Fatty Acyl-CoA Inhibits 1-Alkyl-sn-Glycero-3-Phosphate Acetyltransferase in Microsomes of Immature Rabbit Cerebral Cortex: Control of the First Committed Step in the De Novo Pathway of Platelet-Activating Factor Synthesis

Abstract: Microsomal fractions of cerebral cortices of 15-day-old rabbits were used to study the 1-alkyl- sn -glycero-3-phosphate (AGP) acetyltransferase that generates 1-alkyl-2-acetyl- sn -glycero-3-phosphate in the de novo path of platelet-activating factor synthesis. The AGP acetyltransferase activity was inhibited by small concentrations of medium-long chain fatty acyl-CoA thioesters. In contrast, the AGP acyltransferase used oleoyl-CoA as substrate and was not inhibited by the presence of acetyl-CoA in high molar excess. The inhibition of AGP acetyltransferase was seen at concentrations of oleoyl-CoA as low as 0.5 µ M using 12.5 µ M AGP and 200 µ M acetyl-CoA. The inhibition by oleoyl-CoA was noncompetitive for the acetyl-CoA substrate. However, there was evidence that the oleoyl-CoA was competing with AGP in the acetyltransferase reaction, as the inhibition was lessened by increasing the AGP substrate concentration. Several acyl-CoA thioesters were effective as inhibitors of the AGP acetyltransferase, including oleoyl-, palmitoyl-, lauroyl-, and octanoyl-CoA. Propionyl- and butyryl-CoA were less effective as inhibitors, and propionyl-CoA was found to be a competitive inhibitor for acetyl-CoA. We have noted earlier that MgATP is an effective inhibitor of the AGP acetyltransferase and here we show that the inhibition by oleoyl-CoA can be increased by the presence of 0.1 m M MgATP. In brain ischemia, a decline in ATP levels would likely lead to a corresponding fall in acyl-CoA concentrations, thereby relieving the inhibition of AGP acetyltransferase and permitting the flow of AGP into the de novo pathway of platelet-activating factor synthesis.     

GTPγS restores nucleophosmin (NPM) localization to nucleoli of GTP-depleted HeLa cells

Previous studies showed that localization of nucleophosmin/B23 (NPM) to nucleoli requires adequate cellular GTP levels (Finchet al., J Biol Chem 268, 5823–5827, 1993). In order to study whether hydrolysis of GTP plays a role in NPM localization, we introduced a nonhydrolyzable GTP analog into HeLa cells. Cells were first depleted of GTP with the IMP dehydrogenase inhibitor, mycophenolic acid (MA), to induce translocation of NPM from the nucleoli to the nucleoplasm. Non-hydrolyzable GTP analogs were then introduced into cells by electroporation. We found that introduction of the non-hydrolyzable analog, GTPS, was effective in restoring NPM localization to nucleoli. Cells incubated in medium containing G-nucleotides without electroporation showed no effect. To reduce the possibility that cells use guanine from degraded nucleotide to supplement GTP pools via salvage pathways, experiments were also performed in the presence of (6-mercaptopurine) 6MP, a competitive inhibitor of the salvage enzyme, HGPRT (hypoxanthine guanine phosphoribosyl transferase), in addition to MA. Under these conditions, introduction of GTPS still effectively restored the localization of NPM into nucleoli. This study demonstrates that electroporation can be used effectively to introduce nucleotides into cultured cells without excessive loss of viability. Our results also indicate that the GTP dependent localization of NPM to the nucleoli may not require GTP hydrolysis.     

Functional Role of Amino-Terminal Serine16 and Serine27 of Gαz in Receptor and Effector Coupling

Abstract: The α subunit of G_z (α_z) harbors two N-terminal serine residues (at positions 16 and 27) that serve as protein kinase C-mediated phosphorylation sites. The cognate residues in the α subunit of G_t1 provide binding surfaces for the β₁ subunit. We used three serine-to-alanine mutants of α_z to investigate the functional importance of the two N-terminal serine residues. Wild-type or mutant α_z was transiently coexpressed with different receptors and adenylyl cyclase isozymes in human embryonic kidney 293 cells, and agonist-dependent regulation of cyclic AMP accumulation was examined in a setting where all endogenous α subunits of G_i were inactivated by pertussis toxin. Replacement of one or both serine residues by alanine did not alter the ability of α_z to interact with δ-opioid, dopamine D₂, or adenosine A₁ receptors. Its capacity to inhibit endogenous and type VI adenylyl cyclases was also unaffected. Functional release of βγ subunits from the mutant α_z subunits was not impaired because they transduced βγ-mediated stimulation of type II adenylyl cyclase. Constitutively active mutants of all four α_z subunits were constructed by the introduction of a Q205L mutation. The activated mutants showed differential abilities to inhibit human choriogonadotropin-mediated cyclic AMP accumulation in luteinizing hormone receptor-transfected cells. Loss of both serine residues, but not either one alone, impaired the receptor-independent inhibition of adenylyl cyclase by the GTPase-deficient mutant. Thus, replacement of the amino-terminal serine residues of α_z has no apparent effect on receptor-mediated responses, but these serine residues may be essential for ensuring transition of α_z into the active conformation.     

The use of luciferase as a reporter for response of plant cells to the fireblight pathogen Erwinia amylovora

Summary We have introduced the gene encoding luciferase from Photinus pyralis into pear and tobacco cells in order to judge the reaction of plant tissue to damaging conditions such as incubation at high temperature or inoculation with a pathogen. The constitutive expression of the luciferase gene via a strong promoter slowly decreased during propagation of the transformed pear cell line. After various stress treatments the resulting luciferase activity and the ATP content of the plant cells were determined by bioluminescence and found to correspond to each other. Inoculation of transformed pear cells with Erwinia amylovora resulted in a continuous decrease of luciferase activity in contrast to tobacco cells, where the enzyme activity was significantly higher in the first period after inoculation with bacteria compared to the untreated control cells. The pattern of the luciferase activity reflected the slow damage of the host-plant cells by E. amylovora and the elevated metabolism of the non-host cells after inoculation with the pathogen.Abbreviations 2,4-D 2,4-dichloro-phenoxyacetic acid - CaMV Cauliflower mosaic virus - DTT dithiothreitol - EDTA ethylenediaminetetraacetate - FDA fluorescein diacetate - HEPES (hydroxyethyl)piperazine(ethanesulfonic acid) - HR hypersensitive reaction - Tris tris (hydroxymethyl)amino-methane     

Cloning and mapping of Saccharomyces cerevisiae photoreactivation gene PHR1.

The yeast Saccharomyces cerevisiae, like most organisms, is able to directly repair pyrimidine dimers by using a photoreactivating enzyme and visible light. Cells carrying the phr1 mutation were shown previously to be unable to photoreactivate dimers, but neither the map position nor the primary gene product of the PHR1 gene has been determined. We have cloned this gene and determined its map position. A plasmid containing a 6.4-kilobase yeast DNA insert has been isolated and shown to restore photoreactivation in a phr1 strain. A 3.1-kilobase subclone has also been shown to complement phr1. The original plasmid was targeted to integrate into chromosomal DNA at a site homologous to the insert by cutting within the insert. Two of these integrants have been mapped on the right arm of chromosome XV; the integrants have been further mapped at ca. 13 centimorgans from prt1. It has also been independently determined that phr1 maps at this location. Thus, we have determined the map position of PHR1 and also have shown that the plasmid contains PHR1 rather than a suppressor of the phr1 mutation.     

Mass spectra of underivatized peptide amides related to substance P

Mass spectra of underivatized hexa- and heptapeptide amides related to Substance P have been obtained with a conventional electron ionization mass spectrometer using sample vaporization from a tungsten wire by the technique of rapid heating, proton transfer ionization using ammonia, and photoplate recording of spectra. These spectra exhibit little evidence of sample pyrolysis and are readily interpreted to yield amino acid sequences.     

Studies on the mechanism of enkephalin receptor down regulation

The association of [3H] [D-Ala2, D-Leu5] enkephalin ([3H]DADLE]) with mouse neuroblastoma cells (N4TG1) was investigated. Under identical conditions the time course, dose response curve and temperature dependence for ligand uptake were similar to those for ligand-induced receptor loss (down regulation). Uptake of [3H]DADLE was inhibited by opiate ligands as well as by the metabolic inhibitors sodium azide and 2,4 dinitrophenol. Comparison of the effects of these inhibitors on receptor binding, ligand uptake and receptor loss indicated that these cells accumulate [3H]DADLE in excess of their surface receptor number. The data suggest that receptor recycling occurs and that ligand is internalized via receptor mediated endocytosis.