Gel permeation chromatography-high performance liquid chromatography combination as an automated clean-up technique for the multiresidue analysis of fats.

The well-known and almost universally utilizable clean-up technique of gel permeation chromatography (GPC) and subsequent conventional silica-gel column chromatography was automated by an on-line solvent evaporation of the GPC fraction, followed by normal-phase HPLC separation. The ternary solvent system n-hexane-toluene-acetone (88:10:2, v/v/v) was used as the mobile phase which resulted in only one HPLC fraction for all relevant analytes. The HPLC column was cleaned automatically after each sample by backflushing with polar solvents. The recoveries and reproducibilities of 35 analytes (mainly organochlorine compounds) were in the range of 77-90% and 3-7%, respectively; the high efficiency of the HPLC separation provide very clean extracts for the GC analysis. This automated clean-up technique is routinely used for the multiresidue analysis of various fat-containing food and biota samples.

The degradation of bioactive peptides and proteins by dipeptidyl peptidase IV from human placenta.

The degradation of several bioactive peptides and proteins by purified human dipeptidyl peptidase IV is reported. It was hitherto unknown that human gastrin-releasing peptide, human chorionic gonadotropin, human pancreatic polypeptide, sheep prolactin, aprotinin, corticotropin-like intermediate lobe peptide and (Tyr-)melanostatin are substrates of this peptidase. Kinetic constants were determined for the degradation of a number of other natural peptides, including substance P, the degradation of which has been described earlier in a qualitative manner. Generally, small peptides are degraded much more rapidly than proteins. However, the Km-values seem to be independent of the peptide chain length. The influence of the action of dipeptidyl peptidase IV on the biological function of peptides and proteins is discussed.

Processing of pro-colipase and trypsinogen by pancreatic dipeptidyl peptidase IV.

Purified dipeptidyl peptidase IV from porcine pancreas or from human placenta cleaves N-terminal dipeptides from two proteins of the pancreatic juice, namely trypsinogen and pro-colipase. Phenylalanyl-proline is very effectively released (Km approximately 50 microM) from bovine or porcine trypsinogen. Both purified dipeptidyl peptidases also rapidly cleave valyl-proline from the N-terminus of porcine pro-colipase. This degradation does not increase the colipase activity of the precursor. However, under certain conditions, which are not fully understandable at present, dipeptidyl peptidase IV releases more slowly a second dipeptide, aspartyl-proline, from pro-colipase, and this results in a partial activation. Dipeptidyl peptidase IV apparently lines the excretory ducts of porcine pancreas and, therefore, is in close contact to the proteins of pancreatic juice in vivo. The possible significance of these degradations is discussed.

Substance P in human plasma is degraded by dipeptidyl peptidase IV, not by cholinesterase.

Human serum cleaves two dipeptides from the N-terminus of the neurohormone substance P. It has been suggested that this degrading activity is inherent to serum cholinesterase. We oppose this, because it turned out that highly purified serum cholinesterase contains traces of dipeptidyl peptidase IV, an enzyme known to attack the N-terminus of substance P. The peptidase is incompletely separated from cholinesterase during the procainamide-gel affinity chromatography as the last step of the usual purification procedure. Physostigmine completely inhibits the hydrolysis of butyrylthiocholine by such purified cholinesterase preparations, but not their substance P-degrading activity. Vice versa, epsilon-carbobenzoxy-lysylproline, an inhibitor of dipeptidyl peptidase IV, inhibits the peptidase activity of these preparations more than their esterase activity. After rechromatography on procainamide gel the peptidase is completely separated and the remaining cholinesterase has lost its substance P-degrading activity. We conclude that the N-terminal region of substance P is not degraded by cholinesterase but by the contaminating dipeptidyl peptidase IV, a different serine enzyme.