A novel opioid peptide, leumorphin, acts as an agonist at the kappa opiate receptor

The primary structure of the common precursor of porcine beta-neo-endorphin and dynorphin (preproenkephalin B) has shown the existence of a third leucine-enkephalin (leu-enkephalin) sequence with a C-terminal extension of 24 amino acids. This nonacosapeptide, named leumorphin, was approximately 70 times more potent than leu-enkephalin in inhibiting the contraction of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum. This action of leumorphin, like those of beta-neo-endorphin and dynorphin, was antagonized less effectively by naloxone than that of leu-enkephalin, but more effectively by Mr2266, an antagonist relatively specific for the kappa type opiate receptor. The inhibitory action of leumorphin or beta-neo-endorphin on the contraction of the guinea pig ileum muscle strip was reduced in a dose-dependent manner by pretreatment with dynorphin and vice versa. Leumorphin as well as beta-neo-endorphin and dynorphin inhibits the contraction of the rabbit vas deferens which is known to have only the kappa type opiate receptor. This action was also effectively antagonized by Mr2266. It is concluded that leumorphin has potent opioid activity and acts at the kappa receptor, like other opioid peptides derived from preproenkephalin B.     

Distribution of sodium-plus-potassium-stimulated adenosine-triphosphatase activity in isolated nerve-ending particles

1. A rapid method for the isolation of nerve-ending particles from brain is described. This involved the centrifugation of the large-granule fraction over a discontinuous density gradient consisting of 3% (w/v) and 13% (w/v) Ficoll dissolved in 0.32m-sucrose. The results of the biochemical as well as morphological identification of nerve-ending particles are given. 2. Approx. 20% of the (Na(+)+K(+))-stimulated adenosine-triphosphatase activity originally present in the cerebral grey-matter suspension was recovered in the fraction consisting principally of large nerve-ending particles (approx. 1mu in diameter). The activity of the adenosine triphosphatase/mg. of protein in the nerve-ending fraction approximated to that in the small-granule fraction after the treatment with glycol ether diamine-tetra-acetic acid. The conclusion was drawn that the synaptic structure, supposedly the limiting membrane of the nerve-ending particle, is one of the feasible sites of localization of the (Na(+)+K(+))-stimulated adenosine-triphosphatase activity in cerebral tissues. Adenosine triphosphatase in purified cerebral mitochondria was not stimulated by Na(+). 3. No qualitative differences were found between the (Na(+)+K(+))-stimulated adenosine-triphosphatase activities exhibited by the nerve-ending particles and by the cerebral small-granule fraction with respect to pH-dependence, cation requirements and susceptibility to ouabain.     

Production and characterization of recombinant human neutrophil chemotactic factor

A putative mature human neutrophil chemotactic factor (NCF) corresponding to the C-terminal 72 amino acids of its precursor was directly produced in Escherichia coli by recombinant DNA technology. Human NCF was present in both the soluble and insoluble protein fractions of the homogenate of host cells, and it was partially purified as a water-soluble polypeptide from both fractions, separately. The partially purified NCF preparation was highly purified to an endotoxin-free homogeneous polypeptide by means of CM-Sepharose CL-6B column chromatography and gel filtration on Toyopearl HW-55. No difference between the human NCF preparations purified from both starting materials could be found concerning purity, primary structure, solubility, molecular weight, and chemotactic activity for human neutrophils. The amino acid sequence of recombinant human NCF was identical to the sequence deduced from the cDNA sequence. A methionine residue due to the translation initiation codon was removed. Recombinant human NCF was found to be biologically active and to exhibit chemotactic activity for human neutrophils in vitro and cause a neutrophil infiltration in vivo in mice.