Isolation of the C-fragment and C′-fragment of lipotropin from pig pituitary and C-fragment from brain

Smyth, Derek G.; Snell, C. R.; Massey, D. E.

doi:10.1042/bj1750261

Cited by 58 publications

(10 citation statements)

References 25 publications

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“…The width of the immunoreactive peak, however, was much greater than that of [3-endorphinrelated marker peptides chromatographed on the same col umn. For comparison, authentic lipotropin isolated on a large scale from porcine pituitary [10] exhibited similar chromatographic behaviour to the immunoreactive lipotro pin obtained from the dissected regions.…”

Section: Limited Trypsin Digestion O F Lipotropins Obtained From Porcmentioning

confidence: 99%

Sequential Formation of Beta-Endorphin-Related Peptides in Porcine Pituitary

Smyth¹,

Darby²,

Maruthainar³

1988

Neuroendocrinology

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Lipotropin and peptides related to β-endorphin were extracted from the anterior pituitary and the pars intermedia of porcine pituitary and were resolved by gel exclusion and ion exchange chromatography. Possible heterogeneity in the structure of the lipotropin was investigated by identifying the C-terminal fragment released by limited proteolysis with trypsin; the cleavage was restricted to the carboxyl group of arginine residues by employing citraconylation to protect the Ε-NH₂ groups of lysine. The lipotropin obained from both regions of the pituitary gave rise to the same C-terminal peptide which contained the 31-residue sequence of β-endorphin; none of the 26- and 27-residue forms was detected. In contrast, the β-endorphin-related peptides that were isolated directly from the pars intermedia exhibited a high degree of C-terminal proteolysis: they were present principally as the 26- and 27-residue peptides. The results demonstrate that lipotropin differs from β-endorphin in that it occurs exclusively in the form that contains the full C-terminal sequence. It is concluded that during biosynthesis lipotropin undergoes conversion to β-endorphin before proteolysis takes place at the C-terminus. The processing reactions that convert lipotropin to β-endorphin 1–31 and β-endorphin 1–31 to β-endorphin 1–27 are thus ordered and not competitive. The results also indicate that glycylglutamine, the bioactive C-terminal dipeptide of lipotropin, is formed from β-endorphin and not from lipotropin.

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Section: Limited Trypsin Digestion O F Lipotropins Obtained From Porcmentioning

confidence: 99%

Sequential Formation of Beta-Endorphin-Related Peptides in Porcine Pituitary

Smyth¹,

Darby²,

Maruthainar³

1988

Neuroendocrinology

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“…LPH, the C-Fragment of LPH, the C'-Fragment, the acetyl derivatives of the C-and C'-Fragments, the nonadecapeptide (LPH-61-79), and corticotropin were obtained by extraction from porcine pituitary (12,15). Methionine enkephalin was prepared by synthesis.…”

Section: Methodsmentioning

confidence: 99%

Distribution of active and inactive forms of endorphins in rat pituitary and brain.

Zakarian¹,

Smyth²

1979

Proc. Natl. Acad. Sci. U.S.A.

143

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The recent isolation and identification of a-N-acetyl forms of the C-Fragment of lipotropin (fl-endorphin, residues 61-91) and the C'-Fragment (residues 61-87) [Smyth, D. G., Massey, D. E., Zakarian, S. & Finnie, M. (1979) Nature (London) 279, 252-254] has led to a study of their distribution in the pituitary and brain of the rat. Regions were mapped by the method of immunofluorescent staining and the reactive peptides were determined by immunoassay after extraction, gel filtration, and ion exchange chromatography. The major immunoreactive peptides in both lobes of the pituitary were found to be C'-Fragment and N-acetyl C'-Fragment, which are weakly active or inactive as opiates; the C-Fragment and its N-acetyl derivative represented minor components. This indicates that in the rat the circulating "endorphins" released from pituitary would have little morphinomimetic activity. The same four immunoreactive peptides were observed in rat brain. In the hippocampus the C'-Fragment was the principal component; in the midbrain there was more C-Fragment but C'-Fragment predominated; in the hypothalamus the C-Fragment was the major peptide, almost to the exclusion of the other peptides. The results demonstrate that the processing of lipotropin is under differential control in anatomically distinct regions of the central nervous system. The processing of lipotropin in the hypothalamus is directed specifically to the production of lipotropin C-Fragment.Understanding of the full physiological role of the endorphins must start from the identification of these peptides and elucidation of their distribution in regions of pituitary and brain. Present in picomolar concentrations, they can be detected and measured by the application of highly sensitive immunological techniques involving the use of specific antibodies. In studies of f3-endorphin [lipotropin (LPH) C-Fragment] the antisera employed have exhibited COOH-terminal specificity (1-4) and would react with the a-N-acetyl derivatives of the C-and C'-Fragments of LPH, as well as with a series of peptides including the 31,000-Mr endorphin prohormone, LPH, the C-Fragment (residues , and the C'-Fragment (residues 61-87). All these peptides occur naturally, but previous studies on the distribution of ,B-endorphin by immunofluorescence (5, 6) or by radioimmunoassay of tissue extracts after gel filtration (7-10) have not differentiated between them. Because only the CFragment exhibits potent opiate activity-the C'-Fragment is '/5ooth as active as an analgesic agent (11) and the acetyl peptides (12) and LPH (13,14) are inert-it is essential that the immunoreactive peptides should be precisely identified. We give here a brief description of our detailed mapping of the pituitary and brain of the rat by immunofluorescence using antibodies raised against homogeneous porcine C-Fragment and we present the results of experiments that have allowed characterization of the fluorescing substances in specific regions. The data reveal strikingly different patterns of distribution of ...

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“…Each of these peptides occurs also in an a-N-acetyl form (1)(2)(3)(4)(5). Studies of the regional distribution and biosynthetic pathways of the 1-EPrelated peptides in the brain and pituitaries have shown distinctive proportions of these forms in specialized areas arising from differential proteolytic cleavages and acetylation of ,3-EP (6)(7)(8).…”

mentioning

confidence: 99%

Beta-endorphin-(1-27) is a naturally occurring antagonist to etorphine-induced analgesia.

Nicolas¹,

Li²

1985

Proc. Natl. Acad. Sci. U.S.A.

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The potent opioid peptide ,-endorphin is found in the brain and pituitary with two related fragments, fi-endorphin-(1-27) and f3-endorphin-(1-26 Recent studies have shown that P-endorphin (p-EP) is found in the pituitaries and the brain of various species together with two related peptides: P-EP-(1-27) and its des-His27 or des-Tyr27 derivative ,-EP-(1-26). Each of these peptides occurs also in an a-N-acetyl form (1-5). Studies of the regional distribution and biosynthetic pathways of the 1-EPrelated peptides in the brain and pituitaries have shown distinctive proportions of these forms in specialized areas arising from differential proteolytic cleavages and acetylation of ,3-EP (6-8). Of various peptides related to P-EP, only the unmodified hormone possesses potent analgesic properties. The formation of its derivatives is accompanied by loss of analgesic activity (9-12).Since no particular role had been reported for these truncated and/or acetylated peptides, it was suggested that post-translational inactivation of,-EP might be an important all-or-none metabolic process maintaining a physiological level of bioactive endorphin in the brain (5, 6). However, the diversity and natural abundance of these inert opioid peptides, in regard to such a simple switch mechanism for inactivation, have given the impetus to search for more specific biological functions. We have recently reported on the inhibition of the analgesic activity of ,-EP by B-EP-(1-27) (13). This particular fragment retains 30% of the binding potency of the parent hormone for brain opioid receptors and <2% of its analgesic potency. It diminishes the analgesic effect of j-EP when coinjected intracerebroventricularly (icv) into mice in doses that are compatible with those found in the brain. To provide further evidence in support of the hypothesis that natural fragments of 8-EP might be implicated in the control of pain perception, we have studied effects of B-EP-(1-27) and 3-EP-(1-26) on the modulation of antinociception induced both by j3-EP and by the highly potent opiate agonist etorphine in mice. Effect of icv administered opiates or peptides on heat escape latency was assessed by the tail-flick method (18), using groups of 10 mice (male Swiss Webster, 20-25 g; Simonsen Laboratories, Gilroy, CA) per dose. Percentage analgesia was calculated as described (19). Median antinociceptive dose (AD50), 95% confidence limits and slope of the logarithm of the dose vs. probit (% analgesia) curves were calculated by a nonlinear least-squares regression to a 2 parameters logistic. AD50 values were calculated for each peptide or opiate alone and in combination with various fixed doses of putative antagonist. The ratio of the AD50 value in the presence of antagonist to that in its absence (dose ratio, x) was then calculated for each dose of antagonist (13,20). MATERIALS AND METHODSCompetitive antagonism was quantified by use of the apparent pA2 (the negative logarithm of the antagonist dose required to reduce the effect of a dose of agonist by one-half) fo...

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Isolation of the C-fragment and C′-fragment of lipotropin from pig pituitary and C-fragment from brain

Cited by 58 publications

References 25 publications

Sequential Formation of Beta-Endorphin-Related Peptides in Porcine Pituitary

Sequential Formation of Beta-Endorphin-Related Peptides in Porcine Pituitary

Distribution of active and inactive forms of endorphins in rat pituitary and brain.

Beta-endorphin-(1-27) is a naturally occurring antagonist to etorphine-induced analgesia.

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