On the mechanism of the peroxidase-catalyzed oxygen-transfer reaction

We reported evidence that horseradish peroxidase (HRP) and chloroperoxidase (CPO) catalyze oxygen transfer from H2O2 to thioanisoles [Kobayashi, S., Nakano, M., Goto, T., Kimura, T., & Schaap, A. P. (1986) Biochem. Biophys. Res. Commun. 135, 166-171]. In the present paper, the reaction mechanism of this oxygen transfer is discussed. The oxidation of para-substituted thioanisoles by HRP compound II showed a large negative rho value of -1.46 vs. the sigma + parameter in a Hammett plot. These results are in accord with the formation of a cation radical intermediate in the rate-determining step. Hammett treatments for HRP- and CPO-dependent S-oxygenations did not provide unequivocal proofs to judge the reaction mechanism, because of the poor correlations for sigma + and sigma p parameters. Different behavior was found in kinetics and stereoselectivity between the two enzymes. Results in the present study and recent studies strongly suggested the formation of a cation radical intermediate. The oxygen atom would transfer by reaction of compound II and the cation radical intermediate. Although involvement of the cation radical was not confirmed in the CPO system, a similar mechanism was proposed for CPO.     

Application of halophilic nuclease H of Micrococcus varians subsp. halophilus to commercial production of flavoring agent 5'-GMP

RNA was degraded at 60 degrees C for 24 h by halophilic nuclease H in supernatants from broth cultures of Micrococcus varians subsp. halophilus containing 12% NaCl. Since contaminating 5'-nucleotidase exhibited almost no activity under these conditions, the 5'-GMP formed could be recovered from the reaction mixture, and the yield was 805 mg from 5 g of RNA.     

Immune deficiency in the X-linked lymphoproliferative syndrome. I. Epstein-Barr virus-specific defects

Eleven males with XLP were evaluated for EBV-specific antibodies during periods of 2 to 7 yr. Variable responses to EBV-specific antigens were found. All 11 patients had subnormal anti-EBNA titers, which probably reflected a T cell deficiency. The patients showed four different patterns in their anti-VCA response: 1) two boys who had experienced malignant lymphoma mounted no antibodies at all; 2) two patients showed intermittent anti-VCA titers; 3) four males had persistently elevated anti-VCA titers; and 4) three patients showed normal anti-VCA titers. ADCC against EBV-infected cells was abnormally low in six patients and was elevated in two patients given gamma-globulin. ADCC titers did not correlate with anti-VCA titers. However, most patients with XLP failed to effect regression of autologous EBV-infected lymphoblastoid cell lines, indicating a deficiency in long-lived T cell-mediated immunity to EBV.     

Structure of the spectrin-actin binding site of erythrocyte protein 4.1

The complete primary structure of the functional site of erythrocyte protein 4.1 involved in spectrin-actin associations has been determined. The sequence of this domain, which contains 67 amino acids and has a molecular mass of 8045 daltons, has been obtained by NH2-terminal sequence analysis of an 8-kDa chymotryptic peptide, three endoproteinase lysine C-cleaved peptides and two peptides obtained by Staphylococcus aureus protease V8 cleavage. All peptides including the 8-kDa domain peptide were purified by reverse-phase high performance liquid chromatography. Antibodies against two different synthetic peptides of the 8-kDa domain are able to inhibit the association between protein 4.1, spectrin, and F-actin, corroborating that the 8-kDa domain is responsible for the formation of a ternary complex. A computer search of the 8-kDa sequence with the National Biomedical Research Foundation database did not detect any significant homologies to known sequences. Protein 4.1 is not related to any known proteins and may represent a new protein superfamily.     

The stimulus-secretion coupling of amino acid-induced insulin release metabolic interaction L-asparagine and L-leucine in pancreatic islets

1. Because L-asparagine augments insulin release evoked by L-leucine, the metabolism of these two amino acids was investigated in rat pancreatic islets. 2. L-Leucine inhibited the uptake and deamidation of L-asparagine, but failed to exert any obvious primary effect upon the further catabolism of aspartate derived from exogenous asparagine. 3. L-Asparagine augmented the oxidation of L-leucine, and effect possibly attributable to activaion of 2-ketoisocaproate dehydrogenase. 4. The association of L-asparagine and L-leucine exerted a sparing action on the utilization of endogenous amino acids, so that the integrated rate of nutrients oxidation was virtually identical in the sole presence of L-leucine and simultaneous presence of L-asparagine and L-leucine, respectively. 5. It is proposed that the enhancing action of L-asparagine upon insulin release evoked by L-leucine is attributable to an increased generation rate of cytosolic NADPH rather than any increase in nutrients oxidation.     

Synaptonemal complexes and meiosis in myxomycetes

Synaptonemal complexes (SC) have been observed in spores 18–24 hr past cleavage in natural fruitings of Physarum cinereum, P. bogoriense, Hemitrichia stipitata, Tubifera ferruginosa, and Arcyria incarnata. Laboratory fruitings of Arcyria cinerea, Stemonitis herbatica, and a homothallic isolate of Physarum pusillum also have SC's present in spores during the same postcleavage period. The presence of these paired chromosomes of meiotic prophase in spores of species collected in nature and in a diversity of taxa suggests that the usual position of meiosis in Myxomycetes is inside the postcleavage spore. Criteria are proposed for evaluating the validity of the SC as an indicator of meiosis.     

Levels of genetic polymorphism: marker loci versus quantitative traits.

Species are the units used to measure ecological diversity and alleles are the units of genetic diversity. Genetic variation within and among species has been documented most extensively using allozyme electrophoresis. This reveals wide differences in genetic variability within, and genetic distances among, species, demonstrating that species are not equivalent units of diversity. The extent to which the pattern observed for allozymes can be used to infer patterns of genetic variation in quantitative traits depends on the forces generating and maintaining variability. Allozyme variation is probably not strictly neutral but, nevertheless, heterozygosity is expected to be influenced by population size and genetic distance will be affected by time since divergence. The same is true for quantitative traits influenced by many genes and under weak stabilizing selection. However, the limited data available suggest that allozyme variability is a poor predictor of genetic variation in quantitative traits within populations. It is a better predictor of general phenotypic divergence and of postzygotic isolation between populations or species, but is only weakly correlated with prezygotic isolation. Studies of grasshopper and planthopper mating signal variation and assortative mating illustrate how these characters evolve independently of general genetic and morphological variation. The role of such traits in prezygotic isolation, and hence speciation, means that they will contribute significantly to the diversity of levels of genetic variation within and among species.