Abstract
Histidyl-proline diketopiperazine is produced in brain as a product of the metabolism of thyrotropin-releasing hormone. A number of the previously observed central nervous system and pituitary activities resulting from an exposure to thyrotropin-releasing hormone appear to involve the conversion of the releasing factor to the cyclic dipeptide. In the present study, the development of a rabbit antiserum that is highly specific for histidyl-proline diketopiperazine is described; the antiserum has essentially no capability to bind thyrotropin-releasing hormone or a number of other related peptides. The antibody can also distinguish between the natural form of the cyclic dipeptide and a diastereomer containing D-proline. A procedure for extraction, with high yield, of histidyl-proline diketopiperazine from brain is described. With the aid of the specific antiserum it was found that the preponderance of the cyclic dipeptide in rat brain is bound to high molecular weight material, mainly in the range of Mr = 70,000; histidyl-proline diketopiperazine can be disassociated from this material by boiling in salt/methanol solution. The concentration of the dipeptide in rat brain is in the range of 275 to 565 pmol/brain, approximately 2.5 times the concentrations determined for thyrotropin-releasing hormone (113 to 210 pmol/brain). A study of the subcellular distribution of histidyl-proline diketopiperazine and thyrotropin-releasing hormone suggests that the releasing factor is concentrated in synaptosomal vesicles while the diketopiperazine is not. A determination of the regional distribution of thyrotropin-releasing hormone and histidyl-proline diketopiperazine indicated that both peptides are found in highest concentrations in pituitary and hypothalamus, but are detectable in other areas of brain as well.
Highlights
A new direction in investigations concerning TRH was hormone appear to involve the conversion of the releasing factor to the cyclic dipeptide
With the aid of volves an inhibition of the sodium pump [6].In thepituitary, the specific antiserum it was found that the preponder- the diketopiperazine inhibits, while TRH promotes, prolactin ance of the cyclic dipeptide in rat brain is bound tohigh release [7, 8]
Histidyl-proline diketopiperazine has a variety of effects in the central nervous system; it plays a role in regulation of body temperature [4], in modulation of cGMP levels [5], and as an anti-depressant factor [3]
Summary
Antigen ZI: CycZo(His-Pro) Coupled to Keyhole Limpet Hemocyanin using a Bridge of Bis-diazotized Benzidine-Sodium nitrite. Four New Zealand White rabbits were immunized with 2.3 mg of Antigen I in 0.9% NaCl solution (saline) emulsified with complete Freund'sadjuvant (l:l, total volume, 1 ml) Injections weregiven both sub- and intracutaneously at multiple sites on the backs. Molar concentrations of the fractions were calculated from their prolinecontent determined by amino aciadnalysis after acid hydrolysis.These fractionswere tested for their capacity to compete with [3H]cyclo(His-Pro)for binding to the antiserum (Fig 1). In this test system, 4pmolofunlabeled cyclo(His-Pro) produced 50% displacement obf indingof [3H]cyclo(His-Pro).Peaks 1,2,5,and 6 showed essentiallyno capabilitytodisplace cyclo(His-Pro) from the antiserum, whilePeak 3 (the 4-azo derivative) produced50% displaceantigen. In the absence of competitors, the binding to antiserum was 710 cpm (correspondingto 9.9%of the input countsper min)
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