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Tyrosine hydroxylase was purified from bovine corpus striatum. The native enzyme had a half-life of 15 +/- 3 min at 50 degrees C. Phosphorylation of tyrosine hydroxylase with protein kinase purified from both corpus striatum and heart activated the enzyme, but activity was rapidly lost with additional preincubation of the enzyme at 30 degrees C. Thermal denaturation studies indicated that phosphorylated tyrosine hydroxylase had a half-life of 5 +/- 2 min at 50 degrees C

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.

A specific and sensitive radioimmunoassay procedure for Met-enkephalin[Arg6,Phe7] which allows its measurement in regions of the rat brain is described. The antiserum as 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.

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.

The properties of purified tyrosine hydroxylase (TH) from bovine corpus striatum, both native and phosphorylated forms of the enzyme, were studied. TH had a tendency toward greater affinity for tetrahydrobiopterin (BH4) than for the synthetic cofactor 6-methyltetrahydropterin (6-MPH4), although the maximal velocity of the TH-catalyzed reaction was greater with 6-MPH4. Phosphorylation increased the affinity of TH for cofactor at pH 6.0, with little change in Vmax. At pH 7.0, phosphorylation caused increased activation of TH by increasing Vmax as well as reducing the Km for cofactor. The K1 for dopamine was increased twofold by phosphorylation at pH 6.0, but eightfold at pH 7.0. Phosphorylation was not associated with a change in Km for tyrosine at any pH or with any cofactor studied, although the Km for tyrosine of TH was cofactor-dependent and seven to eight times greater with 6-MPH4 than with BH4 as cofactor. Heparin and NaCl activated native TH at pH 6.0, but not at pH 7.0. Phosphorylated TH was unaffected by heparin or salt at pH 6.0, but was relatively inhibited at pH 7.0. The data are presented in the context of the physiological environment of TH.