Selective Oxidation of 2′-Deoxyguanosine to Imidazolone by the Chemical Nuclease MnTMPyP Associated to KHSO5 or Sulfite/O2.

Abstract

Mn TMPyP in the presence of sulfite/O₂ catalyses the oxidation of dG into dIz as selectively but slower and less efficiently than in the presence of KHSO_5.     

Proline hydroxylation of collagens synthesized at different temperatures in vivo by two poikilothermic species

Extent of prolyl hydroxylation in newly synthesized viper collagen is decreased at 10 degrees C to approximately 23% of normal on skin and to approximately 57% of normal in bone collagen. At 20 degrees C, prolyl hydroxylation is approximately 50% of normal in skin and normal in bone. At 10 degrees C and 20 degrees C, prolyl hydroxylation is decreased approximately 32% in the skin collagen of carp. In contrast, prolyl hydroxylation is unchanged at 10 and 20 degrees C in bone, scale and lepidotrichia. Prolyl hydroxylation of cartilaginous endoskeleton showed an approximately 25% decrease at 20 degrees C.     

DNA breaks generated by the bleomycin-iron III complex in the presence of KHSO5, a single oxygen donor

KHSO5, a water soluble single oxygen donor, is shown to be capable of activating bleomycin-FeIII complex for DNA cleavage. DNA breaks mediated by bleomycin-FeIII in the presence of H2O2 or KHSO5 are compared and the P450-like activation of metallobleomycins is discussed.     

Chondrogenesis in mouse limb buds in vitro: Effects of dibutyryl cyclic AMP treatment

Abstract. We studied the effects of dibutyryl cyclic AMP (dbcAMP) on mouse limb-bud chondrogenesis at three stages of embryonic development. After 24 h of culture, limb buds with or without a covering of ectoderm were treated with 1 mM dbcAMP for 48 h and were then compared with untreated cultured limb buds. Treatment with dbcAMP enhanced cartilaginous differentiation in organ cultures of stage-17 and -19 (according to Theiler's) limb buds, although the presence of ectoderm reduced the level of dbcAMP stimulation. By stage 20, treatment with dbcAMP irreversibly inhibited cartilaginous differentiation. These results suggest that the responsiveness of mesenchymal limb-bud cells to dbcAMP is stage related. The results of histological studies as well as of analyses of DNA content and sulphated glycosaminoglycan accumulation supported the hypothesis that dbcAMP treatment induces recruitment of initially non-chondrogenic cells whose commitment explains the enhancement of cartilaginous differentiation. Limb-bud competence for chondrogenesis throughout the three developmental stages studied is also discussed.     

Demonstration of the O-demethylation reaction catalyzed by a peroxidase: application to 9-methoxyellipticine

9-methoxy ellipticine, an antitumor compound, is O-demethylated in presence of the system peroxidase-H2O2; this reaction yields the corresponding electrophilic quinone-imine and methanol. This O-demethylation reaction is reported for the first time and might be possibly extended to some other antitumor drugs.     

Purification and properties of factor XIII from human placenta.

1. FXIII was isolated and purified over 4000 fold from human placenta to apparent electrophoretic homogeneity by a new procedure including ethanol precipitation. DEAE-Cellulose, molecular sieving on Sephacryl S-300 and Phenyl-Sepharose chromatography. 2. Its pI was about 5.1. Under appropriate conditions, the incubation of FXIII in the presence of thrombin did not lead to inactivation cut in the polypeptidic chain. 3. FXIII was also activated by CaCl2 and, in a lesser extent, by other divalent cations like SrCl2, BaCl2 or MgCl2. 4. The binding of calcium to FXIII exhibited a negative cooperativity. 5. The activity-pH curve of the calcium-activated enzyme did not appear very different from that of the thrombin-activated enzyme.     

On the chemical nature of DNA and RNA modification by a hemin model system.

In order to model the interaction of hemin with DNA and other polynucleotides, we have studied the degradation of DNA, RNA, and polynucleotides of defined structure by [meso-tetrakis(N-methyl-4-pyridyl)porphinato]manganese(III) (MnTMPP) + KHSO5. The activated porphyrin was shown to release adenine, thymine, and cytosine from DNA; RNA degradation afforded adenine, uracil, and cytosine. The same products were obtained from single- and double-stranded DNA oligonucleotides of defined sequence, and also from single-stranded DNA and RNA homopolymers. The overall yield of bases from the dode-canucleotide d(CGCT3A3GCG) was equal to 14% of the nucleotides present initially, indicating that each porphyrin catalyzed the release of approximately 4 bases. Although no guanine was detected as a product from any of the substrates studied, the ability of MnTMPP + KHSO5 to degrade guanine nucleotides was verified by the destruction of pGp, and by the appearance of bands corresponding to guanosine cleavage following treatment of 32P end labeled DNA restriction fragments with activated MnTMPP. Inspection of a number of sites of MnTMPP-promoted cleavage indicated that the process was sequence-selective, occurring primarily at G residues that were part of 5'-TG-3' or 5'-AG-3' sequences, or at T residues. Also formed in much greater abundance were alkali-labile lesions; these were formed largely at guanosine residues. Also studied was the degradation of a 47-nucleotide RNA molecule containing two hairpins. Degradation of the 5'-32P end labeled RNA substrate afforded no distinct, individual bands, suggesting that multiple modes of degradation may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and purification of chloroplastic spinach (Spinacia oleracea) sedoheptulose-1,7-bisphosphatase.

Higher-plant sedoheptulose-1,7-bisphosphatase was isolated and purified over 200-fold from spinach (Spinacia oleracea) chloroplast stromal extracts to apparent electrophoretic homogeneity by DEAE-Fractogel, molecular sieving on Sephadex G-200 and Blue B dye-matrix affinity chromatography. It is a protein of Mr 66,000, made up of two apparently identical subunits (Mr 35,000). The enzyme is activated by reduced thioredoxin fb in the presence of dithiothreitol. Its specificity towards sedoheptulose 1,7-bisphosphate versus fructose 1,6-bisphosphate is high, but not absolute.     

Calcium-Induced Desensitization of Acetylcholine Release from Synaptosomes or Proteoliposomes Equipped with Mediatophore, a Presynaptic Membrane Protein

A "fatigue" of acetylcholine (ACh) release is described in cholinergic synaptosomes stimulated with the calcium ionophore A23187 or gramicidin. A small conditioning calcium entry, which did not trigger a large ACh release, led to a decrease of transmitter release elicited by a second large calcium influx. This fatigue was half-maximal at approximately 30 microM external calcium and developed in a few minutes. In contrast, activation of release by calcium was very rapid and was half-maximal at approximately 0.5 mM external calcium. Activation and desensitization of release could be attributed to the recently identified presynaptic membrane protein, the "mediatophore." Proteoliposomes equipped with purified mediatophore showed a calcium-dependent activation and "fatigue" of ACh release similar to that of synaptosomes. It was found that the ionophore A23187 rapidly equilibrated internal and external calcium concentrations in proteoliposomes. Thus, the external calcium concentration gave the internal concentration required for activation or desensitization of proteoliposomal ACh release. The mediatophore showed remarkable calcium binding properties (20 sites/molecule) with a KD of 25 microM. The physiological implications of desensitization on the organization of release sites are discussed.     

Effects of pH and fructose 2,6-bisphosphate on oxidized and reduced spinach chloroplastic fructose-1,6-bisphosphatase

This report describes the effects of pH and fructose 2,6-bisphosphate (an analog of fructose 1,6-bisphosphate) on the activity of oxidized and reduced fructose-1,6-bisphosphatase from spinach chloroplasts. Studies were carried out with either fructose 1,6-bisphosphate, the usual substrate, or sedoheptulose 1,7-bisphosphate, an alternative substrate. The reduction of the oxidized enzyme is achieved by a thiol/disulfide interchange. The pK values relative to each redox form for the same substrate (either fructose 1,6-bisphosphate or sedoheptulose 1,7-bisphosphate) are identical, suggesting the same site for both substrates on the active molecule. The finding that the analog (fructose 2,6-bisphosphate) behaves like a competitive inhibitor for both substrates also favours this hypothesis. The inhibitory effect of this sugar is more important when the enzyme is reduced than when it is oxidized. The shift in the optimum pH observed when [Mg2+] was raised is interpreted as a conformational change of oxidized enzyme demonstrated by a change in fluorescence. The reduced and oxidized forms have the same theoretical rates relative to both substrates, but the reduced form has an observed Vmax which is 60% of the theoretical Vmax while that of the oxidized form is only 37% of the theoretical Vmax. The reduced enzyme appears more efficient than the oxidized one in catalysis.