Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies.

Three peptides corresponding to residues 354-367, 364-374, 373-387 of the acetylcholine receptor (AChR) delta subunit were synthesized. These peptides represent the proposed phosphorylation sites of the cAMP-dependent protein kinase, the tyrosine-specific protein kinase and the calcium/phospholipid-dependent protein kinase respectively. Using these peptides as substrates for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase it was shown that only peptides 354-367 was phosphorylated whereas the other two were not. These results verify the location of the cAMP-dependent protein kinase phosphorylation site within the AChR delta subunit. Antibodies elicited against these peptides reacted with the delta subunit. The antipeptide antibodies and two monoclonal antibodies (7F2, 5.46) specific for the delta subunit were tested for their binding to non-phosphorylated receptor and to receptor phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Antibodies to peptide 354-367 were found to react preferentially with non-phosphorylated receptor whereas the two other anti-peptide antibodies bound equally to phosphorylated and non-phosphorylated receptors. Monoclonal antibody 7F2 reacted preferentially with the phosphorylated form of the receptor whereas monoclonal antibody 5.46 did not distinguish between the two forms.     

Transfer of coreopsis congregata (Compositae: Heliantheae) to coreocarpus

Coreopsis congregata Blake is out of place in the genusCoreopsis. Its achene morphology, pubescence, leaf cutting and geographical range suggest that it would be better treated in the genusCoreocarpus (Coreocarpus congregatus) and that transfer is proposed.     

Aminoacylation of tRNA and Protein Turnover in Nitrateand Sulphate-Deficient Spirodela polyrhiza

Cultures of Spirodela polyrhiza were maintained in completeHoagland's medium at 25°C in continuous light. Nitrate-and sulphate-deficient plants were cultured in media containing1/20 Hoagland's nitrate and 1/200 Hoagland's sulphate respectively.After 10 days of growth the plants were examined for total aminoacyltRNA levels. Turnover of leucyl-tRNA and rates of protein synthesiswere assessed by pulse feeding [³H]leucine. Control and nutrient-deficientplants had similar levels of tRNA-associated amino acids. Howeverthe amounts of tRNA, expressed on a fresh weight basis, weresignificantly lower in nitrate- and sulphate-deficient plants.Although the specific radioactivities of leucyltRNA were highestin deficient cultures the rate of turnover of this pool wasless than non-deficient control or nitrate- and sulphate-supplementedplants. Calculation of the average rate constants for proteinsynthesis and degradation showed that nitrate deficiency, althoughnot affecting rates of synthesis, supported rates of proteindegradation that were higher than control cultures. ¹ Present address: The Biological Laboratories, Harvard University,16 Divinity Avenue, Cambridge, Massachusetts 02138, U.S.A. (Received March 7, 1983; Accepted August 16, 1983)     

Reassessment of Brain Free Fatty Acid Liberation During Global Ischemia and Its Attenuation by Barbiturate Anesthesia

Abstract: We previously reported that whole-brain free fatty acids (FFA) rose almost linearly for up to 1 h after decapitation of unanesthetized rats and was significantly attenuated by pentobarbital anesthesia. However, our values for total FFA and arachidonic, stearic, oleic, and palmitic acids were severalfold higher than those obtained by previous investigators. Based upon the suggestion that this may be due to FFAs released from di- and triglycerides in the quantitation of FFAs, we have now analyzed and improved our procedures for TLC separation of FFA and reassessed the accumulation of FFA in whole brain during decapitation ischemia in unanesthetized and pentobarbital-anesthetized rats. FFA levels in whole brain after 0.5 min of ischemia were one-half to one- fourth the levels previously reported after 1 min of ischemia. The rise in FFA between 0.5 and 60 min of ischemia was 9-fold for total FFA, and between 7 and 12-fold for each of the FFAs quantitated. Pentobarbital significantly attenuated the rise of all FFAs with, however, greater effects on oleic and palmitic acids than previously reported.     

Conformational flexibility of membrane proteins in electric fields. I. Ultraviolet absorbance and light scattering of bacteriorhodopsin in purple membranes

Bacteriorhodopsin of halobacterial purple membranes exhibits conformational flexibility in high electric field pulses (1-30 x 10(5) V m(-1), 1-100 micros). High-field electric dichroism data of purple membrane suspensions indicate two kinetically different structural transitions within the protein; involving a rapid (approximately 1 micros) concerted change in the orientation of both retinal and tyrosine and/or tryptophan side chains concomitant with alterations in the local protein environment of these chromophores. as well as slower changes (approximately 100 micros) of the microenvironment of aromatic amino acid residues concomitant with pK changes in at least two types of proton-binding sites. Light scattering data are consistent with the maintenance of the random distribution of the membrane discs within the short duration of the applied electric fields. The kinetics of the electro-optic signals and the steep dependence of the relaxation amplitudes on the electric field strength suggest a saturable induced-dipole mechanism and a rather large reaction dipole moment of 1.1 x 10(-25) C m ( = 3.3 x 10(4) debye) per cooperative unit at E = 1.3 x 10(5) V m(-1), which is indicative of appreciable cooperativity in the probably unidirectional transversal displacement of ionic groups on the surfaces of and within the bacteriorhodopsin proteins of the membrane lattice. The electro-optic data of bacteriorhodopsin are suggestive of a possibly general, induced-dipole mechanism for electric field-dependent structural changes in membrane transport proteins such as the gating proteins in excitable membranes or the ATP synthetases.     

Conformation and dynamic interactions in hyaluronate solutions

In hyaluronate solutions, the polysaccharide chains behave as random coils with minor but important deviations; (1) there is an extra degree of stiffening that could have its origin in inter-residue hydrogen bonding, (2) chain-chain contact induces the transient development of tertiary and higher levels of structure, which are enhanced by suppression of electrostatic repulsions on increasing ionic strength or lowering pH, melted out on heating, and inhibited by competition with short chain segments. The deviations are important because they account for viscoelastic properties that are believed to be relevant to the role in biological fluids and the intercellular matrix; thus the chain-chain interactions are favoured entropically by the inherent stiffness of isolated segments and contribute appreciably to the dynamic network structure.Viscoelastic and flow properties have been measured over a wide range of conditions using both oscillatory and steady shear techniques, and systematised in terms of recent rheological theory. The onset of intermolecular coupling occurs at an unusually low degree of coil overlap, and shows no systematic variation with pH, molecular weight or ionic strength. At higher concentrations, however, dynamic intermolecular interactions are enhanced by increasing ionic strength and suppression of molecular charge on lowering pH. Breakdown of the resulting transient intermolecular network on shearing is systematised in terms of the generalised shear parameter $\text{β = γ(η}{}_0\text{? η}{}_{\mathrm{<mtext>solvent</mtext>}}\text{) ×}\text{M}\text{cRT}$. The large anomalous decrease in intrinsic viscosity under alkaline conditions that parallels similar effects seen in nuclear magnetic resonance relaxation, small-angle X-ray scattering and viscoelastic behaviour, is ascribed to an increase in chain flexibility, which may result in part from the ionisation of hydroxy groups involved in hydrogen bonding.     

Mathematische beschreibung der reaktion farbempfindlicher nervennetze auf intensitäts- und farbsprünge

The reaction of color sensitive neural networks to intensity and color steps on logarithmic transformation of the input signals is calculated mathematically. The networks consist of opponent-color cells respectively with (duple system 1) or without a surround (duple system 2) or of double opponent-color cells (quadruple system). The output signals are independent of the intensity level. Both duple systems are able to code the color of homogeneous areas on a dichromatic level. The hue corresponds to the sign, the saturation to the absolute value of the output signal. The coding of saturation becomes incorrect at intensity borders only with duple system 1 (due to a Mach band response) at color borders however with duple system 1 and 2 (due to low-pass properties). The quadruple system (like duple system 2) is insensitive to intensity differences. It only responds to color differences, which are transferred according to a band-pass filter. The system therefore is able to function as a detector of color borders. The results are used in a new model for the processing of color and color borders. A linear transformation has been found to be less suited for color coding.     

Glycine synthesis in a Neurospora mutant deficient in serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT), commonly implicatedin the glycine synthesis of eucaryotes, was examined in Neurosporacrassa, wild type (FGSC 853) and a formate-requiring mutant(FGSC 9). The mutant was SHMT-deficient, containing only 15%of the total activity found in the wild type. Differential anddensity gradient centrifugations showed the mutant to be deficientin soluble SHMT activity. Both strains contained particulateSHMT which sedimented with mitochondrial marker enzymes. The origins of glycine were examined by a combination of enzyme,growth and ¹⁴C feeding experiments. Growth of the mutant wasstrongly inhibited by the isocitrate lyase-directed inhibitoritaconate. This inhibition was reduced when exogenous glycinewas supplied. Itaconate (up to 30 mM) did not inhibit growthof the wild type but in both strains isocitrate lyase activitieswere reduced. The mutant contained more lyase and glyoxylateaminotransferase than the wild type. In feeding experiments,[2-¹⁴C]acetate and [l-¹⁴C]glyoxylate were more readily incorporatedinto glycine in the mutant than the wild type. Itaconate (30mM) reduced the flow of acetate carbon into glycine by up to70% in the mutant. It is concluded that deficiency in solubleSHMT necessitates glycine synthesis via an isocitrateglyoxylateglycinesequence. (Received December 24, 1979; )