Lester C. E. Taylor

A novel 1745-dalton pyroglutamyl peptide derived from chromogranin B is in the bovine adrenomedullary chromaffin vesicle

Summary1.Following the recent demonstration of a glutaminyl cyclase activity localized in adrenomedullary chromaffin vesicles, an assay was developed to isolate and characterize posttranslationally modified peptides from this tissue which contain pyroglutamate. This assay consisted of spectrometric identification of peptides before and after enzymatic removal of pyroglutamyl residues.2.Using this procedure, a pyroglutamyl peptide (BAM-1745) was isolated and sequenced and was shown to be a significant component of adrenomedullary secretory vesicles.3.A computer search through the Swiss-Prot protein sequence database revealed a 93% identity of BAM-1745 and a fragment of human chromogranin B (Gln580-Tyr593).

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.

In vitro metabolism of a potent HIV-protease inhibitor (141W94) using rat, monkey and human liver S9.

Compound 141W94 (Vertex VX478) (3S)-tetrahydro-3-furyl N-[((S,2R)-3-(4-amino-N-isobutylbenzenesulfonamido)-1-benzyl- 2-hydroxypropyl] carbamate, is a potent HIV-protease inhibitor and is currently undergoing clinical trials. The purpose of this study was the rapid identification of the phase I and II in vitro metabolite of 141W94 using mass spectrometry. Four different sources of liver S9 fractions were used for studying comparative in vitro metabolism of 141W94. They were obtained from Arochlor-induced rat, normal (untreated) rat, cynomolgus monkey and human livers. Selected incubations were supplemented with uridine diphosphate glucuronic acid and the reduced form of glutathione. The predominant species seen in the incubation mixture was the parent compound 141W94. Metabolites arising from ring opening to form the diol and carboxylic acid and oxidation of the tetrahydrofurran ring (formation of dihydrofuran) were identified. In addition, of the two monohydroxylated products identified, one resulted from hydroxylation on the aniline ring and the other from hydroxylation at the benzylic position. Two different glucuronides were also observed. Comparing the three species, very little metabolism was seen in the normal (non-induced) rat. The metabolic profile and extent of metabolism with induced rat, monkey and human S9 was similar. Induced rat S9 incubation showed the formation of two unique metabolites that were not seen in non-induced rat, monkey and human S9 fractions. They were the monohydroxylated glucuronide and a carbamate cleavage product. The metabolites were identified using mass spectrometry based on their molecular masses and fragmentation patterns.

Identification of a 7B2-derived tridecapeptide from bovine adrenal medulla chromaffin vesicles

Summary1.A novel tridecapeptide was isolated from extracts of bovine adrenal medulla chromaffin vesicles and the primary structure determined to be SVPHFSDEDKDPE.2.This peptide is identical to the C termini of human and porcine 7B2 and is highly homologous to the same region of the mouse andXenopus lavis protein.3.In all these species the homologous peptide is preceded by a pair of lysine residues, a potential proteolytic processing site.4.Ser6 is part of a well-conserved casein kinase II consensus phosphorylation sequence. Evidence for phosphorylation of this residue was obtained during Edman sequencing.5.Thus, this novel adrenal medullary probably arises from the posttranslational processing of the bovine 7B2 protein.

A novel tetradecapeptide isolated from bovine adrenal medulla chromaffin vesicles with strong homology to an internal sequence coded by the rat 1B1075 (Preprosecretogranin III) gene.

A novel tetradecapeptide, PheProLysProAlaGlySerGlnAspLysProLeuHisAsn, was isolated from boiling water extracts of bovine adrenal medulla chromaffin vesicles. The primary structure of the peptide was characterized by amino acid analysis, fast atom bombardment mass spectrometry, and gas-phase sequencing. The synthetic and native peptides comigrated on reversed-phase high-performance liquid chromatography, supporting the proposed sequence. This peptide shares 86% homology to residues 67-80 of the recently reported rat 1B1075 gene product secretogranin III and probably represents a processed product derived from the bovine equivalent.

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.