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Biomedical subjects

E Hazum

Publications and source records attributed to E Hazum.

90 records · Page 5Linked to original sources
PubMed1979

Insulin receptor: covalent labeling and identification of subunits.

Two methods were used to label insulin receptors covalently with 125I. In the first, an aryl azide derivative of insulin, 125I-labeled 4-azido-2-nitrophenyl-insulin, was synthesized and used to photolabel the binding region of the insulin receptor in rat liver membranes and human placenta membranes. In the second, insulin receptors were purified from rat liver membranes and labeled with 125I by use of chloramine-T; this method presumably has no specificity for the binding region of the receptor. The proteins labeled by both methods were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis after or without reduction by dithiothreitol. The photoaffinity label specifically labeled a single band in both liver and placenta that had an apparent molecular weight of 135,000 after reduction. A band with similar mobility was present in the chloramine-T-labeled preparation, which also contained a second major band with an apparent molecular weight of 45,000. Without reduction, both methods resulted in a single labeled band with an apparent molecular weight of about 310,000. These results indicate that the insulin receptor of both liver and placenta has a subunit of molecular weight 135,000 that binds insulin and that the receptor may be composed of at least two different subunits that are linked together or greatly stabilized by disulfide bonds.

Affinity Labels↗
PubMed1977

Gonadotropin-releasing hormone in milk.

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) has been found in milk of man, cow, and rat. Radioimmunoassays of acidified milk indicate concentrations of GnRH ranging between 0.1 and 3 nanograms per milliliter. Multistep extractions, followed by electrophoresis, reveal gonadotropin-releasing activity in the fraction that comigrates with the GnRH-marker. A second hypothalamic hormone, thyrotropin-releasing hormone, is present in milk at a much lower concentration. "Milk -GnRH" may influence the secretion of the gonadotropic hormones in neonates.

Animals↗
PubMed1977

On the role of tryptophan in luteinizing-hormone-releasing hormone (luliberin).

The tryptophan residue of luteinizing-hormone-releasing hormone (luliberin) was chemically modified to produce the following analogs: [Trp(o)3]luliberin, Trp-(2,4-dinitrophenylsulfenyl)-luliberin, Trp-(2-hydroxy-5-nitrobenzyl)luliberin, (Trp-S-luliberin)2, Trp-CH3S-luliberin and Trp-formyl-luliberin. The luteinizing-hormone-releasing activity of those analogs was determined by bioassay in vitro and found to be 0.2%, 0.2%, 0.6%, 1.5%, 1.7% and 7% of that of the natural hormone, respectively. These results demonstrate that alterations in the indole moiety of tryptophan-3, which lead to a reduction in its electron density or sterically restrict its electron avaliability, are associated with a dramatic loss of biological activity.

Animals↗
PubMed1977

A photolabile protecting group for the phenolic hydroxyl function of tyrosine.

The ortho-nitrobenzyl group (ONBzl) was utilized to protect the phenolic functions of the side chain of tyrosine. Optimal conditions for its mild photolyti removal were established. This paper describes the preparation and properties of some (O-ONBzl)-tyrosine derivatives of potential value in the synthesis of tyrosine-containing peptides. Their use in the synthesis of the dipeptide, N-benzyloxycarbonyl-L-tyrosyl-glycine ethyl ester is presented as an example.

Benzyl Compounds↗
PubMed1977

Purification of the gonadotropin-releasing hormone-degrading enzyme by affinity chromatography.

A crude preparation of Kallikrein inactivator, which inhibits the gonadotropin-releasing hormone (GnRH)-degrading enzyme(s) from rat hypothalamus and anterior pituitary, was fractionated by passage through an ion-exchange column. The enzyme-inhibiting fraction was coupled to Sepharose and the resin obtained was used for, affinity-chromatography purification of the GnRH-degrading enzyme. The enzyme from crude tissue preparations was retained on this column and eluted by 0.05 M phosphate buffer. A 9-12 fold increase in the specific activity of the enzyme was achieved. Bacitracin, an effective peptide inhibitor of the degradation of GnRH, was also coupled to Sepharose. Three different such Sepharose-bacitracin conjugates were synthesized, two of which inhibited the degradation of GnRH by hypothalamic and pituitary extracts. They all failed, however, to separate the active enzymic fraction from the bulk of accompanying proteins, using affinity chromatographic techniques.

Animals↗
PubMed1976

Structure-activity relationships of luliberin substituted at position 8.

Substitution of arginine at position 8 of luliberin by the basic amino acids homoarginine, lysine and diaminobutyric acid resulted in analogues in which the luteinizing hormone-releasing activity is markedly reduced, whereas the cross reactivity with specific antibodies to luliberin is preserved. Fluorimetric titrations of these analogues, carried out as with luliberin, revealed pK values of 6.00 +/- 0.05 and of 9.75 +/- 0.15 for His 2 and Try 5 respectively which are essentially the same as in luliberin. However, the rate of collisions between the side chains of His 2 and Trp 3 in these analogues was found to decrease by 36-39%. Substitution at position 8 with the non-basic amino acid omega-nitro arginine yielded an analogue possessing a very low hormonal activity as well as poor recognition of antibodies specific to luliberin. The fluorescence properties of this peptide are markedly different from those of luliberin and its three basic analogues. These results indicate that the functional integrity of the active unit His 2 . . . Tyr 5 . . . Arg 8 in luliberin depends both on size and basicity of the amino acid side chain at position 8.

Amino Acids, Diamino↗
PubMedDate not supplied

Characterization of Leydig cell gonadotropin-releasing hormone binding sites utilizing radiolabeled agonist and antagonist.

Gonadotropin-releasing hormone (GnRH) binding sites in intact Leydig cells and in membrane preparations were investigated using 125I-labeled GnRH agonist and antagonist. Binding was saturable and involved a single class of high affinity sites. Intact Leydig cells and a membrane preparation had a higher affinity for GnRH agonist (Kd 3.0 +/- 1.7 X 10(-10) M) than for GnRH antagonist (Kd 10.0 +/- 1.8 X 10(-10) M). With anterior pituitary membranes the Kd was 2.8 +/- 0.7 X 10(-10) M for the agonist and 2.4 +/- 1.4 X 10(-10) M for the antagonist. The Kd for GnRH was similar for Leydig cells and the anterior pituitary. Chymotrypsin and trypsin digestion decreased receptor binding, but neuraminidase increased Leydig cell binding in contrast to the decrease in binding observed with pituitary receptors. The results suggest that the Leydig cell GnRH binding sites may differ from the pituitary receptor which may be related to structural differences in GnRH-like peptides recently described in extracts of rat testis.

Animals↗
PubMedDate not supplied

Cytochemical characterization of pituitary target cells for biotinylated gonadotropin releasing hormone.

These studies describe the application of new cytochemical stains that co-localize a biotin-labeled gonadotropin releasing hormone (GnRH) analog and FSH or LH in the same field or cell. Pituitary monolayer cells were stimulated with the [D-Lys6] GnRH analog or the same analog labeled with biotin. Biotinylated [D-Lys6] GnRH exhibited a higher affinity and was 7-10 X more potent than unlabeled [D-Lys6] GnRH. The avidin-biotin peroxidase complex technique (ABC) was applied to localize the biotinylated GnRH on the cells with the use of a dense black peroxidase substrate. Specificity tests showed that the stain could be eliminated by competition with unlabeled [D-Lys6] GnRH. The GnRH stain was followed by immunocytochemical stains for LH beta, FSH beta or 25-39ACTH with a different peroxidase substrate (amber or orange-red). Stain for GnRH was found on the surfaces of 16% of the cells and 60-90% of the GnRH stained cells also stained for one of the gonadotropins. Most (90-100%) of the gonadotropes showed stain for GnRH. Our studies demonstrate that a potent biotinylated GnRH analog binds cells that can be identified specifically as gonadotropes.

Animals↗
PubMedDate not supplied

Testicular GnRH receptors: photoaffinity labeling and fluorescence distribution studies.

Specific GnRH receptor proteins of purified rat Leydig cells and membrane preparations were identified using an 125I-labeled bioactive photoaffinity derivative of GnRH. Sodium dodecyl sulfate polyacrylamide gel electrophoresis resulted in the identification of two specific components with apparent molecular weights of 60,000 and 54,000 daltons. Fluorescent visualization of GnRH receptors in these cells, utilizing a bioactive rhodamine derivative of the hormone, indicated that the fluorescently labeled receptors were initially distributed uniformly on the cell surface and then formed clusters which subsequently internalized (at 37 degrees C) into endocytic vesicles. These processes were dependent on specific binding sites for the rhodamine-labeled peptide on Leydig cells. These findings indicate further characterization of the testicular GnRH receptors and may have important implications towards the understanding of the molecular events involved in the action of the hormone in the testis.

Affinity Labels↗
PubMedDate not supplied

Biotinylated endothelin as a probe for the endothelin receptor.

Biotinylated derivatives of endothelin (ET)-1 were prepared by chemical modification of ET-1 with sulfosuccinimidyl 6-(biotinamido) hexanoate. Two major biotinylated ET analogs were purified by reversed-phase high performance liquid chromatography. Edman degradation indicated that the first eluting peptide contains one biotin residue on lysine at position 9, while the second derivative contains an additional biotin residue at position 1. Competition binding studies to mouse osteoblastic cell line MC3T3-E1 using 125I-labeled ET-1 revealed IC50 values of 5, 30 and 600 nM for native ET, the mono- and the dibiotinylated ET analog, respectively. A similar order of potency was obtained when these ET derivatives were examined for stimulation of DNA synthesis in MC3T3-E1 cells. In addition, incubation of MC3T3-E1 cells with the monobiotinylated ET and subsequent addition of rhodamine-avidin resulted in an evenly distributed fluorescence over the cell surface. The fluorescence observed was completely abolished in the presence of an excess of native ET. Thus the monobiotinylated ET proves to be useful for localization of the ET receptors.

Amino Acid Sequence↗
PubMedDate not supplied

Endothelin rapidly stimulates tyrosine phosphorylation in osteoblast-like cells.

The mitogenic activity of endothelin (ET) was studied in osteoblast-like cells, MC3T3-E1. [3H] Thymidine incorporation induced by ET was markedly lower than that of platelet-derived growth factor (PDGF). ET synergistically stimulated [3H] thymidine incorporation induced by PDGF with an apparent ED50 value of 2.5 nM. Treatment of MC3T3-E1 cells with ET and subsequent immunoblotting of the cell extracts with antiphosphotyrosine antibodies followed by labeling with [125I] protein A resulted in the identification of several phosphotyrosine-containing proteins. The intensity of these labeled phosphoproteins significantly increased when the cells were treated with a combination of ET and PDGF. Genistein, an inhibitor of tyrosine kinases, blocked [3H] thymidine incorporation as well as protein tyrosine phosphorylation stimulated by either ET, PDGF or the combination of ET and PDGF. These findings suggest that tyrosine phosphorylation could play a role in the comitogenic activity of ET in osteoblast-like cells.

Animals↗