Workshop proceeding - final.pdf - Faculty of Information and ...
Workshop proceeding - final.pdf - Faculty of Information and ...
Workshop proceeding - final.pdf - Faculty of Information and ...
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Distribution <strong>of</strong> three forms alpha-melanocyte stimulating hormone in the<br />
brain <strong>of</strong> male <strong>and</strong> female sheep by using HPLC <strong>and</strong> Mass Spectrometry<br />
Analysis<br />
Rui Zeng 1,2 * , A.Ian.Smith 2 , Iain.J.Clarke 3<br />
* Presenter<br />
1. Department <strong>of</strong> Cardiovascular diseases, West China Hospital, School <strong>of</strong> Clinic Medicine,<br />
Sichuan University, Chengdu 610041, China<br />
2. Department <strong>of</strong> Biochemistry & Molecular Biology, <strong>Faculty</strong> <strong>of</strong> Medicine, Nursing <strong>and</strong><br />
Health Sciences, Monash University, Vic 3800,Australia<br />
3. Department <strong>of</strong> Physiology, <strong>Faculty</strong> <strong>of</strong> Medicine, Nursing <strong>and</strong> Health Sciences, Monash<br />
University, Vic 3800, Australia<br />
To compare des-acetyl α-MSH(des-α-MSH), acetyl α-MSH(act-α-MSH) <strong>and</strong> di-acetyl α-<br />
MSH(di-act-α-MSH) between the male <strong>and</strong> female sheep in ten special brain tissues. All <strong>of</strong><br />
this will let us ascertain whether different gonadal steroids causes any change in acetylated<br />
peptide. Mass spectrometry (MS) was used for molecular weight determination, HPLC/EIA<br />
was also used for quantitation. Before MS, Immunomagnetic protein isolation using<br />
Dynabeads Protein A also provides a fast <strong>and</strong> reliable method for capturing Ig for small scale<br />
purification. Mass spectrometry analysis indicated that: In female sheep, all three forms <strong>of</strong> α-<br />
MSH were detected in hypothalamus, neurointermediate lobe, cortex, pre-optic area <strong>and</strong><br />
median eminence. In arcuate nucleus, des- <strong>and</strong> act-α-MSH were present, <strong>and</strong> only acetylated<br />
forms(including mono-act <strong>and</strong> di-act) were detected in anterior pituitary. For male sheep, only<br />
in neurointermediate lobe <strong>and</strong> pre-optic area were present all three forms <strong>of</strong> α-MSH. In<br />
hypothalamus, median eminence, cortex, anterior pituitary, des- <strong>and</strong> act-α-MSH were detected.<br />
We can only find des-α-MSH in arcuate nucleus. Cerebellum, spinal cord <strong>and</strong> brain stem have<br />
no any forms <strong>of</strong> α-MSH both in male <strong>and</strong> female sheep. HPLC/EIA results showed that in<br />
hypothalamus, neurointermediate lobe, pre-optic area <strong>and</strong> median eminence, there is no any<br />
difference in des-α-MSH between male <strong>and</strong> female sheep, but female sheep has more act-α-<br />
MSH <strong>and</strong> di-act-α-MSH compared with male sheep.P < 0.05. In arcuate nucleus, there is no<br />
any difference in des-α-MSH between male <strong>and</strong> female sheep, but female sheep has more actα-MSH<br />
only compared with male sheep.P < 0.05. In anterior pituitary, there is more des-α-<br />
MSH in male sheep, but female sheep has more act-α-MSH <strong>and</strong> di-act-α-MSH compared with<br />
male sheep. P < 0.05. Finally, in cerebral cortex, female sheep has more des-α-MSH, act-α-<br />
MSH <strong>and</strong> di-act-α-MSH compared with male sheep.P < 0.05. These data suggest that N-<br />
acetylation <strong>of</strong> products formed by the processing <strong>of</strong> α-MSH can markedly adapt to their<br />
biological properties. The results also illustrated that the distribution <strong>of</strong> α-MSH may be<br />
affected by the sex steroid hormone.<br />
Keywords<br />
α-MSH, POMC, sheep, HPLC, Mass Spectrometry<br />
Introduction<br />
Proopiomelanocortin (POMC) is a precursor protein that contains the sequence for several<br />
bioactive peptides including adrenocorticotropin (ACTH), β-endorphin(β-EP), <strong>and</strong> melanocytestimulating<br />
hormone (α-, β- <strong>and</strong> γ- MSH). The functional peptides have been detected in a variety <strong>of</strong><br />
tissues.<br />
Post-translational processing <strong>of</strong> prohormones is an important biochemical mechanism employed<br />
by many secretory cells to generate a diversity <strong>of</strong> biological peptides from the same gene product, a<br />
process that occurs in a tissue-specific manner (1). Similarly to many prohormones, POMC is<br />
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