A family of genes coding for proteins homologous to the a subunit of the muscle nicotinic acetylcholine receptor has been identified in the rat genome. These genes are transcribed in the central and peripheral nervous systems in areas known to contain functional nicotinic receptors. In this paper, we demonstrate that three of these genes, which we call alpha3, alpha4, and beta2, encode proteins that form functional nicotinic acetylcholine receptors when expressed in Xenopus oocytes. Oocytes expressing either alpha3 or alpha4 protein in combination with the beta2 protein produced a strong response to acetylcholine. Oocytes expressing only the alpha4 protein gave a weak response to acetylcholine. These receptors are activated by acetylcholine and nicotine and are blocked by Bungarus toxin 3.1. They are not blocked by a-bungarotoxin, which blocks the muscle nicotinic acetylcholine receptor. Thus, the receptors formed by the alpha3, alpha4, and beta2 subunits are pharmacologically similar to the ganglionic-type neuronal nicotinic acetylcholine receptor. These results indicate that the alpha3, alpha4, and beta2 genes encode functional nicotinic acetylcholine receptor subunits that are expressed in the brain and peripheral nervous system. It seems likely that complex brain functions, such as learning and memory, involve changes in the efficiency of synaptic transmission. One way in which synaptic efficiency might be modified is through a change in the availability or properties of neurotransmitter receptors in the postsynaptic membrane. Testing this idea, and understanding mechanisms that might accomplish such a modification, requires means of detecting and quantifying receptors at synapses in the central nervous system. However, the low abundance and great diversity of neurotransmitter receptors in the central nervous system have made their analysis difficult.We therefore chose first to study neurotransmitter receptors at the more accessible neuromuscular junction and were able to obtain and express cDNA clones encoding the subunits of the muscle-type nicotinic acetylcholine receptor of the rat. We subsequently used these cDNA clones to identify homologous genes that code for acetylcholine receptor a subunits found in the central nervous system. This approach led to the isolation of two new cDNA clones (1, 2) that represent gene transcripts found in different regions of the brain and that encode proteins with the general structural features of muscle nicotinic acetylcholine receptor a subunits. We proposed that these genes, called alpha3 and alpha4, code for the a subunits of functional nicotinic acetylcholine receptors expressed in the central and peripheral nervous systems. We have tested this hypothesis and in this paper report that RNA transcribed from either the clone derived from the alpha3 gene or the clone derived from the alpha4 gene, in concert with RNA transcribed from a new clone, PCX49, will direct the synthesis offunctional neuronal nicotinic acetylcholine receptors in Xenopus oocytes.

We have isolated a complementary DNA clone containing sequences homologous to those encoding the alpha-subunit of a mouse muscle nicotinic acetylcholine receptor. Based on the structural similarities between the encoded protein and the muscle acetylcholine receptor alpha-subunit, and the presence of hybridizing RNA species in the brain, we propose that this clone codes for a neural nicotinic acetylcholine receptor alpha-subunit.

Lignocellulosic biomass, the most abundant polymer on Earth, is typically composed of three major constituents: cellulose, hemicellulose, and lignin. The crystallinity of cellulose, hydrophobicity of lignin, and encapsulation of cellulose by the lignin-hemicellulose matrix are three major factors that contribute to the observed recalcitrance of lignocellulose. By means of designer cellulosome technology, we can overcome the recalcitrant properties of lignocellulosic substrates and thus increase the level of native enzymatic degradation. In this context, we have integrated six dockerin-bearing cellulases and xylanases from the highly cellulolytic bacterium, Thermobifida fusca, into a chimeric scaffoldin engineered to bear a cellulose-binding module and the appropriate matching cohesin modules. The resultant hexavalent designer cellulosome represents the most elaborate artificial enzyme composite yet constructed, and the fully functional complex achieved enhanced levels (up to 1.6-fold) of degradation of untreated wheat straw compared to those of the wild-type free enzymes. The action of these designer cellulosomes on wheat straw was 33 to 42% as efficient as the natural cellulosomes of Clostridium thermocellum. In contrast, the reduction of substrate complexity by chemical or biological pretreatment of the substrate removed the advantage of the designer cellulosomes, as the free enzymes displayed higher levels of activity, indicating that enzyme proximity between these selected enzymes was less significant on pretreated substrates. Pretreatment of the substrate caused an increase in activity for all the systems, and the native cellulosome completely converted the substrate into soluble saccharides.

Fructansucrases, members of glycoside hydrolase family 68, catalyze both sucrose hydrolysis and the polymerization of fructose to ␤-D-fructofuranose polymers. The resulting fructan polymers are distinguished by the nature of the glycosidic bond: inulin (␤-(2-1)-fructofuranose) and levan (␤-(2-6)-fructofuranose). In this study we demonstrate that Zymomonas mobilis levansucrase exists in two active forms, depending on the pH and ionic strength. At pH values above 7.0, the enzyme is mainly a dimer, whereas at pH values below 6.0, the protein forms well ordered microfibrils that precipitate out of the solution. These two forms are readily interchangeable simply by changing the pH. Surprisingly the manner in which the enzyme is arranged strongly affects its product specificity and kinetic properties. At pH values above 7.0, the activity of the enzyme as a dimer is mainly sucrose hydrolysis and the synthesis of short fructosaccharides (degree of polymerization, 3). At pH values below 6.0, in its microfibril form, the enzyme catalyzes almost exclusively the synthesis of levan (a degree of polymerization greater than 20,000). This difference in product specificity appears to depend on the form of the enzyme, dimer versus microfibril, and not directly on the pH. Images made by negative stain transmission electron microscopy reveal that the enzyme forms a very ordered structure of long fibrils that appear to be composed of repeating rings of six to eight protein units. A single amino acid replacement of H296R abolished the ability of the enzyme to form microfibrils with organized fibril networks and to synthesize levan at pH 6.0.

Abstract. We have analyzed two genetic variants of C2 muscle cells that have reduced levels of binding activity for a-bungarotoxin and have found that both synthesize only low levels of the a-subunit of the acetylcholine receptor. In both variants the uptake of 22Na in response to carbachol is diminished in proportion to the reduction in toxin-binding activity. In addition, the kinetic and sedimentation properties of the residual toxin-binding activity in both is indistinguishable from that seen in wild-type cells. Immunoblotting experiments on extracts of the variants using subunit-specific antibodies to a-and 13-subunits of the acetylcholine receptor demonstrated that the 13-subunit was present, but failed to detect a-subunit. In both variants, the amount of a-subunit accumulated after a 5-min period of labeling with [35S]methionine was reduced by over 90%, leading to the conclusion that the a-subunit is synthesized at greatly reduced rates. Northern blot and S1 nuclease analysis showed no differences between the a-subunit mRNA in wild-type and variant cells.