A functional analysis of the repeated methionine initiator tRNA genes (IMT) in yeast

Summary Standard laboratory yeast strains have from four to five genes encoding the methionine initiator tRNA (IMT). Strain S288C has four IMT genes with identical coding sequences that are colinear with the RNA sequence of tRNA _I ^Met . Each of the four IMT genes from strain S288C is located on a different chromosome. A fifth IMT gene with the same coding sequence is present in strain A364A but not in S288C. By making combinations of null alleles in strain S288C, we show that each of the four IMT genes is functional and that tRNA _I ^Met is not limiting in yeast strains with three or more intact genes. Strains containing a single IMT2, 3 or 4 gene grow only after amplification of the remaining IMT gene. Strains with only the IMT1 gene intact are viable but grow extremely slowly; normal growth is restored by the addition of another IMT gene by transformation, providing a direct test for IMT function.Abbreviations IMT and imt (imt=initiator methionine tRNA), designate the genotype of the wild-type and the mutant alleles respectively, of the initiator methionine transfer RNA gene - met-tRNA _I ^Met methionylated initiator methionine transfer RNA - eIF-2 eukaryotic initiation factor two - GTP guanosine 5-triphosphate The calculation of Td values (the temperature at which half of the duplex is dissociated) for oligonucleotides used as probes in hybridizations was based on the assumption that the increase in Td value was 4° C for each G:C base pair and 2° C for each A:T base pair (Wallace et al. 1981)     

Methionine sulfoxide formation in proteins: NMR study

Summary ¹H-NMR spectra of bovine pancreatic trypsin inhibitor (BPTI) both native and oxidized by chloramine T, are reported. The spectrum of the oxidized form is characterized by the appearance of two singlets for methyl group shifted 0.60 and 0.46 ppm downfield with respect to the native form.     

Fermentation parameters of Peptostreptococcus productus on gaseous substrates (CO,H2/CO2)

Intrinsic growth and substrate uptake parameters were obtained for Peptostreptococcus productus, strain U-1, using carbon monoxide as the limiting substrate. A modified Monod model with substrate inhibition was used for modeling. In addition, a product yield of 0.25 mol acetate/mol CO and a cell yield of 0.034 g cells/g CO were obtained. While CO was found to be the primary substrate, P. productus is able to produce acetate from CO₂ and H₂, although this substrate could not sustain growth. Yeast extract was found to also be a growth substrate. A yield of 0.017 g cell/g yeast extract and a product yield of 0.14 g acetate/g yeast extract were obtained. In the presence of acetate, the maximum specific CO uptake rate was increased by 40% compared to the maximum without acetate present. Cell replication was inhibited at acetate concentrations of 30 g/l. Methionine was found to be an essential nutrient for growth and CO uptake by P. productus. A minimum amount of a complex medium such as yeast extract (0.01%) is, however, required.     

Influence of δ-Opioid Receptors on Production of Labeled Methionine5-Enkephalin in Murine Neuroblastoma Cells

Synthesis of methionine5-enkephalin by intact cells of murine neuroblastoma clone N1E-115 has been demonstrated both immunocytochemically and biochemically. In addition, N1E-115 cells possess homogeneous enkephalin (delta) receptors which inhibit prostaglandin E1-induced intracellular cyclic AMP formation. An assay was developed for measuring de novo synthesis of methionine5-enkephalin by pulsing cells in culture with radioactive methionine and isolating this pentapeptide to radiochemical purity by a procedure that included immunoaffinity chromatography specific for oxidized methionine5-enkephalin. This assay indicated that production of radiolabeled-methionine5-enkephalin was increased upon lengthy exposure of intact N1E-115 cells in the late logarithmic phase of growth to a nonproteolyzable analog of methionine5-enkephalin. This increase in synthesis of intracellular methionine5-enkephalin relative to control cells was prevented by prior incubation of the clone with naloxone, indicating that the response was mediated by the delta receptor.     

Evidence for [D-Ala2,D-Leu5]Enkephalin-Induced Supersensitivity to 5-Hydroxytryptamine in a Neurotumor × Brain Hybrid Cell Line (NCB-20)

A neuroblastoma X Chinese hamster embryonic brain explant hybrid cell line (NCB-20) expressed 5-hydroxytryptamine (5-HT1) receptors, linked to adenylate cyclase, which closely resembled 5-HT1 receptors previously characterized in central nervous tissue. However, the affinity of the receptors for 5-HT was only 150 nM compared to 5 nM in membranes prepared from cerebral cortex. The elevation of cyclic AMP levels in NCB-20 cells produced by 5-HT was found additive to that produced by cholera toxin but synergistic with that produced by either prostaglandin E1 (PGE1) or forskolin, suggesting that these latter two agents elevate cyclic AMP levels by a different mechanism than 5-HT. The elevation of cyclic AMP levels by either 5-HT or PGE1 was reversed by [D-Ala2,D-Leu5]enkephalin (DADLE), morphine, clonidine, and 3,4-dihydroxyphenylethylamine (dopamine) on a short (30 min) time scale. However, continued exposure to DADLE resulted in loss of the initial inhibitory effects of DADLE after 6 h and return of cyclic AMP levels to that seen with either 5-HT or PGE1 alone. When the DADLE exposure time was increased to 48 h, 5-HT produced a further twofold increase in cyclic AMP levels, but there was no increase in the responsiveness of the cells to PGE1 unless naloxone was added 1 h prior to treatment with PGE1. Scatchard analysis showed that the increased potency of 5-HT resulted from an increase in receptor affinity for 5-HT (from a KD of 150 +/- 20 nM to one of 20 +/- 7 nM), with a reduction in the number of apparent binding sites. The 5-HT supersensitivity observed in NCB-20 cells may be a good model for neurotransmitter interactions that produce desensitization or facilitation in the intact nervous system.     

Enkephalin pseudopeptides: resistance to in vitro proteolytic degradation afforded by amide bond replacements extends to remote sites

Five analogs of leucine enkephalin containing the CH2S group as an amide bond replacement were evaluated with respect to resistance toward degradation by human serum in an HPLC-based assay using both ultraviolet and electrochemical detection. Analogs with the modification at the 1-2, 2-3, 3-4, or 4-5 peptide linkages demonstrated half-lives of 118, 85, 134, and 318 min vs. 12 min for the parent peptide. A pseudopeptide analog with additional D-Ala2 protection had a half-life of greater than 1000 min, while the potent [D-Ala2]-leucine enkephalin analog showed approximately a 10-fold increase in stability. The significant increase in stability for a compound with protection only at the C-terminus suggests that serum enzymes may have greater specificity toward backbone changes than previously realized.     

Immunoelectrophoretic approach to the metabolic regulation of glutamine synthetase in Rhodopseudomonas capsulata E1F1: role of glutamine

Anti-glutamine synthetase serum was raised in rabbits by injecting purified glutamine synthetase (GS) of the phototrophic bacterium Rhodopseudomonas capsulata E1F1. The antibodies were purified to monospecificity by immunoaffinity chromatography in GS-sepharose gel. These anti-GS antibodies were used to measure the antigen levels in crude extracts from bacteria, grown phototrophically with dinitrogen, nitrate, nitrite, ammonia, glutamate, glutamine or alanine as nitrogen sources. The amount of GS detected by rocket immunoelectrophoresis was proportional to Mn²⁺-dependent transferase activity measured in the crude extracts. Addition of GS inhibitor l-methionine-d,l-sulfoximine (MSX) to the actively growing cells promoted increased antigen levels, that were not found in the presence of glutamine or chloramphenicol. The ammonia-induced decrease in GS relative levels was reverted by MSX. GS levels remained constant when phototrophically growing cells were kept in the dark.Abbreviations GS glutamine synthetase - MOPS 2-(N-morpholine) propane sulfonate - MSX l-methionine-d,l-sulfoximine     

Alteration of Striatal Glutamate Release After Glutamine Synthetase Inhibition

The effect of the glutamine synthetase (GS) inhibitor, methionine sulfoximine (MSO), on glutamate levels in, and glutamate release from, rat striatal tissue was examined. Tissue levels of glutamate were unchanged 24 h after an intraventricular injection of MSO, but tissue glutamine levels were decreased 50%. Calcium-dependent, potassium-stimulated glutamate release was diminished in tissue prisms from animals pretreated with MSO compared to controls. The decreased release of glutamate correlated over time with the inhibition of GS following an intraventricular injection of MSO. The maximum diminution of calcium-dependent, potassium-stimulated glutamate release (50%) and the maximum inhibition of GS activity (51%) were observed 24 h after MSO. The addition of 0.5 mM glutamine to the perfusion medium completely reversed the effects of MSO pretreatment on calcium-dependent, potassium-stimulated glutamate release. Since GS is localized in glial cells and the measured glutamate release is presumed to occur from neurons, the data support the contention that astroglial glutamine synthesis is an important contributor to normal neuronal neurotransmitter release.     

Distal Retinal Pigment of the Fiddler Crab,Uca pugilator: Release of the Dark-Adapting Hormone by Methionine Enkephalin and FMRFamide

The neuropeptides methionine enkephalin and FMRFamide, when injected into intact fiddler crabs, Uca pugilator, produce dark adaptation of the distal retinal pigment. Furthermore, both neuropeptides stimulate release of distal retinal pigment dark-adapting hormone activity from the isolated eyestalk neuroendocrine complex. It is hypothesized that both neuropeptides, when injected into intact fiddler crabs, act only indirectly on the distal retinal pigment, by stimulating release of this dark-adapting hormone.