Uridine diphosphate xylosyltransferase activity in cartilage from manganese-deficient chicks. (9/4482)

The glycosaminoglycan content of cartilage is decreased in manganese deficiency in the chick (perosis). The activity of xylosyltransferase, the first enzyme in the biosynthetic pathway of sulphated glycosaminoglycans, was studied in the epiphysial cartilage of 4-week-old chicks which had been maintained since hatching on a manganese-deficient diet. Enzymic activity was measured by the incorporation of [14C]xylose from UDP-[14C]xylose into trichloroacetic acid precipitates. Optimal conditions for the xylosyltransferase assay were established and shown to be the same for both control and manganese-deficient cartilage. Assay of the enzyme by using an exogenous xylose acceptor showed no difference in xylosyltransferase activity between control and manganese-deficient tissue. Further, the extent of xylose incorporation was greater in manganese-deficient than in control cartilage preparations, suggesting an increase in xylose-acceptor sites on the endogenous acceptor protein in the deficient cartilage. 35S turnover in the manganese-deficient cartilage was also increased. The data suggest that the decreased glycosaminoglycan content in manganese-deficient cartilage is due to decreased xylosylation of the acceptor protein plus increased degradation of glycosaminoglycan.  (+info)

Purification and characterization of a detergent-requiring membrane-bound metalloendopeptidase from porcine brain. (10/4482)

A detergent-requiring metalloendopeptidase cleaving a progastrin-C-terminal peptide (progastrin-(88-101)) mainly at the Arg95-Gly96 bond was solubilized from porcine cerebral vesicular membranes and purified to homogeneity as examined by PAGE. The purified enzyme had a molecular mass of approximately 76 kDa as estimated by both SDS/PAGE and Sephacryl S-300 gel filtration. It hydrolyzed progastrin-(88-101) peptide, BAM-12P, and bradykinin fairly specifically, and more efficiently than various other neuropeptides and related oligopeptides examined as substrates. It was inactive in the absence of detergents, and required certain detergents such as Triton X-100 or Lubrol PX for activity. Its optimum pH was about 6.5 and was strongly inhibited by metal-chelating agents such as EDTA, EGTA, and o-phenanthroline. It was extremely sensitive to EDTA and was completely inhibited even by 0.3 microM EDTA; the activity was fully restored by addition of a 10-fold higher concentration of Zn2+, CO2+, or Mn2+ ions over EDTA. On the other hand, dynorphin A-(1-13) peptide, a strong inhibitor of neurolysin, failed to inhibit the enzyme. The various characteristics indicated that the present enzyme is a unique membrane-bound metalloendopeptidase.  (+info)

Studies on the mechanism of collagen glucosyltransferase reaction. (11/4482)

The mechanism of collagen glucosyltransferase reaction was studied with enzyme preparations purified about 2500-5000-fold from extract of homogenate of whole chick embryos. Data obtained in experiments on initial velocity and inhibition kinetics of the reaction were consistent with an ordered mechanism in which the substrates are bound to the enzyme in the following order: Mn2+, UDP-glucose and collagen substrate, the addition of Mn2+ being at thermodynamic equilibrium and the binding site of the UDP-glucose to the enzyme not being the same as that for Mn2+ and collagen substrate. Only one metal co-factor seems to be involved in the reaction. The collagen substrate can probably also react in some conditions with enzyme-Mn2+ and with enzyme-Mn2+-UDP, and the UDP with the free enzyme, but in all these instances dead-end complexes are formed. Evidence is presented for an ordered release of the products in the following order: glucosylated collagen, UDP and Mn2+, in which Mn2+ need not leave the enzyme during each catalytic cycle.  (+info)

Endoribonuclease IV. A poly(A)-specific ribonuclease from chick oviduct. 1. Purification of the enzyme. (12/4482)

A new endoribonuclease, termed endoribonuclease IV, has been described. This enzyme has been isolated from chick oviducts and purified 15 000-fold in a 25% yield nearly to homogeneity. The nuclease, which specifically degrades poly(A), forms oligonucleotides of an average chain length of 10. These (A)-10 fragments are terminated by 3'-hydroxyl and 5'-phosphate groups. The enzyme has a pH optimum at 8.7, requires Mn2+ or Mg2+ as a cofactor, and has a molecular weight of about 45 000.  (+info)

A novel endonuclease of human cells specific for single-stranded DNA. (13/4482)

We have fractionated from human aneuploid cell cultures three different enzyme fractions degrading single-stranded DNA. We have purified and characterized one of these DNases; this is an endonuclease working at alkaline pH (around 9.5) and requiring Mg2+ for its activity. The enzyme degrades denatured DNA over 100 times more efficiently than native DNA in optimal conditions. The termini produced by the enzyme have 5'P and 3'OH ends. The enzyme can attack, though at reduced rate, the supertwisted circular molecule of Simian virus 40 DNA, whereas it is inactive on the nicked circular molecule. The ultraviolet irradiation of DNA, whether native or denatured, does not affect its efficiency as substrate of the DNase. The properties of this endonuclease distinguish it from those of the other DNases described previously in mammalian cells; the denomination DNase VI is therefore proposed. Its properties are similar to those of DNases described in Ustilago maydis and Bacillus subtilis, for which an essential role in recombination seems likely.  (+info)

cumA, a gene encoding a multicopper oxidase, is involved in Mn2+ oxidation in Pseudomonas putida GB-1. (14/4482)

Pseudomonas putida GB-1-002 catalyzes the oxidation of Mn2+. Nucleotide sequence analysis of the transposon insertion site of a nonoxidizing mutant revealed a gene (designated cumA) encoding a protein homologous to multicopper oxidases. Addition of Cu2+ increased the Mn2+-oxidizing activity of the P. putida wild type by a factor of approximately 5. The growth rates of the wild type and the mutant were not affected by added Cu2+. A second open reading frame (designated cumB) is located downstream from cumA. Both cumA and cumB probably are part of a single operon. The translation product of cumB was homologous (level of identity, 45%) to that of orf74 of Bradyrhizobium japonicum. A mutation in orf74 resulted in an extended lag phase and lower cell densities. Similar growth-related observations were made for the cumA mutant, suggesting that the cumA mutation may have a polar effect on cumB. This was confirmed by site-specific gene replacement in cumB. The cumB mutation did not affect the Mn2+-oxidizing ability of the organism but resulted in decreased growth. In summary, our data indicate that the multicopper oxidase CumA is involved in the oxidation of Mn2+ and that CumB is required for optimal growth of P. putida GB-1-002.  (+info)

A simple manganous chloride and Congo red disc method for differentiating Neisseria gonorrhoeae from Neisseria meningitidis. (15/4482)

Manganous chloride and Congo red incorporated into blotting paper discs have been used to differentiate gonococci from meningococci. The new technique is simple and reliable; the materials for the test are inexpensive. The method will increase the efficiency of distinguishing between the pathogenic Neisseria in any clinical bacteriology laboratory and especially in those in the tropical areas.  (+info)

Assay of intercellular adhesiveness using cell-coated Sephadex beads as collecting particles. (16/4482)

A simple, rapid and precise method, based on a previous method, for measuring relative rates of intercellular adhesion is described. DEAE-Sephadex beads were treated with nitrocellulose in order to allow cells to grow on their surfaces. Balb/c 3T3 and Balb/c 3T12 cells were used to characterize the assay. They formed confluent cell layers on nitrocellulose-treated DEAE-Sephadex. These cell-coated beads were employed to collect 32P-labelled cells from single cell suspensions. Since they formed statistically uniform, large collecting surfaces, the collection of labelled cells was markedly improved as compared to the original assay. The cell-coated beads collected a large percentage of the labelled cells in a short time. The percentage of cells collected was independent of the concentration of labelled cells in the assay mixture, and the collection was linear for approximately 60 min. The variability between replicate assays was usually +/- 5%. The assay allows the rapid and precise determination of intercellular adhesion in large numbers of individual samples. These features make it useful to screen for effects of different treatments on intercellular adhesions.  (+info)