The role of the epidermal growth factor-1 and hydrophobic stack domains of human factor IX in binding to endothelial cells.

To determine the function and specificity in factor IX of the first epidermal growth factor (EGF)-like domain and the eight-amino acid hydrophobic stack encoded by exon C (residues 39-46), these domains were replaced by the corresponding polypeptide regions of factor X and chimeric proteins were produced in human embryo kidney cells. Both chimeras were activated by factor XIa at a rate similar to plasma factor IX and exhibited calcium-dependent fluorescence quenching similar to plasma factor IX. Both chimeras competed equally for binding to the endothelial cell receptor. Our findings make it unlikely that the first EGF-like domain or the hydrophobic stack of factor IX are responsible for the specific binding of factor IX to its endothelial cell receptor.     

Recognition of complex oligosaccharides by the multi-subunit asialoglycoprotein receptor.

The hepatic asialoglycoprotein receptor, a galactose lectin, is an oligomer of two types of similar polypeptide chains, each of which weakly binds galactose. High-affinity binding of complex oligosaccharides requires a precise geometric arrangement of receptor subunits. The two subunits have different functions in receptor assembly, ligand binding and endocytosis.     

Mu-opioid-receptor-mediated inhibition of the N-type calcium-channel current.

The predominant consequences of mu-opioid-receptor activation are depression of both neuronal activity and transmitter release. Mu-Opioid agonists have previously been observed to increase a potassium conductance and to inhibit adenylate cyclase. We now report that activation of mu-opioid receptors directly decreases the N-type calcium-channel current in a differentiated, human neuroblastoma cell line (SH-SY5Y). The coupling between the mu-opioid receptor and the calcium channel involves a pertussis toxin-sensitive G protein and is independent of changes in adenylate cyclase activity. The inhibition of the calcium-channel current is voltage dependent because it is largely overcome by strong membrane depolarization. It is not associated with changes in the kinetics of current inactivation. Therefore, the mu-receptor belongs to the superfamily of G-protein-coupled, inhibitory neurotransmitter receptors which modulate the activity of calcium and potassium channels and adenylate cyclase.     

Depalmitylation with hydroxylamine alters the functional properties of rhodopsin

Rhodopsin, the photosensitive protein found in rod photoreceptors, has two covalently attached palmitates that are thought to anchor a portion of the C terminus to the disc membrane, forming a fourth cytoplasmic loop. Using hydroxylamine (NH2OH) to cleave the thioester linkage, we have characterized the effect of depalmitylation on certain functional properties of rhodopsin. Treatment of rod outer segment membranes (prepared from rat retinas previously labeled in vivo with [3H]palmitate) with 1 M NH2OH typically removed greater than or equal to 75% of the [3H]palmitate initially bound to rhodopsin. Spectrophotometry of rod outer segment membranes that had been treated with 1 M NH2OH indicated preservation of 85% of the native rhodopsin and no effect on the shape of the absorbance spectrum of rhodopsin. In vivo labeled rhodopsin that had been treated with 1 M NH2OH did not reincorporate free endogenous [3H] palmitate over a 2-h incubation period. Both NH2OH-treated and untreated rhodopsin incorporated [14C]palmitate from exogenously added [14C]palmitoyl-CoA. This incorporation was substantially greater in the NH2OH-treated sample. The removal of palmitate by NH2OH inhibited rhodopsin regeneration by 44% and increased the ability of rhodopsin to activate transducin's light-dependent GTPase activity by 61%. However, the removal of palmitate from rhodopsin did not affect the light-dependent binding of transducin (T alpha and T beta gamma).     

Displacement of hepatic ornithine carbamoyltransferase from mitochondria to cytosol in Reye's syndrome.

In two patients with fatal Reye's Syndrome, total ornithine carbamoyltransferase (OCTase) activity in the liver was 50 and 75% of that found in three control livers. The levels of enzymatic activity would not be expected to have resulted in the 7- and 17-fold elevations in plasma ammonia levels found in the patients. Levels of 47 and 60% of the OCTase activity, however, were found in the cytosolic fraction compared to an average of 7% for control livers. Thus, the amount of enzymatic activity in the mitochondrial fractions was only 20 and 30% of that found in control mitochondrial fractions. This study suggests that, if only mitochondrial OCTase is active in the urea cycle, the decreases in functional enzyme found in Reye's Syndrome may be considerably greater than that reflected in total enzyme assays.     

Genes newly identified as regulated by glucocorticoids in murine thymocytes

Glucocorticoids induce dramatic biochemical and morphological changes in lymphocytes through an unknown process that requires RNA and protein synthesis. In order to identify genes involved in this response, we previously isolated 11 cDNA clones from the murine WEHI-7TG thymoma cell line that correspond to mRNAs induced by glucocorticoids. We now report the isolation of two new cDNA clones whose gene expression is regulated by glucocorticoids in WEHI-7TG cells. We further characterize the two new cDNA clones, as well as those described previously, by examining the response of each of the corresponding mRNAs to glucocorticoids in murine thymocytes. With the exception of two, all cDNAs correspond to genes that are induced by glucocorticoids in murine thymocytes within 4 h of treatment. We previously identified two of the cDNAs as the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein. We have now identified four additional cDNA clones that correspond to the genes for calmodulin, mitochondrial phosphate carrier protein, immunoglobulin (Ig)-related glycoprotein (GP-70), and the 70 kilodalton autoantigen for Lupus and Graves diseases. Two other cDNA clones represent previously undescribed genes: one shares a high similarity to known sequences for the family of G-protein-coupled receptors and the other to a human placental-specific protein, PP11. Another cDNA appears to contain sequences for an unknown gene and the remnants of a mouse transposon. ETn. The remaining clones represent new, unidentified genes induced by glucocorticoids in murine thymocytes and in the WEHI-7TG cell line.     

Translation and ribosome assembly in extremely thermophilic archaebacteria.

Several features of translation and ribosome structure in extremely thermophilic, sulfur-dependent archaebacteria are described, including: i) a peculiar mechanism of transfer RNA-mediated 70S ribosome formation from free subunits; ii) poly(U)translation by hybrid ribosomes composed by one archaebacterial and one eucaryotic subunit; iii) ribosome assembly and homologous and heterologous RNA/protein recognition.