Terbium(III) luminescence study of tyrosine emission from Escherichia coli glutamine synthetase.

Radiationless energy transfer from tyrosine to Tb(III) in Escherichia coli glutamine synthetase and its two mutants (W57L and W158S) has been utilized to assess the tyrosine residue(s) responsible for the observed tyrosine emission and to investigate its spatial relationships to the two metal binding sites of GS. The interference from tryptophan fluorescence was removed by chemical modification of the tryptophan residues by N-bromosuccinimide (NBS). The Tyr-Tb(III) distances measured by using F?rster energy-transfer theory were in good agreement among the three enzymes with average distances of 10.7 and 11.2 A from Tyr to the two metal binding sites. The pKa value for the ionization of tyrosine was determined from fluorescence titration experiments to be approximately 10 for both mutant enzymes. The similarities in pKa values and Tyr-Tb(III) distances observed for all three enzymes lead to the conclusion that the same tyrosine residue(s), is (are) most likely responsible for the Tyr emission. According to the crystal structure distances from tyrosine residues to the two metal binding sites of GS, it is believed that Tyr-179 is the main contributor to the observed Tyr emission. The fact that an intense Tyr emission was observed for W57L GS but not for W158S GS indicates that Trp-57 is much more effective than Trp-158 in quenching the Tyr-179 emission probably through a F?rster-type energy transfer. Furthermore, modification of Trp-57 by NBS causes no significant increase in Tyr-179 emission while replacement of Trp-57 by leucine does. This may indicate that oxidized Trp-57 is also an effective quencher for Tyr-179 emission.     

Solid-state 13C-NMR studies of the effects of sodium ions on the gramicidin A ion channel.

End-to-end helical dimers of gramicidin A form transmembrane pores in lipid bilayers, through which monovalent ions may pass. The groups within the peptide that interact with these ions have been studied by application of solid-state spectroscopic methods to a series of gramicidin A analogues synthesized with 13C in selected peptide carbonyl groups. The resonances of D-Leu10, D-Leu12 and D-Leu14 analogues were perturbed in the presence of 0.16 M sodium ions, whereas the resonances of the carbonyls of Gly2, Ala3, D-Leu4 and Val7, which are closer to the formylated N-terminal end of the peptide, were unaffected. The observed changes in chemical shift anisotropy are indicative of a change in orientation of the abovementioned leucine carbonyls.     

Aniridia-Wilms' tumor association: evidence for specific deletion of 11p13.

A 7-year-old boy with aniridia, Wilms' tumor, and mental retardation, previously reported as having an interstitial deletion of the short arm of chromosome 8 resulting from a t(8p+;11q-) translocation (Ladda et al., 1974), has been restudied using high-resolution trypsin-Giemsa banding of prometaphase chromsomes. The results revealed a complex rearrangement with four break points in 8p, 11p, and 11q, leading to a net loss of an interstitial segment of 11p (region p1407 yields p1304) but not of 8p. His red blood cells contained normal activities of glutathione reductase (gene on 8p) and lactate dehydrogeanse A (gene on 11p12), indicating a gene dosage consistent with the chromosomal findings. The revised interpretation of this case agrees with seven others reported as having aniridia and interstitial 11p deletions in establishing the distal half of band 11p13 as the site of gene(s) which lead to aniridia and predispose to Wilms' tumor if present in a hemizygous state. Possible relationships between heterozygous deletion of specific chromosomal bands 11p13 and 13q14 and the autosomal dominant disorders aniridia, Wilms' tumor, and retinoblastoma, respectively, are discussed.     

Location and identification of the genes for adenovirus type 2 early polypeptides

Virus-specific RNA was prepared from cells early after adenovirus type 2 infection and fractionated by hybridization to specific fragments of viral DNA. The viral mRNA was used to program cell-free protein synthesis, and the products were analyzed by electrophoresis. The genes for the early polypeptides of apparent molecular weight 44,000, 15,000, 72,000, 15,500, 19,000, and 11,000 daltons were located, respectively, between positions 0–4.1, 4.1–16.7, 58.5–70.7, 75.9–83.4, 89.7–98.6, and 89.7–98.6 of the conventional adenovirus DNA map. The polypeptide of molecular weight 72,000 daltons was shown to be the single-strand DNA-binding protein described by others. RNAs from three different adeno-transformed cell lines each program the synthesis in vitro of predominantly the 15K polypeptide, as well as variable amounts of the polypeptide of molecular weight 44,000 daltons. The genes for these two polypeptides are located in the portion of DNA known to be required for transformation of rodent cells by adenovirus.     

Left ventricular dynamics during recovery from exercise.

Left ventricular dynamics during recovery were measured in dogs, 3 min after brief periods of mild, moderate, and severe treadmill exercise. As compared with resting values, stroke volume was unchanged, and the maximum first derivative of the left ventricular pressure was either unchanged or slightly elevated. Increases in heart rate of 20, 26, and 46 beats/min for mild, moderate, and severe exercise appear to be the major factor in augmenting cardiac output during recovery. With moderate and severe exercise, left ventricular end-diastolic diameter increased and continued to be elevated during recovery, whereas end-systolic diameter decreased during exercise but was elevated above resting values during recovery. Therefore, with strenuous exercise, a sympathetic-mediated increase in contractility recedes promptly during the postexercise period but the Frank-Starling mechanism continues to be a factor.     

X-ray diffraction studies on the connective tissue polysaccharides. Two-dimensional packing schemes for threefold hyaluronate chains.

An extended conformation of sodium and calcium hyaluronate with helical parameters n (number of residues per turn) = 3 and h (axial rise per disaccharide) = 0.95 ± 0.01 nm has been found to pack in a number of two-dimensional arrays. Each of the structures exhibits a variant of a motif consisting of six chains surrounding a “vacant” site. The distribution of chains around this site, together with certain aspects of the periodicity, suggests that there might be a special relationship between this conformation of hyaluronic acid and water.     

Asparagine metabolism-key to the nitrogen nutrition of developing legume seeds

Asparagine accounted for 50 to 70% of the nitrogen carried in translocatory channels serving fruit and seed of white lupin (Lupinus albus L.). Rates of supply of the amide always greatly exceeded its incorporation as such into protein. An asparaginase (l-asparagine amido hydrolase EC 3.5.1.1) was demonstrated in crude extracts of seeds. In vitro activity was up to 5 mumoles of aspartate formed per hour per gram fresh weight at the apparent Km(Asn) value of 10 mM, and this more than accounted for the estimated rates of asparagine utilization in vivo. Asparaginase activity per seed increased 10-fold in the period 5 to 7 weeks after anthesis, coinciding with early stages of storage protein synthesis in the cotyledons.Double labeled ((14)C (U), (15)N (amide)) asparagine was fed to fruiting shoots through the transpiration steram. Fruit phloem sap analysis indicated that virtually all of the label was translocated to seeds in the form of asparagine. In young seeds (15)N from asparagine breakdown was traced to the ammonia, glutamine, and alanine of endospermic fluid, the (14)C appearing mainly in nonamino compounds. In the cotyledon-filling stage the C and N of asparagine was contributed to a variety of amino acid residues of protein.