Penicilloic Acid Research Articles

Screening bacterial colonies for penicillinase production.

RECENT syntheses of novel penicillins1,2 have led to revived interest in the detection of species and strains of bacteria able to destroy penicillin; it has also become important to identify the destructive agent. Screening methods based on substantial diminution of the antibiotic activity of penicillin against Gram-positive bacteria3,4 or on changes in pH associated with the formation of extra carboxyl groups4,5 have always had a range of application limited by their susceptibility to interference from general metabolic products. It has now become evident that such methods are also unsuitable for distinguishing penicillinase from penicillin acylase6–8. But penicillinase converts penicillin to penicilloic acid which, being a thiazolidine with a non-acylated imino group, reacts with iodine; whereas the 6-amino-penicillanic acid formed by penicillin acylase has an intact β-lactam ring and is as inert towards iodine as penicillin itself.

Microbial resistance to penicillin as related to penicillinase or penicillin acylase activity.

Summary1. The penicillin moiety, 6-aminopenicillanic acid, has antibacterial activity but is considerably less active than penicillin. 2. Resistance to penicillin in one group of bacteria could be explained logically by presence of penicillinase activity, i.e., conversion of penicillin to the inactive penicilloic acid. 3. Resistance to penicillin in a second group of bacteria could be explained logically by presence of penicillin acylase activity, i.e., conversion of penicillin to the relatively inactive 6-aminopenicillanic acid. 4. In a third group of bacteria resistant to penicillin, neither penicillinase nor penicillin acylase activity could be detected. Still another metabolic mechanism other than penicillinase or penicillin acylase activity must be present in selected bacteria.

The development of antibodies to penicilin in rabbits.

A method for the production of antibodies specifically directed against penicillin is described. The inability of this antibody to significantly reduce the antibiotic activity of penicillin is noted. Evidence to show the variability of specificities of various sera, some directed for the most part against the side chain, others against the nucleus is presented. Studies on serum fractions separated electrophorectically indicate that the antibody migrates in the fast gamma-globulin and beta-globulin fractions and requires dextran or albumin to effect agglutination. The inability of penicilloic acid, an hydrolysis product of penicillin to provoke antibody formation despite its ability to inhibit the antibody is shown and the implications of this observations are discussed.

The mechanism of inactivation of penicillin by cysteine and other mercaptoamines

The kinetics of penicillin inactivation by various mercaptoamines have been followed by polarimetry. In the case of primary mereaptoamines the reactions are of second order with respect to time and concentration of the reactants. By determining the appropriate rate constants under different conditions of pH and ionic strength the conclusion is reached that the rate limiting step is a bimolecular reaction involving the mercaptide ion. The mechanism probably involves the formation of a semimercaptol. Several possible mechanisms for the role of the amino group in the opening of the β-lactam ring are discussed. The inactivation of penicillin by the tertiary mercaptoamine N:N-dimethylcysteamine nearly follows first order kinetics and gives as end product a partially racemized penicilloic acid.

家兎肝臓を用いてのペニシリンGの分解

Decomposition of penicillin G by rabbit liver was examined. By perfusion through surviving rabbit liver, it was found that penicillin G is hydrolyzed in rabbit liver to penicilloic acid. Perfusion for 2hours, with initial concentration of the penicillin in flowing fluid at 900γ/cc., resulted in the decrease of penicillin concentration to 250γ/cc. and formation of ca. 400γ/cc. of penicilloic acid. Formation of penicilloic acid was identified from paper electrophoresis and from Rf values in paper chromatography using butanol, isobutanol, or isoamyl alcohol, saturated with 0.2M phosphate buffer (pH 6.0), as a developing solvent. Coloration of the chromatogram utilized the reduction of arsenomolybdic acid to blue color and coloration of the spot to brown by spraying 0.1N ammonia water and silver nitrate.Penicillin also changed to penicilloic acid on application of liver homogenate but the penicillin-decomposing enzyme is so rapidly inactivated that the liver must be homogenized under ice-cooling immediately after killing of the animal and reacted at once with penicillin. Optimal pH of this reaction is 6.9 and the reaction rate is proportional to the concentration of the homogenate used. No decomposition of penicillin occurred when the homogenate was heated at 100° for 3minutes. When liver homogenate and penicillin G were reacted, 3.2mg. of penicillin per 1g. wet weight of the liver was hydrolyzed and 3mg. of penicilloic acid was formed. Excess of penicillin did not inhibit this reaction. Ferric nitrate and calcium nitrate, which inhibit penicillinase of bacteria, do not inhibit hydrolysis of penicillin by liver homogenate and, therefore, penicillin-decomposing enzyme present in the liver is different in nature from bacterial penicillinase.

Simultaneous Determination of Penicillin and Penicilloic Acid in Fermentation Samples by Colorimetric Method

Simultaneous Determination of Penicillin and Penicilloic Acid in Fermentation Samples by Colorimetric Method

The binding of penicillin in relation to its cytotoxic action. III. The binding of penicillin by mammalian cells in tissue culture (HeLa and L strains).

1. (a) Mammalian cells in tissue culture (mouse fibroblasts and malignant human uterine epithelium) did not concentrate penicillin from the culture medium. Even at low concentrations, the cellular accumulation was usually less than that in the surrounding fluid, and most of it was removed by washing. The radioactive material in such eluates was actively bactericidal, and was presumably in large part unchanged penicillin. (b) Penicilloic acid, produced by the action of penicillinase, was bound to the same (limited) extent as the active antibiotic. In both these respects mammalian cells behaved like naturally penicillin-resistant bacteria, and unlike such penicillin-sensitive bacteria as Streptococcus pyogenes or Diplococcus pneumoniae. 2. Cell-free sonic extracts of the L strain had the same limited reactivity with penicillin as the intact cells. The relatively minute amounts bound by the cells are therefore not due to their impermeability, but instead reflect the inherently low reactivity of the cellular constituents with penicillin. 3. It is suggested that the relative non-toxicity of penicillin for the mammalian host, and for mammalian cells in tissue culture, may be related to this low order of reactivity with the antibiotic.

THE BINDING OF PENICILLIN IN RELATION TO ITS CYTOTOXIC ACTION

1. Bacteria exposed to C(14)- or S(35)-labelled penicillin bound and concentrated the antibiotic. The amount bound from low concentrations (0.001 to 0.01 microg. per ml.) was related to the penicillin sensitivity of the strain; and highly sensitive organisms such as Streptococcus pyogenes concentrated the antibiotic as much as 200-fold. The material bound by highly sensitive strains from these low concentrations of penicillin was removed to only a minor extent by washing. Penicillin inactivated with penicillinase, by hot H(2)SO(4) or by cold HCl was not similarly concentrated. 2. Despite wide differences in their sensitivity to penicillin, at equieffective (LD(99.9)) levels, four of the five strains here studied had bound comparable amounts of antibiotic. That lethal intracellular concentration averaged 1.7 to 4 microg. per gm., and 1600 to 3300 molecules per cell. 3. Bacteria in the logarithmic phase of growth, "resting" organisms, and cell-free sonic extracts, had roughly the same reactivity with penicillin. The differences in the amount of penicillin bound by bacteria of varying sensitivity therefore do not rest on differences in the permeability of the cell. 4. Escherichia coli (K12)-inactivated penicillin within the cell; and that inactivation adequately explains its low binding affinity, and its relative resistance. The other species here studied inactivated the diffusible cellular penicillin to only a minor degree, and not enough to explain the differences in their penicillin-sensitivity. The working hypothesis is suggested that their varying sensitivity is instead determined by differences in the reactivity of vital cell components with the antibiotic. 5. At high concentrations of antibiotic (1 to 1000 microg. per ml.), there was non-specific binding by all the cell strains and cell extracts here studied, unrelated to their sensitivity, and masking the specific differences in reactivity noted at low concentrations. At these high levels, penicilloic acid was bound to the same degree as the active antibiotic.

A method for the determination of penicillinase.

Henry and Housewright1 described a manometric method for the assay of penicillinase which was based on the formation of penicilloic acid from penicillin in the presence of this enzyme. It has been found that a convenient way of following this reaction without the use of a manometric apparatus is to estimate penicilloic acid iodometrically. The uptake of iodine by penicilloic acid is not stoichiometric, 6–9 equivalents of iodine being absorbed per mole of penicilloic acid according to conditions2. However, with a standardized procedure, reproducible results can be obtained.

抗生物質の理化学的研究 (第2報)

Decomposition velocity of procaine penicillin in aqueous solution was measured at various temperatures and pH. All showed primary reaction and the velocity was found to be the smallest at around pH 6.0. Absorption spectra during the decomposition were measured and it was assumed that procaine penicillin underwent decomposition to penicilloic acid above pH 6.0.