Peptide fragments, activity

At the time of the discovery of Met-enkephalin, its sequence was observed to be identical to that of residues 61—65 contained in the C-fragment of the pituitary hormone p-Hpotropin [12584-99-5] (p-LPH) (see Hormones), first isolated in 1964 (11). In 1976, the isolation of a larger peptide fragment, P-endorphin [60617-12-1] that also displayed opiate-like activity was reported (12). This peptide s 31-amino-acid sequence comprised residues 61—91 of P-LPH. Subsequentiy, another potent opioid peptide, dynorphin [72957-38-17, was isolated from pituitary (13). The first five amino acids (qv) of this 17-amino-acid peptide are identical to the Leu-enkephalin sequence (see Table 1). 

Another subclass of proteases attacks internal peptide bonds and Hberates large peptide fragments. Bromelain, a plant protease derived from the stem of the pineapple plant, can even produce detectable semm proteolysis after oral adrninistration (180). Oral therapy with bromelain significantly reduces bmising that stems from obstetrical manipulations (181). Bromelain—pancreatin combinations have been more effective in digestive insufficiency compared to either pancreatin or placebo (182,183). Bromelain may also enhance the activity of antibiotics, especially tetracycline, when adrninistered concurrently (184). 

S., Grzesiek, S., litman, B. J., Bax, A. Measurement of dipolar couplings in a transdudn peptide fragment weakly bound to oriented photo-activated rhodopsin. f. Biomol. NMR 2000, 16, 121-125. 

A superspiral consisting of two spirals (coiled coil), known as the leucine zip, is formed in this sequence via dimerisation. The condensation reaction, carried out in the aqueous phase, involves two peptide fragments which contain 15 and 17 amino acid residues respectively. Activation takes place via thioester formation (see Sect. 5.3.1). The ligation to a complete GCN4 matrix gives a new 32 amino acid peptide, which can itself serve as a matrix. The autocatalytic reaction exhibits a parabolic increase in the peptide concentration (caused by product inhibition see Section 6.4). 

The earliest published example of microwave-assisted SPOS involved diisopropyl-carbodiimide (DlC)-mediated solid-phase peptide couplings [24], Numerous Fmoc-protected amino acids and peptide fragments were coupled with glycine-preloaded polystyrene Wang resin (PS-Wang) in DMF, using either the symmetric anhydride or preformed N-hydroxybenzotriazole active esters (HOBt) as precursors (Scheme 12.1). 

The concentration of Ca2+ required for half-maximal activation of AC1 is approximately 150mmol/l, while AC8 is five times less sensitive. The Clb domain of adenylyl cyclase appears to mediate the activation of the enzyme by Ca2+/calmodulin (see Fig. 21-3). Activation of AC1 by Ca2+/calmodulin can be blocked by a peptide fragment of the Clb portion of this enzyme. This peptide is also capable of binding Ca27calmodulin itself. 

No tandem MS experiment can be successful if the precursor ions fail to fragment (at the right time and place). The ion activation step is crucial to the experiment and ultimately defines what types of products result. Hence, the ion activation method that is appropriate for a specific application depends on the MS instrument configuration as well as on the analyzed compounds and the structural information that is wanted. Various, more or less complementary, ion activation methods have been developed during the history of tandem MS. Below we give brief descriptions of several of these approaches. A more detailed description of peptide fragmentation mles and nomenclature is provided in Chapter 2. An excellent review of ion activation methods for tandem mass spectrometry is written by Sleno and Volmer, see Reference 12, and for a more detailed review on slow heating methods in tandem MS, see Reference 13. 

In moths, it was discovered in Helicoverpa zea that a peptide produced in the subesophageal ganglion portion of the brain complex regulates pheromone production in female moths (19). This factor has been purified and characterized in three species, Helicoverpa zea (20), Bombyx mori (21, 22), and Lymantria dispar (23). They are all a 33- or 34-amino acid peptide (named pheromone biosynthesis activating neuropeptide, PBAN) and have in common an amidated C-terminal 5-amino acid sequence (FXPRL-amide), which is the minimum peptide fragment required for pheromon-tropic activity. In the redbanded leafroller moth, it was shown that PBAN from the brain stimulates the release of a different peptide from the bursae copulatrix that is used to stimulate pheromone production in the pheromone gland found at the posterior tip of the abdomen (24). 

Figure 10.10 Stability studies analysis by LC-MS. Long-term stability studies (3 months, 30°C) are evaluated by LC-MS analysis of a C-terminal peptide fragment. Various degradation mechanisms are visualized, removal of C-terminal residues due to proteolytic activities, isomerization and deamidation of specific asparagine residues. Future development efforts will allow the use of this methodology to assess progress toward a stable formulation.

Fig. 8.10 Sequential functional characterization and structural identification of an enzyme. Initially, information about the activity is obtained by assessing substrate consumption and product formation. Afterwards, the enzyme is digested on the plate, and the formed peptide fragments (F1-F4) are determined by means of mass spectrometry.

The injector, columns and valves reside in a low temperature chamber to minimize the loss of deuterium by back exchange (Fig. 12.2). The quenched protein solution is pumped in series through a column containing an immobilized protease and a trap column to capture the peptide fragments. The gradient pump is activated following digestion and the peptides captured on the trap column are eluted and separated over an analytical reverse-phase HPLC column directly into the mass spectrometer. 

Xiao XQ, Zhang HY, Tang XC, Huperzine A attenuates amyloid fl-peptide fragment 25-35-induced apoptosis in rat cortical neutons via inhibiting reactive oxygen species formation and caspase-3 activation, J Neurosci Res 67 30—36, 2002. 

Substmcture search of this backbone (smiles O = C(N(C)C1C)CN(C)C1=0) reveals several hundreds commercially available compounds, which likely have been synthesized by the above synthetic route [1]. This backbone can also be considered as a peptide mimetic by using a-amino acid derived isocyanide and amine components and will be of value for biological studies and for the discovery of hydrolysis resistant and biologically active peptide fragments (Fig. 6). 

HeLa cell nuclear extracts were incubated at different concentrations with a radiolabeled acety-lated Histone 4 peptide fragment as substrate. HDAC activity was assessed measuring release of free acetyl groups. 

---