Bruce L. Currie

Biological evidence that separate hypothalamic hormones release the follicle stimulating and luteinizing hormones

A preparation from porcine hypothalami essentially free of the decapeptide releasing hormone, pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, releases FSH and LH. It released, invitro, a greater amount of FSH than the decapeptide. A certain dose stimulated an unequivocal release of FSH but virtually no LH. Because this activity is chemically separate from that of the decapeptide, and because the relative FSHLH releasing ratio is greater than that of the decapeptide, it is concluded to be due to FSH-RH. Thus, the decapeptide is LH-RH, and FSH-RH is a separate hypothalamic releasing hormone.

High-pressure liquid chromatography of peptides.

Peptides varying in size from di- to decapeptide have been subjected to high-pressure liquid chromatography on Phenyl-Corasil, Poragel PN, and Poragel PS under reversed-phase conditions with acetonitrile-water mixtures. It has been found that residual silanol groups in the Phenyl-Corasil and the functional groups in the Poragels significantly influence retention. Peptides were also chromatographed on a Hydrogel IV gel filtration packing with aqueous eluants, and it was found that effects other than gel filtration play a major role in determining retention.

Synthesis of the luteinizing releasing hormone of the hypothalamus and its hormonal activity

The decapeptide amide and acid of LH-RH/FSH-RH were synthesized and bioassayed in rats. The structure pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH3 was made by conventional synthesis of pGlu-His-Trp coupled with the heptapeptide synthesized on solid state resin. Bioassays were performed in ovariectomized rats given 50 mcg estradiol benzoate and 25 mg progesterone 72 hours before by injecting the peptides and sampling the jugular vein then assaying LH by double antibody radioimmunoassay. The decapeptide amide was effective from 25 ng to 10 mcg. At 1 or 10 mcg LH level was elevated about 25 fold for 30-60 minutes and decreased to about 10 fold in 2 hours. The decapeptide-OH was active but only at about 1000 times the amide dose. Considering prior work with the tetrapeptide pGlu-Tyr-Arg-Trp-NG2 the authors proposed that the conformation of Tyr and Arg are fundamental in its function.

Synthesis and relationship of L-glutaminyl-L-histidyl-L-prolinamide to the thyrotropin releasing hormone

Abstract The new L-glutaminyl-L-histidyl-L-prolinamide (Gln-His-Pro(NH 2 )) has been synthesized. This open glutaminyl-form has been compared with the cyclized pyro-form of (pyro)Glu-His-Pro(NH 2 ), thyrotropin releasing hormone (TRH). Gln-His-Pro(NH 2 ) is converted into (pyro)Glu-His-Pro(NH 2 ) under very mild conditions some of which are analogous to steps used in the isolation of TRH. Gln-His-Pro(NH 2 ) had up to five per cent of the activity of TRH although it is unknown whether this activity is intrinsic or due to contamination or conversion. There is no evidence that Gln-His-Pro(NH 2 ) is present in the hypothalamus, but if it were it could be a moiety of a larger molecule which is convertible into Gln- or (pyro)Glu-His-Pro(NH 2 ) or it could be a biosynthetic precursor or a metabolic transformation product of TRH. (Pyro)Glu-His-Pro(NH 2 ) shows the functions expected of the hormone.

Discovery of a new synthetic tetrapeptide having luteinizing releasing hormone (LRH) activity

Abstract A new synthetic tetrapeptide, pGlu-Tyr-Arg-Trp-NH 2 , has been discovered which has the activity of the luteinizing releasing hormone (LRH) of the hypothalamus, and is, to our knowledge, the first one reported with LRH-activity. Studies indicated the presence of pGlu, Arg, Tyr, and Trp in bovine LRH and Trp in porcine LRH. Of the six possible tetrapeptides, only pGlu-Tyr-Arg-Trp-NH 2 , shows LRH activity. This tetrapeptide is less active than LRH, and is not the hormone, but it is relatively potent. A part or all of the sequence of pGlu-Tyr-Arg-Trp-NH 2 may be identical to part(s) of the sequence of LRH. All available data on LRH indicate it apparently is a decapeptide and not a nonapeptide reported by others.

On the chemical existence and partial purification of the hypothalamic follicle stimulating hormone releasing hormone.

Abstract Others have stated that the hypothalamic hormone, pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 , in the “gonadotropin-releasing hormone” which regulates both the luteinizing and follicle stimulating hormones (LH and FSH). Such dual regulation is contradictory to a long considered “unitary” interrelationship for hormones of the hypothalamus and pituitary. Four fractions at stage SP and thirteen at stage P-2 released, in vitro , FSH at levels as high as 40,000->128,000 ng/ml. The release of FSH by synthetic LRH averaged about 16,700 (range 6150–28000) at 500 ng/ml. These data are basic and have priority, because they reveal the unknown FSHRH by differentiation of its activity from that of LHRH; this was not feasible before the availability of LHRH.

PURIFICATION OF A PROLACTIN INHIBITING HORMONE AND THE REVEALING OF HORMONE D-GHIH WHICH INHIBITS THE RELEASE OF GROWTH HORMONE

Summary A prolactin inhibiting factor (hormone, PIF, PIH) has been purified from porcine hypothalami. Inhibition, in vitro , of prolactin release was observed at a dosage of approx. 10–100 ng, estimated on the basis of uv absorption. The same fraction at the same dosage also inhibited the release of the growth hormone. The entities behaving like PIH and GHIH were separated by high pressure liquid chromatography. The PIH activity was inactivated by pronase. The entity showing GHIH-activity and synthetic somatostatin appear to be different. Since hypothalamic hormones frequently show multiple activities, this entity is provisionally named hormone D-GHIH until it is chemically and biologically characterized. The D-GHIH designation merely defines the assay guiding isolation.

Biosynthesis and evidence for the existence of the follicle stimulating hormone releasing hormone

Abstract Biosynthesis with 14C-glutamic acid and 14C-glutamine in a hypothalamic system followed by fraction yielded fractions after defatting, Bio-Gel P2, CMC, and Sephadex G25 partition chromatography which released 40,000 to >128,000 ng/ml of FSH. The decapeptide, pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 released about 18,000 and rarely up to 35,000 ng/ml of FSH. Radioactivity was associated with these exceptionally active FSH-fractions. The apparent new hypothalamic hormone releases both FSH and LH and can be FSHRH which is different from the decapeptide which is apparently LHRH and not LHRH FSHRH The associated radioactivity and FSH-releasing activity indicate that FSHRH may have a pGlu-moiety.

Biosynthesis in,vitro of the luteinizing releasing hormone by hypothalamic tissue

The biosynthesis, in,vitro, of the luteinizing releasing hormone (LRH) from 14C-glutamic acid by hypothalamic tissue has been demonstrated. After fractionations of the incubation mixture, the use of synthetic LRH as a carrier made it possible to isolate by electrophoresis a Pauly-reactive area which exhibited radioactivity. The areas in front of and behind this spot were not radioactive. The radioactive and Pauly-reactive area was further purified by tlc. Acid hydrolysis of the apparently homogeneous Pauly-reactive area, which contained all the expected radioactivity, gave about 75% of the expected 14C-glutamic acid. Assays, in,vivo, showed release of LH and FSH at three appropriate stages of fractionation.

Biosynthesis of the luteinizing hormone releasing hormone in mitochondrial preparations and by a possible pantetheine-template mechanism

Abstract Data show that the luteinizing hormone releasing hormone (LHRH) is biosynthesized by mitochondrial preparations from hypothalami which were obtained by centrifugation of a Ficoll gradient system. Incubation, extraction, use of carrier synthetic LHRH, and chromatography, yielded fractions showing associated hormonal activity and radioactivity. Other investigators reported biosynthesis of the thyrotropin and the growth hormone releasing hormones by soluble enzymes, but these enzymes might have been derived from mitochondria and/or synaptosomes. These particles appear implicated, but the true site of biosynthesis of these hormones is unknown. The mechanism at the site could possibly involve a pantetheine-protein template.