Alfred Jonczyk

COUPLING OF PROTON TRANSLOCATION THROUGH ATPASE INCORPORATED INTO SUPPORTED LIPID BILAYERS TO AN ELECTROCHEMICAL PROCESS

Abstract H+-ATPase is incorporated into solid-supported lipid bilayers separated from the gold support by a peptide spacer. The translocation of protons across the lipid film to the inner side is coupled to the discharge of protons at the gold surface. The overall process is investigated by square wave voltammetry (SWV) and double potential-pulse chronoamperometry (CA). As a result, the formation of a proton gradient is monitored by SWV whereas currents measured by CA monitor the stationary state when the enzyme activity is directly coupled to the charge transfer at the electrode. These currents markedly depend on the number of ATPases present in the bilayer.

Substrate Analogue Renin Inhibitors Containing Replacements of Histidine in P2 or Isosteres of the Amide Bond Between P3 and P2 Sites

The search for orally active renin inhibitors as therapeutic agents continues to represent a challenging target for medicinal chemists.1 Analogues of the angiotensinogen region flanking the bond split by renin have turned out to be very potent and specific inhibitors of renin. However, the high affinity of these angiotensinogen analogues for human renin is often associated with fast hydrolysis between P3 and P2 sites by the intestinal serine protease chymotrypsin.2

Evaluation of absorption enhancement for a potent cyclopeptidic ανβ3-antagonist in a human intestinal cell line (Caco-2)

Abstract Different absorption enhancing principles for a potent cyclopeptidic ανβ3-antagonist (EMD 121974) were investigated in monolayers of a human intestinal cell line (Caco-2). Transepithelial transport was quantitated by reversed-phase high-performance liquid chromatography. Cytotoxic effects were characterized by determination of transepithelial electrical resistances (TEERs), propidium iodide (PI)-influx, FITC-phalloidin staining and the release of cytosolic lactate dehydrogenase (LDH). Medium chain fatty acids (MCFAs, NaC10, NaC12) and taurocholate (NaTC) were the most efficient enhancers of cyclopeptide and FITC-dextran 4400 permeability coefficients, displaying different time profiles of activity. Whereas NaTC (15 mM) showed almost a constant permeation enhancing effect from 20 min up to 120 min (ca. 12-fold), MCFA absorption enhancement was markedly dependent on incubation time (NaC10, 20 min: 1.2-fold, 120 min: 17-fold; NaC12, 20 min: 4.3-fold, 120 min: 13-fold). All cytotoxicity assays demonstrated that MCFAs were significantly more cytotoxic than NaTC. Ion pairing with hydrophobic amino acids and heptane sulfonate distinctly increased octanol–buffer partition coefficients of the cationic cyclopeptide but did not enhance its transepithelial permeability. Nanoparticles as well as β-cyclodextrin neither affected integrity of the cells nor transport properties of the cyclopeptide. In summary, significant absorption enhancement was only observed with NaTC or MCFAs. Increase in permeability coefficients using NaTC occurred rapidly with acceptable cytotoxicities and merits further investigations.

Multiple molecular forms of tachykinins in rat spinal cord: a study comparing different extraction methods.

Various procedures for extraction at acid, neutral and alkaline pH were compared with regard to the yield of different tachykinins and tachykinin-like substances from rat spinal cord. Reverse phase high performance liquid chromatography (RP-HPLC) and radioimmunoassay with various C-terminally directed tachykinin antisera and a newly developed N-terminally directed substance P (SP)-antiserum (SPN 1) were used. Antiserum SPN 1 fully reacts with SP-analogues modified at the C-terminal end (SP free acid and SP-Gly-Lys) and also (77%) with SP(1-9) but not with C-terminal SP-fragments lacking 2 or more N-terminal amino acids. The highest levels of SP-like immunoreactivity (LI) and neurokinin A (NKA)-LI were measured after combined water and acetic acid extraction procedures. Also when measuring cholecystokinin-like immunoreactivity the highest level was obtained following this extraction procedure. RP-HPLC revealed a major component of SP-LI at the position of synthetic SP irrespectively of the extraction method and if the C- or N-terminally directed antiserum was used. Neutral water extracts contained a late eluting component detected with the C-terminally, but not with the N-terminally, directed antiserum. Acid and alkaline extracts, in contrast, contained components which could be detected with the N-terminally, but not with the C-terminally, directed SP-antiserum. Immunoreactive components eluting at the position of NKA and NKB were found in all types of extracts with NKA-, kassinin- and eledoisin-antisera. The NKB- and neuropeptide K (NPK)-components were more prominent in acid than in neutral and alkaline extracts. In conclusion, the present results indicate that rat spinal cord may contain molecular forms of tachykinin-like immunoreactivity in addition to those previously described and illustrate the importance of the choice of extraction method in immunochemical studies. Combined extraction in water and acetic acid appears to be a suitable method when the content of peptides with different chemical properties are to be measured in a tissue sample.