High level, context dependent misincorporation of lysine for arginine in Saccharomyces cerevisiae a1 homeodomain expressed in Escherichia coli.

The Saccharomyces cerevisiae a1 homeodomain is expressed as a soluble protein in Escherichia coli when cultured in minimal medium. Nuclear magnetic resonance (NMR) spectra of previously prepared a1 homeodomain samples contained a subset of doubled and broadened resonances. Mass spectroscopic and NMR analysis demonstrates that the heterogeneity is largely due to a lysine misincorporation at the arginine (Arg) 115 site. Arg 115 is coded by the 5'-AGA-3' sequence, which is quite rare in E. coli genes. Lower level mistranslation at three other rare arginine codons also occurs. The percentage of lysine for arginine misincorporation in a1 homeodomain production is dependent on media composition. The dnaY gene, which encodes the rare 5'-AGA-3' tRNA(ARG), was co-expressed in E. coli with the a1-encoding plasmid to produce a homogeneous recombinant a1 homeodomain. Co-expression of the dnaY gene completely blocks mistranslation of arginine to lysine during a1 overexpression in minimal media, and homogeneous protein is produced.     

2,5-Hexanedione Alters Elemental Composition and Water Content of Rat Peripheral Nerve Myelinated Axons

Abstract: Effects of 2,5-hexanedione on elemental concentrations and water content of peripheral nerve myelinated axons were determined using electron probe x-ray microanalysis. Axons (small, medium, and large) were analyzed in unfixed cryosections from rat tibial and proximal sciatic nerve samples. Animals were intoxicated with 2,5-hexanedione by two dosing paradigms: intraperitoneal or oral. Regardless of the route of exposure, internodal axoplasm of small and medium axons from both nerve regions exhibited selective, progressive reductions in dry weight K concentrations and water content. When calculated on a wet weight basis, K levels were comparable to or slightly above control values in tibial nerve, whereas in sciatic nerve, small transient decreases in wet weight K were evident. These changes in K and water correlated with the development of axonal atrophy. The wet and dry weight internodal elemental changes reported here do not suggest a metabolic or axolemmal defect, but rather imply a homeostatic response possibly related to the process of axonal atrophy. Giant axonal swellings were primarily associated with oral 2,5-hexane-dione intoxication, and corresponding analyses revealed few changes in element or water content compared with control. The absence of significant alterations in these swellings is consistent with mechanical expansion of the axon probably as a function of accumulating neurofilaments.     

An acyl-thioesterase from yeast mitochondria

A previously unstudied acyl-coenzyme A thioesterase activity has been demonstrated in submitochondrial particles from Saccharomyces cerevisiae. The preferred substrate for the enzyme activity is oleoyl-coenzyme A. Tests with inhibitors of the thioesterase showed that, in addition to common thiol inhibitors, the oxidative phosphorylation inhibitors oligomycin and venturicidin also blocked thioesterase activity. Purification of the enzyme catalyzing this activity revealed that thioesterase copurified with mitochondrial ATPase. When thioesterase was isolated from oxidative phosphorylation mutants selected for resistance to these two inhibitors, thioesterase activity was also resistant. The results suggest that thioester hydrolysis may be catalyzed by components associated with the isolated ATPase complex. Further attempts to link this activity to in vivo function of ATPase were not successful.     

Discrimination between oral streptococci by pyrolysis gas-liquid chromatography.

Washed organisms, including strains of Streptococcus mitior, S. mutans, and S. sanguis, were examined by curie-point pyrolysis gas-liquid chromatography. A linear discriminant function based upon three items from the output data was adequate for segregating the strains according to species. Strains with intermediate properties were also encountered. Sources of variability in cultures were evaluated, chromatographic performance was maintained throughout the investigation, and matching performance from a duplicate pair of chromatographic columns was demonstrated.     

Cloning, expression and tissue distribution of the gene encoding rat fibroblast growth factor receptor subtype 4

The rat homologue of the gene encoding the fibroblast growth factor receptor subtype 4 (FGFR4) was cloned from rat lung mRNA, and the cDNA sequence was found to be 95% similar and 92% identical to the human homologue. Northern blot analysis of adult rat tissues demonstrated that a 3.1-kb mRNA encoding FGFR4 is detectable only in the lung and kidney. The receptor variant described here encodes two potential immunoglobulin-like domains, 21 hydrophobic amino acids encoding a potential transmembrane domain, and a split tyrosine kinase motif. However, the acidic box and hydrophobic signal peptide domains are not present in this cDNA isolate.     

The effect of pH, temperature, and D2O on the activity of porcine synovial collagenase and gelatinase

The pH dependence of Vmax and Vmax/Km for hydrolysis of Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond by porcine synovial collagenase and gelatinase was determined in the pH range 5-10. Both enzymes exhibited bell-shaped dependencies on pH for these two kinetic parameters, indicating that activity is dependent on at least two ionizable groups, one of which must be unprotonated and the other protonated. For collagenase, Vmax/Km data indicate that in the substrate-free enzyme, these groups have apparent pK values of 7.0 and 9.5, while the Vmax profile indicates similar pK values of 6.8 and 10.1 for the enzyme-substrate complex. The corresponding pH profiles of gelatinase were similar to those of collagenase, indicating the importance of groups with apparent pK values of 5.9 and 10.0 for the free enzyme and 5.9 and 11.1 for the enzyme-substrate complex. When these kinetic constants were determined in D2O using the peptide substrate, there was no significant effect on Vmax or Km for collagenase or Km for gelatinase. However, there was a deuterium isotope effect of approximately 1.5 on Vmax for gelatinase. These results indicate that a proton transfer step is not involved in the rate-limiting step for collagenase, but may be limiting with gelatinase. The Arrhenius activation energies for peptide bond hydrolysis of the synthetic peptide as well as the natural substrates were also determined for both enzymes. The activation energy (81 kcal) for hydrolysis of collagen by collagenase was nine times greater than that determined for the synthetic substrate (9.2 kcal). In contrast, the activation energy for hydrolysis of gelatin by gelatinase (26.3 kcal) was only 2.4 times greater than that for the synthetic substrate (11 kcal).     

Regulation of plasminogen activation by components of the extracellular matrix

The kinetics of activation of Glu-plasminogen (Glu-Pg) and Lys77-Pg by two-chain recombinant tissue plasminogen activator (t-PA) were determined in the presence of isolated protein components of the extracellular matrix (ECM) and compared to activation in the presence of fibrinogen and fibrinogen fragments and in the absence of added protein. Several ECM protein components were as effective as fibrinogen fragments at stimulating Pg activation. Stimulation of Glu-Pg activation resulted from both a decrease in Km and an increase in Vmax, whereas stimulation of Lys77-Pg was due primarily to increases in Vmax. The most effective stimulators of activation were basement membrane type IV collagen and gelatin which resulted in a 21- and 55-fold increase, respectively, in the kcat/Km of Glu-Pg (relative to a 10-fold increase observed with fibrinogen fragments). Amidolytic activity of t-PA was also enhanced up to 12-fold by ECM proteins. However, plasmin amidolytic activity was unaffected by the presence of added proteins. These data suggest that several ECM-associated proteins can enhanced the activation of Pg in the absence of fibrin.     

An investigation of the basis of a current hypothesis for the lack of G-banding in plant chromosomes

A current hypothesis for the lack of G-banding in plant chromosomes is based on the assumption that plant chromosomes are more compact than animal chromosomes. We tested the basis of the hypothesis by measuring lengths and volumes of plant and animal chromosomes that had been similarly treated. We conclude there is no consistent difference in the compaction of plant vs animal chromosomes.