PubMed Health⌕ Search

Biomedical subjects

M Sonenberg

Publications and source records attributed to M Sonenberg.

15 recordsLinked to original sources
PubMed1992

A growth hormone (GH) analog can antagonize the ability of native GH to promote differentiation of 3T3-F442A preadipocytes and stimulate insulin-like and lipolytic activities in primary rat adipocytes.

The effect of amino acid substitutions introduced to the third alpha-helix in bovine GH (bGH) was investigated. A GH analog (bGH-M8), in which three amino acids were substituted to form an idealized amphiphilic alpha-helix, possessed the same specific binding affinity as wild-type bGH to cell membranes prepared from 3T3-F442A cells or rat adipocytes. However, bGH-M8 failed to stimulate preadipocyte differentiation, as measured by the level of glycerol-3-phosphate dehydrogenase activity. An equimolar concentration of bGH-M8 was inhibitory for this adipogenic effect caused by bGH at a concentration of 30 pM. bGH-M8 also failed to induce an insulin-like response and reduced lipolytic potency in rat primary adipocytes. A 10-fold excess of bGH-M8 abolished the effect of wild-type bGH in the insulin-like and lipolytic assays. Thus, bGH-M8 inhibited these actions of wild-type bGH and, therefore, appears to be a competitive antagonist. These results suggest that a major biologically active domain resides in the third alpha-helix of bGH, which is independent of amino acids important in the initial interaction of GH with its receptor.

3T3 Cells↗
PubMed1992

Growth hormone and fibronectin expression in 3T3 preadipose cells.

In the present study we focused on the relationship between GH action and the extracellular matrix in 3T3-F442A preadipose cells. Results from Northern blotting indicated that in serum-free medium, the presence of 2 nM met-human GH down-regulated levels of fibronectin messenger RNA by approximately 40, 60, and 70% as compared with control levels on days 1, 2, and 4, respectively. GH-dependent reduction of levels of collagen alpha 1(I) mRNA expression occurred later and was less pronounced than effects on levels of fibronectin mRNA, suggesting a specificity in the matrix-altering function of GH. Western blot analyses and immunoprecipitation studies revealed that between 2 and 5 days of culture, matrix-associated fibronectin protein was reduced 70 to 90% by GH treatment. Down-regulation of fibronectin protein expression by met-human GH was dose-dependent between 2 and 0.02 nM. The presence of 2 nM insulin or insulin-like growth factor-1 promoted a 30-40% increase in fibronectin levels compared to control cells. The GH-promoted down-regulation of fibronectin expression was eliminated by concomitant addition of insulin. These data demonstrated that GH effects on matrix-associated fibronectin expression were independent of, and in opposition to, effects promoted by insulin and insulin-like growth factor-1. Treatment of culture dishes with fibronectin or collagen inhibited GH-stimulated adipogenesis 50 and 80%, respectively, compared with controls, as judged by levels of glycerol-3-phosphate dehydrogenase activity. Thus, composition of the extracellular matrix was a critical factor in GH-induced adipogenesis of 3T3-F442A fibroblasts. Our results demonstrate that GH action in 3T3 preadipose cells is intimately coupled to the biology of extracellular matrix.

3T3 Cells↗
PubMed1979

A method for measuring membrane microviscosity using pyrene excimer formation. Application to human erythrocyte ghosts.

In order to determine the microviscosity of human erythrocyte membrane suspensions, a method has been developed which is based on pyrene excimer formation. First, measurements of partitioning of pyrene into membranes, in conjunction with known values for the volume of the lipid compartment of erythrocyte ghosts are used to determine the concentration of pyrene in the membrane lipid. Secondly, reported measurements of the diffusion constants of aromatic hydrocarbons similar in structure to pyrene, are used to derive an empirical equation relating solvent viscosity and the diffusion constant of pyrene. Then, measurements of pyrene excimer formation in a series of solvents ranging up to several poise in viscosity are used to determine that the interaction diameter of the excimer formation reaction is 3 +/- 1 A. Finally all these data are brought together in order to conclude that the viscosity of the lipid in the human erythrocyte ghost is 8.0, 4.0 and 1.6 P at 10, 25 and 40 degrees C, respectively.

Erythrocyte Membrane↗
PubMed1979

Catecholamine regulation of human erythrocyte membrane protein kinase.

The effect of catecholamines on membrane-associated protein kinase in the mature human erythrocyte was investigated. Protein kinase activity was assayed after isolation of membranes from intact erythrocytes incubated with and without catecholamines. Activation of the enzyme is expressed as the ratio of the extent of phosphorylation of exogenous protein substrate in the absence to that in the presence of 2.5 microM cyclic AMP (cAMP). The potent beta-adrenergic agonist, (-)isoproterenol (2 microM), (-)epinephrine (10 microM) and (-)norepinephrine (10 microM) stimulated the cAMP-dependent protein kinase in membranes, 38 +/- 7%, 31 +/- 6%, and 30 +/- 6%, respectively. Maximal stimulation of membrane protein kinase by 10 microM (-)epinephrine was obtained approximately equal to 30 min after initiation of the incubation of erythrocytes with the hormone. The concentrations of (-)catecholamines that gave half-maximal stimulation of the membrane protein kinase were 0.17 microM for isoproterenol, 0.35 microM for epinephrine, and 0.63 microM for norepinephrine. The membrane protein kinase response to beta-adrenergic agonists was found to be stereospecific. The stimulation of membrane protein kinase by 10 microM (-)epinephrine was inhibited by the beta-adrenergic antagonist, (-)propranolol with EC50 = 0.60 microM, and the inhibition of agonist stimulation of the cAMP-dependent protein kinase by propranolol was stereospecific. These studies suggest that a functional beta-adrenergic receptor exists in the mature human erythrocyte.

Catecholamines↗
PubMed1979

1,N6-Etheno-2-aza-adenosine 3', 5'-cyclic phosphate: human erythrocyte membrane binding and activation of membrane protein kinase.

The relative efficiency of 1,N6-etheno-2aza-adenosine 3', 5'-monophosphate (cyclic 2-aza-epsilon AMP), 1,N6-etenoadenosine 3', 5'-monophosphate (cyclic epsilon AMP) and cyclic AMP in activation of membrane protein kinase and binding to membrane was examined using isolated membranes from human erythrocytes. Cyclic 2-aza-epsilon AMP was 81% as active as cyclic AMP in erythrocyte membrane binding and activation of membrane protein kinase. On the other hand, cyclic epsilon AMP was 37% as active toward membrane protein kinase and 29% toward membrane cyclic AMP binding. Since we have previously shown that the fluorescence of cyclic 2-aza-epsilon AMP is highly sensitive to the polarity of solvents, the high efficiency of cyclic 2-aza-epsilon AMP to substitute for cyclic amp suggests that it may be a suitable microenvironmental fluorescent probe for cyclic AMP binding sites.

Aza Compounds↗
PubMed1978

The effects of bioregulators upon amino acid transport and protein synthesis in isolated rat hepatocytes.

Isolated rat hepatocytes prepared by an enzyme perfusion technique possess a functional amino acid transport system and retain the capacity to synthesize protein. Amino acid transport was studied using the non-metabolizable amino acid analog alpha-aminoisobutyric acid. The transport process was time, temperature and concentration dependent. Similarly, leucine incorporation into protein was time and temperature dependent being optimal at 3m degrees C. Amino acid, fetal calf serum, growth hormone and glucose all produced small, reproducible increases in protein synthesis rates. Bovine serum albumin diminished the uptake of alpha-aminoisobutyric acid and leucine incorporation into protein. The amino acid content on either side of the cell membrane was found to affect transport into or out of the cellular compartment (transconcentration effects). High cell concentrations decreased transport and protein synthesis as a result of isotopic dilution of labelled amino acids with those released by the hepatocytes. This was consistent with the capacity of naturally occurring amino aicds to compete with alpha-aminoisobutyric acid for uptake into the hepatocyte. In order to define more precisely the effects of bioregulators on transport and protein synthesis it will be necessary to define and subfractionate cellular compartments and proteins which are the specific targets of cellular regulation.

Amino Acids↗
PubMed1978

Recombination of the biologically active peptides from a tryptic digest of bovine growth hormone.

Two bovine growth hormone peptides containing residues 96 to 133 in one and a larger peptide corresponding to residues 1 to 95 and 151 to 191 linked by a disulfide bond have been recombined. Recombination was performed in 1.0 N acetic acid without and with 8 M urea and the recombined peptides were purified by gel filtration. Evidence of recombination of the peptides has been established by gel filtration, polyacrylamide gel disc electrophoresis, amino acid composition, ultraviolet absorption, circular dichroism, and intrinsic fluorescence. The molar growth promoting activity of the recombined peptides measured by tibial width assay in hypophysectomized rats was 10% that of the native hormone and greater than the sum (1%) of the growth promoting activities of the individual peptides. These studies suggest that some contribution of the larger peptide is necessary for more complete expression of the growth promoting activity of the bovine growth peptide corresponding to residues 96 to 133.

Amino Acids↗
PubMed1978

Accumulation of a slowly dissociable peptide hormone binding component by isolated target cells.

The overall rate of dissociation and the fraction of bound radioiodinated human growth hormone that dissociated from hepatocytes varied with time of association. A smaller fraction of bound hormone was dissociable from isolated target cells with increased receptor occupancy and increased incubation time prior to the onset of dissociation. The inability of bound label to reequilibrate completely with the medium was demonstrated further by preincubating cells with labeled hormone prior to the initiation of saturation experiments. In such experiments, time-dependent changes in the binding properties of bound label were observed in Scatchard plots, as a result of the inability of prebound label to reequilibrate rapidly with the medium over the time course of such experiments. These data suggest that bound hormone may be distributed between at least two kinetic components. This phenomenon could be interpreted in terms of heterogeneity of sites, a slow conformational change in the receptor, or a model incorporating spatial compartmentalization of sites.

Cells, Cultured↗
PubMed1977

Polyalanylation of bovine somatotropin peptide 96-133.

Polyalanylation of bovine somatotropin peptide 96-133 has been investigated. Polyalanylated peptides contained an average of 3-50 additional alanines per mole of peptide. As indicated by circular dichroism, the parent peptide and the polyalanylated peptides were helical at pH 4.0. The biological activity per mole of peptide with 50 additional alanies was approximately the same as that of the parent peptide.

Alanine↗
PubMed1977

Conformation studies of biologically active fragments of bovine growth hormone.

Conformations of bovine growth hormone active fragments were studied using far ultraviolet circular dichroism and intrinsic fluorescence emission spectroscopy. The small fragment, A-II (segment 96-133 of bovine growth hormone), undergoes a helix to random coil structural transition between pH 5 and 10 (pKa = 7.15). At pH9, the random coil state of A-II reverts back to helix conformation as ionic strength increases from 0.01 to 1. The A-II fluorophore, Tyr-110, is quenched by a neighboring carboxyl group of Glu-111, but is only slightly affected by the secondary structural transition. The large fragment, A-I (segments 1-95 and 134-191, connected via a disulfide linkage, of bovine growth hormone), is a rigidly structured molecule with a large amount of beta-sheet structure. Trp-86 of A-I was found to reside in an aromatic and hydrophobic amino acid cluster which is only destroyed by a high concentration of denaturant. Based on the primary sequence of bovine growth hormone, conformation predictions were made using the Chou-Fasman method ((1974) Biochemistry 13, 222). Bovine growth hormone helical structures are predicted to be in segments 10-34, 66-87, 111-127, and 186-191, beta-Sheet structures are predicted to be in segments 45-54, 90-94, 101-105, 136-142, 161-165, and 174-179. Tetrapeptides 37-40, 41-44, 60-63, 129-132, 146-149, and 156-159 were predicted to be beta turns. The prediction scheme confirmed several spectroscopic observations, but it did not completely explain the behavior of bovine growth hormone peptide fragments.

Amino Acid Sequence↗
PubMed1975

Studies on the common active site of growth hormone. Revision of the amino acid sequence of an active fragment of bovine growth hormone.

A fragment, A-II, isolated from a component of a tryptic digest of bovine growth hormone has growth-promoting activity in rats and metabolic activity in humans similar to human growth hormone. The amino acid sequence of this peptide has been reinvestigated and revised. The 38-amino acid peptide was cleaved with cyanogen bromide, chymotrypsin, and trypsin. The amino acid sequences were then established by Edman degradation as well as with overlapping peptides; Homology in the sequence was good between this bovine growth hormone fragment and peptides occurring in ovine growth hormone, human growth hormone, and human chorionic somatomammotropin.

Amino Acid Sequence↗
PubMed1975

In vitro DNA synthesis on smooth membranes observed by fluorescence.

Smooth membranes have been isolated from a human diploid line of lymphocytes. These membranes exhibit an endogenous DNA-synthesizing capability which is partially destroyed by prior treatment with RNase. In order to ascertain the role of the membranes in the DNA synthesis we have examined the conformation of the membrane proteins by observing fluorescence changes of the intrinsic probe, tryptophan. We have observed that on addition of the deoxynucleoside-5'-triphosphates, which permits DNA synthesis, there are fluorescence changes due to the tryptophan residue; when DNA synthesis is prevented by omitting some of the precursor triphosphates, fluorescence changes are absent. These effects have been observed with plasma and nuclear membrane fractions; the former may contain a small fraction of the latter. Similar membrane preparations from non-lymphoid cells do not process the endogenous DNA-synthesizing system, as shown by the lack of incorporation of radioactive precursors of fluorescence changes.

Cell Line↗
PubMed1975

Beneficial effect of human growth hormone on stress ulcers.

Human growth hormone was effective in healing erosions and controlling hemorrhage in six of eight patients with stress ulcers. This approach was based on known beneficial effects of growth hormone on nucleic acid and protein synthesis, demonstration of deleterious effects of stress on nucleic acid and protein synthesis, and demonstrated protective effects of growth hormone on animals subjected to stress. Bleeding cessation within 24 hours of its administration in two patients suggests the possible role of additional mechanisms involved in hemostasis. In a comparable group of eight patients with stress ulcer hemorrhage not treated with this agent, six died with continued bleeding. This high mortality represented the usual outcome in our patients with stress ulcer hemorrhage. These observations need to be extended to additional patients with stress ulcers in a larger randomized study.

Blood Chemical Analysis↗