Michael R. Boarder

Measurement of Methionine-Enkephalin [Arg6,Phe7] in Rat Brain by Specific Radioimmunoassay Directed at Methionine Sulphoxide Enkephalin[Arg6,Phe7]

Abstract: A specific and sensitive radioimmunoassay procedure for Metenkephalin[Arg6,Phe7] which allows its measurement in regions of the rat brain is described. The antiserum was raised against the methionine sulphoxide derivative of the peptide, and all samples and standards were oxidized with hydrogen peroxide prior to use in the assay with chloramine T‐oxidized 125I‐labelled Met(O)‐enkephalin[Arg6,Phe7]. The only significant cross‐reactivity was 30% with the reduced heptapeptide Met‐enkephalin[Arg6,Phe7]. The assay showed less than 0.15% cross‐reactivity with fragments of the heptapeptide and with leucine‐enkephalin‐containing peptides. Acid acetone extraction of rat striatum followed by Sephadex G‐50 chromatography and reverse‐phase high pressure liquid chromatography showed that essentially all immunoreactivity co‐chromatographed with Met‐enkephalin[Arg6,Phe7]. This confirmed the specificity of the assay and showed that the striatum does not contain a high concentration of larger molecular weight forms with the heptapeptide at the COOH terminus. Distribution of the heptapeptide followed that of methionine enkephalin, with highest concentrations in the globus pallidus, intermediate levels in caudate‐putamen and hypothalamus, and low levels in cortex and cerebellum.

Met-Enkephalin [Arg6,Phe7] Immunoreactivity in Bovine Caudate and Bovine Adrenal Medulla

Abstract: A radioimmunoassay specific for the COOH‐terminus of Met‐enkephalin [Arg6,Phe7] and a separate assay specific for the COOH‐terminus of Met‐enkephalin are described. Immunoreactivity by these two assays was compared in bovine caudate and bovine chromaffin granule preparation after Sephadex G75 chromatography in 50% acetic acid. When the assays were applied to the chromatography fractions of the bovine caudate extract, the majority of the immunoreactivity was found in the fractions corresponding to the heptapeptide and the pentapeptide respectively. When the chromaffin granule chromatography fractions were assayed, both of the radioimmunoassays showed that most reactivity was in several peaks in the larger molecular weight fractions. The major peak for the Met‐enkephalin [Arg6,Phe7] assay had an apparent molecular weight of 2800, while with the Met‐enkephalin assay the dominant peak of immunoreactivity had an apparent molecular weight of 10,000. The presence of authentic Met‐enkephalin [Arg6,Phe7] in both caudate and chromaffin granule extracts was confirmed by reverse‐phase chromatography of the previously sized fractions. It appears then that the processing of precursors of opioid peptides is directed, in the caudate, to the synthesis and storage of the enkephalins and of Met‐enkephalin [Arg6,Phe7]; in the adrenal medulla the major products of precursor processing are a variety of polypeptides of larger sizes.

Absence of increased tyrosine hydroxylation after induction of brain tyrosine hydroxylase following reserpine administration

Abstract Reserpine was administered to rats by i.p. injection, and seven days later, with a control group, crude synaptosomal suspensions were prepared from hippocampus and cerebellum for the estimation of rates of synaptosomal tyrosine hydroxylation; additionally, samples of hippocampus, cerebellum and hypothala- mus were homogenized in a hypotonic buffer containing Triton for the 171 in vitro estimation of tyrosine hydroxylase. In the samples from reserpine pretreated animals, in vitro tyrosine hydroxylase activities were considerably elevated compared to controls (234 per cent of controls in the cerebellum, 154 per cent in hippocampus and 181 per cent in hypothalamus). However, the synaptosomal tyrosine hydroxylation rates were not elevated in the drug-treated group. The rate of product formation with time was linear in preparations from both control and reserpine pretreated animals. The results suggests that enzyme induction leads to an increased potential for tyrosine hydroxylation which may not be expressed due to interaction with regulatory mechanisms operating on the enzyme.

Measurement of Total Opioid Peptides in Rat Brain and Pituitary by Radioimmunoassay Directed at the α‐N‐Acetyl Derivative

A sensitive assay, which cross‐reacts with and is specific for diverse opioid peptides, is described. This is based on the prior acetylation of samples and subsequent radioimmunoassay with an antiserum highly specific for the acetylated NH2 terminus of opioid peptides. The result is a procedure that can be used to investigate multiple forms of opioid peptides in extracts of biological material. The sensitivity of the assay is ˜15 fmol of β‐endorphin per incubation tube, i.e., ˜ 100‐fold greater sensitivity than the radioreceptor assay used in our laboratory. The peptide concentration required for 50% displacement of trace ranged from 0.65 nM (β‐endorphin) to 1.6 nM (Met‐enkephalin). The assay apparently shows an absolute requirement for a free (or acetylated) NH2 terminus corresponding to either a Leu‐ or Met‐enkephalin sequence. Use of the assay with and without prior acetylation of sample provides a method for estimation of the ratio of acetylated:nonacetylated opioid peptides in crude or fractionated extracts. The procedure is used to investigate the forms of opioid peptide found in rat brain and pituitary.

Opioid peptides as neuroregulators: Potential areas for the study of genetic-behavioral mechanisms

The opioid peptides have been related to behavior in both animal and human studies. Further investigation can be anticipated which could lead to the elucidation of genetic controls over enzymes which process these peptides and the receptors upon which the peptides act. The enzymes, both synthetic and degradative, can lead to the formation of different forms of the opiate peptides. Differential control of these enzymes or of the multiple forms of opiate receptors could lead to discrete changes in opiate status and subsequent behavioral changes. Conversely, genetically regulated behavioral modification could also lead secondarily to opiate changes.

Peptide E and Its Products, BAM 18 and Leu-Enkephalin, in Bovine Adrenal Medulla and Cultured Chromaffin Cells: Release in Response to Stimulation

Peptide E is a 25 amino acid opioid peptide which, if cleaved at the sole double basic (Lys‐Arg) typical processing site, would generate two opioid fragments, the amino‐terminal fragment BAM 18 and the carboxy‐terminal fragment Leu‐enkephalin. We have analysed extracts of bovine adrenal medulla in order to quantify these three opioid peptides (peptide E, BAM 18, and Leu‐enkephalin). Here we present evidence that BAM 18 and Leu‐enkephalin were present in similar amounts, whereas peptide E was present at a higher concentration. This is consistent with previous observations showing a preferential accumulation of larger peptides in the bovine adrenal, and also with the Lys‐Arg bond being the principal site of cleavage of peptide E. However, when bovine adrenal chromaffin cells were maintained in culture for several days, Leu‐enkephalin was found to be present in much greater amounts than was BAM 18‐like immunoreactivity. The molar amounts of peptide E still exceeded the estimated levels of BAM 18 and Leu‐enkephalin. We provide evidence that under conditions of basal release BAM 18 and peptide E were released, whereas Leu‐enkephalin was released in much smaller amounts, if at all. On stimulation with nicotine results were consistent with an increased release of all three peptides with a preferential stimulation of Leu‐enkephalin release. Under all conditions, the molar amounts of peptide E released apparently exceeded that of the other peptides. The results are discussed in terms of the regulation of partial proteolysis and the fate of peptide E.

Synthetic N-dimethyl β-endorphin, a stabilized opioid peptide

Abstract A procedure for the dimethylation of the amino groups of human β-endorphin by reductive methylation is described. A single product with the theoretical maximum degree of methylation was produced and the consequences of dimethylation on proteolytic attack are reported. The derivative was shown to be resistant to tryptic digestion and to attack by leucine aminopeptidase. The nonmethylated β-endorphin was rapidly degraded by incubation with pituitary homogenate; under these conditions methylated β-endorphin was degraded at a slower rate. In the presence of bacitracin, the methylated peptide was essentially resistant to degradation by the pituitary homogenate. The methylated peptide may be expected to have a longer in vivo half-life.

MEASUREMENT OF MET-ENKEPHALIN(ARG6, PHE7) IN RAT BRAIN BY SPECIFIC RADIOIMMUNOASSAY DIRECTED AT METHIONINE SULPHOXIDE ENKEPHALIN (ARG6, PHE7)

An antiserum was raised in rabbits against the methoxide form of methionine enkephalin (arg6, phe7) and this was used to provide a sensitive and specific assay for the oxidised heptapeptide. Rat brain samples were extracted, oxidised with H2O2 and measured in this assay. Chromatography of extracts prior to assay confirmed that this procedure provides a sensitive method for the measurement of endogenous met-enkephalin (arg6, phe7). Assay of selected rat brain regions shows that the distribution of this heptapeptide follows that of met-enkephalin.

β-LIPOTROPIN, CORTICOTROPIN AND β-ENDORPHIN: PHOSPHORYLATION BY CYCLIC AMP-DEPENDENT AND INDEPENDENT PROTEIN KINASES

The biologically active peptides human β-endorphin and corticotropin as well as the β-endorphin precursor β-lipotropin could be phosphorylated by either the purified catalytic subunit of cyclic AMP-dependent protein kinase or a cyclic nucleotide-independent kinase partially purified from rat brain. Parathyroid hormone and secretin served also as substrates for either kinase suggesting that phosphorylation may be a commonly occurring covalent modification of peptides. The potential role of phosphorylation for peptide processing and function is discussed.

PHOSPHORYLATION OF ENDOGENOUS β-LIPOTROPIN AND OTHER PEPTIDES BY SLICES OF PITUITARY, ADULT BRAIN AND NEONATE BRAIN OF THE RAT

Pituitary slices incubated with H332PO4 yielded phosphorylated peptides which were analyzed on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Several phosphorylated peptides were apparent; one was identified as β-lipotropin (LPH) by comigration and peptide mapping. Incubation of adult and neonate brain slices yielded different patterns of peptide phosphorylation.