David R. Quigley

β(1→3)Glucan synthase activity ofNeurospora crassa: Stabilization and partial characterization

Abstract Neurospora crassa β(1→3)glucan synthase activity was localized to low-speed particulate fractions consisting primarily of membranes. The particulate enzyme activity was stabilized by inhibitors of proteinases (phenylmethylsulfonyl fluoride and EDTA) and of phosphatases (sodium fluoride); glycerol and bovine serum albumin were also found to result in increases in enzyme activity. Enzyme activity was specific for UDP-glucose as substrate, did not require a metal ion, and was inhibited by UDP. The reaction product appeared to be a linear,β(1→3)-linked glucan.

β(1–3)Glucan synthase activity ofNeurospora crassa: Kinetic analysis of negative effectors

Abstract β(1–3)Glucan synthase activity of Neurospora crassa was inhibited by a number of compounds. Uridine nucleotides were linear competitive inhibitors of enzyme activity. Hill plots for the competitive inhibitors and for the substrate (UDP-glucose) resulted in straight lines with Hill numbers near unity suggesting a single substrate binding site. Tunicamycin, dolichol, or dolichol phosphate did not affect enzyme activity and a lipid-linked intermediate was not detected. Sorbose, gluconolactone, echinocandin B, and papulacandin B were uncompetitive inhibitors. Mixed inhibitor studies revealed that the binding of one uncompetitive inhibitor blocked completely the binding of each of the other uncompetitive inhibitors.

Optimal conditions for the release of protoplasts ofNeurospora using Novozym 234

Abstract The optimal conditions for the release of protoplasts from 18-h-hyphae of Neurospora crassa using Novozym 234 were 120-minute incubations at 25°C and 10 mg/ml Novozym. Protoplasts were released from all parts of hyphae and were found not to contain surface-bound cell wall material. About 10% of Novozym-derived protoplasts were viable.

(1–3)-β-Glucan synthesis ofNeurospora crassa

Abstract(1–3)-β-d-Glucan synthase activity ofNeurospora crassa was localized to the plasma membrane by autoradiography of colloidal gold-labeled plasma membranes. The active site of glucan synthase for substrate hydrolysis was determined to be cytoplasmic facing. However, glucan synthase activity present in intact protoplasts was partially sensitive to Novozym 234 and to glutaraldehyde treatments, suggestive that enzyme activity is transmembrane. Enzyme activity also directed the formation of microfibrils in vitro. Taken together, these and previous results support the following scheme for glucan synthesis: 1. The sequential addition of glucose residues from UDP-glucose to glucan chains occurs on the cytoplasmically facing portion of glucan synthase. 2. As each glucan chain is synthesized, it is extruded to the extracytoplasmic side of the plasma membrane. 3. As each chain is extruded, it forms interchain hydrogen bonds with adjacent chains, resulting in glucan microfibril assembly.

β(1-3)Glucan synthase of Neurospora crassa: Solubilization and partial characterization

Abstract β(1-3)Glucan synthase activity of Neurospora crassa was rendered soluble by treatment of protoplast membranes from either a protoplast-forming strain ( os-1 ) or wild-type with 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 m M 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid buffer, pH 7.4, containing 5 m M EDTA, 100 m M glycerol, 1 m M phenylmethylsulfonyl fluoride, and 10 m M sodium fluoride. Additions of 1 m M dithiothreitol, 200 m M inorganic phosphate, 10 μ M GTP, and 0.5 M glycerol increased the amount of enzyme activity solubilized (∼50% of recovered enzyme activity was soluble). Solubilized glucan synthase from both os-1 and wild-type catalyzed the formation of linear, unbranched β(1-3)glucan; K mapp and V max were similar to those of the particulate enzyme activity.

Protoplasts of Neurospora crassa: α-amylase improves yield from wild-type hyphae

Treatment of wild-type hyphae of Neurospora crassa with a mixture of chitinase (4 units/ml), Zymolyase 20T (5 units/ml), cellulysin (100 units/ml), lytic enzyme L-1 (1 mg/ml), and β-glucuronidase (1500 units/ml) to which 5000 units/ml α-amylase had been added resulted in the quantitative conversion of hyphae to protoplasts which did not aggregate or clump. Overall, α-amylase addition improved protoplast yield by over twofold.

β(1–3)Glucan synthase ofNeurospora crassa: Reaction sequence based on kinetic evidence

On the basis of the kinetic effects of substrate, activator, and inhibitors on β(1–3) glucan synthase activity ofNeurospora crassa, we propose the following reaction sequence for glucan synthesis. First, enzyme binds laminaribiose (activator), forming an enzyme-laminaribiose complex. Substrate (UDP-Glc) binding follows. UDP-Glc is hydrolyzed, releasing UDP, while the glucose residue remains associated with glucan synthase. The resulting enzyme-activator-glucose complex binds another UDP-Glc. It is likely that linear competitive inhibitors act at this step. Initial polymerization occurs, forming a disaccharide (which remains bound to glucan synthase) and UDP, which is released. The resulting enzyme-activator-disaccharide binds another UDP-Glc, and Glc is covalently added; further polymerization occurs by addition of Glc (from UDP-Glc) to the growing glucan chain, which remains associated with glucan synthase. Uncompetitive inhibitors are likely to affect enzyme activity at this step.

β-Linked disaccharides stimulate, but do not act as primer for,β(1–3)glucan synthase activity ofNeurospora crassa

Abstractβ-Linked disaccharides (laminaribiose and cellobiose) stimulatedβ(1–3)glucan synthase activity ofNeurospora crassa by reducing the Km app for the substrate while not changing the Vmax. Laminaribiose and cellobiose werelinear activators with a Ka app of 0.32 mM and Ka app of 1.7 mM, respectively. Laminaribiose was not found to be incorporated into product, i.e., did not act as a primer covalently bound to product.

Permeabilization ofNeurospora crassa hyphae with toluene-ethanol and filipin

Young hyphae ofNeurospora crassa were made permeable to UDP-glucose and trypan blue by treatment with toluene-ethanol and filipin. Less than 2% of treated cells survived treatment with 8% and 16% toluene-ethanol, while 25% survived treatment with 4% toluene-ethanol. Similarly, 98% of treated cells were killed by treatment with 16 μg/ml filipin. Electron microscopy revealed that toluene-ethanol-treated cells lost pieces of plasma membrane and contained a number of vacuole-like structures; filipin-treated cells were less affected. Both filipin- and toluene-ethanol-treated cells were able to incorporate UDP-glucose into insoluble material (likely glycogen and glucan).

Sorbose-resistant mutants ofNeurospora crassa do not have alteredβ(1–3)glucan synthase activity

A number of mutants ofNeurospora crassa (sor-1, sor-3, sor-4, sor-5, sor-6, sor-15, sor-T9, andpatch) were found to be resistant to the growth-inhibiting effect of sorbose.β(1–3)-Glucan synthase activity from each strain was found to be as sensitive to sorbose as wild-type enzyme activity. Four of these strains (sor-1, sor-4, sor-5, sor-T9) had altered sorbose transport; the remaining strains tested had normal sorbose transport. All of these strains (except forsor-3) were found to metabolize sorbose to glucose (and other compounds). This may explain their sorbose resistance.