Insulin-like growth factor-binding protein-3 (IGFBP-3) is a member of a family of structurally conserved proteins (IGFBP-1 to -6) which act as carriers and regulators of the mitogenic peptide hormones IGF-I and IGF-II. Members of the IGFBP family share conserved cysteine-rich aminoand carboxyl-terminal regions. The amino-terminal domain of these proteins is recognised to contain an IGF-binding determinant, but evidence to support a binding site in the carboxyl-terminal region of the protein is less rigorous. To further investigate this, we have synthesised both the amino-terminal (residues 1-88; N-88) and carboxyl-terminal (residues 165-264; C-165) domains of human IGFBP-3 in bacteria, as fusion proteins with a carboxyl-terminal FLAG peptide. Although only C-165 showed binding to IGF-I and -II by solutionbinding assays, both N-88 and C-165 demonstrated binding to IGF-I and -II by biosensor analysis albeit with reduced affinities compared with full-length IGFBP-3. Only the carboxyl-terminal fragment (C-165) was able to form hetero-trimeric complexes with IGF-I and the acid-labile subunit (ALS). We conclude that the carboxylterminal domain of IGFBP-3 contains an IGF-binding determinant and can form ternary complexes with ALS.
Genes encoding the basic protease of Dichelobacter nodosus (bprV) and the subtilisin of Bacillus subtilis (aprE) were cloned and expressed in Corynebacterium glutamicum. In each case, enzymatically active protein was detected in the supernatants of liquid cultures. While the secretion of subtilisin was directed by its own signal peptide, the natural signal peptide of the bprV basic protease did not facilitate secretion. A hybrid aprE-bprV gene in which the promoter and signal peptide coding sequences of subtilisin replaced those of bprV could be expressed, and basic protease was secreted by C. glutamicum. Expression of these proteases in C. glutamicum provides an opportunity to compare protein secretion from this gram-positive host with that from other gram-positive and gram-negative bacteria.
The crude protein content (amino acid Nx6.25) of six varieties of Psophocarpus tetragonolobus tuber varied from 12.7 to 16.9%. Free amino acids and low molecular weight peptides account for about 40% of this crude protein value. The non-dialysable protein content of winged bean tuber is therefore less than previously assumed, but is still relatively high for a tuber crop. The amino acid contents of the tuber varieties were similar and nutritionally acceptable, except for the sulphur containing amino acids. Fresh tubers showed trypsin and chymotrypsin inhibitory activity and haemagglutinin activity. The inhibitor levels showed little variation but differences in the levels of haemagglutinin activity were found. On SDS-polyacrylamide gels the tuber proteins revealed a simple subunit pattern of two major polypeptides of M, 30000 and 20000. Fractionation of the tuber proteins showed that the inhibitors and haemagglutinins comprise about 35% of the tuber protein. In addition, three acidic proteins (about 12% of tuber protein), three basic proteins (ca. 34% of tuber protein) and a minor component (M,-10000) with a high cystine content (11 residue %) were identified.
In an attempt to differentiate virulent and benign strains of B. nodosus, the extracellular proteolytic activity of these cultures was assayed with elastin, casein and hide powder azure, and the stability to heating at 55°C was determined. Broth cultures of both strains hydrolysed 125I-labelled elastin, indicating that this activity is not a unique marker of virulence. When cultures were grown in Trypticase-arginine-serine broth medium modified by omitting Na2C03 and thioglycollic acid, the total proteolytic activity and its stability at 55°C could be used to differentiate isolates causing virulent or benign footrot lesions. However, when other broth cultures were used, these parameters could no longer be used to make such a distinction. The proteases of a virulent and benign strain of B. nodosus were partially purified and characterized. Four to five closely related proteases were detected by polyacrylamide gel electrophoresis at pH 8·8 in both types of isolates. The proteases are serine-type enzymes requiring a divalent metal ion such as calcium for activity. The proteases of the benign strain were somewhat less stable to heat than the enzymes of the virulent strain. Differences in the relative mobilities of the proteases of virulent and benign strains of B. nodosus, on electrophoresis at pH 8'8, suggest that this property may be used to distinguish virulent and benign strains.
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