Proteolysis of Human Calcitonin in Excised Bovine Nasal Mucosa: Elucidation of the Metabolic Pathway by Liquid Secondary lonization Mass Spectrometry (LSIMS) and Matrix Assisted Laser Desorption lonization Mass Spectrometry (MALDI)

Purpose. Two calcitonins, i.e. human calcitonin (hCT) and, for comparison, salmon calcitonin (sCT), were chosen as peptide models to investigate nasal mucosal metabolism. Methods. The susceptibility of hCT and sCT to nasal mucosal enzymes was assessed by in-and-out reflection kinetics experiments in an in vitro model based on the use of freshly excised bovine nasal mucosa, with the mucosal surface of the mucosa facing the peptide solution. The kinetics of CT degradation in the bulk solution was monitored by HPLC. Peptide sequences of the main nasal metabolites of hCT were analyzed by using both liquid secondary ionization mass spectrometry (LSIMS), following HPLC fractionation of the metabolites, and matrix-assisted laser desorption ionization mass (MALDI) spectrometry. For sCT, the molecular weights of two major metabolites were determined by LC-MS with electrospray ionization. Results. Both CTs were readily metabolized by nasal mucosal enzymes. In the concentration range studied metabolic rates were higher with hCT than with sCT. Presence of endopeptidase activities in the nasal mucosa was crucial, cleaving both calcitonins in the central domain of the molecules. Conclusions. Typically, initial metabolic cleavage of hCT in nasal mucosa is due to both chymotryptic- and tryptic-like endopeptidases. The subsequent metabolic break-down follows the sequential pattern of aminopeptidase activity. Tryptic endopeptidase activity is characteristic of nasal sCT cleavage.     

beta-Lactamase binds to GroEL in a conformation highly protected against hydrogen/deuterium exchange.

Escherichia coli RTEM beta-lactamase reversibly forms a stable complex with GroEL, devoid of any enzymatic activity, at 48 degrees C. When beta-lactamase is diluted from this complex into denaturant solution, its unfolding rate is identical to that from the native state, while the unfolding rate from the molten globule state is too fast to be measured. Electrospray mass spectrometry shows that the rate of proton exchange in beta-lactamase in the complex at 48 degrees C is slower than in the absence of GroEL at the same temperature, and resembles the exchange of the native state at 25 degrees C. Similarly, the final number of protected deuterons is higher in the presence of GroEL than in its absence. We conclude that, for beta-lactamase, a state with significant native structure is bound to GroEL. Thus, different proteins are recognized by GroEL in very different states, ranging from totally unfolded to native-like, and this recognition may depend on which state can provide sufficient accessible hydrophobic amino acids in a suitably clustered arrangement. Reversible binding of native-like states with hydrophobic patches may be an important property of GroEL to protect the cell from aggregating protein after heat-shock.