Albert Tseng

Pre- and postjunctional actions of neuropeptide Y and related peptides.

The effects of neuropeptide Y (NPY) and related peptide fragments on blood pressure and vagal action at the heart were compared in the anaesthetized rat. A change in vagal action was taken as a measure of presynaptic activity and a change in blood pressure was taken as a measure of postsynaptic activity. NPY, NPY-(13-36), PYY-(13-36), des-Ser22-NPY-(13-36) and a stabilized 13-36 analogue of NPY (ANA NPY) all exerted pressor actions and attenuated vagal action at the heart. The maximum vagal inhibitory or presynaptic action in order of potency was NPY, ANA-NPY, PYY-(13-36) significantly greater than NPY-(13-36), des-Ser22-NPY-(13-36). The order of potency for the half time of this effect was NPY, ANA-NPY significantly longer than PYY-(13-36) and NPY-(13-36), which were significantly longer than des Ser22-NPY-(13-36). For the pressor or postsynaptic effects, NPY increased blood pressure significantly more and for a longer duration than all the 13-36 fragments, which were not demonstrably different in this respect. These results are consistent with the proposal that there are two populations of NPY receptors. The C-terminal flanking peptide of NPY (CPON) and desamido-NPY had no effect on either vagal action at the heart or on blood pressure.

A novel neuropeptide Y analog, N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36), with functional specificity for the presynaptic (Y2) receptor.

We have carried out functional and in vitro studies on a novel analog of neuropeptide Y which shows selectivity for the prejunctional or neuropeptide Y Y2 receptor. In anaesthetised rats N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36) attenuates cardiac vagal action (a prejunctional or neuropeptide Y Y2 action) and has no significant pressor effects (postjunctional or neuropeptide Y Y1 action). In the human neuroblastoma cell line (SMS-KAN) which expresses and endogenous Y2-like neuropeptide Y receptor, N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36) competes with peptide YY for binding sites with an IC50 of 0.5 +/- 0.1 nM. In contrast in a fibroblast Chinese hamster ovary cell line which expresses the cloned human neuropeptide Y Y1 receptor and is used to study changes in cytosolic calcium evoked by (a neuropeptide Y Y1 effect), N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36) showed no activity even at high concentrations. The steric structure for this novel compound has been determined using proton nuclear magnetic resonance (NMR) spectroscopy and it is consistent with the C-terminal end of published structures of neuropeptide Y. We suggest acetylation and amino acid substitutions stabilise the molecule and allow it to bind only to the neuropeptide Y Y2 receptor.

Functional Characteristics of a Phospholipase A2Inhibitor from Notechis ater Serum

A phospholipase A2 inhibitor has been purified from the serum of Notechis ater using DEAE-Sephacel chromatography. The inhibitor was found to be composed of two protein subunits (α and β) that form the intact complex of approximately 110 kDa. The α-chain is a 30-kDa glycoprotein and the β-chain a nonglycosylated, 25-kDa protein. N-terminal sequence analysis reveals a high level of homology to other snake phospholipase A2 inhibitors. The inhibitor was shown to be extremely pH and temperature stable. The inhibitor was tested against a wide variety of phospholipase A2 enzymes and inhibited the enzymatic activity of all phospholipase A2 enzymes tested, binding with micromole to nanomole affinity. Furthermore, the inhibitor was compared with the Eli-Lilly compound LY311727 and found to have a higher affinity for human secretory nonpancreatic phospholipase A2 than this chemical inhibitor. The role of the carbohydrate moiety was investigated and found not to affect thein vitro function of the inhibitor.

Neuropeptide Y analog with selective antagonism of effects mediated by postjunctional Y1 receptors

Neuropeptide, a 36 amino acid peptide, is one of the most ubiquitous neuropeptides in the nervous system. It is released during stimulation of sympathetic nerves and is implicated as an important neurotransmitter regulating cardiovascular activity. Administration of neuropeptide Y results in vasoconstriction and inhibition of neurotransmitter release. However, the absence of any effective inhibitors of neuropeptide Y action have precluded the examination of its possible role in hypertension. Here we describe a synthetic hexapeptide (BRC 672), corresponding to residues 22-27 of neuropeptide Y. Following the administration of BRC 672 (6.7 mumol/kg), neuropeptide Y-induced pressor responses were reduced by 32-48% in a dose-dependent fashion. The inhibition was specific for neuropeptide Y, as the pressor response to phenylephrine, an alpha-adrenoceptor agonist, was unchanged. It was selective for the postsynaptic (neuropeptide Y Y1 receptor-mediated) vasoconstrictor activity, because the presynaptic (neuropeptide Y Y2 receptor-mediated) cardiac vagal inhibition evoked by injection of neuropeptide Y to rats was not affected. The hexapeptide inhibited the neuropeptide Y-induced increase in cytosolic free Ca2+ in mammalian cells expressing the cloned human neuropeptide Y Y1 receptor. Injections of BRC 672 significantly reduced blood pressure in anaesthetised rats and in conscious spontaneously hypertensive rats. Resting arterial blood pressure decreased from 136 +/- 4 mm Hg to 122 +/- 3 mm Hg and remained depressed 2 h after the administration of the hexapeptide in anaesthetised rats. In spontaneously hypertensive rats blood pressure was decreased for up to 4 h.

Stabilized structure of the presynaptic (Y2) receptor-specific neuropeptide Y analog N-acetyl[Leu-28Leu-31]NPY(24-36)

Neuropeptide Y analog N-acetyl[Leu-28,Leu-31]NPY(24-36)-amide binds specifically to prejunctional or Y2 receptors acting to inhibit neurotransmitter release. The structure of this biologically active mutant was studied by two-dimensional proton nuclear magnetic resonance spectroscopy. Assignments of all backbone and side chain hydrogens were made by using totally correlated spectroscopy (TOCSY) experiments providing through-bond 1H-1H connectivities, and nuclear Overhauser effect spectroscopy (NOESY), which provided through-space and sequential backbone connectivities. Structure analysis of the peptide was performed using distance geometry and dynamic simulated annealing revealing the presence of a helical structure exhibiting an amphiphilic character and slight constriction in the segment 24-29.

A strategy for obtaining active mammalian enzyme from a fusion protein expressed in bacteria using phospholipase A2 as a model

An active preparation of human phospholipase A2 (PLA2) was made after expression as an insoluble fusion protein in Escherichia coli. The new key elements required for PLA2 isolation were the maintenance of the fusion protein in solution after the initial solubilization and the use of a tryptophan cleavage procedure for regeneration of native PLA2 from the fusion protein. The fusion protein was composed of a beta-galactosidase leader peptide incorporating six consecutive threonine residues to aid in insoluble inclusion body formation, followed by a tryptophan adjacent to the N-terminus of PLA2. The fusion protein was purified from cell lysates, and the leader peptide was cleaved on the C-terminal side of the tryptophan residue with N-chlorosuccinimide. The released PLA2 was refolded and renatured to produce an enzyme with activity comparable to that of other phospholipases A2.

Formation of proline thiohydantoin with ammonium thiocyanate: progress towards a viable C-terminal amino-acid-sequencing procedure

Pure amino acid thiohydantoins are required as reference standards for development of C-terminal-sequencing procedures based on thiohydantoin formation of the C-terminal amino acids of peptides and proteins. Proline thiohydantoin was prepared using a straightforward method involving reaction of acetylproline with ammonium thiocyanate. It was characterized by UV spectrophotometry, mass spectrometry and back-hydrolysis to the free amino acid. These data establish unequivocally that the thiocyanate procedure is applicable to proline as well as to the other common amino acids. This work also validates earlier claims that proline thiohydantoin can be prepared by reaction with thiocyanic acid.