Effect of starvation and diabetes on the activity of the eukaryotic initiation factor eIF-2 in rat skeletal muscle

The ability of the initiation factor eIF-2 in skeletal muscle extracts to form ternary initiation complexes ([Met-tRNA(f).eIF-2.GDP]) is decreased by either starvation or diabetes. These conditions also impair the ability of muscle extracts to dissociate [eIF-2.GDP], suggesting inhibition of the guanine nucleotide exchange reaction essential for eIF-2 recycling. We could not, however, detect any change in the phosphorylation state of the alpha subunit of eIF-2. This suggests that eIF-2 activity may be regulated in this system by a mechanism not involving its phosphorylation.     

Amyloid-beta peptide, substance P, and bombesin bind to the serpin-enzyme complex receptor.

During the formation of an inhibitory complex with neutrophil elastase, alpha 1 antitrypsin (alpha 1 AT) undergoes a structural rearrangement and the resulting alpha 1 AT-elastase complex becomes endowed with chemoattractant activities, mediates an increase in synthesis of alpha 1 AT, and is rapidly cleared from the circulation. In previous studies we have provided evidence that these biological activities involve the recognition of a conformation-specific domain in the alpha 1 AT molecule by a cell surface receptor on human hepatoma HepG2 cells and human monocytes. The receptor has been termed the serpin-enzyme complex (SEC) receptor because it also recognizes complex of serpins antithrombin III, alpha 1 anti-chymotrypsin, and C1 inhibitor with their cognate enzymes. Because a pentapeptide domain of alpha 1 AT (amino acids 370-374, Phe-Val-Phe-Leu-Met) is sufficient for binding to the SEC receptor and the sequence of this domain is remarkably similar to those of substance P, several other tachykinins, bombesin, and the amyloid-beta peptide, we have examined the possibility that these other ligands bind to the SEC receptor. The results indicate that substance P, several other tachykinins, and bombesin compete for binding to, and cross-linking of, the SEC receptor. The SEC receptor is distinct from the substance P receptor by several criteria. There is no substance P receptor mRNA in HepG2 cells; the SEC receptor is present in much higher density on receptor-bearing cells and binds its ligands at lower affinity than the substance P receptor; the SEC receptor is much less restricted in the specificity with which it recognizes ligand; ligands for the SEC receptor including peptide 105Y (based on alpha 1 AT sequence 359-374), alpha 1 AT-protease complexes, and bombesin do not compete for binding of substance P to a stable transfected cell line expressing the substance P receptor. Finally, we show here that the amyloid-beta peptide competes for binding to the SEC receptor but does not bind to the substance P receptor, therein raising the possibility that the SEC receptor is involved in certain biological activities, including the recently described neurotrophic and neurotoxic effects ascribed to the amyloid-beta peptide.     

The 39-kilodalton subunit of eukaryotic translation initiation factor 3 is essential for the complex's integrity and for cell viability in Saccharomyces cerevisiae.

Eukaryotic translation initiation factor 3 (eIF3) in the yeast Saccharomyces cerevisiae comprises about eight polypeptides and plays a central role in the binding of methionyl-tRNAi and mRNA to the 40S ribosomal subunit. The fourth largest subunit, eIF3-p39, was gel purified, and a 12-amino-acid tryptic peptide was sequenced, enabling the cloning of the TIF34 gene. TIF34 encodes a 38,753-Da protein that corresponds to eIF3-p39 in size and antigenicity. Disruption of TIF34 is lethal, and depletion of eIF3-p39 by glucose repression of TIF34 expressed from a GAL promoter results in cessation of cell growth. As eIF3-p39 levels fall, polysomes become smaller, indicating a role for eIF3-p39 in the initiation phase of protein synthesis. Unexpectedly, depletion results in degradation of all of the subunit proteins of eIF3 at a rate much faster than the normal turnover rates of these proteins. eIF3-p39 has 46% sequence identity with the p36 subunit of human eIF3. Both proteins are members of the WD-repeat family of proteins, possessing five to seven repeat elements. Taken together, the results indicate that eIF3-p39 plays an important, although not necessarily direct, role in the initiation phase of protein synthesis and suggest that it may be required for the assembly and maintenance of the eIF3 complex in eukaryotic cells.     

Sedimentation Rate as a Measure of Molecular Weight of DNA

Zone centrifugation of mixtures of two labeled DNA's at low concentrations in density gradients of sucrose permits accurate measurement of relative sedimentation rates. The individual rates are constant during the run. Measurements with DNA's from phages T2, T5, and lambda conform to the relation D₂/D₁ = (M₂/M₁)^0.35, where D and M refer to distances sedimented and molecular weights of the DNA pair. The results show that high molecular weight DNA's sediment artificially fast in the optical centrifuge, owing to a hitherto unknown effect of molecular interactions. The molecular weight of lambda DNA is 31 million, measured either from sedimentation rate or from tests of fragility under shear.     

Cloning and functional expression of the small subunit of acetolactate synthase from Nicotiana plumbaginifolia

Acetolactate synthase (ALS) is the first committed step of branched-chain amino acid biosynthesis in plants and bacteria. The bacterial holoenzyme has been well characterized and is a tetramer of two identical large subunits (LSUs) of 60 kDa and two identical small subunits (SSUs) ranging in molecular mass from 9 to 17 kDa depending on the isozyme. The enzyme from plants is much less well characterized. Attempts to purify the protein have yielded an enzyme which appears to be an oligomer of LSUs, with the potential existence of a SSU for the plant enzyme remaining a matter of considerable speculation. We report here the discovery of a cDNA clone that encodes a SSU of plant ALS based upon the homology of the encoded peptide with various bacterial ALS SSUs. The plant ALS SSU is more than twice as large as any of its prokaryotic homologues and contains two domains that each encode a full-length copy of the prokaryotic SSU polypeptide. The cDNA clone was used to express Nicotiana plumbaginifolia SSU in Escherichia coli. Mixing a partially purified preparation of this SSU with the LSU of ALS from either N. plumbaginifolia or Arabidopsis thaliana results in both increased specific activity and increased stability of the enzymic activity. These results are consistent with those observed for the bacterial enzyme in similar experiments and represent the first functional demonstration of the existence of a SSU for plant ALS.     

A 110-kilodalton subunit of translation initiation factor eIF3 and an associated 135-kilodalton protein are encoded by the Saccharomyces cerevisiae TIF32 and TIF31 genes.

Translation initiation factor eIF3 is a multisubunit protein complex required for initiation of protein biosynthesis in eukaryotic cells. The complex promotes ribosome dissociation, the binding of the initiator methionyl-tRNA to the 40 S ribosomal subunit, and mRNA recruitment to the ribosome. In the yeast Saccharomyces cerevisiae eIF3 comprises up to 8 subunits. Using partial peptide sequences generated from proteins in purified eIF3, we cloned the TIF31 and TIF32 genes encoding 135- (p135) and 110-kDa (p110) proteins. Deletion/disruption of TIF31 results in no change in growth rate, whereas deletion of TIF32 is lethal. Depletion of p110 causes a severe reduction in cell growth and protein synthesis rates as well as runoff of ribosomes from polysomes, indicative of inhibition of the initiation phase. In addition, p110 depletion leads to p90 co-depletion, whereas other eIF3 subunit levels are not affected. Immunoprecipitation or nickel affinity chromatography from strains expressing (His)6-tagged p110 or p33 results in the co-purification of the well characterized p39 and p90 subunits of eIF3 as well as p110 and p33. This establishes p110 as an authentic subunit of eIF3. In similar experiments, p135 and other eIF3 subunits sometimes, but not always, co-purify, making assignment of p135 as an eIF3 subunit uncertain. Far Western blotting and two-hybrid analyses detect a direct interaction of p110 with p90, p135 with p33, and p33 with eIF4B. Our results, together with those from other laboratories, complete the cloning and characterization of all of the yeast eIF3 subunits.     

Regulation of initiation factors during translational repression caused by serum depletion. Abundance, synthesis, and turnover rates

During growth in unreplenished medium, the fraction of active, polysomal ribosomes progressively decreases about 3-fold from 80-90% to only 20-40% due to a reduced rate of initiation. To assess whether the abundance of initiation factors could be involved in this repression of translational activity. HeLa cell cytoplasmic lysates were resolved by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and spots corresponding to the initiation factor proteins were quantitated. The relative abundance of most of the initiation factor proteins only decreases by 10-40% and roughly parallels that of the ribosomes. Measurement of the rates of synthesis and turnover of the initiation factor proteins establishes that during periods of active growth, synthesis and degradation occur coordinately with total cell protein. As growth rate decreases, the synthesis of some initiation factor proteins, particularly eukaryotic initiation factor (eIF)-3 subunits, becomes depressed. Serum stimulation of serum-depleted cells recruits most inactive ribosomes and mRNAs into polysomes, but most initiation factor mRNAs are not selectively recruited. The principal exceptions are eIF-3p24 which exhibits 4-5 fold enhanced synthesis and eIF-3p44 and eIF-4A whose syntheses are moderately stimulated.     

Two translational initiation sites in the infB gene are used to express initiation factor IF2 alpha and IF2 beta in Escherichia coli.

The gene infB codes for the two forms of translational initiation factor IF2: IF2 alpha (97 300 daltons) and IF2 beta (79 700 daltons). To determine whether the two forms differ at their N terminus, purified IF2 alpha and IF2 beta were subjected to 11 or more steps of Edman degradation. The N-terminal amino acid sequences are completely different, but match perfectly the DNA sequences at the beginning of the infB open reading frame and an in-phase region 471 bp downstream. A fusion was constructed between the proximal half of the infB gene and the lacZ gene lacking the region coding for the first eight amino acids. The fused gene expresses two products of 170 000 and 150 000 daltons, corresponding to the fused proteins IF2 alpha-beta-galactosidase and IF2 beta-beta-galactosidase, which confirms in vivo that the IF2 forms differ at their N terminus. A deletion of the 5'-non-translated region of the fused gene, including the Shine/Dalgarno ribosomal binding site, results in the expression of IF2 beta-beta-galactosidase but not IF2 alpha-beta-galactosidase. This strongly suggests that IF2 beta results from independent translation rather than from a precise proteolytic cleavage of IF2 alpha. Further evidence for initiation of protein synthesis at the putative IF2 alpha and IF2 beta start sites was sought by using an in vitro dipeptide synthesis assay. A DNA fragment containing the entire infB gene was cloned into three plasmid vectors and the resulting recombinant DNAs were used as templates in assays containing fMet-tRNA and various labelled aminoacyl-tRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)     

The biogeochemistry and zoogeography of lakes and rivers in arctic Alaska

Water samples from 45 lakes and 8 rivers in arctic Alaska were analyzed for major anions, cations, nutrients, chlorophyll, zooplankton, and benthos. The waters were dilute (conductivities of 30 to 843 μS cm⁻¹), and their composition varied from Na-Ca-Cl waters near the Arctic Ocean to Ca-Mg-HCO₃ waters further inland. Sea salt input in precipitation was important in determining the chemistry of coastal lakes, partly because of low groundwater flow and less time for water to react with shallow unfrozen soils. Further inland, variations in water chemistry among sites were related mainly to differences in bedrock, the age of associated glacial drift, and the input of wind blown sediment. Variations in zooplankton species composition among the lakes were related more to latitude, lake morphometery, and biotic interactions than to water chemistry. The presence of fish as predators mostly determined the overall size structure of the zooplankton community. The chironomid taxa identified have been previously reported from the Neararctic, except for Corynocera oliveri which is a new record. The abundance of the widely distributed chironomid Procladius appears to be controlled by sculpin predation.