Facile measurement of polypeptide JHNHα coupling constants from HMQC-J spectra

A method, based on linewidth measurements, is described which permits the rapid and facile determination of J_HNH coupling constants from ¹⁵N labeled proteins. Using appropriately processed HMQC-J data, we have found that a simple linear relationship exists between the half-height linewidth (_1/2) of ¹⁵N–¹H cross peaks and their corresponding J_HNH coupling constants. Tests indicate that this technique permits the accurate measurement of up to 100 J_HNH coupling constants in less than 30 min. Furthermore, the J_HNH measurements can be done manually – without the need of any computer-based curve-fitting or minimization. Comparisons between J_HNH values predicted from high resolution X-ray structures and those determined using this technique indicate that the method is both accurate and precise (correlation coefficient = 0.90, rmsd = 0.75 Hz).     

Rapid Measurement of Scalar Three-bond 1HN-1Hα spin coupling constants in 15N-labelled proteins

Two new 2D NMR experiments, CT-HMQC-HA and CT-HMQC-HN, are proposed for the rapid measurement of homonuclear 3JHNH coupling constants of uniformly 15N-enriched proteins in solution. The experiments are based on the comparison of the signal intensities in a pair of constant-time [15N,1H]-HMQC spectra recorded with and without decoupling of the amide proton - proton coupling. Experimental data recorded with the 78-residue N-terminal domain of the E. coli arginine repressor (ArgR-N) and with oxidized E. coli flavodoxin (176 residues) showed good agreement with 3JHNH coupling constants obtained by fitting of the multiplet fine structure of the amide proton resonances or from a 3D HNHA-J experiment, respectively. Quantitative estimates for the effects from different relaxation rates of in-phase and antiphase magnetization are given.     

A New Experiment for the Measurement of nJ(C,P) Coupling Constants Including 3J(C4′i,Pi) and 3J(C4′i,Pi+1) in Oligonucleotides

A new experiment for the measurement of nJ(C,P) coupling constants along the phosphodiester backbone in RNA and DNA based on a quantitative-J HCP experiment is presented. In addition to coupling constants, in which a carbon atom couples to only one phosphorus atom, both the intraresidual 3J(C4i,Pi) and the sequential 3J(C4i,Pi+1) for the C4 resonances that couple to two phosphorus atoms can be obtained. Coupling constants obtained by this new method are compared to values obtained from the P-FIDS experiment. Together with 3J(H,P) coupling constants measured using the P-FIDS experiment, the backbone angles and can be determined.     

Rapid and Efficient Synthesis of Peptides Containing α,α-dialkylamino acids employing KOBt

Summary Several Fmoc-α,α-dialkylamino acids and their acid chlorides have been prepared, isolated and characterised. The synthesis of peptides containing sterically hindered dialkylamino acids has been accomplished using acid chloride/KOBt in dichloromethane. The yields as well as the purity of the peptides were satistactory.     

Evidence for C---C coupling between two mutually cis carbon ligands, η-crotyl and aryl in organopalladium(II) complexes

Some (η³-crotyl) (2,5-dichlorophenyl)palladium(II) complexes containing phosphine and phosphite ligands Pd(η³-CH₂CH-CHMe)(Ar)(PR₃) (Ar = C₆H₃Cl₂-2,5) were isolated as crystalline solids or generated in solution. These existed as a mixture of two geometrical isomers arising from a different way of risposition of the crotyl-methyl group and the aryl ligand. The electronic nature of the PR₃ ligand controlled the relative rates of the interconversion between the two isomers and the reductive elimination of the complexes which released MeCH=CHCH₂Ar and CH₂=CHCH(Me)(Ar). Electron-withdrawing phosphite ligands were particularly effective in enhancing the reductive elimination rate, making the contribution of the isomerization path almost negligible and allowing the formation of two coupling products to be followed separately by spectroscopic means. The observations demonstrated the occurrence of C---C coupling between mutually cis carbon ligands in (η³-allyl)(hydrocarbyl)palladium(II) complexes. The η¹-crotyl complex, (Pd(η¹-CH₂CH=CHMe)(Ar) (dppen) (dppen = cis-Ph₂PCH=CHPPh₂) was isolated and shown to exist as a sole regio-isomer in solution. Reductive elimination of this η¹-crotyl complex gave MeCH=CHCH₂Ar exclusively.     

Calmodulin and Ca2+- and Calmodulin-Dependent Protein Kinase in Rat Anterior Pituitary Gland

Calmodulin and Ca2+- and calmodulin-dependent protein kinase were identified in the rat anterior pituitary gland. The concentration of calmodulin was 1.18 +/- 0.11 microgram/mg protein (n = 7) in the cytosol fraction. The calmodulin of the anterior pituitary gland co-migrated with brain calmodulin on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The Ka value of the partially purified enzyme for Ca2+ was 3.3 microM in the presence of 0.30 microM calmodulin. Trifluoperazine and chlorpromazine, calmodulin-interacting agents, inhibited enzyme activity, with Ki values of 1.3 and 2.6 X 10(-5) M, respectively. The enzyme was resolved into two peaks of activity, with sedimentation coefficients of 5.5 S and 16.5 S, by sucrose density gradient centrifugation. At least nine proteins were phosphorylated by the enzyme in a Ca2+- and calmodulin-dependent manner. In light of these results, the possibility that calmodulin and the calmodulin-activatable protein kinase system are involved in the mediation of the Ca2+ effect on hormone release from the anterior pituitary gland must be given consideration.     

In vitro synthesis of photosynthetic membranes: I. Development of photosystem I activity and cyclic photophosphorylation

First successful $\text{in vitro}$ synthesis of functional photosynthetic phosphorylating membrane is reported. Etioplasts, highly enriched in cytoplasmic and plastid proteins, isolated from etiolated Cucumber cotyledons pretreated with kinetin and gibberellic acid, and illuminated in a cofactor fortified medium showed commencement of chlorophyll (Chl) synthesis immediately after illumination from exogenous δ-aminolevulinic acid, while photosystem I (PS I) activity commenced 15 min after the onset of illumination. When cotyledons pretreated with kinetin and gibberellic acid were illuminated directly, there was a lag phase of 30 min before the commencement of Chl synthesis and PS I activity developed after 1 h of illumination. In plastids developed both $\text{in vivo}$ and $\text{in vitro}$, the electron flow from dichlorophenolindophenol to methyl-viologen was coupled to phosphorylation as observed by an increase in the electron transport rate on the addition of uncouplers. Analysis of polypeptide profiles of the greening plastids $\text{in vitro}$ showed the disappearance of many higher molecular weight proteins during greening. Polypeptides of molecular weight 32, 20.5, 19.5 K absent in etioplasts appeared as distinct bands after 4 h of greening $\text{in vitro}$.     

Equilibration of methionine into the Met-tRNA pool of rabbit reticulocyte lysates: Additional evidence that deacylation of ribosomal subunit-bound Met-tRNAf does occur in heme-deficiency

We have measured the rate of equilibration of [³⁵S]methionine into the Met-tRNA pool of rabbit reticulocyte lysate as in [FEBS Lett. (1982) 143, 301–305]. Our results indicate that hemin-deficiency inhibits the equilibration of methionine into the tRNA pool much less than protein synthesis or the equilibration of alanine into the tRNA pool, whereas cycloheximide inhibits these processes similarly. This finding is consistent with our previous data and supports the hypothesis that with hemin-deficiency much of the Met-tRNA_f that becomes bound to 40 S subunits subsequently undergoes enzymatic deacylation.     

Role of voltage-dependent ionic currents in coupling glucose stimulation to insulin secretion in canine pancreatic islet B-Cells

Summary Glucose-induced electrical activity in canine pancreatic islet B cells is distinct from that in rodent islets, though both display Ca²⁺-dependent insulin secretion. Rodent islet B cells undergo regular bursts of Ca²⁺-dependent action potentials, while canine islet B cells generate isolated Na⁺-dependent action potentials which often give way to a plateau depolarization. Here we present evidence to reconcile the species difference in electrical activity with the similarity of Ca²⁺ dependence of secretion. (i) In canine B cells increasing glucose concentrations produce membrane depolarization and increasing frequency of Na_o-dependent action potentials until a background membrane potential (-40mV) is reached where Na⁺ currents are inactivated. (ii) Voltage-dependent Ca²⁺ currents are present which are activated over the voltage excursion of the action potential (–50 to +20 mV) and inactivate slowly, (over seconds) in the range of the plateau depolarization (–40 to –25 mV). Hence, they are available to contribute to both phases of depolarization. (iii) Tetrodotoxin (TTX) reduces by half an early transient phase of glucosestimulated insulin secretion but not a subsequent prolonged plateau phase. The transient phase of secretion often corresponds well in time to the period of initial high frequency action potential activity. These latter results suggest that in canine B cells voltagedependent Na⁺ and Ca²⁺ currents mediate biphasic glucose-induced insulin secretion. The early train of Na⁺-dependent action potentials, by transiently activating Ca²⁺ channels and allowing pulsatile Ca²⁺ entry, may promote an early transient phase of insulin secretion. The subsequent sustained plateau depolarization, by allowing sustained Ca²⁺ entry, may permit steady insulin release.