Binding of oxytocin and 8-arginine-vasopressin to neurophysin studied by 15N NMR using magnetization transfer and indirect detection via protons

NMR was used to monitor the binding to neurophysin of oxytocin and 8-arginine-vasopressin, 15N labeling being used to identify specific backbone 15N and 1H signals. The most significant effects of binding were large downfield shifts in the amino nitrogen resonance of Phe-3 of vasopressin and in its associated proton, providing evidence that the peptide bond between residues 2 and 3 of the hormones is hydrogen-bonded to the protein within hormone-neurophysin complexes. Suggestive evidence of hydrogen bonding of the amino nitrogen of Tyr-2 was also obtained in the form of decreased proton exchange rates on binding; however, the chemical shift changes of this nitrogen and its associated proton indicated that such hydrogen bonding, if present, is probably weak. Shifts in the amino nitrogen of Asn-5 and in the -NH protons of both Asn-5 and Cys-6 demonstrated that these residues are significantly perturbed by binding, suggesting conformational changes of the ring on binding and/or the presence of binding sites on the hormone outside the 1-3 region. No support was obtained for the thesis that there is a significant second binding site for vasopressin on each neurophysin chain. The behavior of both oxytocin and vasopressin on binding was consistent with formation of 1:1 complexes in slow exchange with the free state under most pH conditions. At low pH there was evidence of an increased exchange rate. Additionally, broadening of 15N resonances in the bound state at low pH occurred without a corresponding change in the resonances of equilibrating free hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

13C NMR assignments of the protonated carbons of [d(TAGCGCTA)]2 by two-dimensional proton-detected heteronuclear correlation

The resonances of the protonated carbons of [d(TAGCGCTA)]2 have been assigned by the two-dimensional proton-detected double-quantum heteronuclear correlation experiment [( 1H-13C]-DQCOSY). 13C-coupled and 13C-decoupled versions of the experiment were used. The assignment method is discussed in detail. The deoxyribose cross peaks segregate into five well-resolved regions, and the base cross peaks have distinct features that are helpful for assignments. The cross peaks from the 1H-13C pairs at the Cyd5, Ado2 and ThdCH3 base positions fall in separate regions of the spectrum from each other; they also are resolved from the closely spaced Ado8, Guo8, Cyd6 and Thd6. Additional parameters for distinction of the base signals are their differing J-coupling values and long-range coupling patterns.     

13C NMR of the bases of three DNA oligonucleotide duplexes: assignment methods and structural features

Natural abundance 13C NMR spectra of three DNA oligomers have been obtained. Most of the base resonances are well resolved from one another. A combination of two independent methods was used in making assignments: a one-dimensional spectral comparison method and a two-dimensional proton-detected 1H-13C correlated experiment for the protonated carbons. There are large shielding changes (between 1.62 and -1.40 ppm) upon thermal dissociation of the duplex. The shapes of the chemical shift vs temperature curves are largely independent of sequence. The base carbon resonance frequencies are sensitive to hydrogen bonding, base stacking, sugar conformation, and changes in the glycosyl torsion angle.     

Enhancement by Potassium of Carbachol-Stimulated Inositol Phospholipid Breakdown in Rat Cerebral Cortical Miniprisms: Comparison with Other Depolarising Agents

Increasing the [K+] in the assay medium from 5.7 to 17.8 mM produces a large enhancement of the inositol phospholipid breakdown response to the muscarinic agonist carbachol in rat cerebral cortical miniprisms, with minor effects on basal inositol phospholipid breakdown. This effect is also found with Rb+. The enhancement by a raised [K+] is not accompanied by a change in the composition of the labelled polyphosphoinositides. The carbachol-stimulated inositol phospholipid breakdown at 17.8 and 42.7 mM K+ was antagonised by veratrine (5-80 microM), 4-aminopyridine (5 mM), and tetraethylammonium (20 mM). These compounds, however, also inhibited the binding of [3H]quinuclidinyl benzilate to cortical membranes. BRL 34915 (0.2-20 microM) was without significant effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+.Mg2+ (10 mM) considerably reduced the carbachol-stimulated inositol phospholipid breakdown at 17.8, but not 42.7, mM K+. Inositol phospholipid breakdown was also stimulated, albeit to a small extent, by L-glutamate (100-3,000 microM) and quisqualate (1-100 microM), with the stimulation being additive to that produced by carbachol at both 5.7 and 17.8 mM K+. N-Methyl-D-aspartate (10-1,000 microM in Mg2+-free medium) had no significant effect on basal inositol phospholipid breakdown and had little or no effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+. It is concluded that it may not be correct to ascribe wholly the enhancement by K+ of carbachol-stimulated inositol phospholipid breakdown to the tissue-depolarising actions of this ion and that other actions of K+ may be involved.     

A preliminary study of the use of Colilert for water quality monitoring

Substrate specificities of proteases produced by two putrefactive marine bacteria, Shewanella putrefaciens and Alteromonas haloplanktis , were surveyed by using peptidyl-7-amino-4-methylcoumarin (MCA-substrates). Shewanella putrefaciens produced trypsin-like enzyme(s) showing broad spectrum specificity and chymotrypsin-like enzyme specifically hydrolysing Glt-Gly-Gly-Phe-MCA. Alteromonas haloplanktis produced high activity of ammopeptidase and trypsin-like enzyme(s) preferring Z-Phe-Arg-MCA, Bz-Arg-MCA and Boc-Leu-Ser-Thr-Arg-MCA. The two organisms would be able to utilize different proteins for their growth.     

Peptide recognition by PTB and PDZ domains

Protein tyrosine binding (PTB) and ‘post synaptic density disc-large zo-1’ (PDZ) domains bind to short peptidic ligands by augmentation of one of the domain's β sheets and other recognition mechanisms. The two domain classes have a superficial resemblance to each other, even though no sequential homology exists. The structural bases of the interactions are well understood for the domains now experimentally determined, and ligand—target pairs can probably be identified in favorable cases by analogy with the known domains. For both PTB and PDZ classes, functional activities are still not fully defined: it is possible that these domain classes, along with pleckstrin homology domains, have multiple roles.     

Apolipoprotein E isoform-specific disruption of phosphoinositide hydrolysis: protection by estrogen and glutathione

The effect of non-esterified myristate (C14:0) or dodecyl sulfate was studied on passive swelling of rat liver mitochondria suspended in hypotonic alkaline KCl medium in the absence of the potassium ionophore valinomycin. Both compounds rapidly initiated large-amplitude swelling. However, they failed to initiate swelling when the mitochondria were suspended in hypotonic alkaline sucrose medium. In contrast to myristate or dodecyl sulfate, the non-ionic detergent Triton X-100 initiated swelling of mitochondria in both of the media. The following findings indicate that the inner mitochondrial membrane (IMM) is permeabilized by myristate to K+ and Cl- in a specific manner. (i) Swelling initiated by myristate did not respond to cyclosporin A, (ii) the protonophoric uncoupler FCCP was unable to mimic the myristate effect on swelling, and (iii) myristate-induced Cl- -permeation (measured with KCl medium plus valinomycin) was inhibited by N,N'-dicyclohexylcarbodiimide, quinine or ATP. Myristate- or dodecyl sulfate-initiated swelling was paralleled by the lowering of endogenous Mg2+ content. Both effects, stimulation of swelling and depletion of endogenous Mg2+ are correlated with each other. Similar effects have been reported previously for the carboxylic divalent cation ionophore calcimycin (A23187). The A23187-induced swelling has identical inhibiting characteristics on Cl- -permeation with respect to N,N'-dicyclohexylcarbodiimide, quinine and ATP as the myristate-stimulated swelling. Therefore, we conclude that non-esterified fatty acids increase the permeability of mitochondria to K+ and Cl- at alkaline pH by activating Mg2+-dependent ion-conducting pathways in IMM.     

The effect of noncollinearity of 15N-1H dipolar and 15N CSA tensors and rotational anisotropy on 15N relaxation, CSA/dipolar cross correlation, and TROSY

Current approaches to 15N relaxation in proteins assume that the 15N-1H dipolar and 15N CSA tensors are collinear. We show theoretically that, when there is significant anisotropy of molecular rotation, different orientations of the two tensors, experimentally observed in proteins, nucleic acids, and small peptides, will result in differences in site- specific correlation functions and spectral densities. The standard treatments of the rates of longitudinal and transverse relaxation of amide 15N nuclei, of the 15N CSA/15N-1H dipolar cross correlation, and of the TROSY experiment are extended to account for the effect of noncollinearity of the 15N-1H dipolar and 15N CSA (chemical shift anisotropy) tensors. This effect, proportional to the degree of anisotropy of the overall motion, (D/D–1), is sensitive to the relative orientation of the two tensors and to the orientation of the peptide plane with respect to the diffusion coordinate frame. The effect is negligible at small degrees of anisotropy, but is predicted to become significant for D/D1.5, and at high magnetic fields. The effect of noncollinearity of 15N CSA and 15N-1H dipolar interaction is sensitive to both gross (hydrodynamic) properties and atomic-level details of protein structure. Incorporation of this effect into relaxation data analysis is likely to improve both precision and accuracy of the derived characteristics of protein dynamics, especially at high magnetic fields and for molecules with a high degree of anisotropy of the overall motion. The effect will also make TROSY efficiency dependent on local orientation in moderately anisotropic systems.     

Presenilin expression during induced differentiation of the human neuroblastoma SH-SY5Y cell line

Human neuroblastoma SH-SY5Y cells stably transfected with both wild-type and exon-9 deleted (deltaE9) presenilin constructs were used to study the role of the presenilin proteins during differentiation. Cells transfected with either wild-type or deltaE9 PS1, of which the latter abolishes normal endoproteolytic cleavage of the protein, showed no obvious differences in their ability to differentiate to a neuronal-like phenotype upon treatment with retinoic acid (RA). A defined pattern of PS1 expression was observed during differentiation with both RA and the phorbol ester TPA. Full-length PS1 was shown to increase dramatically within 5-24 h of RA treatment. TPA gave an earlier and longer lasting increase in full-length PS1 levels. The intracellular distribution pattern of PS1 was markedly altered following RA treatment. Within 24h PS1 was highly up-regulated throughout the cell body around the nucleus. Between 2 and 4 weeks PS1 staining appeared punctate and also localised to the nucleus. Increases in PS1 expression upon treatment with RA and TPA were blocked by treatment with cycloheximide, indicating a role of de-novo protein synthesis in this effect. PS2 expression remained unchanged during differentiation. Levels of full-length PS1 were also seen to increase during neurogenesis and neuronal differentiation in the forebrain of first trimester human foetuses between 6.5 and 11 weeks. These combined observations support the idea that PS1 is involved in neuronal differentiation by a mechanism likely independent of endoproteolysis of the protein.