ABSTRACT
Purified preparations of chitin synthetase (EC 2.4.1.16; UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxyglucosyltransferase), capable of forming microfibrils in vitro, were isolated from yeast cells of Mucor rouxii. Chitin synthetase was obtained either by substrate-induced liberation of bound enzyme (54,000 x g pellet) or by isolation of unbound enzyme present in the 54,000 x g supernatant of a cell-free extract. Both preparations contained ellipsoidal granules from about 350 to 1000 A diameter. Many granules exhibited a marked depression. No typical unit membrane profiles appeared in thin sections of glutaraldehyde/OsO4-fixed samples. Upon incubation with substrate and activators, chitin microfibrils were produced. The microfibrils were often found intimately associated with granules. The most common configurations were: a microfibril with a granule at one end, or two microfibrils "arising" from the same granule. These findings lend support to the granule hypothesis for the elaboration of cell wall microfibrils by end-synthesis.
Subject(s)
Cell Wall/physiology , Chitin Synthase/metabolism , Hexosyltransferases/metabolism , Mucor/enzymology , Cell-Free System , Centrifugation, Density Gradient , Cytoplasmic Granules/enzymology , Cytoplasmic Granules/ultrastructure , Membranes/enzymology , Microscopy, Electron , Mucor/ultrastructure , SolubilityABSTRACT
Biosynthesis of glucans occurred in cell-free fractions isolated from onion stem (Allium cepa L.) enriched in either dictyosomes or plasma membranes. beta-1,3- and beta-1, 4-Glucans were synthesized in differing proportions and at different rates as the concentration of uridine diphosphoglucose or the proportion of dictyosomes or plasma membrane varied. At low (1.5 mum) UDP-glucose concentrations synthesis of alkali-insoluble glucan was correlated with abundance of dicytosomes; most of the substrate utilized by plasma membrane was for glycolipid synthesis. At high (1 mm) UDP-glucose concentration, the synthesis of alkali-insoluble glucans correlated with the abundance of plasma membrane. Substrate enhancement of beta-1, 4-glucan synthesis in dictyosome fractions was less than proportional to increases in substrate concentration. In contrast, beta-1, 4-glucan synthesis by plasma membrane was more than proportionately increased. At high substrate concentrations the synthesis of beta-1, 3-glucans predominated in both dictyosome and plasma membrane fractions. The results show that the capacity to synthesize glucans resides in both Golgi apparatus and plasma membranes of onion stem, but that the plasma membrane has the greatest capacity for synthesis of alkali-insoluble glucans at high UDP-glucose concentrations.
