Vicky Clement-Jones

Iodination of proteins, glycoproteins, and peptides using a solid-phase oxidizing agent, 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen)

Abstract A new, commercially available oxidizing agent, 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen) was compared with chloramine-T and solid-phase lactoperoxidase in the radioiodination of proteins, glycoproteins, and peptides. A method for performing low-level iodinations is described and was used to determine maximum 125I incorporation. Iodinated proteins were purified on analytical gel filtration columns and peptides by reverse-phase high-performance liquid chromatography. Both methods were designed to analyze the tracers for the presence of aggregate and breakdown products caused by the iodination. All tracers prepared were tested in antibody dilution and dose-response curves in their respective radioimmunoassays. Results indicate that Iodogen can be used for a wide range of proteins and peptides, can permit theoretical iodine incorporation with minimal oxidation damage, and can produce tracer stable for up to 3 months.

INCREASED β-ENDORPHIN BUT NOT MET-ENKEPHALIN LEVELS IN HUMAN CEREBROSPINAL FLUID AFTER ACUPUNCTURE FOR RECURRENT PAIN

Low-frequency electroacupuncture effectively alleviated recurrent pain in 10 patients. Basal levels of beta-endorphin and met-enkephalin in the lumbar cerebrospinal fluid (CSF) of these patients were not different from those in pain-free control subjects. After electroacupuncture in the patients with pain CSF beta-endorphin levels rose significantly in all subjects, but met-enkephalin levels were unchanged. These results suggest that the analgesia observed after electroacupuncture in patients with recurrent pain may be mediated by the release into the CSF of the endogenous opiate, beta-endorphin.

Regional distribution of methionine-enkephalin and substance P-like immunoreactivity in normal human brain and in Huntington's disease.

The regional distributions of substance P and Methionine-enkephalin (Met-enkephalin) were determined in normal human brains and in Huntingtons disease using sensitive radioimmunoassays. Model experiments showed that both Met-enkephalin- and substance P-like immunoreactivities were stable for up to 72 h post-mortem in mouse brain. The results of high pressure liquid chromatography (HPLC) analyses indicated that the majority of the immunoreactivity detected in human globus pallidus corresponded to the native peptides, substance P or Met-enkephalin. In Huntingtons disease the present results confirm that there is a substantial drop (> 80%) in the substance P content of the globus pallidus (both medial and lateral segments) and substantia nigra, and there was also a reduction (> 50%) in the Met-enkephalin content of these areas. This result suggests the loss of striato-pallidal and striato-nigral substance P and enkephalin-containing projections in Huntingtons disease.

ACUPUNCTURE IN HEROIN ADDICTS: CHANGES IN MET-ENKEPHALIN AND β-ENDORPHIN IN BLOOD AND CEREBROSPINAL FLUID

In heroin addicts showing features of heroin withdrawal basal beta-endorphin levels were elevated in both blood and cerebrospinal fluid (CSF) and did not change during electroacupuncture, although this therapy suppressed the clinical features of withdrawal. Met-enkephalin levels were not elevated in blood or CSF before treatment. However, successful electroacupuncture was associated with a rise in CSF met-enkephalin levels in all patients studied, although concentrations in blood did not alter.

STUDIES ON CIRCULATING MET-ENKEPHALIN AND β-ENDORPHIN: NORMAL SUBJECTS AND PATIENTS WITH RENAL AND ADRENAL DISEASE

Studies were performed to define the responses of plasma met‐enkephalin to various endocrine and pathological stimuli and to determine the relationship between plasma β‐endorphin and met‐enkephalin. During insulin‐induced hypoglycaemia ACTH, β‐LPH and β‐endorphin immunoreactivity rose in parallel, but plasma met‐enkephalin did not change significantly. Sephadex G75 chromatography of samples taken at the time of the peak response (45 min) confirmed the rise in both β‐LPH and β‐endorphin. During administration of dexamethasone, 0·5mg 6 hourly for 48 h, plasma cortisol and ACTH became undetectable at 24 h, and β‐LPH and β‐endorphin fell significantly by 24 h and were undetectable by 48 h; plasma met‐enkephalin, however, showed no significant change. Nine adrenalectomized patients with Cushings disease and four patients with Addisons disease had elevated plasma ACTH, β‐LPH and β‐endorphin but normal plasma met‐enkephalin levels. Each of ten patients with renal failure had markedly elevated plasma met‐enkephalin immunoreactivity which co‐eluted with synthetic met‐enkephalin on BioGel P4 chromatography. Trypsin and carboxypeptidase‐B digestion of the P4 chromatography fractions generated met‐enkephalin immunoreactivity in earlier fractions, indicating the presence of a potential high molecular weight met‐enkephalin precursor.

Simultaneous release of neurotensin, somatostatin, enkephalins and catechol-amines from perfused cat adrenal glands

Acid extracts of cat adrenal medullae were found to contain neurotensin-like (0.71 +/- 0.21nmol/gm), met-enkephalin-like (11.71 +/- 2.87nmol/gm), leu-enkephalin-like (1.95 +/- 0.11nmol/gm) and somatostatin-like (1.57 +/- 0.63pmol/gm) immunoreactivities. Using isolated retrogradely perfused cat adrenal glands the secretory responses to acetylcholine (ACh), nicotine and potassium ions (K+) were studied. Spontaneous secretion of catecholamines, neurotensin-like, met-enkephalin-like, leu-enkephalin-like and somatostatin-like immunoreactivities was negligible. However, ACh (5.5 X 10(-5)M), nicotine (2.2 X 10(-5)M) and 55mM K+ all evoked the simultaneous release of noradrenaline, adrenaline and the four neuropeptide immunoreactivities. The ACh stimulated secretion of the four neuropeptides could be prevented by perfusion with hexamethonium (C6, 2.8 X 10(-4)M). The concomitant release of these neuropeptides with catecholamines suggests that they may have a role in the modulation of stress responses.

Isolation of human met-enkephalin and two groups of putative precursors (2K-pro-met-enkephalin) from an adrenal medullary tumour

Abstract Human met-enkephalin from an adrenal medullary tumour has been purified and characterised. Two groups of putative human met-enkephalin precursors (MW ∼ 2000) were also isolated and were only opiate active after trypsinisation and yielded met-enkephalin on further treatment with carboxypeptidase B. Amino acid analyses of these peptides show they are not related to β-LPH nor any other reported putative enkephalin precursors. Their amino acid compositions suggest they are structurally closely related and may represent two groups of heterologous peptides.

Ectopic production of methionine enkephalin and beta-endorphin.

Immunoreactive methionine enkephalin and beta-endorphin were sought by serial dilution of tissue extracts and assay of chromatographic fractions in non-endocrine tumour tissue from three patients with the ectopic adrenocorticotrophin syndrome associated with carcinoid tumours and in normal lung tissue and thymic tissue from a patient with myasthenia gravis. In all cases serial dilution of extracts showed parellelism to standard radioimmunoassay curves. The two peptides were found in high concentration in the three tumours but were undetectable in the control tissues. In a single case tested the methionine enkephalin concentration in a vein draining the tumour was twice that in a peripheral vein. In view of their profound effect on behaviour in animals and potent analgesic activity in animals and man the ectopic secretion of methionine enkephalin and beta-endorphin may modify the clinical features of a wide variety of tumours and produce some of the diverse clinical syndromes associated with malignancy.

A study of 24-hour profiles of plasma met-enkephalin in man

Met-enkephalin has recently been demonstrated to circulate in human plasma and using this highly specific extracted radioimmunoassay the fluctuations of plasma Met-enkephalin in man were studied over 24 h. The subjects were 6 healthy volunteers. Following a 24 h adaptation period in the metabolic ward and sleep laboratory, an i.v. catheter was inserted. Blood samples were taken at hourly intervals through the day and at 30 min intervals between 23.00 h and 07.00 h. Sleep was monitored polygraphically. There was no regular rhythm discernible in plasma Met-enkephalin levels throughout the 24 h, nor was there any relationship with sleep or food intake. In a further 3 subjects beta-LPH and beta-endorphin levels as estimated by N- and C-terminal beta-LPH radioimmunoassay were elevated on waking compared with 01.00 h, suggesting a nyctohemeral rhythm. In contrast to the correlated circadian fluctuations in beta-LPH, ACTH and beta-endorphin levels therefore, the lack of circadian rhythmicity and dissociation of plasma Met-enkephalin from plasma levels of the former group of peptides suggests control mechanisms for the secretion of Met-enkephalin are quite different and adds support to the concept of separate Met-enkephalin precursors.

ACTH, LPH AND RELATED PEPTIDES IN THE ECTOPIC ACTH SYNDROME

Adrenocorticotrophin. lipotrophin and the related peptides α‐MSH, CLIP, β‐endorphin and met‐enkephalin have been measured, and characterized chromatographically in tumour extracts from seven patients with the ectopic ACTH syndrome. Four of the seven tumours contained the complete family of peptides, although the proportion of one to another varied between tumours. In addition, large molecular weight forms of ACTH and met‐enkephalin were seen. The potential clinical importance of these observations is discussed.