Determination of 3J(H infi supN,C infi sup′ ) coupling constants in proteins with the C′-FIDS method

Summary We introduce the C-FIDS-¹H,¹⁵N-HSQC experiment, a new method for the determination of ³J(H _infi ^supN ,C _infi ^sup ) coupling constants in proteins, yielding information about the torsional angle . It relies on the ¹H,¹⁵N-HSQC or HNCO experiment, two of the the most sensitive heteronuclear correlation experiments for isotopically labeled proteins. A set of three ¹H,¹⁵N-HSQC or HNCO spectra are recorded: a reference experiment in which the carbonyl spins are decoupled during t₁ and t₂, a second experiment in which they are decoupled exclusively during t₁ and a third one in which they are coupled in t₁ as well as t₂. The last experiment yields an E.COSY-type pattern from which the ²J(H _infi ^supN ,C _infi-1 ^sup ) and ¹J(N_i,C _infi-1 ^sup ) coupling constants can be extracted. By comparison of the coupled multiplet (obtained from the second experiment) with the decoupled multiplet (obtained from the first experiment) convoluted with the ²J(H _infi ^supN ,C _infi-1 ^sup ) coupling, the ³J(H _infi ^supN ,C _infi ^sup ) coupling can be found in a one-parameter fitting procedure. The method is demonstrated for the protein rhodniin, containing 103 amino acids. Systematic errors due to differential relaxation are small for ⁿJ(H^N,C) couplings in biomacromolecules of the size currently under NMR spectroscopic investigation.     

Degradation products of myelin proteins in a light CNS subcellular fraction

The fraction floating on 0.32 M sucrose when normal mammalian spinal cord homogenate is submitted to discontinuous density gradient centrifugation is highly enriched in Marchi-positive material. In situ this material is located along paranodal myelin sheath segments. We here show by immunoblotting that degradation products of the myelin-associated glycoprotein (MAG) and of the enzyme 2,3-cyclic nucleotide 3-phosphodiesterase (CNP) is present in the Marchi-positive floating fraction but is not found in the myelin fraction. Since previous biochemical analyses of the floating fraction show a gross composition closely resembling myelin and since metabolic studies show the specific activity of incorporated amino acids to proceed with time from beavier to lighter myelin subfractions the results strongly suggest that normally occurring Marchi-positive bodies represents an intermediate stage in myelin catabolism.     

Polymerization of nucleotide analogues I: Reaction of nucleoside 5′-phosphorimidazolides with 2′-amino-2′-deoxyuridine

Summary 2-Amino-2-deoxyuridine reacts efficiently with nucleoside 5-phosphorimidazolides in aqueous solution. The dinucleoside monophosphate analogues were obtained in yields exceeding 80% under conditions in which little reaction occurs with the natural nucleosides.In a similar way, the 5-phosphorimidazolide of 2-amino-2-deoxyuridine undergoes self-condensation in aqueous solution to give a complex mixture of oligomers.The phosphoramidate bond in the dinucleoside monophosphate analogues is stable for several days at room temperature and pH 7. The mechanisms of their hydrolysis under acidic and alkaline conditions are described.Abbreviations A adenosine - C cytidine - G guanosine - U uridine - T thymidine - U_{N 3} 2-azido-2-deoxyuridine - U_{NH 2} 2-amino-2-deoxyuridine - ImpA adenosine 5-phosphorimidazolide - ImpU uridine 5-phosphorimidazolide - ImpU_{N 3} 2-azido-2-deoxyuridine 5-phosphorimidazolide - ImpU_{NH 2} 2-amino-2-deoxyuridine 5-phosphorimidazolide - pA adenosine 5-phosphate - pU uridine 5-phosphate - pU_{N 3} 2-azido-2-deoxyuridine 5-phosphate - pU_{NH 2} 2-amino-2-deoxyuridine 5-phosphate - UpA uridylyl-[35]-adenosine - UpU uridylyl-[35]-uridine - U_NpA adenylyl-[52]-2-amino-2-deoxy-uridine - U_NpU uridylyl-[52]-2-amino-2-deoxyuridine (pU_N)_n n=2,3,4 [25]-linked oligomers of pU_{NH 2} poly(A) polyadenylic acid - Im imidazole - MeIm l-methylimidazole     

Preparation of a human lung purified plasma membrane fraction: Confirmation by enzyme markers,electron microscopy,and histamine H1 receptor binding

Summary A simple and rapid method of isolating plasma membranes from human peripheral lung tissue is described. The method involves homogenization of tissue in 0.25m sucrose-buffered medium followed by differential and sucrose density gradient centrifugation. Enzymatic and morphological characterization of the plasma membrane fraction revealed minimal contamination by nonplasma membrane fragments. The isolated plasma membranes showed an 18-fold purification of 5-nucleotidase activity compared to the original homogenate. Electronmicroscopic studies of the plasma membrane fraction revealed the presence of small membrane vesicles having a trilaminar membrane structure. To further examine the purity of the plasma membrane preparation, the binding of the H₁ receptor antagonist,³H pyrilamine, to the plasma membrane-enriched fraction was compared to the binding to crude membrane preparations. Both the plasma membrane-enriched fraction and the crude membrane preparation had similar K_d's for the histamine antagonist, but the plasma membrane-enriched fraction had a threefold greater binding capacity, reflecting the relative enrichment of plasma membranes of the preparation. Thus, a method has been developed for the isolation of plasma membranes from human peripheral lung which should provide material for a variety of biochemical and pharmacological studies.     

Catalysts for the self-polymerization of adenosine cyclic 2′,3′-phosphate

Summary When adenosine cyclic 2,3-phosphate is evaporated from solution in the presence of simple catalysts such as aliphatic diamines at alkaline pH, and maintained in a dry state at moderate temperatures (25-85°C), self-polymerization to give oligonucleotides of chainlength up to at least 6 is observed. The products contain an excess of [35]-linkages over [25]-linkages. The effects of different catalysts and reaction conditions on the efficiency of the reaction are described. The prebiological relevance of these reactions is discussed.     

Isolation of trace amounts of 3′,4′-dehydro-4′-deoxydothistromin from peanut plants naturally infected with Cercospora personata

Trace amounts of the anthraquinonoid toxic metabolite, 3,4-dehydro-4-deoxydothistromin have been identified for the first time, from peanut tissues naturally infected by Cercospora personata. Characteristic uv spectral and Chromatographic properties of the metabolite and its tetraacetate as well as the mass spectrum of the latter have established its identity.     

Normal coordinate analysis of 2′-deoxythymidine and 2′-deoxyadenosine

The proposed valence force field allows us to reproduce the vibration modes of 2-deoxythymidine and 2-deoxyadenosine. The present calculations are based on the Wilson GF-method and a non-redundant set of symmetrical coordinates. The calculated wavenumbers have been compared to the available Raman and infrared peak positions observed in solid, amorphous or aqueous samples. Moreover, the results obtained with the present force field allow us to assign some of the characteristic vibration modes for the thymidine and adenosine residues involved in DNA double-helical chains.     

Cofactor interactions and the regulation of glutamate decarboxylase activity

More than 50% of glutamate decarboxylase (GAD) in brain is present as apoenzyme. Recent work has opened the possibility that apoGAD can be studied in brain by labeling with radioactive cofactor. Such studies would be aided by a compound that inhibits specific binding. One possibility is 4-deoxy-pyridoxine 5-phosphate, a close structural analog of the cofactor pyridoxal 5-phosphate. The effects of deoxypyridoxine-P on the cyclic series of reactions that interconverts apo- and holoGAD was investigated and found to be consistent with simple competitive inhibition of the activation of apoGAD by pyridoxal-P. As expected from the cycle GAD was inactivated when incubated with glutamate and deoxypyridoxine-P even though cofactor was present, but no inactivation was observed with deoxypyridoxine-P in the absence of glutamate. Deoxypyridoxine-P also stabilized apoGAD against heat denaturation. These effects were quantitatively accounted for by a kinetic model of the apo-holoGAD cycle. Deoxypyridoxine-P inhibited the labeling by [³²P]pyridoxal-P of GAD isolated from rat brain. Hippocampal extracts were labeled with [³²P]pyridoxal-P and analyzed by SDS-polyacrylamide gel electrophoresis. Remarkably few bands were strongly labeled. The major labeled band (at 63 kDa) corresponded to one of the forms of GAD. Other strongly-labeled bands were observed at 65 kDa (corresponding to the higher molecular weight form of GAD) and at 69–72 kDa. Labeling of the 63- and 65-kDa bands was inhibited by deoxypyridoxine-P, but the 69–72 kDa bands were unaffected, suggesting that the latter were non-specifically labeled. The results suggest that the 63-kDa form of GAD makes up the majority of apoGAD in hippocampus.Special issue dedicated to Dr. Eugene Roberts.     

Volume-sensitive taurine transport in fish erythrocytes

Summary Taurine plays an important role in cell volume regulation in both vertebrates and invertebrates. Erythrocytes from two euryhaline fish species, the eel (Anguilla japonica) and the starry flounder (Platichthys stellatus) were found to contain high intracellular concentrations of this amino acid ( 30 mmol per liter of cell water). Kinetic studies established that the cells possessed a saturable high-affinity Na⁺-dependent -amino-acid transport system which also required Cl^– for activity (apparentK _m (taurine) 75 and 80 m;V _max 0.85 and 0.29 mol/g Hb per hr for eel (20°C) and flounder cells (10°C), respectively. This -system operated with an apparent Na⁺/Cl^–/taurine coupling ratio of 211. A reduction in extracellular osmolarity, leading to an increase in cell volume, reversibly decreased the activity of the transporter. In contrast, low medium osmolarity stimulated the activity of a Na⁺-independent nonsaturable transport route selective for taurine, -amino-n-butyric acid and small neutral amino acids, producing a net efflux of taurine from the cells. Neither component of taurine transport was detected in human erythrocytes. It is suggested that these functionally distinct transport routes participate in the osmotic regulation of intracellular taurine levels and hence contribute to the homeostatic regulation of cell volume. Volume-induced increases in Na⁺-independent taurine transport activity were suppressed by noradrenaline and 8-bromoadenosine-3, 5-cyclic monophosphate, but unaffected by the anticalmodulin drug, pimozide.     

Atrial natriuretic peptide inhibits the phosphoinositide hydrolysis in murine Leydig tumor cells

The ability of ANP to inhibit the hydrolysis of phosphoinositides was examined in [³H] myoinositol-labeled intact murine Leydig tumor (MA-10) cells. Arginine vasopressin (AVP) stimulated the formation of inositol monophosphate (IP₁), inositol bisphosphate (IP₂), and inositol trisphosphate (IP₃) both in a time- and dose- dependent manner in MA-10 cells. ANP inhibited the AVP-induced formation of IP₁, IP₂, and IP₃ in these cells. The inhibitory effect of ANP on the AVP-stimulated formation of IP₁, IP₂, and IP₃ accounted for 30%, 38% and 42%, respectively, which was observed at the varying concentrations of AVP. ANP caused a dose-dependent attenuation in AVP-stimulated production of IP₁, IP₂ and IP₃ with maximum inhibition at 100 nM concentration of ANP. The production of inositol phosphates was inhibited in the presence of 8- bromo cGMP in a dose-dependent manner, whereas dibutyryl-cAMP had no effect on the generation of these metabolites. The LY 83583, an inhibitor of guanylyl cyclase and cGMP production, abolished the inhibitory effect of ANP on the AVP-stimulated production of inositol phosphates. Furthermore, 10 M LY 83583 also inhibited the ANP-stimulated guanylyl cyclase activity and the intracellular accumulation of cGMP by more than 65–70%. The inhibition of eGMP-dependent protein kinase by H-8, significantly restored the levels of AVP-stimulated inositol phosphates in the presence of either ANP or exogenous 8-bromo cGMP. The results of this study suggest that ANP exerts an inhibitory effect on the production of inositol phosphates in murine Leydig tumor (MA-10) cells by mechanisms involving cGMP and cGMP-dependent protein kinase.Established Investigator of the American Heart Association