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L L Spremulli

Publications and source records attributed to L L Spremulli.

At least 91 records · Page 5Linked to original sources
PubMed1980

Purification of Euglena gracilis chloroplast elongation factor G and comparison with other prokaryotic and eukaryotic translocases.

Euglena gracilis chloroplast protein synthesis elongation factor G (EF-Gchl) has been purified to about 80% homogeneity by a two-step procedure which removes all traces of the cytoplasmic and mitochondrial translocases (EF-2 and EF-Gmt). The purification scheme generally results in approximately 130-fold purification with 20% recovery of the total EF-G activity present in whole cell extracts. The Euglena EF-Gchl is a monomeric protein with a molecular weight of approximately 85,000. As observed for all translocases to date, the activity of Euglena EF-Gchl is inhibited by treatment with low concentrations of N-ethylmaleimide, suggesting that a free sulfhydryl group is required for catalytic activity. Treatment with 3 microM fusidic acid results in a 50% inhibition of Euglena EF-Gchl activity and of the EF-G activity present in Chlamydomonas reinhardtii. About 10-fold higher concentrations of this antibiotic are required to inhibit the mitochondrial EF-G of Euglena and Escherichia coli EF-G to the same extent. Yeast mitochondrial EF-G is clearly distinguishable from the other organellar translocases tested, requiring 1 mM fusidic acid for 50% inhibition. Fusidic acid also inhibits the cytoplasmic translocases from yeast, wheat germ, and Euglena, although a wide range of sensitivities is observed. When antiserum raised against highly purified Euglena EF-Gchl is used to inhibit enzymatic translocation, a low degree of cross-reaction of the antiserum with Chlamydomonas EF-G and with E. coli EF-G is observed. The EF-G activity present in spinach is very slightly inhibited by the antiserum, whereas that of yeast is not affected. The mitochondrial and cytoplasmic translocases of Euglena are also unaffected by the antiserum against EF-Gchl. The evolutionary implications of these observations are discussed.

Chlamydomonas↗
PubMed1980

Reversible dissociation of wheat germ ribosomal subunits: cation-dependent equilibria and thermodynamic parameters.

The influence of magnesium ion concentration upon the equilibrium between the wheat germ ribosome and its subunits has been studied by light scattering. The curves obtained for ribosome dissociation and subunit reassociation were identical and were independent of the origin and direction of the magnesium ion titration, suggesting that the wheat germ ribosomes are behaving as a homogeneous population equivalent to Escherichia coli type A ribosomes. Increasing K+ concentrations in the presence of Mg2+ favored ribosome dissociation. Polyamines favored subunit reassociation, with 0.1 mM spermine acting as effectively as 1 mM spermidine. Thermodynamic parameters have been determined from the temperature-dependent equilibria and have been compared to those of E. coli type A ribosomes. The association of the 40S and 60S subunits is exothermic. At 1.5 mM Mg2+ and 100 mM K+, the entropy term is negative, favoring ribosome dissociation, and contributes less to the free energy than the enthalpy term. The determination of these thermodynamic parameters was based on molecular weights of 1.2 x 10(6) and 2.3 x 10(6) for the 40S and 60S subunits, respectively, calculated from laser light scattering data.

Kinetics↗
PubMed1979

Purification and characterization of a ribosome dissociation factor (eukaryotic initiation factor 6) from wheat germ.

A wheat germ ribosome dissociation factor, eukaryotic initiation factor 6 (eIF-6), has been purified almost to homogeneity from the 25 to 40% ammonium sulfate fraction of the postribosomal supernatant. This dissociation factor is distinct from initiation factor eIF-3 and its chromatographic properties permit its separation from the known wheat germ initiation factors. Under certain conditions, eIF-6 stimulates the incorporation of amino acids into polypeptides in a partially fractionated wheat germ cell-free system. The eight-step purification procedure developed includes chromatography on DEAE-cellulose, phosphocellulose, Sephadex G-75, and hydroxyapatite and yields a dissociation factor more than 80% pure. The purified factor is composed of a single polypeptide chain with a molecular weight of approximately 23,000 as determined by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is an acidic protein which is heat labile and is inactivated by treatment with N-ethylmaleimide. The dissociation factor is much more effective in preventing the reassociation of 40 S and 60 S ribosomal subunits than in directly dissociating 80 S ribosomes. Like Escherichia coli IF-3, about 10 pmol of the dissociation factor are required to dissociate 1 pmol of ribosomes.

Ethylmaleimide↗
PubMed1978

Identification of a wheat germ ribosome dissociation factor distinct from initiation factor eIF-3.

A ribosome dissociation factor(s) has been partially purified from extracts of wheat germ. This factor can be detected by its ability to prevent the reassociation of ribosomal subunits when the magnesium concentration is raised from 1 mM to 5 mN. The majority of the dissociation factor activity is found in the postribosomal supernatant of the cell and can be precipitated between 0 and 40% ammonium sulfate. Chromatography of this material on DEAE-cellulose yields a fraction which contains both the dissociation factor activity and initiation factor eLF-3. However, the majority of the dissociation factor activity can be readily separated from eIF-3 by chromatography on phosphocellulose. The dissociation factor appears to be distinct from any of the othe known wheat germ initiation factors.

Peptide Initiation Factors↗
PubMed1977

Initiation of protein synthesis without formylation in a mutant of Escherichia coli that grows in the absence of tetrahydrofolate.

Starting from a p-aminobenzoate-requiring strain of Escherichia coli (E. coli K-12 AB3292), we have isolated mutants that can grow in the absence of p-aminobenzoate (and thus tetrahydrofolate). The following lines of evidence suggest that at least one of these mutants is capable of initiating protein synthesis without formylation of methionyl-transfer ribonucleic acid (methionyl-tRNA(fMet)). (i) tRNA isolated (and charged in vivo with [(35)S]methionine) from this mutant grown in a p-aminobenzoate-free medium contained less than 0.4% of the total methionine charged to the tRNA as formylmethionine. However, when the mutant was grown in the presence of p-aminobenzoate, 40 to 50% of the total [(35)S]methionine was detected as formylmethionine. (ii) Extracts of the mutant grown in the absence of p-aminobenzoate contained no formyl-tetrahydrofolate, but such extracts did contain formylatable methionyl-tRNA and a functional transformylase. (iii) Tetrahydrofolate-free extracts of the mutant were capable of supporting protein synthesis with viral RNA (from f2) as messenger, but the resulting synthesized proteins contained no formylmethionine, and methionine residues were detected where formylmethionine residues are normally found. In the presence of formyl-tetrahydrofolate, use of a similar extract resulted in the detection of 30 to 40% of the total polypeptide methionine as formylmethionine. (iv) Initiation of protein synthesis in vitro occurred more readily with formyl-tetrahydrofolate-free extracts of the mutant than with similar extracts prepared from the parent strain. However, in the presence of formyl-tetrahydrofolate, initiation of protein synthesis proceeded equally well with both kinds of extracts. tRNA from this mutant and another spontaneously derived mutant was found to be partially deficient in the modified nucleoside ribothymidine (rT). Analysis of extracts showed that the mutants contained decreased levels of the methylase that results in the formation of ribothymidine. In vivo studies with an independently isolated rT(-) strain suggest that the lack of rT in tRNA facilitates the growth of E. coli under conditions where protein synthesis is forced to take place without formylation.

Bacterial Proteins↗
PubMed1977

Repression of the tyrosine, lysine, and methionine biosynthetic pathways in a hisT mutant of Salmonella typhimurium.

A comparison was made of the repressibility of certain enzymes in the tyrosine, methionine, and lysine biosynthetic pathways in wild-type Salmonella typhimurium and a hisT mutant. The results show that (i) tyrosine represses the synthesis of the tyrosine-sensitive 3-deoxy-D-arabino-heptulsonic acid 7-phosphate synthetase and the tyrosine aminotransferase to the same extent in a hisT mutant as in wild type and (ii) there is no detectable alteration in the extent to which methionine represses O-succinylhomoserine synthetase or in the extent to which lysine represses the lysine-sensitive beta-aspartokinase as a result of the hisT mutation.

2-Isopropylmalate Synthase↗