Biochemical mechanism of nitrofurantoin resistance in Vibrio el tor.

Vibrio el tor cells contain a constitutive reductase enzyme which converts nitrofurantoin to an active principle that is responsible for the observed antibacterial activity of the drug. Acquisition of resistance of this strain towards nitrofurantoin is associated with the loss of this reductase. This enzyme is located in the periplasmic region of the nitrofurantoin-sensitive cells, and seems to play an important role in transporting the drug into the cells.

Epidermal growth factor and epidermal growth factor receptor-dependent phosphorylation of a Mr = 34,000 protein substrate for pp60src.

A Mr = 34,000 protein present in the 100,000 X g supernatant fraction from A431 human epidermoid carcinoma cells is the major radiolabeled phosphate acceptor from [gamma-32P]ATP in a cell-free system requiring epidermal growth factor (EGF) and EGF receptor kinase. This protein is immunoprecipitated by IgG directed against avian Mr = 34,000 cellular substrate for pp60src. Phosphoamino acid analysis of the Mr = 34,000 protein labeled with 32Pi from [gamma-32P]ATP in a cell-free system requiring EGF and EGF receptor kinase yielded radiolabeled phosphotyrosine with no detectable radioactivity in phosphoserine or phosphothreonine.

cAMP-dependent protein kinase stimulates epidermal growth factor-dependent phosphorylation of epidermal growth factor receptors.

Epidermal growth factor (EGF)-dependent transfer of radiolabeled phosphate from [gamma-32P]ATP to 160-kDa EGF receptor solubilized from human epidermoid carcinoma A431 cell surface membranes was stimulated up to 3-fold by addition of 3',5'-cAMP and purified cAMP-dependent protein kinase. Phosphorylation of EGF receptors was stimulated to the same extent when cAMP-dependent protein kinase catalytic subunit was substituted for 3',5'-cAMP and cAMP-dependent protein kinase. Phosphoamino acid analysis revealed that the extent of phosphorylation of EGF receptor at tyrosine residues was the same regardless of whether cAMP-dependent protein kinase catalytic subunit was present in or omitted from the system. Increased EGF receptor phosphorylation occurring in response to cAMP-dependent protein kinase catalytic subunit was accounted for by phosphorylation at serine or threonine residues. In samples phosphorylated in the presence of cAMP-dependent protein kinase catalytic subunit, phosphate was present in tyrosine, serine, and threonine in a ratio of 32:60:8. Two-dimensional mapping of radiolabeled phosphopeptides produced from EGF receptors by digestion with trypsin revealed the generation of one additional major phosphoserine-containing peptide when cAMP-dependent protein kinase was present with EGF in the EGF receptor kinase system. Degradation of 160-kDa EGF receptors to a 145-kDa form by purified Ca2+-activated neutral protease produced a 145-kDa fragment with phosphoserine content increased over that present initially in the 160-kDa precursor.

Protein synthesis in rabbit reticulocytes. Demonstration of the requirements for eIF-2 and Co-eIF-2A for peptide chain initiation using immune sera.

Antisera to eIF-2 and Co-eIF-2A have been prepared by immunizing two chickens separately with homogeneous preparations of either eIF-2 or Co-eIF-2A. Addition of anti-eIF-2 or anti-Co-eIF-2A to reticulocyte lysates strongly inhibits protein synthesis and, in each case, protein synthesis inhibition is reversed by the addition of the corresponding homogeneous factor. Protein synthesis inhibition by anti-Co-eIF-2A is not reversed by eIF-2 at any concentration tested indicating an absolute requirement for Co-eIF-2A in protein synthesis. Also, in partial peptide chain initiation reactions, the addition of anti-Co-eIF-2A inhibits Co-eIF-2A stimulation of ternary complex formation by eIF-2 and Co-eIF-2A protection of ternary complexes in the presence of aurintricarboxylic acid.

Protein synthesis in rabbit reticulocytes. Preparation of homogeneous Met-tRNAf deacylase and studies of its role in protein synthesis.

Met-tRNAf deacylase from reticulocyte ribosomes has been purified to homogeneity. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the homogeneous preparation gives a single protein band corresponding to a molecular weight of approximately 67,000. Purified Met-tRNAf deacylase degrades free Met-tRNAf and also Met-tRNAf bound to 40 S ribosomes in the presence of AUG codon but does not degrade Met-tRNAf in the ternary complex, Met-tRNAf.eIF-2.GTP. Purified Met-tRNAf deacylase does not inhibit protein synthesis in reticulocyte lysates at any concentration tested indicating that Met-tRNAf deacylase is not a protein synthesis inhibitor. Antibodies against Met-tRNAf deacylase have been prepared by immunizing a chicken with homogeneous preparation of Met-tRNAf deacylase. Addition of anti-Met-tRNAf deacylase does not have any effect on protein synthesis in reticulocyte lysates indicating that Met-tRNAf deacylase is not required for protein synthesis.

Protein synthesis in rabbit reticulocytes. Co-eIF-2A reverses mRNA inhibition of ternary complex (Met-tRNAf.eIF-2.GTP) formation by eIF-2.

mRNAs, at low concentrations, drastically inhibit ternary complex formation by eIF-2 (Met-tRNAf.eIF-2.GTP) and, when added to the preformed ternary complex, cause extensive dissociation of the complex. Co-eIF-2A stimulates (2- to 4-fold) Met-tRNAf binding to eIF-2 and, in the presence of excess Co-eIF-2A, the stimulated Met-tRNAf binding to eIF-2 is fully resistant to mRNAs. Other cofactors tested such as Co-eIF-2B and Co-eIF-2C do not reverse mRNA inhibition of ternary complex formation.

Protein synthesis in rabbit reticulocytes. A study of the mechanism of interreaction of fluorescently labeled co-eIF-2A with eIF-2 using fluorescence polarization.

5-Dimethylaminonaphthalene-1-sulfonyl (dansyl)-Co-eIF-2A was prepared using homogeneous Co-eIF-2A. Dansyl-Co-eIF-2A was as active as untreated Co-eIF-2A when assayed for stimulation of ternary complex formation and also for protection of the ternary complex from dissociation by aurintricarboxylic acid. The mechanism of interaction of dansyl-Co-eIF-2A with eIF-2 was studied by measuring changes in fluorescence polarization. These studies indicate that dansyl-Co-eIF-2A interacts specifically with the ternary complex and does not interact with free eIF-2 or with two other high molecular weight protein complexes, Co-eIF-2B and Co-eIF-2C. Mg2+ inhibits ternary complex formation by eIF-2 and Co-eIF-2C relieves this Mg2+ inhibition of ternary complex formation. In both cases, the changes in fluorescence polarization of dansyl-Co-eIF-2A correlate well with the extent of ternary complex formed.

Protein synthesis in rabbit reticulocytes. Purification and characterization of a double-stranded RNA-dependent protein synthesis inhibitor from reticulocyte lysates.

Reticulocyte lysates contain a latent form of eukaryotic peptide chain initiation factor 2 (eIF-2) kinase (dsI) which becomes activated in the presence of double-stranded RNA and ATP and inhibits protein synthesis. The latent form of dsI has been partially purified from reticulocyte ribosomal salt wash. The purified dsI has been activated by incubation in the presence of poly(rI).poly(rC) and [gamma 32P]ATP and the activated dsI has been further purified to near homogeneity. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, purified [32P]dsI shows an intensely staining 67,000-dalton polypeptide band which corresponds to a single 67,000-dalton radioactive band. During Sephadex (G-200) gel filtration, both the latent form of dsI and the activated dsI elute similarly with a peak corresponding to a molecular weight of 67,000. Purified dsI phosphorylates the 38,000-dalton subunit of eIF-2 and, under conditions of eIF-2 phosphorylation, dsI strongly inhibits AUG-dependent Met-tRNAf binding to 40 S ribosomes. Also, in partial reactions, eIF-2 alpha (P) formed by phosphorylation of eIF-2 using dsI and ATP, is not recognized by two eIF-2 ancillary factors, Co-eIF-2B and Co-eIF-2C. These results are similar to those reported previously for the heme-regulated eIF-2 kinase (Das, A., Ralston, R. O., Grace, M., Roy, R., Ghosh-Dastidar, P., Das H. K., Yaghmai, B., Palmieri, S., and Gupta, N. K. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,5076-5079) and suggest that dsI, like the heme-regulated eIF-2 kinase phosphorylates eIF-2 and eIF-2 alpha (P) thus formed, in both cases, is not recognized by Co-eIF-2B and Co-eIF-2C, and is inactive in some step(s) of Met-tRNAf.40 S initiation complex formation.

Progesterone receptor subunits are high-affinity substrates for phosphorylation by epidermal growth factor receptor.

Purified preparations of epidermal growth factor (EGF) receptor were used to test hen oviduct progesterone receptor subunits as substrates for phosphorylation catalyzed by EGF receptor. Both the 80-kilodalton (kDa) (A) and the 105-kDa (B) progesterone receptor subunits were phosphorylated in a reaction that required EGF and EGF receptor. No phosphorylation of progesterone receptor subunits was observed in the absence of EGF receptor, even when Ca2+ was substituted for Mg2+ and Mn2+. Phospho amino acid analysis revealed phosphorylation at tyrosine residues, with no phosphorylation detectable at serine or threonine residues. Two-dimensional maps of phosphopeptides generated from phosphorylated 80- or 105-kDa subunits by tryptic digestion revealed similar patterns, with resolution of two major, several minor, and a number of very minor phosphopeptides. The Km of progesterone receptor for phosphorylation by EGF-activated EGF receptor was 100 nM and the Vmax was 2.5 nmol/min per mg of EGF receptor protein at 0 degrees C. The stoichiometry of phosphorylation/hormone binding for progesterone receptor subunits was 0.31 at ice-bath temperature and approximately 1.0 at 22 degrees C.

Protein synthesis in rabbit reticulocytes: mechanism of protein synthesis inhibition by heme-regulated inhibitor.

Partially purified Met-tRNAf binding factor, eIF-2, was phosphorylated by using heme-regulated inhibitor (HRI). Phosphorylated eIF-2 was freed from HRI by phosphocellulose column chromatography. Analysis by isoelectric focusing showed 100% phosphorylation of the 38,000-dalton subunit of eIF-2. Both eIF-2 and eIF-2(P) formed ternary complexes with Met-tRNAf and GTP with almost the same efficiency, and in both cases the ternary complex formation was drastically inhibited by prior addition of Mg2+. However, whereas the ternary complexes formed with eIF-2 could be stimulated by Co-eIF-2C at 1 mM Mg2+ and dissociated by Co-eIF-2B at 5 mM Mg2+, the ternary complexes formed with eIF-2(P) were unresponsive to both Co-eIF-2B and Co-e-IF-2C. Also, under conditions of eIF-2 phosphorylation, HRI drastically inhibited AUG-dependent Met-tRNAf binding to 40S ribosomes. However, HRI (in the presence of ATP) had no effect on the joining of preformed Met-tRNAf . 40S . AUG complex to the 60S ribosomal subunit to form Met-tRNAf-80S . AUG complex. These studies suggest that HRI inhibits protein synthesis initiation by phosphorylation of the 38,000-dalton subunit of eIF-2. HRI-phosphorylated eIF-2 does not interact with at least two other protein factors, Co-eIF-2B and Co-eIF-2C, and is thus inactive in protein synthesis initiation.

Expression of synthetic thaumatin genes in yeast.

Thaumatin is a plant protein that contains 8 disulfides and 207 amino acids in the mature form. The protein is of potential commercial interest since microgram quantities elicit an intense sweetness sensation. Two major variants of thaumatin have been identified in our laboratory by using sequence data obtained from thaumatin tryptic peptides. These differ by one amino acid at position 46 (asparagine or lysine), and both proteins differ from previously published sequences. We have synthesized DNA-coding sequences for three of these thaumatin variants using yeast preferred codons. The genes were inserted into an expression vector that contained a yeast 3-phosphoglycerate kinase promoter and terminator, and the vectors were transformed into yeast for expression of the recombinant protein. Upon lysis of the yeast cells, all thaumatin was localized in the insoluble cell fraction. Analysis of the sodium dodecyl sulfate solubilized yeast extracts by gel electrophoresis and Western blotting showed that thaumatin represented about 20% of the insoluble yeast protein. Although expressed at high levels, none of the thaumatins was biologically active (sweet). Preliminary protein folding experiments showed that two of three thaumatin variants could be folded to the sweet conformation.

Specificity of cotranslational amino-terminal processing of proteins in yeast.

Polypeptides synthesized in the cytoplasm of eukaryotes are generally initiated with methionine, but N-terminal methionine is absent from most mature proteins. Many proteins are also N alpha-acetylated. The removal of N-terminal methionine and N alpha-acetylation are catalyzed by two enzymes during translation. The substrate preferences of the methionine aminopeptidase (EC 3.4.11.x) and N alpha-acetyltransferase (EC 2.3.1.x) have been partially inferred from the distribution of amino-terminal residues and/or mutations found for appropriate mature proteins, but with some contradictions. In this study, a synthetic gene corresponding to the mature amino acid sequence of the plant protein thaumatin, expressed in yeast as a nonexported protein, i.e., lacking a signal peptide, has been used to delineate the specificities of these enzymes with respect to the penultimate amino acid. Site-directed mutagenesis, employing synthetic oligonucleotides, was utilized to construct genes encoding each of the 20 amino acids following the initiation methionine codon, and each protein derivative was isolated and characterized with respect to its amino-terminal structure. All four possible N-terminal variants--those with and without methionine and those with and without N alpha-acetylation--were obtained. These results define the specificity of these enzymes in situ and suggest that the nature of the penultimate amino-terminal residue is the major determinant of their selectivity.