Di (2-ethylhexyl)phthalate content of plasma fractions prepared by low temperature ethanol (Cohn) fractionation.

Plasma pools, prepared from time-expired blood stored in plastic packs, were analysed for DEHP content and levels ranging from 0.78 to 7.42 mh DEHP/100 ml plasma obtained. The DEHP content of the Cohn fractions prepared from such plasma pools was estimated. Only very low levels of DEHP were found in the four therapeutically useful fractions and high levels in the discard fractions III and IV.

Preparation of metabolically active Staphylococcus aureus protoplasts by use of the Aeromonas hydrophila lytic enzyme.

Stable, metabolically active protoplasts of Staphylococcus aureus have been prepared by the use of a staphylolytic enzyme produced by Aeromonas hydrophila. Respiratory and glycolytic rates and synthesis of nucleic acids, protein, and lipid in these protoplasts, stabilized variously in 1.1 M sucrose, 0.37 M sodium succinate, or 0.37 M sodium sulfate, have been shown to be comparable with the same parameters in intact cells under the same conditions.

Formation and secretion of induced penicillinase in protoplasts of Staphylococcus aureus.

Conditions suitable for induced formation and secretion of penicillinase (EC 3.5.2.6) in protoplasts of Staphylococcus aureus were determined. No requirement was found for cells to be exposed to inducer prior to formation of protoplasts. Neither cell wall components nor mesosomes appeared to be necessary for induction or secretion. In medium containing 1.1 M sucrose about half of the formed enzyme was soluble, whereas in medium containing 0.37 M sodium succinate only about 10% of the penicillinase remained protoplast-bound. Low concentrations of polyanions (dextran sulfate and potassium polyvinyl-sulfonate) inhibited the formation of induced penicillinase, as did 4-acetamido-4'-isothiocyano-stilbene 2,2'-disulfonic acid. None of these compounds inhibited the activity of native penicillinase, and none would be expected to pass through the protoplast membrane. Penicillinase, denatured in 4 M urea, could be renatured by dilution in the presence of benzylpenicillin, and the above three inhibitors interfered strongly with this process. The results are taken as evidence that penicillinase may be secreted through the protoplast membrane in an incompletely folded form.

Exopenicillinase synthesis in Staphylococcus aureus.

In Staphylococcus aureus, penicillinase remaining cell-bound (60 to 75% of original total) after treatment with citrate does not become exopenicillinase. Exopenicillinase in these cells appears only under conditions permitting de novo penicillinase synthesis. By use of (14)C-labeled cells, it was shown that exopenicillinase consists of newly synthesized molecules which have not equilibrated with preformed membrane-bound enzyme.

Purification, properties and mechanism of action of a staphylolytic enzyme produced by Aeromonas hydrophila.

1. An enzyme produced by Aeromonas hydrophila and capable of lysing Staphylococcus aureus cells was purified 180-fold by gel filtration and chromatography on columns of AG-50 W resin. 2. Physical measurements on the purified enzyme suggest that it is a small basic protein with an isoelectric point between pH9.0 and pH9.5. 3. Maximum lytic activity was obtained in 20mm-tris-glycine buffer, pH8.5, at 45 degrees , with no detectable activity in the absence of a nitrogenous base. 4. The enzyme is active in the above buffer containing 1.5m-sucrose, and is useful for the preparation of protoplasts of Staphylococcus aureus. 5. Purified cell wall peptidoglycans of two strains of Staphylococcus aureus, differing in amino acid composition, were hydrolysed by the enzyme with the liberation of glycine oligopeptides, principally diglycine and triglycine. 6. Synthetic glycine oligopeptides larger than triglycine, but not polyglycine, were hydrolysed, as were a number of leucine-containing dipeptides and tripeptides, but no proteolytic activity could be demonstrated. 7. It is concluded that the enzyme is lytic towards Staphylococcus aureus because it splits the pentaglycine cross-links of the cell-wall peptidoglycan.

Adenosine triphosphatase in isolated membranes of Staphylococcus aureus.

The preparation of cytoplasmic membranes from suspensions of Staphylococcus aureus lysed by an enzyme recently isolated in these laboratories is described. These membranes contained: protein, 34.4%; ribonucleic acid, 6.6%; lipids, 34.5%; and total phosphorus, 1.4%. Such membranes exhibited adenosine 5'-triphosphatase (E.C. 3.6.1.3) activity, liberating orthophosphate at an initial rate of 0.53 mumole per min per mg of protein under optimal conditions. The enzyme was Mg(++)-dependent and K(+)- or Na(+)-stimulated. Maximal activity was observed with a molar adenosine 5'-triphosphate (ATP) to Mg(++) ratio of 1. One mole of orthophosphate was liberated per mole of ATP; the other product of digestion was adenosine 5'-diphosphate. Inorganic pyrophosphate and the 5'-triphosphates of guanosine, uridine, and cytidine were also attacked by membrane preparations, but more slowly than ATP. Ouabain, p-chloromercuribenzoate, and 2,4-dinitrophenol did not alter adenosine triphosphatase activity, whereas both Atebrine and chlorpromazine were inhibitory.

Electron microscopy of the lysis of Staphylococcus aureus cell walls by Aeromonas lytic factor.

Isolated and purified cell walls of Staphylococcus aureus were treated with a purified fraction of the culture supernatant fluid of a species of Aeromonas. The course of lysis of the cell walls was followed over a period of time by examination of samples under an electron microscope. The undifferentiated cell wall was rapidly digested, but the equatorial rings were more resistant. The undifferentiated cell wall became a very thin sheet before completely dissolving, leaving a series of equatorial rings of various widths. As digestion proceeded, solubilization of the entire cell wall occurred. Analogous findings were obtained with purified S. aureus mucopeptide. It is concluded that the Aeromonas lytic principle is an enzyme, and that susceptible bonds are more concentrated in the undifferentiated cell wall mucopeptide.

Liberation of surface-located penicillinase from Staphylococcus aureus.

1. Growth of Staphylococcus aureus (8325; alphai(-)p(+)), constitutive for the production of penicillinase, in CY medium results in about 40% of the enzyme being free in the medium. By modifying the medium, 98% of the enzyme remains cell-bound. 2. Part of this is bound ionically to the surface of the cell wall and may be liberated instantaneously by certain inorganic anions. Maximum liberation was achieved with either phosphate or arsenate, both of which showed marked pH-dependence. 3. Polyanions that do not penetrate the cell wall, such as heparin, RNA and dextran sulphate, are also effective in liberating penicillinase. 4. Polyanions added to the growth medium prevent the appearance of ionically bound penicillinase owing to their strong affinity for the sites on the cell wall required for binding of the enzyme.

Influence of organic anions on the liberation of penicillinase from Staphylococcus aureus.

1. A further fraction of ;cell-bound exopenicillinase', in addition to that bound ionically to the surface of the cell wall, has been identified in Staphylococcus aureus. 2. Treatment of cells with low concentrations (0.01m) of organic anions liberates part of this exopenicillinase, such liberation being dependent on both time and temperature, but independent of an external supply of energy. 3. Tricarboxylic acids tend to be more effective than dicarboxylic or monocarboxylic, although there are large differences between ionic species even when tested at the same normalities.