Robert L. Johnson

Hepatocyte Nuclear Factor 4 Is a Transcription Factor that Constitutively Binds Fatty Acids.

The 2.7 A X-ray crystal structure of the HNF4gamma ligand binding domain (LBD) revealed the presence of a fatty acid within the pocket, with the AF2 helix in a conformation characteristic of a transcriptionally active nuclear receptor. GC/MS and NMR analysis of chloroform/methanol extracts from purified HNF4alpha and HNF4gamma LBDs identified mixtures of saturated and cis-monounsaturated C14-18 fatty acids. The purified HNF4 LBDs interacted with nuclear receptor coactivators, and both HNF4 subtypes show high constitutive activity in transient transfection assays, which was reduced by mutations designed to interfere with fatty acid binding. The endogenous fatty acids did not readily exchange with radiolabeled palmitic acid, and all attempts to displace them without denaturing the protein failed. Our results suggest that the HNF4s may be transcription factors that are constitutively bound to fatty acids.

Open access atmospheric pressure chemical ionization mass spectrometry for routine sample analysis

We report the introduction and use of an atmospheric pressure chemical ionization liquid chromatography-mass spectrometry instrument that has been designed specifically for use by the synthetic chemist on an open access, walk-in basis. This instrument has been configured with an easy-to-use sample log-in terminal that requires the user to provide only a sample identification number and a user name. Sample analysis takes approximately 4 min and provides the synthetic and medicinal chemist with rapid and reliable mass spectrometry analysis. Since installation of the system, it has analyzed an average of about 80 samples per day and has the capacity to run over 100 samples per day without the intervention of a specialist operator. This capability has eliminated the need for an operator to analyze routine samples and allows the mass spectroscopist more time to deal with problem solving.

Initial characterization of multiple endopeptidases in bovine adrenal chromaffin vesicles

All regulatory peptides are synthesized as large, inactive precursor proproteins that must undergo specific endoproteolytic processing to yield bioactive peptides. In most cases, enzymatic release of the biologically active peptides occurs by endoproteolytic cleavage at doublets of basic amino acid residues that precede and/or follow that particular sequence [1,2]. The catecholamine-containing chromaffin vesicles of the adrenal medulla are enriched in a great variety of regulatory peptides (i.e. enkephalins, neurotensin, neuropeptide Y, etc.) and thus are good sources for the isolation and characterization of peptide processing enzymes [3,4,5]. To isolate putative endopeptidases of the thioland serineprotease families, the dialysed lysate of purified bovine chromaffin vesicles was consecutively fractionated through p-chloro-mercuribenzoateagarose (PCMB-agarose), p-aminobenzamidineagarose (p-ABZ-agarose) and soybean trypsin inhibitor-agarose (STI-agarose) affinity columns. Three intermediate proenkephalin precursor peptides (BAM12P, BAM22P and amidorphin) were used as substrates for the assay of endopeptidase activities. These peptides contain pairs of basic amino acids, Arg-Arg, Lys-Arg and Lys-Lys, which have putative cleavage sites for the endopeptidases. Degradation peptide fragments were separated by reverse phase HPLC and identified by FAB mass spectrometry, amino acid analysis and sequence analysis, which was performed with an Applied Biosynthesis model 477a protein sequencer using Edman degradation chemistry. The fraction retained and eluted from PCMB-agarose affinity chromatography hydrolyzed the Arg-Arg sequence of BAM12P, resulting in the generation of Met-enkephalin and Met-enkephalin-Arg at pH 5.7. However, this enzyme preparation was unable to hydrolyze amidorphin at the Lys-Lys pair of basic residues. This activity was inhibited by PCMB and E64, indicating that a thiol protease is involved. The dialysate fraction that was not retained by the PCMB-agarose column was subsequently retained and eluted from the p-ABZagarose affinity column. This dialysate fraction contained enzyme activity which cleaved at the Lys-Arg of BAM22P and at the Lys-Lys of amidorphin at pH 7.4. BAM12P was however a poor substrate for this fraction. This activity was not inhibited by ST1, which is indicative of a non-trypsin-like endopeptidase. Additionally, a separate endopeptidase cleaving at Glu-Trp of BAM22P, resulting in the generation of BAM12P, was also found in this preparation. The dialysate fraction not retained in the first two columns but retained and eluted from the STI-agarose affinity column had an enzyme activity capable of hydrolyzing amidorphin at the carboxy side of Lys-Lys. This activity was completely inhibited by STI which is indicative of a trypsin-like endopeptidase. BAM12P however was poorly cleaved by this preparation. This study demonstrates that a variety of different endopeptidase activities is found in soluble lysates of adrenal medulla chromaffin vesicles. A multiplicity of peptide processing enzymes with different specificities suggests the possibility that modification of a particular processing enzyme may result in specific changes in the cocktail of regulatory peptides.