A protein-glucan intermediate during paramylon synthesis.
(1/2513)
A sodium deoxycholate extract containing glucosyltransferase activity was obtained from a particulate preparation from Euglena gracilis. It transferred glucose from UDP-[14C]glucose into material that was precipitated by trichloroacetic acid. This material released beta-(1 leads to 3)-glucan oligosaccharides into solution on incubation with weak acid, weak alkali and beta-(1 leads to 3)-glucosidase. The products of the incubation of the deoxycholate extract with UDP-[14C]glucose were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Radioactive bands were obtained that had the properties of beta-(1 leads to 3)-glucan covalently linked to protein by a bond labile to weak acid. High-molecular-weight material containing a beta-(1 leads to 3)-glucan was also shown to be present by gel filtration. The bond linking glucan to aglycone is possibly a pyrophosphate linkage. It is proposed that in Euglena gracilis beta-(1 leads to 3)-glucan (paramylon) is synthesized on a protein primer. (+info)
Patterns of evolutionary rate variation among genes of the anthocyanin biosynthetic pathway.
(2/2513)
The anthocyanin biosynthetic pathway is responsible for the production of anthocyanin pigments in plant tissues and shares a number of enzymes with other biochemical pathways. The six core structural genes of this pathway have been cloned and characterized in two taxonomically diverse plant species (maize and snapdragon). We have recently cloned these genes for a third species, the common morning glory, Ipomoea purpurea. This additional information provides an opportunity to examine patterns of evolution among genes within a single biochemical pathway. We report here that upstream genes in the anthocyanin pathway have evolved substantially more slowly than downstream genes and suggest that this difference in evolutionary rates may be explained by upstream genes being more constrained because they participate in several different biochemical pathways. In addition, regulatory genes associated with the anthocyanin pathway tend to evolve more rapidly than the structural genes they regulate, suggesting that adaptive evolution of flower color may be mediated more by regulatory than by structural genes. Finally, for individual anthocyanin genes, we found an absence of rate heterogeneity among three major angiosperm lineages. This rate constancy contrasts with an accelerated rate of evolution of three CHS-like genes in the Ipomoea lineage, indicating that these three genes have diverged without coordinated adjustment by other pathway genes. (+info)
Molecular cloning and biochemical characterization of a novel anthocyanin 5-O-glucosyltransferase by mRNA differential display for plant forms regarding anthocyanin.
(3/2513)
UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT) is responsible for the modification of anthocyanins to more stable molecules in complexes for co-pigmentation, supposedly resulting in a purple hue. The cDNA encoding 5-GT was isolated by a differential display applied to two different forms of anthocyanin production in Perilla frutescens var. crispa. Differential display was carried out for mRNA from the leaves of reddish-purple and green forms of P. frutescens, resulting in the isolation of five cDNA clones predominantly expressed in the red form. The cDNA encoded a polypeptide of 460 amino acids, exhibiting a low homology with the sequences of several glucosyltransferases including UDP-glucose: anthocyanidin 3-O-glucosyltransferase. By using this cDNA as the probe, we also isolated a homologous cDNA clone from a petal cDNA library of Verbena hybrida. To identify the biochemical function of the encoded proteins, these cDNAs were expressed in Saccharomyces cerevisiae cells. The recombinant proteins in the yeast extracts catalyzed the conversion of anthocyanidin 3-O-glucosides into the corresponding anthocyanidin 3,5-di-O-glucosides using UDP-glucose as a cofactor, indicating the identity of the cDNAs encoding 5-GT. Several biochemical properties (optimum pH, Km values, and sensitivity to inhibitors) were similar to those reported previously for 5-GTs. Southern blot analysis indicated the presence of two copies of 5-GT genes in the genome of both red and green forms of P. frutescens. The mRNA accumulation of the 5-GT gene was detected in the leaves of the red form but not in those of the green form and was induced by illumination of light, as observed for other structural genes for anthocyanin biosynthesis in P. frutescens. (+info)
GTPase activity and biochemical characterization of a recombinant cotton fiber annexin.
(4/2513)
A cDNA encoding annexin was isolated from a cotton (Gossypium hirsutum) fiber cDNA library. The cDNA was expressed in Escherichia coli, and the resultant recombinant protein was purified. We then investigated some biochemical properties of the recombinant annexin based on the current understanding of plant annexins. An "add-back experiment" was performed to study the effect of the recombinant annexin on beta-glucan synthase activity, but no effect was found. However, it was found that the recombinant annexin could display ATPase/GTPase activities. The recombinant annexin showed much higher GTPase than ATPase activity. Mg2+ was essential for these activities, whereas a high concentration of Ca2+ was inhibitory. A photolabeling assay showed that this annexin could bind GTP more specifically than ATP. The GTP-binding site on the annexin was mapped into the carboxyl-terminal fourth repeat of annexin from the photolabeling experiment using domain-deletion mutants of this annexin. Northern-blot analysis showed that the annexin gene was highly expressed in the elongation stages of cotton fiber differentiation, suggesting a role of this annexin in cell elongation. (+info)
Expression and structural characterization of a baculovirus ecdysteroid UDP-glucosyltransferase.
(5/2513)
The baculovirus enzyme ecdysteroid UDP-glucosyltransferase (EGT) disrupts the hormonal balance of the insect host by catalysing the conjugation of ecdysteroids, the moulting hormones, with the sugar moiety from UDP-glucose or UDP-galactose. In this study, EGT has been overproduced using a recombinant Autographa californica nucleopolyhedrovirus and an antiserum has been raised against the purified protein. This antiserum was used to visualize the kinetics of expression of EGT by wild-type AcMNPVL-1 and by the overproducing recombinant virus. The inclusion of tunicamycin during these time-course experiments suggested that EGT is glycosylated. This was confirmed by Endo F treatment, which showed that glycosylation increased the apparent subunit molecular mass by approximately 11 kDa. These sugars do not appear to be required for enzyme activity. EGT activity invariantly elutes from gel-filtration columns as a single peak corresponding to a 260 kDa (+/-50 kDa) protein. This suggests that the enzyme is an oligomer of three to five subunits, since the subunit molecular mass is 56 kDa. (+info)
Manganese sulfate-dependent glycosylation of endogenous glycoproteins in human skeletal muscle is catalyzed by a nonglucose 6-P-dependent glycogen synthase and not glycogenin.
(6/2513)
Glycogenin, a Mn2+-dependent, self-glucosylating protein, is considered to catalyze the initial glucosyl transfer steps in glycogen biogenesis. To study the physiologic significance of this enzyme, measurements of glycogenin mediated glucose transfer to endogenous trichloroacetic acid precipitable material (protein-bound glycogen, i.e., glycoproteins) in human skeletal muscle were attempted. Although glycogenin protein was detected in muscle extracts, activity was not, even after exercise that resulted in marked glycogen depletion. Instead, a MnSO4-dependent glucose transfer to glycoproteins, inhibited by glycogen and UDP-pyridoxal (which do not affect glycogenin), and unaffected by CDP (a potent inhibitor of glycogenin), was consistently detected. MnSO4-dependent activity increased in concert with glycogen synthase fractional activity after prolonged exercise, and the MnSO4-dependent enzyme stimulated glucosylation of glycoproteins with molecular masses lower than those glucosylated by glucose 6-P-dependent glycogen synthase. Addition of purified glucose 6-P-dependent glycogen synthase to the muscle extract did not affect MnSO4-dependent glucose transfer, whereas glycogen synthase antibody completely abolished MnSO4-dependent activity. It is concluded that: (1) MnSO4-dependent glucose transfer to glycoproteins is catalyzed by a nonglucose 6-P-dependent form of glycogen synthase; (2) MnSO4-dependent glycogen synthase has a greater affinity for low molecular mass glycoproteins and may thus play a more important role than glucose 6-P-dependent glycogen synthase in the initial stages of glycogen biogenesis; and (3) glycogenin is generally inactive in human muscle in vivo. (+info)
Induction of selected lipid metabolic enzymes and differentiation-linked structural proteins by air exposure in fetal rat skin explants.
(7/2513)
The epidermal permeability barrier of premature infants matures rapidly following birth. Previous studies suggest that air exposure could contribute to this acceleration, because: (i) development of a structurally and functionally mature barrier accelerates when fetal rat skin explants are incubated at an air-medium interface, and (ii) occlusion with a water-impermeable membrane prevents this acceleration. To investigate further the effects of air exposure on epidermal barrier ontogenesis, we compared the activities of several key enzymes of lipid metabolism and gene expression of protein markers of epidermal differentiation in fetal rat skin explants grown immersed versus air exposed. The rate-limiting enzymes of cholesterol (HMG CoA reductase) and ceramide (serine palmitoyl transferase) synthesis were not affected. In contrast, the normal developmental increases in activities of glucosylceramide synthase and cholesterol sulfotransferase, responsible for the synthesis of glucosylceramides and cholesterol sulfate, respectively, were accelerated further by air exposure. Additionally, two enzymes required for the final stages of barrier maturation and essential for normal stratum corneum function, beta-glucocerebrosidase, which converts glucosylceramide to ceramide, and steroid sulfatase, which desulfates cholesterol sulfate, also increased with air exposure. Furthermore, filaggrin and loricrin mRNA levels, and filaggrin, loricrin, and involucrin protein levels all increased with air exposure. Finally, occlusion with a water-impermeable membrane prevented both the air-exposure-induced increase in lipid enzyme activity, and the expression of loricrin, filaggrin, and involucrin. Thus, air exposure stimulates selected lipid metabolic enzymes and the gene expression of key structural proteins in fetal epidermis, providing a biochemical basis for air-induced acceleration of permeability barrier maturation in premature infants. (+info)
Regulation of intracellular ceramide content in B16 melanoma cells. Biological implications of ceramide glycosylation.
(8/2513)
We previously reported that ceramide released from glycosphingolipids (GSLs) by endoglycoceramidase was directly metabolized to GSLs, and thus the content of GSLs was constantly maintained in B16 melanoma cells (Ito, M., and Komori, H. (1996) J. Biol. Chem. 271, 12655-12660). In this study, the metabolism of ceramide released from sphingomyelin (SM) by bacterial sphingomyelinase (SMase) was examined using B16 cells and their GSL-deficient mutant counterpart GM95 cells. Treatment of B16 melanoma cells with bacterial SMase effectively hydrolyzed SM on the plasma membrane. Under these conditions, NeuAcalpha2,3Galbeta1, 4Glcbeta1,1ceramide was significantly increased. Interestingly, UDP-glucose:ceramide glucosyltransferase-1 (GlcT-1) activity and GSL synthesis, but not SM synthesis or sphingosine generation, were found to be up-regulated by SMase treatment. The up-regulation of GSL synthesis seemed to occur at both the transcriptional and post-translational steps of GlcT-1 synthesis. Accumulation of ceramide by bacterial SMase was much higher in GM95 cells than in the parental cells. When the enzyme was removed from the culture medium, the intracellular ceramide level in B16 cells, but not that in the mutant cells, normalized. No rapid restoration of SM in either of the cell lines was observed after removal of the enzyme. SMase treatment strongly inhibited DNA synthesis in GM95 cells but not that in B16 cells. In the presence of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of GlcT-1, SMase treatment markedly increased the ceramide content and thus inhibited DNA synthesis in B16 cells. Our study provides the first evidence that GlcT-1 functions to regulate the level of intracellular ceramide by glycosylation of the ceramide when it is present in excess. (+info)