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P Carter

Publications and source records attributed to P Carter.

At least 127 records · Page 7Linked to original sources
PubMed1986

Construction of heterodimer tyrosyl-tRNA synthetase shows tRNATyr interacts with both subunits.

The tyrosyl-tRNA synthetase (EC 6.1.1.1) from Bacillus stearothermophilus is a dimer of two identical subunits. The dimer shows "half-of-the-sites" reactivity in that only one molecule of tyrosyladenylate is formed and one molecule of tRNATyr binds per dimer. To identify whether the tRNATyr binds to a single subunit in the dimer, or to both subunits, heterodimers were constructed by mixing two variant dimers together in 8 M urea. As the unfolded protein is electrophoresed into a native polyacrylamide gel, it refolds and reassociates, and heterodimers can be purified from the parental dimers. Kinetic analysis of heterodimers formed between variant enzymes with defective tyrosine activation or tRNA aminoacylation shows that a molecule of tRNATyr interacts with the N-terminal region of one subunit and the C-terminal region of the other subunit in the dimer.

Amino Acyl-tRNA Synthetases↗
PubMed1985

Fine structure-activity analysis of mutations at position 51 of tyrosyl-tRNA synthetase.

Residue Thr-51 at the active site of tyrosyl-tRNA synthetase (Bacillus stearothermophilus) has been replaced with all the smaller amino acids by protein engineering to investigate direct and indirect effects of mutation on substrate binding and catalysis. The gamma-hydroxyl group of Thr-51 was thought to be 0.5 A too far from the ribose ring oxygen of ATP to form a hydrogen bond. Consistent with this, it is found that mutation of Thr-51----Cys-51, which should place the gamma-thiol group within its correct distance for hydrogen bonding, increases the affinity of the enzyme for ATP. Other mutations (Ser-51, Ala-51, and Gly-51) show the contributions to binding of the other atoms in the side chain of Thr-51. A family of enzymes has been produced, TyrTS(Thr-51) (wild type), TyrTS(Ala-51), TyrTS(Cys-51), and TyrTS(Pro-51), in which the value of kcat/KM for ATP in aminoacylation increases along the series. This is achieved by the value of KM decreasing significantly (2.5, 1.25, 0.29, and 0.019 mM, respectively) while there are smaller decreases in kcat (4.7, 4.0, 2.9, and 1.8 s-1, respectively). These variations cause each one of the enzymes to be more active than the others at particular concentrations of ATP. For example, at concentrations of ATP greater than 5.9 mM, TyrTS(Thr-51) is the most active, while TyrTS(Ala-51), TyrTS(Cys-51), and TyrTS(Pro-51) are the most active at 5.9-2.2, 2.2-0.42, and less than 0.42 mM ATP, respectively. Interestingly, position 51 shows variation in tyrosyl-tRNA synthetases isolated from different organisms.

Adenosine Triphosphate↗
PubMed1985

Probing histidine-substrate interactions in tyrosyl-tRNA synthetase using asparagine and glutamine replacements.

We have analyzed the interactions of a histidine residue with a substrate using site-directed mutagenesis. Previous studies on tyrosyl-tRNA synthetase from Bacillus stearothermophilus have shown that a histidine residue (His-48) makes an interaction with ATP, which is improved on mutating Thr-51----Pro-51. We find on replacing His-48 in wild-type enzyme with either asparagine or glutamine that Asn-48 is equally as good as His-48 but His-48----Gln-48 leads to a far lower activity. The side chain of an asparagine residue may be superimposed on that of a histidine so that the amide-NH2 group of asparagine occupies the same position as the pi-N of histidine, whereas the equivalent -NH2 group of glutamine may be superimposed upon the tau-N. This suggests that it is the pi-N of histidine that hydrogen bonds with ATP and that there is no significant electrostatic interaction between the histidine and ATP. Incorporating the Pro-51 mutation into each of the Asn-48 and Gln-48 mutants gives an improvement in the affinity of the enzyme for ATP, but this improvement is less than that seen with the wild-type enzyme.

Amino Acid Sequence↗
PubMed1985

Improved oligonucleotide site-directed mutagenesis using M13 vectors.

An improved method is described for the construction of mutations in M13 vectors using synthetic oligonucleotides. The DNA is first cloned into a novel M13 vector (based upon M13mp18 or M13mp19), which carries a genetic marker that can be selected against, such as an EcoK or EcoB site, or an amber mutation in an essential phage gene. In this "coupled priming" technique, one primer is used to construct the silent mutation of interest, and a second primer is used to eliminate the selectable marker on the minus strand. After primer extension and ligation, the heteroduplex DNA is transfected into a strain of E. coli which is repair deficient and selects against the plus strand marker. Over 50 mutants have been constructed with this approach, and the yields can be excellent (up to 70%). For the stepwise construction of mutations using separate rounds of mutagenesis, the EcoK and EcoB markers offer a particular advantage over the amber marker. They permit selection in each round, as it is possible to cycle between the two markers. However for construction of multiple mutations over a short region, long synthetic oligonucleotides with multiple mismatches to the template can offer an alternative strategy.

Base Sequence↗
PubMed1985

Renal abnormalities and spondylometaphyseal dysplasia.

A renal lesion has not been previously described in association with spondylometaphyseal dysplasia. A case of nephrotic syndrome with progression to renal failure from focal segmental glomerulosclerosis is described.

Child↗
PubMed1983

Algorithms for processing spatial information.

Pairs of stimuli taken from a psychometric measure of spatial aptitude were shown to 9-year-olds, 13-year-olds, and adults. The stimuli in pairs were (a) either identical or mirror images, and (b) presented in orientations that differed by 0-150 degrees. Individuals judged, as rapidly as possible, if the stimuli in a pair would be identical or mirror images if presented at the same orientation. In Experiment 1, in which the stimuli were letter-like characters, at all ages most persons solved the problems using an algorithm in which an individual encodes the stimuli in working memory, mentally rotates one stimulus to the orientation of the other, compares them to determine if they are identical, and responds. In Experiment 2, the stimuli were multielement flags; here, the modal algorithm for both 9- and 13-year-olds differed from the previously described algorithm in that if the comparison process revealed that the stimuli were dissimilar, individuals did not respond immediately, but continued processing until a self-imposed deadline was reached. Among adults, the modal algorithm was the same one used in Experiment 1. Results are discussed in terms of the roles of encoding in contributing to the use of a particular algorithm.

Adolescent↗
PubMed1983

Postlaryngectomy voice reconstruction.

A simple one staged technique for voice reconstruction after total laryngectomy is described. A section of posterior tracheal wall is preserved and converted into a tracheo-oesophageal shunt. Results in nine patients are described.

Esophagus↗