Paul Elliott Correa

Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells.

The bone morphogenetic proteins (BMPs) are a group of transforming growth factor beta (TGF-beta)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57- to 62-kDa) and type II (75- to 82-kDa) receptor components for TGF-beta and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-beta and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP-4 binding and the appearance of a major affinity-labeled product of approximately 64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs. Images

Characterization of the RDC1 gene which encodes the canine omolog of a proposed human VIP receptor Expression does not correlate with an increase in VIP binding sites

We have isolated a portion of the canine gene encoding the orphan receptor RDC1 [1]. The complete coding sequence is contained in a single exon, and an intron divides the 5′ untranslated region of RDC1 mRNA. The RDC1 protein is 94% homologous to the gene product of GPRN1, which has been proposed to serve as a VIP receptor when expressed in CHO‐K1 and COS‐7 cells (Sreedharan, S.P. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 4986–4990). Northern analysis indicates that CHO‐K1 cells endogenously express a 2.1 kb RDC1 mRNA. However, while CHO‐K1 cells possess detectable low affinity [125I]VIP binding sites, VIP binding is not altered in membranes of CHO‐K1 cells expressing varying amounts of the RDC1 gene construct. Further, endogenous VIP binding is not increased by transient expression of RDC1 in COS‐7 cells. Taken together, the data suggest that RDC1 is not a canine homolog of the proposed VIP receptor.

Synthesis of potent and selective inhibitors of human plasma kallikrein.

The synthesis and in vitro enzyme inhibition profile of a series of novel trifluoromethylketone (TFMK) inhibitors of human plasma kallikrein (PK) are described. We have developed an efficient method for the construction of peptide TFMKs that provides the final product devoid of compromised stereochemical integrity. Many of these compounds are potent inhibitors of PK and exhibit reduced inhibition of tissue kallikrein (TK) and plasmin (HP).

Selective molecular oxygen cleavage of alkynes catalyzed by Ce(IV)

Abstract The selective molecular oxygen oxidation of alkynes is catalyzed in homogeneous systems (CH 3 CN and H 2 O) by soluble salts of Ce(IV); e.g., ceric ammonium nitrate CAN [1]. The rate enhancements observed with catalytic amounts of cerium are often dramatic compared to the background autoxidation of alkynes. For example, aryl alkynes, such as phenylacetylene, exhibit no autoxidation at 115 °C and 70 bar O 2 pressure in CH 3 CN, but with a sub/cat ratio = 20 under similar conditions CAN catalyzes the reaction such that complete substrate conversion is achieved in 90%) the cleavage products of the alkyne under mild conditions (70–100 °C and 100psig O 2 pressure) with very high rates (T.N. > 100min −1 ) in many cases. Mechanistic studies (including kinetic, gas uptake, solvent labeling and autocatalysis experiments) reveal that a novel oxygen-driven Ce(IV)/Ce(III) redox cycle gives rise to catalysis of the hydrocarbon oxidation. Cerium(IV) oxidizes an alkyne-dioxygen adduct generating a dioxetene radical cation which, it is proposed, reoxidizes Ce(III) to Ce(IV). The dioxetene so produced rearranges to an α-diketone intermediate whose presence was demonstrated by GC/mass spectroscopy and whose competence as an intermediate was confirmed in separate addition experiments.

Data requirements for protein identification using chemically-assisted fragmentation and tandem mass spectrometry

Many laboratories identify proteins by searching tandem mass spectrometry data against genomic or protein sequence databases. These database searches typically use the measured peptide masses or the derived peptide sequence and, in this paper, we focus on the latter. We study the minimum peptide sequence data requirements for definitive protein identification from protein sequence databases. Accurate mass measurements are not needed for definitive protein identification, even when a limited amount of sequence data is available for searching. This information has implications for the mass spectrometry performance (and cost), data base search strategies and proteomics research.