László Gráf

The human insulin receptor cDNA: The structural basis for hormone-activated transmembrane signalling

A cloned approximately 5 kb cDNA (human placenta) contains the coding sequences for the insulin receptor. The nucleotide sequence predicts a 1382 amino acid precursor. The alpha subunit comprises the N-terminal portion of the precursor and contains a striking cysteine-rich cross-linking domain. The beta-subunit (the C-terminal portion of the precursor) contains a transmembrane domain and, in the intracellular region, the elements of a tyrosine phosphokinase: an ATP-binding site and a possible tyrosine autophosphorylation site or sites. The overall structure is reminiscent of the EGF receptor; the cross-linking domain of the alpha subunit and several regions of the beta subunit exhibit sequence homology with the EGF receptor. The phosphokinase domain also exhibits homology with some oncogenic proteins that have tyrosine phosphokinase activity, in particular, a striking homology with v-ros. Southern blotting experiments suggest that the coding region spans more than 45 kb. The insulin receptor gene is located on chromosome 19.

Attenuation of morphine tolerance and dependence by α-melanocyte stimulating hormone (α-MSH)

Abstract Repeated administration of morphine resulted in significant reduction of its analgesic potency. If 0.1 mg/kg α-MSH was coadministered, the tolerance development was attenuated, 1 mg/kg MIF (MSH release inhibiting factor), given simultaneously with morphine, did not affect tolerance. Injecting, however, MIF 1 hour prior to the daily opiate treatment resulted in accelerated development of tolerance supposedly by lowering the plasma α-MSH level at the time of morphine administration. Of the morphine abstinence symptoms the naloxone-induced jumping in morphine pretreated mice could not be modified either by α-MSH coadministration or by MIF pretreament, but the withdrawal body weight loss was found to be diminished by the former and increased by the latter peptide. The possible role of α-MSH in preventing the development of tolerance to the analgesic effect of endogenous opioid peptides is discussed.

Effect of bacitracin on the biodegradation of β-endorphin

Abstract β-endorphin was incubated with rat brain homogenate, and the amino acids released were measured by amino acid analysis. Phe, Leu, Tyr, and Lys were liberated in the greatest amount indicating that the cleavage of Leu77-Phe78 and some Lys-X peptide bonds with endopeptidases followed by the removal of the terminal residues by exopeptidases are the main routes of β-endorphin degradation in the brain. Bacitracin considerably reduced the amino acid release from β-endorphin incubated with rat brain homogenate, and its action is suggested to be due to the inhibition of brain amino- and carboxypeptidases. Bacitracin also potentiated and prolonged the in vivo analgesic activity of β-endorphin.

On the primary structure of pituitary bovine growth hormone

Summary The primary structure of the tryptophan containing tryptic fragment obtained from bovine growth hormone has been determined. This sequence was found to be identical with that of the corresponding portion of sheep growth hormone. Together with other data, the corrected structure of bovine growth hormone is proposed herein.

Action of plasmin on ovine beta-lipotropin: Revision of the car☐yl terminal sequence

Human plasmin was found to cleave the lysylasparagine bond specifically in positions 79 and 80 of the ovine beta-lipotropin molecule. Structural studies of the COOH-terminal fragment revealed the amino acid sequence to be: Asn-Ala-Ile-Ile-Lys-Asn-Ala-His-Lys-Lys-Gly-Gln which is slightly different from the structure originally proposed. Together with an earlier revision of the NH2-terminal sequence, the corrected structure of ovine beta-lipotropin is shown in Figure 2.

Cross tolerance between morphine and β-endorphin in vivo

Experiments were performed to establish the development of cross tolerance between morphine and the C-terminal fragment of porcine β-lipotropin /LPH61–91, β-endorphin/ in rats. Repeated intracerebroventricular /ICV/ or peripheral administration of morphine induced tolerance both to morphine and β-endorphin. The tolerance induced by ICV administration of morphine was more pronounced than in the case of peripheral application. The results give support to the theory that at least in part similar sites and mechanisms are involved in the analgesic activity of morphine and the endorphins.

Cathepsin D generates γ-endorphin from β-endorphin

Abstract A crude extract of porcine anterior pituitary was found to contain endopeptidase activity that splits the Leu77-Phe78 peptide bond of β-lipotropin in a pH range 3.0–7.0. The specificity and susceptibility to pepstatin of pituitary extract were the same as those of cathepsin D (E.C. 3.4.23.5) isolated from calf brain by affinity chromatography. Cathepsin D was shown to split the same Leu-Phe peptide bond of β-endorphin, leading to the formation of γ-endorphin. Based on the above data it is suggested that cathepsin D is a major factor in the generation of endorphins of intermediate size.

STRUCTURE-FUNCTION RELATIONSHIPS IN LIPOTROPINS

Twelve years ago Li and co-workers’, 2 reported the discovery of a new lipolytic polypeptide of adenohypophysis. Because of the considerable fatmobilizing effect of this polypeptide it was designated as lipotropic hormone (LPH) or 1ipotropin.l The lipolytic activity however, has not been a specific property of lipotropin; on the contrary almost all the adenohypophyseal hormones have lipolytic side-effect in addition to their main biologic function. This fact may, at least in part, explain why comparatively less attention has been paid to lipotropin in the last ten years. During this period of time only Dr. C. H. Li’s laboratory and few additional ones, namely the authors’, Dr. M. Chretien’s, Dr. Y. Pankov’s and more recently Dr. P. J. Lowry’s group, made efforts to elucidate the structure and to understand the physiologic role of these curious polypeptides. After the discovery of enkephalins-two brain pentapeptides structurally related to lipotropin-at the end of 1975,s lipotropin, this so far “useless” polypeptide, suddenly became the focus of interest and excitement, and in turn, the knowledge of the chemistry and biology of lipotropins acquired a special value. This paper reviews the most important results achieved in our institute during the last few years, but mostly in 1976, as regards the relationships between the structure and different biologic functions of lipotropins.

Studies on the conformation of β-endorphin and its constituent fragments in water and trifluoroethanol by CD spectroscopy

Structure-function studies on P-LPH fragments have revealed that the in vitro opiate agonist activity is an exclusive property of fragments containing the complete structure of Met-enkephalin (LPH (61-65)) [ 1 ] at their NH*-terminus and that the biological potency is a function of the chain length of the peptides [2-61. Peptides intermediate between Metenkephalin and LPH (61-9 1) (named fl-endorphin by Li and Chung, [7] ) were found to be less potent than Met-enkephalin in different in vitro bioassays [2-61. Elongation of the peptide chain of LPH (61-79) by addition of the 12 COOH-terminal residues of fl-LPH [4-61 to form P-endorphin caused a dramatic change in the biological properties of the molecule. Among all the fragments studied so far, /3-endorphin is the most active in receptor binding [2,7] in guinea-pig ileum bioassays [3-61 and the least active in mouse vas deferens test [5,6]. In addition, &endorphin exerts the most profound analgesic [2,4,8,9] and behavioral effects [ 10,l l] when administered intraventricularly [2,4,8,10,11] or intravenously [9] to different animals. From the above relationships the impression has

Effect of two synthetic α-gliadin peptides on lymphocytes in celiac disease: Identification of a novel class of opioid receptors

Two synthetic peptides containing residues 43-47 and 43-49 of alpha-gliadin were tested for inhibition of leukocyte migration in 47 patients with celiac disease. In nineteen patients, all on a normal diet, leukocyte migration was inhibited by the peptides and naloxone blocked this effect. In twenty-eight patients (24 of whom were on strict gluten-free diet) leukocyte migration was not affected by the peptides. Our results suggest that alpha-gliadin-(43-49), Tyr-Pro-Gln-Pro-Gln-Pro-Phe, is closely related to the active fragment, or to one of the active fragments of alpha-gliadin, and that it interacts with receptors that are similar to but not identical with the known opiate receptors.