Structure of polymerizable lipid bilayers. V. Synthesis, bilayer structure and properties of diacetylenic ether and ester lipids.

Four diacetylenic phosphatidylcholines (PC's) have been synthesized and the structures of bilayers of these lipids have been determined at low resolution by low-angle X-ray diffraction. The PC's all have 18-carbon chains but differ with respect to the ether/ester linkage at the sn-1 and sn-2 positions and the relative position of the diacetylene moiety: diester-PC (1): 1,2-bis(octadeca-4',6'-diynoyl)-sn-glycero-3-phosphocholine diester-PC (2): 1-(octadeca-4',6'-diynoyl)-2-(octadeca-5',7'-diynoy l)-sn- glycero-3-phosphocholine diester-PC (3): 1,2-bis(octadeca-8',10'-diynoyl)-sn-glycerol-3-phosphocholin e diether-PC (4): 1-O-(octadeca-4',6'-diynyl)-2-O-(octadeca-5",7"-din yl)-sn- glycero-3-phosphocholine Only (1) exhibits the typical bilayer profile, whereas (2), (3) and (4) show evidence of interdigitation and/or significant disorder. Only (1) polymerized effectively upon illumination with 254 nm light, turning deep blue in seconds, indicating the formation of long, well-ordered polydiacetylenic structures. Liposomes of these derivatives were tested for permeability by osmotic swelling. Polymerized liposomes of (1) underwent osmotic swelling with urea, glycerol, and acetamide more rapidly than did liposomes of stearoyl-oleoyl-PC, but the initial rates of osmotic swelling of polymerized liposomes of (1) were 3-10-times lower than those of unpolymerized liposomes of (1). Blue polymerized multilayer samples of (1) exhibited an irreversible thermochromic transition to red at approx. 40 degrees C. Differential scanning calorimetry with liposome suspensions of (1) revealed an endotherm at 28.3 degrees C with a transition enthalpy of 40 J/g. PC (1) is a potentially useful diacetylenic lipid which exhibits facile, complete polymerization and a bilayer thickness comparable to that of biomembrane lipids.     

Studies on the biosynthesis of tropane alkaloids by Datura stramonium L. transformed root cultures

Transformed root cultures of Datura stramonium, competent in tropane-alkaloid biosynthesis, have been treated with exogenous plant growth regulators. It was found that combinations of -naphthalene-acetic acid, kinetin (N⁶-furfurylaminopurine) and 2,4-dichlorophenoxyacetic acid induced de-differentiation, causing both the rooty phenotype and the hyoscyamine-biosynthetic capacity to be lost. Alkaloid biosynthesis disappeared rapidly and prior to the loss of morphological integrity. It was observed that the enzymes ornithine decarboxylase (EC 4.1.1.17), arginine decarboxylase (EC 4.1.1.19) and N-methylputrescine oxidase did not show the increase in level normally associated with subculturing the roots. The level of putrescine N-methyltransferase (EC 2.1.1.53) activity, the first enzyme fully committed to hyoscyamine biosynthesis, rapidly declined, about 80% being lost from the roots within 12h. This activity, although showing some temporary restoration, declined further after a few days, and was totally absent from fully dispersed cultures. N-Methylputrescine oxidase persisted at a low level. Following sub-culture of established de-differentiated lines to plant-growth-regulator-free medium, limited root regeneration occurred. The roots formed showed renewed competence in alkaloid biosynthesis and putrescine N-methyltransferase and N-methylputrescine oxidase activities were restored to their normal levels. The relationship between the morphological state and alkaloid-biosynthetic capacity of the cultures is discussed in relation to the overall control of alkaloid biosynthesis.Abbreviations ADC arginine decarboxylase - FW fresh weight - MPO N-methylputrescine oxidase - NAA -naphthalineacetic acid - ODC ornithine decarboxylase - pgr plant growth regulator - PMT putrescine N-methyltransferase We are most grateful to Abigael Peerless and Bridget Chapman for assistance with various part of this work.     

The formation of 3 alpha- and 3 beta-acetoxytropanes by Datura stramonium transformed root cultures involves two acetyl-CoA-dependent acyltransferases

Tropine (tropan-3 alpha-ol) is an intermediate in the formation of hyoscyamine. An acyltransferase activity that can acetylate tropine using acetylcoenzyme A as cosubstrate has been found in transformed root cultures of Datura stramonium. A further acyltransferase activity that acetylates pseudotropine (tropan-3 beta-ol) with acetyl-coenzyme A is also present. These two activities can be partially resolved by anion-exchange chromatography, some fractions containing only the pseudotropine-utilizing activity. The basic properties of these two enzymes are reported and their roles in forming the observed alkaloid spectrum of D. stramonium roots discussed.     

Characterization of the carboxyl-terminal sequences responsible for protein retention in the endoplasmic reticulum.

The COOH-terminal sequence KDEL has been shown to be essential for the retention of several proteins in the lumen of the endoplasmic reticulum (Munro S., and Pelham, H. R. B. (1987) Cell 48, 899-907; Pelham, H. R. B. (1988) EMBO J. 7, 913-918; Mazzarella; R. A., Srinivasan, M., Haugejorden, S. M., and Green, M. (1990) J. Biol. Chem. 265, 1092-1101). We have previously demonstrated that variants to the KDEL retention signal, particularly at the initial two positions of the tetrapeptide, can be made without affecting its ability to direct intracellular retention when appended to the neuropeptide Y precursor (pro-NPY) (Andres, D. A., Dickerson, I. M., and Dixon, J. E. (1990) J. Biol. Chem. 265, 5952-5955). To further investigate the nature of the KDEL retention signal, oligonucleotide-directed mutagenesis and transfection was used to generate stable mouse anterior pituitary AtT-20 cell lines expressing pro-NPY mutants with variants of the KDEL sequence added to their direct carboxyl terminus. Analyses of dibasic processing and indirect immunofluorescent microscopy of AtT-20 subclones were consistent with the retention of the pro-NPY mutants bearing the COOH-terminal extensions QDEL, KEDL, or KDEI within the endoplasmic reticulum. A change in the final amino acid of the tetrapeptide from Leu to Val abolished retention completely, and the peptide hormone was processed and secreted. These results indicate that only a limited number of conservative changes can be made to the final two positions of the tetrapeptide without abolishing activity and suggest a highly specific interaction of the retention signal and the KDEL receptor.     

Structure of polymerizable lipid bilayers: water profile of a diacetylenic lipid bilayer using elastic neutron scattering

Elastic neutron scattering experiments have been used to study the hydration of multibilayers of 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC). Previously published FTIR spectroscopic data had suggested, based on shifts in the carbonyl (C = O) stretch frequencies, that the phosphocholine headgroup in these polymerizable lipid bilayers was much less hydrated than that of saturated phosphatidylcholines. Our results demonstrate that the DC8,9PC headgroup is at least as well hydrated as that of dipalmitoylphosphatidylcholine (DPPC), a saturated lipid, under the same conditions.