Biosynthesis of glucans occurred in cell-free fractions isolated from onion stem (Allium cepa L.) enriched in either dictyosomes or plasma membranes. l8-1,3-and p-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 uM) UDP-glucose concentrations synthesis of alkaliinsoluble 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 g-1,4-glucan synthesis in dictyosome fractions was less than proportional to increases in substrate concentration. In contrast, ,-1,4-glucan synthesis by plasma membrane was more than proportionately increased. At high substrate concentrations the synthesis of 6-1 ,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.In vivo studies of cell wall biogenesis indicate that cellulose synthesis occurs at the cell surface, possibly at the surface of the plasma membrane (28,34,39,44,52), while pectic and hemicellulosic polysaccharides are synthesized by the Golgi apparatus (1,5,10,18,27,40). In contrast, Ray et al. (36) found UDP-glucose: /3-1,4-glucan glucosyltransferase (3-1,4-glucan synthetase) activity to be localized in a membrane frac-

Purified preparations of chitin synthetase (EC 2.4.1.16; UDP-2-acetamido2-eoxy--glucose:chitin 4-flacetamidodeoxyglucosyltransferase), capable of forming microfibrils in vitro, were isolated from yeast cells of Mucor rouxii. Chitin synthetase was obtained either by substrateinduced liberation of bound enzyme (54,000 X g pellet) or by isolation of unbound enzyme present in the 54,000 X g su ernatant 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/Os04-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 endsynthesis.The mechanism of formation of microfibrillar cell walls of plants and fungi has been the subject of various investigations. This topic has been recently reviewed (1, 2). Most studies have been concerned with the formation of cellulose and chitin. It is generally agreed that the nonfibrillar, amorphous components of the wall pose fewer spatial problems of biosynthesis. As to microfibrils, their shape, size, and orientation present a number of spatial demands for their elaboration. Among the organelles commonly suggested as the site of elaboration of microfibrillar polysaccharides are: plasmalemma (3, 4), Golgi apparatus (5, 6), and granules present between wall and plasmalemma (1, 2, 7). We have recently shown (8) that chitin microfibrils can be assembled in vitro by a "solubilized" form of chitin synthetase (EC 2.4.1.16; UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-,B-acetamidodeoxyglucosyltransferase) prepared from yeast cells of Mucor rouxii. The enzyme was "solubilized" from a crude membrane-rich preparation (35,000 X g pellet) by incubation with the enzyme substrate, uridine diphosphate N-acetyl-D-glucosamine (UDP-GlcNAc), and activator, N-acetyl-D-glucosamine (GlcNAc), at 0°C. The present communication examines the electron microscopic morphology of chitin synthetase isolated by this technique and also of unbound enzyme separated from a crude cellfree extract of the yeast form of M. rouxii. MATERIALS AND METHODSCultivation Techniques. Spores (1.5 X 108) of Mucor rouxii, IM-80, were inoculated into 0.5 liter of liquid YPG (yeast extract/peptone/glucose) medium (9) in 2-liter Erlenmeyer flasks and incubated in a reciprocating shaker at 280C for 12 hr. A mixed gas stream (30% CO2 + 70% N2) was bubbled through the medium during the entire incubation period.Cell-Free Extract-Preparation. Yeast cells from 1.5 liters of medium were harvested on sintered-glass filters, washed twice with 200 ml of ice-cold 0.05 M phosphate buffer, pH 6.0, containing 10 mM MgCl2, resuspended in ...

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Secretory vesicles containing polysaccharide were isolated from germinated pollen of Lilium longiflorum and characterized by biochemical and ultrastructural investigation. Pollen tubes exhibit a secretory pathway in which the vesicles concentrated in the tube apex are produced by the Golgi apparatus and contributed to the cell wall at the apex upon fusion of the vesicle membrane with the plasma membrane. Secretory vesicles were isolated by a method involving the size discrimination of cytoplasmic components using Millipore filters. Cells were disrupted under conditions which minimized membrane vesiculation. Identification was made by electron-microscopic comparison of the periodic acid-silver hexamine (PASH) reactivities of in situ and isolated secretory vesicles. The secretory vesicles contained polysaccharides which were high in galacturonic acid and similar in sugar composition to those of the hot-water-soluble fraction of pollen tube cell wall. A hot-water-insoluble, non-cellulosic glucan was the major component of the cell wall. Less than 7% of the wall was cellulosic. Chitin was absent. Similarities in the ultrastructure and PASH staining of apical secretory vesicles and an amorphous component of the cell wall support a precursor-product relationship between these 2 cell components. Ultrastructural investigations revealed complexes of the endoplasmic reticulum (ER) associated with electron-translucent regions of cytoplasm, suggesting a possible function of the ER in cell wall formation. Additionally, patterns of PASH staining show that changes in polysaccharides occur in secretory vesicles after vesicles have been formed by dictyosomes. Therefore, secretory vesicles may have a role in polysaccharide synthesis as well as in membrane and product transport.

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