Louise I. Lowney

Partial Purification of an Opiate Receptor from Mouse Brain

A proteolipid isolated from a lipid extract of mouse brain demonstrates stereospecific binding properties for levorphanol. It is present only in neuronal tissue and most abundant in the rhombencephalon. One component saturates at a concentration corresponding to maximum pharmacologic effect in vivo. The estimated mass is 60,000 daltons per bound opiate molecule.

Use of trifluoroacetic acid in the separation of opiates and opioid peptides by reversed-phase high-performance liquid chromatography

Abstract Dilute trifluoroacetic acid (TFA) was observed to enhance partition of opioid peptides into butanol during the course of aqueous-organic phase extractions aimed at further purification of pituitary endorphins found in commercial ACTH preparations. Since TFA is volatile and easily removed by lyophilization, it was decided to study TFA as a counter ion for ion-pair partition reversed-phase liquid chromatography. It was found, that in addition to ion-pair formation, TFA appeared to decrease non-specific adsorption of opiates and opioid peptides to column matrices. Elution time of opiates and opioid peptides was decreased without loss of resolution when TFA was employed. Peak sharpening and elimination of tailing was observed for several compounds eluted with solvent systems containing TFA.

Protein synthesis at 0°C in Escherichia coli*

Leucine-starved cells of an Escherichia coli auxotroph will incorporate radioactive leucine into protein at 0°C. The incorporation is linear for the first 10 to 15 min. It appears that all the essential steps in protein synthesis go on at 0°C, and a wide variety of proteins is synthesized. The rate of synthesis is about 350 times slower than at 37°C. In this system the polarity of assembly of polypeptide chains, from N-terminal to C-terminal, can be demonstrated by treating the proteins with carboxy-peptidase. A quantitative analysis of the release of incorporated leucine by carboxypeptidase permits estimation of the average time required to add a single amino acid residue to a nascent protein chain at 0°C, whence the number of nascent chains can be derived. This number has also been estimated by direct measurement of nascent protein in sucrose density-gradient centrifugation. A cell contains about 5000 chains of nascent protein, and 18,000 70 s ribo-somes. The assembly time of a protein of 400 residues is 5 sec at 37°C.

Attachment of ribosomes to nascent messenger RNA in Escherichia coli

Abstract In intact cells of Escherichia coli at 0 °C, radioactive uridine is incorporated into messenger RNA so slowly that cell lysates can be prepared after the addition of fewer than 30 nucleotides to the growing 3′ ends. Under these conditions, nearly half of the radioactivity is associated with ribosomes in a magnesium-dependent binding. The findings are interpreted to mean that polysomes are formed on messenger strands before transcription is completed.

A pituitary endorphin with novel properties

Abstract We describe the further purification of an opioid peptide from a porcine pituitary concentrate. The peptide has typical naloxone-reversible opioid activity in the guinea pig ileum myenteric-plexus preparation and mouse vas deferens, and it inhibits stereospecific binding at opiate receptors. It is distinguished from β-endorphin and the enkephalins by its apparent molecular weight, its slow reversal with washing in the guinea pig ileum preparation, and the resistance of its biologic activity to cyanogen bromide treatment. In beef pituitary, slow-reversing, cyanogen bromide resistant activity is found principally in neurointermediate lobe.

CHARACTERISTICS OF OPIOID PEPTIDES FROM BRAIN AND PITUITARY

Abstract 1 Fragments of the pituitary peptide, s-lipotropin, exert opioid effects in in vitro assays. 2 Most of the opioid activity in pituitary gland is associated with an endorphin of similar size and distribution to the largest active opioid fragment from s-lipotropin. 3 An endorphin with similar characteristics is present in brain, as well as a smaller trypsin-sensitive endorphin. 4 The use of trypsin in classifying endorphins and in revealing potential endorphin precursors is discussed.