Regulation of Formation and Proposed Structure of the Factor Inhibiting the Release of Melanocyte-Stimulating Hormone

Abstract

Microsomal preparations from the stalk median eminence of female rats are shown to contain an enzymic activity that is responsible for the formation of MSH-release-inhibiting factor (MSH-R-IF). The amount of this activity remains constant throughout the estrous cycle. The corresponding mitochondrial preparations from the stalk median eminence contain another enzymic principle, estrous cycle-dependent, which competes with the enzyme present in the microsomal preparation for the same "substrate", and can thereby prevent the formation of MSH-R-IF.Several neurohypophyseal hormones, analogs, and peptide intermediates have been tested for their intrinsic MSH-R-IF activity and for their ability to be transformed into MSH-R-IF by incubation with microsomal preparations of stalk median eminence from male rats; it is concluded that the enzyme responsible for the formation of MSH-R-IF is an exopeptidase and that the release-inhibiting factor itself is a tripeptide. Oxytocin is converted by the incubation to L-prolyl-L-leucylglycinamide; nanogram amounts of this tripeptide inhibit the release of MSH from the pituitary both in vivo and in vitro.Rat hypothalamus contains a factor (MSH-R-IF) that inhibits the release of melanocyte-stimulating hormone (1, 2). Evidence for the enzymic formation of this factor has been presented (3). In male rats the pituitary content of MSH remains constant, whereas in females it fluctuates as a function of the estrous cycle (4).This last-mentioned finding was the point of departure for investigating the manner in which pituitary MSH content reflects a change in the enzymic activities responsible for the control of MSH-R-IF formation. We show that a microsomal exopeptidase in the hypothalamus degrades oxytocin, liberating the hormonal C-terminal tripeptide, prolyl-leucylThis work has been performed in C6rdoba and New York. Therefore, different strains of rats and of toads (Bufo arenarum, Bufo marinus) were employed. Since the results obtained in the two laboratories showed no discrepancies, combined publication was agreed upon.Abbreviations of amino-acid derivatives and peptides are in accordance with the IUPAC-IUB Tentative Rules on Biochemical Nomenclature, Biochemistry, 5, 2485Biochemistry, 5, , 1445Biochemistry, 5, (1966 6, 362 (1967

Characterization of melanotropin-release-inhibiting factor (melanostatin) from frog brain: homology with human neuropeptide Y.

“…In contrast, porcine neuropeptide Y (NPY) appears to be a potent inhibitor of a-melanotropin release in frog (18) and toad (19). Several other regulatory peptides characterized from hypothalamic extracts have been proposed as potential regulators of melanotropin release (20)(21)(22). However, many studies failed to demonstrate that these peptides meet the criteria expected of a physiological melanotropin-release-inhibiting factor (melanostatin) (23)(24)(25).…”

“…Five families of hypothalamic neuropeptides involved in the control of anterior pituitary hormone secretion have now been characterized, namely, thyrotropin-releasing hormone (3, 4), gonadotropinreleasing hormone (5), somatostatin (6), corticotropinreleasing hormone (7), and growth hormone-releasing hormone (8,9 (20)(21)(22). However, many studies failed to demonstrate that these peptides meet the criteria expected of a physiological melanotropin-release-inhibiting factor (melanostatin) (23)(24)(25).…”

Intramolecular motion in peptides determined by 13C NMR: A spin‐lattice relaxation time‐study on MSH‐release‐inhibiting factor

“…Peptide samples were run at 37°C in 12 mm tubes in D20 or deuterated dimethylsulfoxide ((Cq) 2 SO) at concentrations of 50 mg/ml with tetramethylsilane as external standard. Crystalline Pro-Leu-Gly-NH2.1/2 HZ0 [28] was the same as used in earlier biological [22] and conformational investigations [26] The Pro-Leu C, 61.9; H, 7.67; N, 12.5.Found: C, 62.0; H, 7.70; N, 12.41 which was subjected to hydrogenolysis (10% Pd/C) in 25 ml methanol and chromatographed on a silica gel column to give the oily and hydroscopic free base in 79% yield. The base was characterized by amino acid analysis and reconversion to the carbobenzoxy derivative.…”

“…the pituitary [22,23] ; the factor is released from oxytocin by enzymes present in the hypothalamus [22,24,251. On the basis of 300 MHz 'H NMR studies in dimethyl sulfoxide and preliminary X-ray crystallographic investigations it was proposed that the preferred conformation of Pro-Leu-Gly-NH2…”

Preparation of oxytocin specifically 14C‐labeled in the tyrosine residue

“…Enzymes which release glycinamide or the C-terminal dipeptides of oxytocin and vasopressin have now been partially purified [ 10,11 ]. Additional enzymatic mechanisms capable of neurohypophyseal hormone inactivation are known, including disulfide reductase in liver [12] as well as enzymes which can degrade the hormones from the N-terminus [13][14][15].…”