Structural requirements of bioactive peptides for interaction with endopeptidase 22.19.

A series of biologically active peptides and related compounds (opioid peptides, neurotensin, and bradykinin) were used as substrates or competitive inhibitors to study the structural requirements for peptide interaction with endopeptidase 22.19. The kinetics of hydrolysis of these peptides indicated that, in contrast to other proteases, the substrate specificity of endopeptidase 22.19 is not determined by the amino acids flanking the sensitive bonds of the substrates. The competition between bioactive peptide analogues and the quenched fluorescence substrate of endopeptidase 22.19 indicated that their length and their flexibility may be the dominant factors to explain their binding specificities. These peculiar features of endopeptidase 22.19 may be of importance to understand the physiological processes of conversion and inactivation of biologically active peptides.

[Tests of some peptides isolated from residue after insulin isolation from the pancreas. II. Establishment of heterogeneity level]. / Badania niektórych peptydów wyodrebnionych z pozostalosci po izolowaniu insuliny z trzustki. II. Ustalanie stopnia heterogenicznosci.

Heterogeneity of the active substance of Lipotropin-Polfa preparation was investigated by the method of gel column chromatography. The isolated polypeptide hormone has been found to possess the average molecular weight nine times smaller than that reported in the literature. This polypeptide was separated into six fractions by chromatography on Sephadex G-10. These fractions were found to differ in respect of the qualitative and quantitative amino acid composition. With the purpose of comparison, an analogous study of Lipormone-Labor. Choay (France) was performed. Similarity of this preparation to fraction I of Lipotropin-Polfa has been shown.

Lipolytic potency of proopiomelanocorticotropin peptides in vitro.

Four lipolytic active centers were localized in proopiomelanocorticotropin (POMC): in the N-terminal part of POMC ("tryptophane rich peptide"), in the N-terminal part of adrenocorticotropic hormone, in the middle portion of beta-lipotropin and in the C-terminal part of beta-lipotropin. The weak lipolytic activity of the "tryptophane rich peptide" is not mediated by its two partial sequences gamma-MSH and delta-MSH. The minimal amino acid sequence for obtaining lipolysis from the N-terminal part of ACTH was ACTH 4-10. Lengthening of this amino acid sequence on the N- or C- terminus resulted in a strong increase of lipolytic potency. The minimal effective sequence from the middle portion of beta-lipotropin was located in the amino acid residues 47-53 which are identical to ACTH 4-10. Additional amino acids on the N- and C-terminus (beta-lipotropin p 41-58 and beta-lipotropin h 35-56) lead also to increased lipolytic activity. The forth center of POMC resides in the C-terminal part of beta-lipotropin (residues 78-91) because sequences from the N-terminal part of beta-lipotropin 61-91 were ineffective. The order of potency of POMC peptides especially in respect to the minimal effective concentration was ACTH 1-13 (alpha-MSH) greater than beta-lipotropin p greater than ACTH greater than ACTH 1-10 greater than beta-lipotropin p 61-91.

Evidence for the existence of [Gln9]-beta-lipotropin in human pituitary glands.

The isolation of two peptides similar in amino acid composition to that of human beta-lipotropin is presented. Peptide patterns after enzymatic digestions of these two peptides by Staphylococcus aureus protease and by trypsin were nearly identical. Paper electrophoresis and amino acid analyses of acidic peptides generated from the enzymatic digestions of these two peptides indicate that there is an amide difference between the two peptides. It is proposed that this amide difference is in amino acid residue number nine, and that one is the human beta-lipotropin and the other its [Gln9] analog.

Behavioral and electrophysiological effects of peptides related to lipotropin (beta-LPH).

Both C-terminal fragments of lipotropin (beta-LPH) (endorphins) and N-terminal fragments (e.g., ACTH 4-10) delayed extinction of pole-jumping avoidance behavior in rats. After subcutaneous injection Met5-enkephalin appeared to be as active as ACTH 4-10 whereas beta-LPH 61-69, alpha- and beta-endorphin were more potent in delaying extinction of pole-jumping avoidance behavior (approximate ED50 of alpha-endorphin 4 x 10(-11) M rat.) However, the potency of beta-LPH 61-69 and alpha-endorphin appeared to be approximately the same whereas that of beta-endorphin was less than that of ACTH 4-10 after intraventricular administration (approximate ED50 of alpha-endorphin 0.2 x 10(-11) M rat). alpha-Endorphin and ACTH 4-10, administered subcutaneously in a dose which markedly delayed extinction of pole-jumping avoidance behavior, had only slight effects on open field behavior and on responsiveness to electric footshock. A 5 times higher dose of both peptides facilitated passive avoidance behavior. Morphine in two doses significantly delayed extinction of pole-jumping avoidance behavior but the effect was not dose dependent. The specific opiate antagonist naltrexone, however, markedly facilitated extinction of the avoidance response. ACTH 4-10, alpha- and beta-endorphin and a behaviorally potent ACTH 4-9 analog (Org 2766) restored pole-jumping avoidance behavior of rats pretreated with naltrexone. Treatment with a similar dose of naltrexone blocked beta-endorphin-induced analgesia. These results suggest that the influence of peptides related to C-terminal and N-terminal fragments of lipotropin on extinction of avoidance behavior may be dissociated from those exerted on opiate receptor sites. Subcutaneously injected beta-LPH 61-69 or intraventricularly administered beta-endorphin induced a shift from lower to higher frequencies of hippocampal theta rhythm during paradoxical sleep in the same way as that found after ACTH 4-10. This effect is interpreted as indicating an increased arousal state in certain midbrain limbic structures. This may, as has been postulated for ACTH 4-10, alter the motivational value of environmental stimuli (e.g., aversive stimulation).

C-fragment of lipotropin--an endogenous potent analgesic peptide.

1 A series of peptides derived from porcine lipotropin was examined for analgesic and other morphine-like properties on infusion into the cannulated third ventricle of cats.2 Lipotropin (LPH 1-91) itself produced no analgesia or other morphine-like effects when infused in a dose of 150 mug.3 C-fragment (LPH 61-91) produced strong long-lasting analgesia when infused in a dose of 10 or 20 mug; on a molar basis the potency was between 90 and 180 times that of morphine. The following morphine-like effects were also produced: shivering leading to fever, vasodilatation of the pinnae, mydriasis, opening of the palpebral fissures, tachypnoea with bouts of panting, vocalization, hyperexcitability, restlessness and catalepsy. All the effects, including analgesia, were abolished by an intraperitoneal injection of naloxone (1 mg/kg).4 Hyperglycaemia, another central effect produced by morphine, was obtained with C-fragment infused in a dose of 60 mug.5 On intravenous injection, C-fragment produced analgesia with a dose of about 200 mug/kg. Administered by this route, C-fragment was again more potent than morphine.6 C'-fragment (LPH 61-87), LPH 61-78 and LPH 61-69, either had no analgesic effect or produced weak short-lasting analgesia when infused in doses up to 100 mug.7 Methionine enkephalin (LPH 61-65) either produced very weak short-lasting analgesia or had no analgesic effect when infused in doses of between 30 and 400 mug.8N-methyl methionine enkephalin amide in which both termini of methionine enkephalin were protected against degradation by exopeptidases produced long-lasting analgesia when infused in doses of 150 to 180 mug; its analgesic potency was approximately 100 times less than that of C-fragment. Blocking only one terminus of methionine enkephalin did not appear to endow the peptide with analgesic properties. The N-methyl pentapeptide amide produced other morphine-like effects of which the most striking was catalepsy. All the effects were abolished by intraperitoneal naloxone (1 mg/kg).