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PubMed1989

Comparative spectral analysis of mammalian, fungal, and bacterial catalases. Resonance Raman evidence for iron-tyrosinate coordination.

Resonance Raman spectra are reported for catalases from bovine liver, the ascomycete fungus Aspergillus niger, and the bacterium Micrococcus luteus. The vibrational frequencies of the oxidation-, spin-, and coordination number-sensitive spectral bands are indicative of high spin pentacoordinate hemes in the resting ferric enzymes of each of these organisms. This result is in accord with the crystal structure of bovine catalase (Fita, I., and Rossmann, M.G. (1985) J. Mol. Biol. 185, 21-37). In contrast, the crystallographic study of catalase from the ascomycete Penicillium vitale (Vainshtein, B. K., Melik-Adamyan, W. R., Barynin, V. V., Vagin, A.A., Grebenko, A. I., Borisov, V. V., Bartels, K. S., Fita, I., and Rossmann, M. G. (1986) J. Mol. Biol. 188, 49-61) showed electron density on the distal side of the heme which could imply the presence of a sixth ligand, possibly a water molecule. However, both of these crystallographic studies showed the proximal ligand in catalase to be a tyrosine. The present study confirms tyrosinate coordination in each of the three catalases from the appearance of selected resonance-enhanced tyrosine vibrational modes. The most characteristic band is the tyrosinate ring mode at approximately 1612 cm-1 which is maximally enhanced with 488.0 nm excitation. The appearance of tyrosinate modes at 1607 and 1245 cm-1 in the resonance Raman spectra of M. luteus cyano catalase serves to identify tyrosine as an axial ligand in bacterial as well as eukaryotic catalases. Unlike non-heme iron tyrosinate proteins, whose resonance Raman spectra are dominated by several intense bands diagnostic of tyrosine ligation, the heme-linked tyrosine modes are not easily distinguished from the large number of porphyrin vibrations.

Animals↗
PubMed1979

Distribution of membrane-bound monoamine oxidase in bacteria.

The distribution of membrane-bound monoamine oxidase in 30 strains of various bacteria was studied. Monoamine oxidase was determined by using an ammonia-selective electrode; analyses were sensitive and easy to perform. The enzyme was found in some strains of the family Enterobacteriaceae, such as Klebsiella, Enterobacter, Escherichia, Salmonella, Serratia, and Proteus. Among strains of other families of bacteria tested, only Pseudomonas aeruginosa IFO 3901, Micrococcus luteus IFO 12708, and Brevibacterium ammoniagenes IAM 1641 had monoamine oxidase activity. In all of these bacteria except B. ammoniagenes, monoamine oxidase was induced by tyramine and was highly specific for tyramine, octopamine, dopamine, and norepinephrine. The enzyme in two strains oxidized histamine or benzylamine. Correlations between the distributions of membrane-bound monoamine oxidase and arylsulfatase synthesized in the presence of tyramine were discussed.

Bacteria↗
PubMedDate not supplied

[Stability of Escherichia coli to the membranotropic antibiotic gramicidin S].

The work was aimed at studying the effect of gramicidin S on the intact cells, spheroplasts and membrane specimens of Escherichia coli K12S with the natural resistance to this antibiotic. The resistance was shown to be caused by the barrier properties of the cell wall: the spheroplasts were highly sensitive to the lytic action of gramicidin S. The differences in the sensitivity to gramicidin S of substrate oxidation carried by the membranes of E. coli and Micrococcus luteus, a sensitive organism, were not of crucial significance for the manifestation of the resistance. The resistance was not associated with the decrease of gramicidin S adsorption: the cells were capable of binding large quantities of the antibiotic and remaining viable. Gramicidin S appeared to be attached to the cell walls (most likely, the outer membranes) rather than the cytoplasmic membranes.

Cell Membrane↗
PubMedDate not supplied

Comparison at the molecular level of uracil-DNA glycosylases from different origins.

A nuclear and a cytoplasmic uracil-DNA glycosylase have been purified from epithelial cells derived from a rat hepatoma (H4 cells) cultured in vitro. They have different optimum pH, molecular weight, isoelectric points, activation energy, Km. Uracil acts as a non competitive inhibitor towards the nuclear enzyme while it is a competitive one for the cytoplasmic enzyme. Comparison of the properties of the two mammalian enzymes with those of the enzymes isolated from Escherichia coli and Micrococcus luteus shows that they all behave differently. The following criteria were studied: molecular weight, optimum pH, isoelectric point, inhibition by uracil analogs, modulation of their activity by polyamines or by intercalating drugs. The only common properties shared by these four enzymes are: an activity twice as high on single stranded DNA than on double stranded DNA and no requirement for divalent cation for maximal activity.

Animals↗
PubMed1979

The excision of N-methyl-N-nitrosourea-induced lesions from the DNA of Chinese hamster cells as measured by the loss of sites sensitive to an enzyme extract that excises 3-methylpurines but not O6-methylguanine.

An enzyme extract from Micrococcus luteus excises 3-methyladenine and 3-methylguanine but not O6-methylguanine, 7-methylguanine, 1-methyladenine or 7-methyladenine from DNA reacted with N-methyl-N-nitrosourea. The extract was used to detect lesions in the DNA of Chinese hamster cells treated in culture with N-methyl-N-nitrosourea. It was concluded that 3-methyladenine is excised from these cells with a half-life of about 2.3 h.

Animals↗
PubMed1981

Stereochemistry of beta-, gamma-, and epsilon-ring formation in bacterial C50.

Cell-free systems from Corynebacterium poinsettiae and Micrococcus luteus incorporated labeled mevalonic acids into acyclic C40 and cyclic C50 carotenoids. When (3R,4R)-[2-14C,4-3H1]mevalonate was used as substrate, the 14C:3H ratios of C.p.450 and sarcinaxanthin showed that the hydrogen atoms at C-2 of both carotenoids, and that at C-6 of sarcinaxanthin, are derived from the 4-pro-R position of mevalonate. The 14C:3H ratios of C.p.450 and sarcinaxanthin synthesized from (2RS,3R)-[2-14C,2-3H2]mevalonate showed that both hydrogen atoms of C-4 are derived from those at C-2 of mevalonate. These results exclude epsilon- and beta-rings as precursors of the gamma-ring. They also exclude the interconversion of the epsilon- and beta-rings. Sarcinaxanthin samples synthesized from (3R,4R)-[2-14C,4-3H1]- and (2RS,3R)-[2-14C,2-3H2]mevalonate by a cell-free system from M. luteus were found to undergo isomerization in strong alkali. The major product of isomerization (85%) was decaprenoxanthin (epsilon-ring) with the beta-ring C.p.450 present in small amounts (3% yield). The 14C:3H ratios of these isomerization products were consistent with the loss of one C-4 hydrogen atom from each epsilon-ring of the former and one C-6 hydrogen atom from each beta-ring of the latter.

Carbon Radioisotopes↗
PubMed1991

One-step purification of bacterial lipid macroamphiphiles by hydrophobic interaction chromatography.

Bacterial lipid macroamphiphiles extracted with phenol/water can be purified in one step by hydrophobic interaction chromatography. Lipids and the major part of protein are separated from macroamphiphiles during phenol/water extraction. Coextracted nucleic acids, polysaccharides, and residual protein are effectively removed by column chromatography on octyl-Sepharose whereby macroamphiphiles are primarily adsorbed and later eluted with a buffered propanol gradient. The procedure is applicable to macroamphiphiles with various lipid structures as was demonstrated using the diacylglycerol-containing lipoglycan of Micrococcus luteus, the lipid A-containing lipopolysaccharide of Salmonella typhimurium, and the diglyceryl tetraether lipoglycans of Thermoplasma acidophilum and Thermoplasma volcanicum. On elution from octyl-Sepharose, separation into molecular species of different compositions was observed with the lipopolysaccharide of S. typhimurium and the lipoglycan of T. volcanicum. It was also shown that, after phenol/water extraction, membrane lipids are completely recoverable from the phenol layer, which makes it possible to isolate lipids along with macroamphiphiles from the same sample of bacteria.

Chromatography, Agarose↗
PubMed1979

Three additional genes involved in pyrimidine dimer removal in Saccharomyces cerevisiae: RAD7, RAD14 and MMS19.

The ability to remove ultraviolet (UV)-induced pyrimidine dimers from the nuclear DNA of yeast was examined in two radiation-sensitive (rad) mutants and one methyl methanesulfonate-sensitive (mms) mutant of the yeast Saccharomyces cerevisiae. The susceptibility of DNA from irradiated cells to nicking by an endonuclease activity prepared from crude extracts of Micrococcus luteus was used to measure the presence of dimers in DNA. The rad7, rad14 and mms19 mutants were found to be defective in their ability to remove UV-induced dimers from nuclear DNA. All three mutants belong to the same epistatic group as the other mutants involved in excision-repair. All three mutants show enhanced UV-induced mutations. The rad14 mutant also shows epistatic interactions with genes in the other two UV repair pathways.

DNA Repair↗
PubMed1979

Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA.

Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of lambda greater than 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 8-(2-hydroxy-2-propyl)adenine and 8-(2-hydroxy-2-propyl)guanine as the only detectable damage in the DNA. Using this specifically photoalkylated DNA as a substrate, we discovered in extracts of Micrococcus luteus an endonucleolytic activity that is directed towards 8-(2-hydroxy-2-propyl) purines in DNA. The activity is not a combination of a DNA-glycosylase and an apurinic site endonuclease. It is not inhibited by single-stranded DNA, by UV- or gamma-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA. however, gamma- or UV-(254 nm) irradiated double-stranded DNAs to inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNAs. Divalent cations are not required for the incising activity, and it is fully active in 1 mM EDTA, whereas caffeine and ATP cause inhibition. Extracts of mutant M. luteus lacking pyrimidine-dimer-directed endonucleases were found to contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, we could demonstrate the specific excision of the 8-alkylated purines from the damaged DNA. The special conformational consequences of the 8-alkylation of purines, at the nucleotide level, namely their nonregular syn conformation, suggest that it is the distortion in the DNA that is recognized by the endonuclease.

Alkylation↗
PubMed1975

Kinetic studies on the phosphorolysis of polynucleotides by polynucleotide phosphorylase.

The kinetics of the phosphorolysis of polynucleotide (as differentiated from oligonucleotide) by polynucleotide phosphorylase of Micrococcus luteus has been investigated. Double reciprocal plots of initial velocity against either inorganic phosphate or polynucleotide concentration are linear, and furthermore, the affinity of the enzyme for either substrate is unaffected by the presence of the other. dADP, an analogue of ADP product, is a competitive inhibitor with respect to Pi and polynucleotidy. (Ap)tA-cyclic-p is a competitive inhibitor with respect to Pi. The results are almost identical with both primer-independent (Form-I) and primer-dependent (Form-T) enzymes, although the various kinetic constants differ. On the vasis of these data a rapid equilibrium random Bi Bi mechanism is proposed. The demonstration of two different inhibitor constants for dADP and the difference between the Michaelis and the inhibitor constant for polyadenylic acid in polynucleotide phosphorolysis indicate at least two binding sites for polyadenylic acid and dADP on M. luteus polynucleotide phosphorylase. Its is suggested that in the phosphorolysis of long chain polymers the second binding site permits the polynucleotide to snap right back into position after removal of I mononucleotide unit and thus leads to the observed processive degradation. A general discussion of oligonucleotide and polynucleotide phosphorolysis and the differences between Form-I and Form-T enzymes in de novo synthesis and degradation of polynucleotides is presented.

Binding Sites↗
PubMed1982

Ovine ill-thrift in Nova Scotia. 9. Production of experimental quantities of isocyanide metabolites of Trichoderma hamatum.

Laboratory cultures of Trichoderma hamatum produce metabolites that are characterized by an isocyanide functionality. Three such metabolites predominate. One is the known compound trichoviridin (I). The other two, described here for the first time, are 3-(3-isocyano-6-oxabicyclo[3,1,0]hex-2-en-5-yl)acrylic acid (II) and a very unstable compound 3-(3-isocyanocyclopent-2-enylidene-)propionic acid (III). Production of these three metabolites by a random sample of wild isolates of the fungus has been examined. At least one of these isocyanides was isolated from all cultures in which the culture broth inhibited the growth of Micrococcus luteus. The relative amounts of the three isocyanides produced by individual isolates were not the same and cultures were found in which I, II, or III was the main product. The isocyanide III was produced by all wild isolates which had antibiotic activity in their culture broth, and it was present in the concentration range 2-40 mg X L-1.

Antibodies, Fungal↗
PubMed1983

[Production and characteristics of bacteria-labeled talc dust for experimental air hygiene studies].

Freeze-drying of suspensions of Micrococcus luteus together with talc yields bacteria-labelled dust. This material can be used in experimental air hygiene. Loss of viability due to drying in air during experiments can be expected to be negligible. A wide range of particle diameters (1 to greater than 23 micron) is available. Scanning electron microscopy shows the bacteria sticking on talc particles after freeze-drying (Fig. 3a + b). Viable counts of the material decreased very slowly on storage.

Air Microbiology↗
PubMedDate not supplied

[The taxonomic structure and nature of the dominance in the skin microbiocenosis of the breasts].

The species composition of skin microbiocenosis of mammary glands in 120 nulliparae, 175 pregnant women and 120 women at the puerperal period has been studied. The dominant species of the most important ecological groups of bacteria have been established: Staphylococcus epidermidis, Micrococcus luteus and Corynebacterium lipophylicum. Permanent and additional species of the microbiocenosis have been found to form its cenotype at different periods of childbirth. They manifest by the change of the absolutely dominating species in the skin biocenosis of pregnant women and the transformation of the structure of the cenotype at the puerperal period. Proofs of the participation of the resident microflora of the skin in the formation of the cenotype of the microbiocenosis under study are presented.

Adolescent↗
PubMed1982

DNA glycosylases.

Various DNA glycosylases exist, which initiate the first step in base-excision repair. A summary of the kinetic and physical characteristics of three classes of DNA glycosylases are presented here. The first class discussed, include glycosylases which recognize alkylated DNA. Various data from enzymes derived from both prokaryotic and eukaryotic sources is discussed. The second class deals with a glycosylase that recognizes and initiates the excision of pyrimidine dimers in DNA. To date, this enzyme has only been uncovered from two sources, Micrococcus luteus and the T4 bacteriophage of E. coli. The third class consists of the most studied of the glycosylases, the uracil-DNA glycosylase enzymes. Various characteristics are presented for the uracil-DNA glycosylases derived from various sources. Recent information from our laboratory is presented implicating that herpes simplex virus may mediate a uracil-DNA glycosylase activity in productivity infected cells.

Chemical Phenomena↗
PubMed1976

Synthesis and biological activity of pyrazolo[3,4,-d]pyrimidine nucleosides and nucleotides related to tubercidin, toyocamycin, and sangivamycin.

The 6-aza analogues of toyocamycin and sangivamycin were prepared as potential cytotoxic agents. The toyocamycin analogue (4-amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine-3-carbonitrile) could not be obtained directly from its O-acetylated precursor but was accessible via 4-amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine-3-thiocarboxamide. The identity of the nitrile was verified by its ultraviolet, infrared, and mass spectra, and by its conversion to the corresponding 3-carboxamide and thiocarboxamide when treated with water or hydrogen sulfide, respectively. Bioassay of the synthetic compounds in comparison with 4-amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (6-azatubercidin) and 4-amino-2-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine revealed that the 3-thiocarboxamido derivative was more cytotoxic to the growth of mouse fibroblasts than 6-azatubercidin, effecting killing of 3T6 cells at less than or equal to 1 mug/ml. 4-Amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (but not its 2-ribofuranosyl isomer) was shown to act as a substrate for adenosine deaminase from calf intestinal mucosa with an apparent Km of 125 (vs. 20 for adenosine) and the corresponding 5'-diphosphate of 6-azatubercidin was polymerized by polynucleotide phosphorylase (Micrococcus luteus) in the presence of Mn2+ to afford a homopolymer and copolymers with adenosine. The copolymers directed the binding of [3H]lysyl-tRNA to the A-site of ribosomes from Escherichia coli, but could not be used for the synthesis of polylsine in a cellfree system. The copolymer consiting of adenosine and 6-azatubercidin in a 2:1 ratio was found to form a 1:1 complex with poly(uridylic acid) at 4degreesC.

Antibiotics, Antineoplastic↗
PubMed1971

Mutagenic action, loss of transforming activity, and inhibition of deoxyribonucleic acid template activity in vitro caused by chemical linkage of carcinogenic polycyclic hydrocarbons to deoxyribonucleic acid.

In a solvent system of 10(-2)m phosphate buffer (pH 6.8)-ethanol (2:1, v/v) and in an iodine-induced reaction, the polycyclic hydrocarbons [(3)H]3,4-benzpyrene and [(3)H]3,4-BP/[(3)H]9, 10-dimethyl-1,2-benzanthracene (DMBA) can be covalently linked to deoxyribonucleic acid (DNA) at room temperature. By stepwise addition of the hydrocarbon and repeating the reaction two to three times after isolating the hydrocarbon DNA adduct, it was possible to introduce as many as one covalently bound hydrocarbon molecule per 100 nucleotide bases. When 3,4-BP and DMBA were linked in this way to biologically active transforming DNA of Bacillus subtilis, they caused (i) reduction of the transforming activity of the DNA accompanied by (ii) significant increases in the frequency of forward mutations. The majority of these hydrocarbon-induced mutations were not able to revert spontaneously. These samples of DNA covalently linked with hydrocarbons showed much lower levels of survival of biological activity when assayed in recipient strains (hcr(-)) which are known to be deficient in the enzymes required for repair of ultraviolet light-induced damage to DNA. 3,4-BP covalently linked to calf thymus DNA at a level of approximately one hydrocarbon molecule per 330 bases was shown to cause up to 80% inhibition of the in vitro transcription of the DNA by highly purified ribonucleic acid polymerase prepared from Micrococcus luteus under the experimental condition of template saturation. The presence of 3,4-BP and DMBA molecules covalently bound to B. subtilis DNA samples was also found to prevent complete denaturation of the bihelical structure of certain DNA molecules and thus appears to effect a cross-link in these DNA molecules.

Animals↗
PubMed1977

Minimal requirements in defined media for improved growth of some radio-resistant pink tetracocci.

Defined media permitting extensive growth of representative pink radio-resistant tetracocci (Micrococcus radiodurans, Micrococcus roseus, and Micrococcus radiophilus) and two controls (an ultraviolet-sensitive mutant of M. radiodurans and Micrococcus luteus) are described. Availability of Fe (especially Fe3+) proved essential for good growth, as evidenced by (i) favorable effects of hydroxamic acids, e.g., salicylhydroxamic acid, and (ii) the growth promotion by hemin when joined with elevated concentrations of Fe. Cobalamin (B12) and methionine were interchangeable as an absolute requirement for methionine not affected by B12. M. luteus required neither. Pink radio-resistant micrococci may form a coherent group. Some divergences among them might be attributable to the method for isolating them, which for ordinary bacteria would be mutagenic to the point of total lethality. The ecology of these tetracocci vis-à-vis other pink-red radio-resistant organisms is discussed in relation to a question: can these bacteria be isolated without dependence on radiation as the cardinal selective factor?

Culture Media↗
PubMed1972

RNA-primed DNA synthesis in vitro.

In vitro DNA synthesis on single-stranded circular DNA can be initiated by RNA primers. RNA chains are covalently extended by DNA polymerase II from KB cells and DNA polymerase I from Micrococcus luteus, but not by an RNA-dependent DNA polymerase from avian myeloblastosis virus. The reaction product consists of DNA chains with a piece of RNA at their 5'-ends, hydrogen bonded to the template DNA. The primer RNA is linked to the product DNA via a 3':5'-phosphodiester bond, and can be specifically removed by ribonuclease H. The possible role of ribonuclease H in RNA-primed DNA synthesis in vivo is discussed.

Avian Leukosis Virus↗