Fluorescence polarization of polypeptides having a fluorescent endgroup

The polarization of fluorescence of a chromophore chemically bound on the NH₂ end of a poly(L -benzyl glutamate) molecule has been studied as a function of temperature, viscosity, and solvent. The relaxation times depend on both the overall rotation of the helix and the local rotation of the endgroup. In m–cresol the endgroup is rigidly bound and the rotational diffusion constant of the molecule is in good agreement with the values obtained by Kerr effect and dielectric relaxation. In other helicogenic solvents (DMF, DCE, etc.) the local rotation is nearly free. In m-cresol-DMF mixtures a sharp decrease of the polarization around a composition of 40% DMF can be interpreted as a change in the freedom of rotation of the endgroup. No discontinuity in the optical rotation is observed in the solvent mixture. The question of how a rapidly rotating endgroup could show an extrinsic Cotton effect as observed by Bloutand Yamaoka for the system Acridine Orange–PBLG in chloroform is then raised. Polarization of fluorescence measurements on this system show a nearly complete freedom of rotation of the dye and OH D measurements show no detectable Cotton effect in the dye absorption band.     

Skeletal muscle beta-receptors and isoproterenol-stimulated vasodilation in canine heart failure

To investigate whether heart failure alters beta-adrenergic receptors on skeletal muscle and its associated vasculature, the density of beta-adrenergic receptors, isoproterenol-stimulated adenylate cyclase activity, and coupling of the guanine nucleotide-binding regulatory protein were compared in 18 control dogs and 16 dogs with heart failure induced by 5-8 wk of ventricular pacing at 260 beats/min. Hindlimb vascular responses to isoproterenol were compared in eight controls and eight of the dogs with heart failure. In dogs with heart failure, the density of beta-receptors on skeletal muscle was reduced in both gastrocnemius (control: 50 +/- 5; heart failure: 33 +/- 8 fmol/mg of protein) and semitendinosus muscle (control: 43 +/- 9; heart failure: 27 +/- 9 fmol/mg of protein, both P less than 0.05). Receptor coupling to the ternary complex, as determined by isoproterenol competition curves with and without guanosine 5'-triphosphate (GTP), was unchanged. Isoproterenol-stimulated adenylate cyclase activity was significantly decreased in semitendinosus muscle (control: 52.4 +/- 4.6; heart failure: 36.5 +/- 9.5 pmol.mg-1.min-1; P less than 0.05) and tended to be decreased in gastrocnemius muscle (control: 40.1 +/- 8.5; heart failure: 33.5 +/- 4.5 pmol.mg-1.min-1; P = NS). Isoproterenol-induced hindlimb vasodilation was not significantly different in controls and in dogs with heart failure. These findings suggest that heart failure causes downregulation of skeletal muscle beta-adrenergic receptors, probably due to receptor exposure to elevated catecholamine levels, but does not reduce beta-receptor-mediated vasodilation in muscle.     

Molecular deletion patterns in Duchenne and Becker type muscular dystrophy

Summary DNA from 80 Duchenne (DMD) and 15 Becker (BMD) index patients was analyzed with 12 genomic probes and the total cDNA. Deletions were detected in 24 DMD (30%) and 10 BMD patients (67%) by genomic probes alone, mostly p20, pXJ, and/or pERT87. All deletions were confirmed by cDNA probes, and an additional 29 DMD deletions were detected, resulting in a total of 63/95 deletions (66%). The majority of the deletions are localized between kb 6.7 and 9.7 of the cDNA; a smaller group, between kb 0.5 and 3.5. Of the deletions, 90% are detected by the three cDNA probes 1–2a, 7, and 8. This can be applied to strategies for carrier detection and prenatal diagnosis. The order of 13 exon-containing HindIII fragments in the region between probes 7 and 9–10, where most of the deletions are found, could be defined. The deletion patterns in DMD and BMD patients are different and well in accordance with the “reading frame theory” of Monaco and coworkers. Thus our findings indicate that a DMD or BMD phenotype may be predicted according to the breakpoint position and the number of deleted exons.     

Lysine 2,3-aminomutase. Support for a mechanism of hydrogen transfer involving S-adenosylmethionine

The conversion of L-lysine to L-beta-lysine is catalyzed by lysine 2,3-aminomutase. The reaction involves the interchange of the 2-amino group of lysine with a hydrogen at carbon 3. As such the reaction is formally analogous to adenosylcobalamin-dependent rearrangements. However, the enzyme does not contain and is not activated by this coenzyme. Instead it contains iron and pyridoxal phosphate and is activated by S-adenosylmethionine. Earlier experiments implicated adenosyl-C-5' of S-adenosylmethionine in the hydrogen transfer mechanism, apparently in a role similar or analogous to that of adenosyl moiety of adenosylcobalamin in the B12-dependent rearrangements. The question of whether both hydrogens or only one hydrogen at adenosyl-C-5' participate in the hydrogen-transfer process has been addressed by carrying out the lysine 2,3-aminomutase reaction with S-[5'-3H] adenosylmethionine in the presence of 10 times its molar concentration of enzyme. Under these conditions all of the tritium appeared in lysine and beta-lysine, showing that C-5'-hydrogens participate. To determine whether hydrogen transfer is compulsorily intermolecular and intramolecular, various molar ratios of [3,3-2H2]lysine and unlabeled lysine were submitted to the action of lysine 2,3-aminomutase under conditions in which 10-15% conversion to beta-lysine occurred. Mass spectral analysis of the beta-lysine for monodeutero and dideutero species showed conclusively that hydrogen transfer is both intramolecular and intermolecular. The results quantitatively support our postulate that activation of the enzyme involves a transformation of S-adenosylmethionine into a form that promotes the generation of an adenosyl-5' free radical, which abstracts hydrogen from lysine to form 5'-deoxyadenosine as an intermediate.     

An organic radical in the lysine 2,3-aminomutase reaction.

Lysine 2,3-aminomutase from Clostridium SB4 has been studied by electron paramagnetic resonance (EPR) spectroscopy at 77 K. Although the reaction catalyzed by this enzyme is similar to rearrangements catalyzed by enzymes requiring adenosylcobalamin, lysine 2,3-aminomutase does not utilize this cofactor. The enzyme instead contains iron-sulfur clusters, cobalt, and pyridoxal phosphate and is activated by S-adenosylmethionine. Subsequent to a reductive incubation procedure that is required to activate the enzyme, EPR studies reveal the appearance of an organic radical signal (g = 2.001) upon addition of both L-lysine and S-adenosylmethionine. The radical signal is complex, having multiple hyperfine transitions. The total radical concentration is proportional to enzyme activity and decreases in parallel with the approach to chemical equilibrium between alpha-lysine and beta-lysine. The signal changes over the time course of the reaction in a way that suggests the presence of more than one radical species, with different relative proportions of species in the steady state and equilibrium state. Isotopic substitution experiments show that unpaired spin density resides on the molecular framework of lysine and that solvent-exchangeable protons do not participate in strong hyperfine coupling to the radical. The results indicate that lysine radicals participate in the rearrangement mechanism.     

Formation of supported planar bilayers by fusion of vesicles to supported phospholipid monolayers.

A technique for the production of supported phospholipid bilayers by adsorption and fusion of small unilamellar vesicles to supported phospholipid monolayers on quartz is described. The physical properties of these supported bilayers are compared with those of supported bilayers which are prepared by Langmuir-Blodgett deposition or by direct vesicle fusion to plain quartz slides. The time courses of vesicle adsorption, fusion and desorption are followed by total internal reflection fluorescence microscopy and the lateral diffusion of the lipids in the adsorbed layers by fluorescence recovery after photobleaching. Complete supported bilayers can be formed with phosphatidylcholine vesicles at concentrations as low as 35 microM. However, the adsorption, fusion and desorption kinetics strongly depend on the used lipid, NaCl and Ca2+ concentrations. Asymmetric negatively charged supported bilayers can be produced by incubating a phosphatidylcholine monolayer with vesicles composed of 80% phosphatidylcholine and 20% phosphatidylglycerol. Adsorbed vesicles can be removed by washing with buffer. The measured fluorescence intensities after washing are consistent with single supported bilayers. The lateral diffusion experiments confirm that continuous extended bilayers are formed by the monolayer-fusion technique. The measured lateral diffusion coefficient of NBD-labeled phosphatidylethanolamine is (3.6 +/- 0.5) x 10(-8) cm2/s in supported phosphatidylcholine bilayers, independent of the method by which the bilayers were prepared.     

Selenium concentrations in the human thyroid gland

Recently, we found that prediagnostic serum selenium concentration was significantly lower for cases developing thyroid cancer (n=43) than for controls. We assumed that redistribution of serum selenium into the affected tissue took place in the prediagnostic period. The present study was carried out to determine the physiological concentration of selenium in the thyroid, since very few data are available in the literature. The concentrations of selenium in the thyroid (n=45) and liver samples from Norwegians who had died because of acute illness or accidents were determined by hydride generation atomic absorption spectrometry. The mean selenium concentration was found to be 0.72±0.44 μg/g in the thyroid and 0.45±0.11 μg/g in the liver tissue. The surprisingly high concentration of selenium in apparently normal thyroids indicates that selenium has important functions in this organ. The remarkably broad range, together with the observation that no significant correlation exists between thyroid and liver concentrations, suggest that factors other than the selenium status are important determinants for the selenium concentration in the thyroid gland. This observation is consistent with our hypothesis that in carcinogenesis, prediagnostic processes influence the serum-/thyroid-ratio of selenium.     

Excision of the En/Spm transposable element of Zea mays requires two element-encoded proteins.

An excision assay system for En/Spm was developed in transgenic tobacco. The characteristics of excision and integration are similar to the natural system of Zea mays. In this transgenic model system two En/Spm encoded trans-acting functions, TNPA and TNPD, are required for excision. A biochemical model for transposition is proposed that might also be applicable to other transposable elements.     

Regulation of CD3- lymphocyte function with an antibody against the IL-2 beta chain receptor: modulation of NK and LAK activity and production of IFN gamma

To elucidate the role of interleukin 2 (IL-2) activation in CD3- lymphocytes, we examined the ability of monoclonal antibody (MAb) TU27, developed against the IL-2 receptor (IL-2R) p75 protein (IL-2R beta), to block lymphocyte activation with exogenous IL-2, as well as its innate ability to activate lymphocytes as a result of its surface ligand interaction. The binding of the TU27 MAb and the results of 125I-IL-2 cross-linking experiments suggest that the IL-2R beta chain is expressed primarily on CD3-, CD56+ lymphocytes; although the protein was also detected in a small portion of CD3+ cells, its expression appeared to be donor dependent. In the present study, we found that TU27 totally blocked natural killer (NK) cell activation in a 4-h assay but had no effect on basal levels of NK activity. When treatment was extended to 24 to 72 h, the MAb was able to block the induction of both NK and lymphokine-activated killer (LAK) activity. Of interest was the observation that MAb treatment alone augmented NK activity and subsequent interferon gamma (IFN gamma) production in CD3- lymphocytes but did not activate LAK activity or induce cell growth. Collectively, these results indicate that TU27 not only reacts with p70-75 IL-2R beta but can abrogate IL-2 binding and subsequent activation events. In addition, some CD3- lymphocyte functions (e.g., NK activity and IFN gamma secretion) are directly induced by the binding of MAb to p70-75 through signals that only partially mimic IL-2.