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Polymer-grafted magnetite (Fe3O4) nanoparticles (diameter about 10 nm) were prepared through a direct polymer grafting reaction from their surfaces. The chemisorbed initiator (TEMPO-based alkoxyamine) for nitroxide-mediated radical polymerization with a phosphoric acid group gave controlled polystyrene (PS) and poly(3-vinylpyridine) (P3VP) graft layers on the surface. The graft densities of polymers on the surfaces of magnetite particles were estimated at 0.12−0.20 chains/nm² by thermogravimetric analysis (TGA). The improvement in the dispersibility of PS-modified magnetite in good solvents was verified by ultraviolet−visible (UV−vis) absorption spectroscopy, transmission electron microscopy (TEM), and dynamic light scattering (DLS). In the case of P3VP-modified magnetite, the particles were stably dispersed in good solvents, by protonation, in acid solution, and by quarternization with iodomethane, in neutral aqueous solution. The magnetic response of the polymer-grafted magnetite against an external magnetic field was confirmed in the stable dispersion.

A method is proposed for predicting the equilibrium conversion for the synthesis of monoacyl hexose through the lipase-catalyzed condensation of a fatty acid and a hexose in a water-miscible solvent in the presence of a molecular sieve, based on the apparent reaction equilibrium constant, the adsorption isotherm of water on the molecular sieve, the solubility of hexose in the solvent, and the mass balance with respect of water. Validity of the model was examined for the syntheses of lauroyl mannose, lauroyl glucose, and myristoyl mannose in acetonitrile, 2-methyl-2-propanol, or 2-methyl-2-butanol with molecular sieves 3A 1/16 and 4A 1/16. The predicted conversions agreed well with the experimental values except for the case where a significant amount of diester was formed as the result of the addition of an excess amount of the molecular sieve to the solvent or the high molar ratio of the fatty acid to the hexose.

N - (benzyloxycarbonyl) - L - aspartyl - L - phenylalanine methyl ester, the precursor of the synthetic sweetener aspartame, was continuously synthesized in an organic solvent with immobilized thermolysin. Ethyl acetate was found to be the most effective organic solvent for the synthesis of this precursor. The plug flow type reactor was found unsuitable, because the immobilized enzyme in it was gradually inactivated even if calcium, the essential stabilizing factor for thermolysin, was added to the substrate. In addition, a severe channeling of the flow occurred in a column. A stirred tank reactor was successfully operated for over 300 hours with a yield of approximately 90 percent and a large space velocity.

Eleven cDNA clones (pra1 to pra9A, pra9B, and pra9C) were isolated from a pea (Pisum sativum) leaf cDNA library which were similar to small GTP-binding proteins. These cDNAs encoded proteins of 22-25 kDa, which exhibited 45-92% identity to one another at the amino acid level. The putative proteins included the characteristic sequences of ras-related small GTP-binding proteins: four conserved domains involved in binding of GTP/GDP; an effector domain; and cysteine residues at the COOH-terminus. Indeed, the pra6 protein, expressed in Escherichia coli, clearly showed GTP-binding activity. Phylogenetic analysis showed that these clones can be classified into two subgroups: proteins encoded by pra1 to pra7 being related to ypt3 and rab11 proteins; and proteins encoded by pra8, pra9A, pra9B, and pra9C being related to YPT1 and rab1 proteins. The effector sequences in these two subgroups were different. RNA gel blot analysis showed that most of the corresponding genes are differentially expressed in pea leaves and roots. Variations in expression were also observed for structurally related genes.

The gene product of an open reading frame of the chloroplast genome, accD, that has sequence similarity with a subunit of acetyl-CoA carboxylase from Escherichia coli was detected immunochemically in pea chloroplasts. The apparent molecular mass of the accD protein was 87 kDa on SDS-polyacrylamide gel electrophoresis. The protein was acidic and had less mobility than the calculated value, 67,116. Acetyl-CoA carboxylase activity solubilized from pea chloroplasts was inhibited by antibodies against recombinant accD protein. The antibodies precipitated a polypeptide of 35 kDa containing biotin and a polypeptide of 91 kDa together with the 87-kDa-accD protein. The accD protein formed a complex with the molecular mass of about 700 kDa, probably with the 35- and 91-kDa proteins. These results indicate that the chloroplast-encoded polypeptide, accD protein, is a component of a functional acetyl-CoA carboxylase in chloroplasts and this enzyme is a multi-subunit complex, like that from E. coli. The synthesis of accD protein was not induced by light.

We have developed a convenient method of cDNA cloning for monomeric GTP-binding proteins. The method consists of a serial dilution of cDNA expression library, a separation of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and a ligand blotting; each group containing the proteins from about 1000 independent colonies was separated by SDS-PAGE, transferred to nitrocellulose filters, incubated with 10−9 M [α-32P]GTP, and autoradiographed. Separation of proteins by electrophoresis resulted in a drastic reduction of nonspecific binding of labeled GTP to the filters. A serial dilution of colonies resulted in a single clone having GTP-binding activity. We checked about 60,000 colonies and detected two signals. This method is dependent on the ligand specificity of the expressed proteins and is different from the conventional methods based on DNA sequence similarity. This method can be used to isolate cDNAs encoding novel GTP-binding proteins.Copyright 1993, 1999 Academic Press