N-terminal sequence analysis of atrial granule serine proteinase purified by affinity chromatography

Atrial granule serine proteinase is considered the leading candidate endoproteolytic processing enzyme of pro-atrial natriuretic factor. Its cleavage specificity is directed toward a monobasic amino acid processing site, and as such, the atrial enzyme is distinguished from the family of prohormone convertases which act at dibasic amino acid processing sites. To delineate the molecular mechanisms which distinguish monobasic from dibasic amino acid-directed processing enzymes, pure atrial enzyme is needed for sequence determination leading to molecular cloning, and for preparation of antisera. An affinity chromatography purification scheme seemed a logical modification of our established procedures to yield suitable amounts of enzyme for further studies. Surprisingly, pseudo-peptide bond inhibitors of the atrial enzyme [Damodaran and Harris (1995),J. Protein Chem., this issue] formed ineffective affinity ligands, even though these compounds contain essential residues on either side of what would be the scissile bond in a peptide substrate. On the other hand, tripeptide aldehydes (based on the substrate recognition sequence of the atrial enzyme) linked to Sepharose formed effective affinity matrices, permitting purification of the enzyme in a single step from a subcellular fraction enriched for atrial granules and lysosomes. Hence, the enzyme was purified 2000-fold in 90% overall yield, and subjected to N-terminal sequence analysis through 26 residues. The sequence determined, XXPEAAGLPG[R, L]GNPVP[F, G]R[Q, I]XY[G, E]XR(N, A]V, indicates that the atrial enzyme is unique, showing little sequence homology to other proteins in the database.Abbreviations AGSP atrial granule serine proteinase - ANF atrial natriuretic factor - BSA bovine serum albumin - Bz benzoyl - EACA 6()-aminocaproic acid - HEPES N-2-hydroxyethylpiperazine-N'-propanesulfonic acid - HPLC high-performance liquid chromatography - PEG polyethylene glycol-3350 - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Single-letter abbreviations are used to denote amino acids     

Preparations of Ψ-peptide bond and peptide-aldehyde inhibitors of atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor

Pseudo-peptide bond inhibitors (-bond inhibitors) and peptide-aldehyde inhibitors of atrial granule serine proteinase, the candidate processing enzyme of pro-atrial natrieuretic factor, are prepared in high yield and purity by novel synthetic routes. The -bond compounds retain essential residues for enzyme binding, but place the enzyme inhibition site in the midst of the peptide sequence. Thus, Bz-APR--LR and Bz-APR--SLRR can be considered readthrough inhibitors of atrial granule serine proteinase. The most potent -peptide, Bz-APR--SLRR (IC₅₀=250 M), is about fivefold less potent than the best peptide-aldehyde inhibitor (EACA-APR-CHO), and both the -bond and peptide-aldehyde compounds are competitive, reversible inhibitors of the enzyme. The -bond peptides containing two C-terminal Arg residues are three-to tenfold more potent than the analogous compounds containing only one C-terminal Arg residue, confirming the importance of both Arg residues in the enzyme processing recognition site. As expected, because of their moderate potencies, the -peptides are not useful affinity ligands for purification of atrial granule serine proteinase, but both peptide aldehydes are effective affinity ligands [Damodaran and Harris (1995),J. Protein Chem., this issue].Abbreviations AGSP atrial granule serine proteinase - ANF atrial natriuretic factor - Bz benzoyl - DIEA diisopropylethylamine - DIPCDI diisopropylcarbodiimide - DMF dimethylformamide - DMSO dimethylsulfoxide - EACA 6(e)-aminocaproic acid - EtOAc ethyl acetate - HEPES N-2-hydroxyethylpiperazine-N-propanesulfonic acid - HOBt N-hydroxybenzotriazole - HPLC high-performance liquid chrornatography - NMR nuclear magnetic resonance - PEG polyethylene glycol-3350 - PyBOP benzotriazole-1-yl-oxy-trispyrrolidino-phosphonium-hexafluorophospate - TEA triethylamine - TFA trifluoroacetic acid - THF tetrahydrofuran - TLC thin-layer chromatography - UV ultraviolet - pseudo-peptide bond -CH₂-NH-. Single-letter abbreviations are used to denote amino acids     

Glutathione disulfide as an index of oxidative stress during postischemic reperfusion in isolated rat hearts

The objectives of this study were to determine 1) whether reactive oxygen species generated upon postischemic reperfusion lead to oxidative stress in rat hearts, and 2) whether an exogenous prooxidant present in the early phase of reperfusion causes additional injury. Isolated buffer-perfused rat hearts were subjected to 30 min of hypothermic no-flow ischemia followed by 30 min of reperfusion. Increased myocardial content of glutathione disulfide (GSSG) and increased active transport of GSSG were used as indices of oxidative stress. To impose a prooxidant load, cumene hydroperoxide (20 M) was administered during the first 10 min of reperfusion to a separate group of postischemic hearts. Reperfusion after 30 min of hypothermic ischemia resulted in a recovery of myocardial ATP from 28% at end-ischemia to 50–60%, a release of 5% of total myocardial LDH, and an almost complete recovery of both coronary flow rate and left ventricular developed pressure. After 5 and 30 min of reperfusion, neither myocardial content of GSSG nor active transport of GSSG were increased. These indices were increased, however, if cumene hydroperoxide was administered during early reperfusion. After stopping the administration of cumene hydroperoxide, myocardial GSSG content returned to control values and GSH content increased, indicating an unimpaired glutathione reductase reaction. Despite the induction of oxidative stress, reperfusion with cumene hydroperoxide did not cause additional metabolic, structural, or functional injury when compared to reperfusion without cumene hydroperoxide. We conclude that reactive oxygen species generated upon postischemic reperfusion did not lead to oxidative stress in isolated rat hearts. Moreover, even a superimposed prooxidant load during early reperfusion did not cause additional injury.     

Inositol lipid-mediated signalling in response to endothelin and ATP in the mammalian testis

Molecular and Cellular Biochemistry - The testis is a complex organ in which local control is achieved by signalling between its constituent cells. Herein we describe the responses of cultured rat...     

Unique and selective mitogenic effects of vanadate on SV40-transformed cells

Vanadate and insulin both function as unique complete mitogens for SV40-transformed 3T3T cells, designated CSV3-1, but not for nontransformed 3T3T cells. The mitogenic effects induced by vanadate and insulin in CSV3-1 cells are mediated by different signaling mechanisms. For example, vanadate does not stimulate the tyrosine phosphorylation of the insulin receptor -subunit nor the 170 kDa insulin receptor substrate-1. Instead, vanadate induces a marked increase in tyrosine phosphorylation of 55 and 64 kDa proteins that is not observed in insulin-stimulated CSV3-1 cells. Perhaps most interestingly, vanadate-induced mitogenesis is associated with the selective induction ofc-jun andjunB expression without significantly inducingc-fos orc-myc. Furthermore, treatment of CSV3-1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation andc-jun induction. These and related data suggest that modulation of protein tyrosine phosphorylation andc-jun andjunB expression may serve the critical roles in mediating vanadate-induced mitogenesis in SV40-transformed cells.     

Physicochemical characterization and in vitro interaction with brain capillary endothelial cells of artificially monoacylated ribonucleases A

Acylated proteins play a crucial role in cellphysiology because of their increased interaction withmembranes. Their isolation is difficult as aconsequence of their low cellular concentration andtheir chemical preparation is problematic due tosolubility problems. Through the use of reversedmicelles, we produced tens of milligrams of acylatedribonucleases A, chosen as a model, purified them bysemi-preparative high performance liquidchromatography (HPLC) and characterized them by analyticalHPLC, capillary electrophoresis, mass spectrometry, peptide mapping, Edman degradation and enzyme activity. We nextscrutinized the interaction with an in vitro blood–brainbarrier model and demonstrated that palmitoylated andstearoylated ribonucleases A are transported from onecompartment to the other across the cellular monolayer,in contrast to the native enzyme.     

Measurement of 15N-1H coupling constants in uniformly 15N-labeled proteins: Application to the photoactive yellow protein

A modified HNHB experiment is presented that allows thedetermination of J(NH) coupling constants directly from the ratio ofcross-peak to diagonal-peak intensities. The experiment was applied to thephotoactive yellow protein (PYP) and yielded the magnitude of 117³J(NH) coupling constants. In addition, 29³J(NH^(i–1)) coupling constantscould be measured, providing information about the backbone angle .These data, in conjunction with the magnitudes of the³J(H^NH) coupling constantsobtained from the HNHA spectrum, effectively discriminate the twopossibilities for the stereospecific assignment of theH resonances in glycine residues. For all eight glycineresidues in PYP that were not subject to conformational averaging and hadnon-degenerate H resonance frequencies, the J-couplingdata, together with limited NOE data, yielded the stereospecific assignmentof the H resonances for these residues. In addition,reliable and precise , dihedral constraints were also derived forthese residues from the J-coupling data.