

Effect of residual moisture content on the survival of freeze-dried bacteria during storage under various conditions

Summary Aqueous suspensions of Escherichia coli were dried under various conditions of freeze-drying to obtain residual moisture contents ranging from approximately 0 to 20%. The effect of the residual moisture content on the survival rate of these cells during relatively short storage periods was investigated. The results showed that a high residual moisture content produced a rapidly lowered survival rate in cells stored under vacuum or nitrogen, and that the reverse was true for cells stored in air. Higher storage temperatures also produced lowered survival rates.

Studies of the effect of drying conditions on residual moisture content and cell viability in the freeze-drying of microorganisms

Summary A series of studies were made to investigate the effect of the dehydration of cellular water upon cell viability during the drying process in freeze-drying of microorganisms. Water suspensions of Escherichia coli cells were used in these experiments, and the drying conditions were controlled in order to determine how to obtain dried cells that retained a high percentage of residual moisture. The influence of the temperature and the drying rate upon the relationship between the residual moisture content and cell survival was investigated. From the results obtained, it was ascertained that the temperature and the drying rate in secondary drying did not greatly influence the survival rate of the dried cells. In general, it was assumed that survival of the dried cells is primarily affected by dehydration of unfreezable cellular water and therefore depends upon the residual moisture content.

Changes in chemical structure and function in Escherichia coli cell membranes caused by freeze-thawing. I. Change of lipid state in bilayer vesicles and in the original membrane fragments depending on rate of freezing

The effect of different rates of freezing on the character of lipids in unilamellar lipid bilayer vesicles and in the original membrane fragments of Escherichia coli B cells was investigated by measuring the temperature-dependent fluorescence polarization ratio changes of cis- and trans-parinaric acids. In lipid bilayer vesicles, both slow and rapid freezing brought about significant alterations in fluorescence polarization ratios in the specimens derived from both logarithmic and stationary-phase cells. In the original membrane fragments derived from logarithmic-phase cells, slow freezing gave rise to a similar alteration in fluorescence polarization ratio change, but no such alteration was found in the case of rapid freezing. Logarithmic-phase cells suffered from a membrane permeability change during slow freezing, which subsequently resulted in low cell viability. The cells suffered only slight impairment in membrane function during rapid freezing, and maintained higher viability. These results suggest that the primary site of damage due to freezing of the cells is the cellular membranes, and this destruction is due to a lipid state change in the membranes brought about by freezing.