In higher eukaryotes, nine aminoacyl-tRNA synthetases are associated within a multienzyme complex which is composed of 11 polypeptides with molecular masses ranging from 18 to 150 kDa. We have cloned and sequenced a cDNA from Drosophila encoding the largest polypeptide of this complex. We demonstrate here that the corresponding protein is a multifunctional aminoacyltRNA synthetase. It is composed of three major domains, two of them specifying distinct synthetase activities. The amino and carboxy-terminal domains were expressed separately in Escherichia coli, and were found to catalyse the aminoacylation of glutamic acid and proline tRNA species, respectively. The central domain is made of six 46 amino acid repeats. In prokaryotes, these two aminoacyl-tRNA synthetases are encoded by distinct genes. The emergence of a multifunctional synthetase by a gene fusion event seems to be a specific, but general attribute of all higher eukaryotic cells. This type of structural organization, in relation to the occurrence of multisynthetase complexes, could be a mechanism to integrate several catalytic domains within the same particle. The involvement of the internal repeats in mediating complex assembly is discussed.
A detailed investigation by ultracentrifugation of the colipase-taurodeoxycholate system showed the formation of well-defined mixed associations with a sedimentation coefficient of about 2.2 S. The fact that these associations were only detectable above the critical micelle concentration of the salt indicated that micelles rather than monomers were bound to the cofactor.Two technical difficulties must be overcome before the weight of the associations could be measured with a reasonable accuracy. Firstly, the partial specific volume of the associations was determined using a digital microdensimeter and the interferometric system of the ultracentrifuge for concentration determinations. Secondly, due to the fact that micelle concentrations could not be equilibrated by dialysis, even after an extended period of time, an appropriate dilution of the ligand in the buffer compartment was necessary in order to compensate for its fixation by colipase in the solution. Then, the ionic strength dependence of the weight of the associations was found to vary in parallel with that of the micelles and to be in each case equal to the sum of the weights of one colipase molecule and one micelle. Therefore, colipase can be expected to contain a single high affinity site for bile salt micelle binding.During the last few years, several low molecular weight proteins designated colipase have been purified from porcine pancreas. The smallest and structurally best known, colipase 11, contains 84 amino acid residues [I]. It is composed of a central "core" crosslinked by four disulfide bridges and of two "tails" loosely bound by a single bridge [2]. Colipase I, which is the form used throughout this and the next work, differs from colipase I1 by the presence of ten additional residues at the end of the C-terminal tail [l]. Little is known so far about the structure of a third colipase characterized in porcine pancreas, except for the existence of seven N-terminal residues not present in colipases I and I1 [3].Flowing into the duodenum with pancreatic juice [4], colipase has been reported to prevent the inhibition of the lipase-catalyzed hydrolysis of dietary triglycerides by physiological concentrations of bile salts [5]. This effect has been assumed to imply in the first place a bile salt-induced dimerization of colipase and an association of the dimer with lipase [ 6 ] .It has been confirmed in this laboratory [7] that the apparent sedimentation coefficient and molecular weight of colipase are approximately doubled in the presence of sodium taurodeoxycholate. However, the present and following paper will show that this increase results from the binding of a bile salt micelle to the colipase molecule rather than from dimerization. MATERIALS AND METHODS ColipasePorcine pancreatic colipase I was purified to homogeneity by a modification of a recently described technique [7]. The starting material was a commercial pancreas powder (Choay, France) which was exhaustively delipidated in the laboratory prior to use. The extracts were submitted as ...
This is the first report on the existence in Drosophila of a protein with properties similar to those of vertebrate fibronectin that we shall refer to as Drosophila fibronectin. Rabbit antibodies against human plasma fibronectin have allowed the detection of this molecule in Drosophila haemolymph; common epitopes are shared by the two proteins. Drosophila fibronectin with a subunit mol. wt of approximately 230 kd is a glycoprotein which binds to denatured mammalian collagen. It is present throughout development and is as abundant in embryos as in larvae and adult flies. Drosophila fibronectin is differentially expressed during embryogenesis, a small amount being present before the blastoderm stage. Its concentration increases at gastrulation and reaches a steady‐state value at the end of organogenesis. Drosophila fibronectin is predominantly detected by immunofluorescence on frozen sections of 16 h embryos in the extracellular spaces lying between the different tissues and organs. In mature third instar larvae, most of the staining is concentrated in fat body and imaginal discs, and the pattern strongly supports an extracellular localization of the protein. In addition, it is shown that Drosophila embryonic cells can functionally utilize vertebrate fibronectin for their spreading and differentiation. Finally, injection of antihuman plasma fibronectin antibodies in early embryos leads to the same phenotype as injection of Arg‐Gly‐Asp‐containing peptides. This result suggests that one of the Arg‐Gly‐Asp‐bearing protein(s) involved in gastrulation might be fibronectin.
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