Apparatus for the Maintenance of Bacterial Cultures in the Steady State : II. Improved turbidity control and culture cell

(1) A more convenient method for maintaining constant turbidity in the culture cell is described. (2) An improved culture cell and cell wipers which do not cause foaming are described

CRYSTALLINE PEPSIN : II. GENERAL PROPERTIES AND EXPERIMENTAL METHODS

A number of properties of crystalline pepsin have been determined and are summarized in Table II. The experimental procedure used is described

INCREASE IN BACTERIOPHAGE AND GELATINASE CONCENTRATION IN CULTURES OF BACILLUS MEGATHERIUM

1. The increase in bacteria, phage concentration, and gelatinase concentration in cultures of B. megatherium has been determined. 2. With lysogenic cultures the phage concentration, gelatinase concentration, and bacteria concentration increase logarithmically at first. The phage and gelatinase concentration then decrease while the bacteria concentration increases to a maximum. 3. The results are the same with sensitive cultures if the ratio of phage to bacteria is small. If the ratio of phage to bacteria is large phage, gelatinase, and bacteria concentration all increase at first and then decrease. The maximum rate of increase coincides approximately with the maximum rate of oxygen consumption of the culture. 4. 60–90 per cent of the phage is free from the cells. 5. The amount of phage produced is determined by the combined phage and not by the total phage. 6. Phage is produced during growth of the cells and not during lysis. 7. In a very narrow range of pH near 5.55 no increase in bacteria occurs but large increases in phage may be obtained

CONCENTRATION AND PURIFICATION OF B. MEGATHERIUM PHAGE

B. megatherium phage, like staphylococcus phage, is inactivated by repeated high speed centrifugation (Northrop, 1938; Hotchin, 1954). This method of purification has the additional disadvantage that large volumes of material cannot be handled. B. megatkerium phage is precipitated from peptone solution by 0.4 saturated ammonium sulfate. The precipitate is filtered off, washed, and dissolved in a small volume of 1 ~ sodium acetate. A few-/of trypsin are added and this solution kept at 0 ° for several days. The protein content of this solution is now nearly equivalent to that calculated from its active phage content. The phage may be precipitated from this solution by the slow addition (with stirring) of an equal volume of saturated magnesium sulfate. A fine silky precipitate forms which consists of highly refractile irregular particles and fibers. This precipitate contains all the phage activity. The number of plaques formed by 1 rag. nitrogen of this precipitate is very nearly equal to the number calculated from the size (Murphy, 1954) of the phage particle. If a solution of this precipitate is heated or made acid so as to inactivate the phage, the protein then appears as a flocculent amorphous precipitate. ExperimentaL--An example of the method is shown in Table I. Experimental Procedures All filtrations, washings, etc., were carried out as described in Crystallin

PEPSIN ACTIVITY UNITS AND METHODS FOR DETERMINING PEPTIC ACTIVITY

Experimental methods are described for determining the activity of pepsin preparations by means of changes in the viscosity of gelatin, casein, edestin, and powdered milk solutions, and by the rate of formation of non-protein nitrogen from casein and edestin solutions, or by the increase in formol titration of casein, edestin or gelatin. Activity units for pepsin are defined in terms of these measurements

A TEST FOR DIFFUSIBLE IONS : II. THE IONIC NATURE OF PEPSIN.

1. The distribution of pepsin between particles of gelatin or coagulated egg albumin and the outside solution has been found to be equal to the distribution of chloride or bromide ion under the same conditions. This is the case from pH 1 to 7, and in the presence of a variety of salts. 2. Pepsin is therefore probably a monovalent anion. 3. Under certain conditions the enzyme may be adsorbed on the surface of the protein particles. This reaction is irreversible and is markedly influenced by the presence of low concentrations of electrolytes

THE KINETICS OF THE DECOMPOSITION OF PEROXIDE BY CATALASE

It has been shown that the experimental results obtained by Morgulis in a study of the decomposition of hydrogen peroxide by liver catalase at 20°C. and in the presence of an excess of a relatively high concentration of peroxide are quantitatively accounted for by the following mechanisms. 1. The rate of formation of oxygen is independent of the peroxide concentration provided this is greater than about 0.10 M. 2. The rate of decomposition of the peroxide is proportional at any time to the concentration of catalase present. 3. The catalase undergoes spontaneous monomolecular decomposition during the reaction. This inactivation is independent of the concentration of catalase and inversely proportional to the original concentration of peroxide up to 0.4 M. In very high concentrations of peroxide the inactivation rate increases. 4. The following equation can be derived from the above assumptions and has been found to fit the experiments accurately. See PDF for Equation in which x is the amount of oxygen liberated at the time t, A is the total amount of oxygen liberated (not the total amount available), and K is the inactivation constant of the enzyme

ABSORPTION OF PEPSIN BY CRYSTALLINE PROTEINS

Crystalline proteins, such as edestin or melon globulin, remove pepsin from solution. The pepsin protein is taken up as such and the quantity of protein taken up by the foreign protein is just equivalent to the peptic activity found in the complex. The formation of the complex depends on the pH and is at a maximum at pH 4.0. An insoluble complex is formed and precipitates when pepsin and edestin solutions are mixed and the maximum precipitation is also at pH 4.0. The composition of the precipitate varies with the relative quantity of pepsin and edestin. It contains a maximum quantity of pepsin when the ratio of pepsin to edestin is about 2 to 1. This complex may consist of 75 per cent pepsin and have three-quarters of the activity of crystalline pepsin itself. The pepsin may be extracted from the complex by washing with cold N/4 sulfuric acid. If the complex is dissolved in acid solution at about pH 2.0 the foreign protein is rapidly digested and the pepsin protein is left and may be isolated. The pepsin protein may be identified by its tyrosine plus tryptophane content, basic nitrogen content, crystalline form and specific activity

THE EFFECT OF TRYPSIN, CHYMOTRYPSIN, RIBONUCLEASE, AND DESOXYRIBONUCLEASE ON ACTIVE, INACTIVE, AND REVERSIBLY INACTIVATED MEGATHERIUM PHAGE

The effect of trypsin, chymotrypsin, and desoxyribonuclease on active, reversibly inactivated, and heat-inactivated B. megatherium phage, and on living and dead B. megatherium and B. coli has been determined. The results are summarized in Table I