Neutral endopeptidase (NEP) is a mammalian zinc metalloprotease involved in the inactivation of a wide variety of regulatory peptides such as enkephalins and atrial natiuretic factor. The soluble extracellular domain of NEP (sNEP) was expressed in the methylotrophic yeast Pichia pastoris. The protein was purified to homogeneity and single crystals have been obtained. Enzymatic deglycosylation of the enzyme was essential for the production of crystals suitable for X-ray analysis for both the NEP-phosphoramidon binary complex and the apo enzyme.
Platelet-derived growth factor (PDGF) occurs as three dimeric isoforms, AA, BB, and AB, which were previously shown to bind to two receptors with different isoform-specificity, the A/B-type (binds all three isoforms) and the B-type (binds only PDGF-BB). Results from competition binding experiments with Swiss 3T3 cells suggest the existence of a third receptor type, which recognizes PDGF-AB and PDGF-BB. Furthermore, Swiss 3T3 cells and human dermal fibroblasts express different relative and absolute levels of these receptor types. In particular, Swiss 3T3 cells express 90,000 PDGF-AA binding sites (A/B-receptors) per cell, whereas human fibroblasts express only 20,000 A/B-receptors per cell. All three PDGF isoforms were tested in either cell type for their effect on DNA synthesis. PDGF-BB and PDGF-AA were also tested in Swiss 3T3 cells for their effect on inositol phospholipid metabolism and chemotaxis. Each isoform promoted all three processes dose-dependently, but there were differences in the maximum cellular responses elicited. These responses reflect the capacity of the cells to bind the individual isoforms. These results demonstrate that the previous distinctions in responsiveness to the different PDGF isoforms are primarily a consequence of the differences in the levels of surface expression of the various isoform-specific receptor types, rather than of the differences in the intrinsic activity of these isoforms. Furthermore, these results suggest that all types of PDGF receptors are capable of responding to their respective ligands by mediating phosphoinositide breakdown, chemotactic responses, and DNA synthesis. Whether they exhibit other functional differences remains to be seen.
With the deciphering of whole genomes, a tremendous amount of data has been made available for research. In order to analyze all this information within a reasonable time frame and at reasonable cost, new technologies in DNA analysis as well as enzyme assays have been developed in the last few years in a symbiosis of biological, automation and miniaturization technologies. Examples are chip technologies for DNA analysis and microplate enzyme assays for high-throughput screening. Applications in biomedical research and in microbial diagnostics were presented.
