CTP:phosphocholine cytidylyltransferase activation by oxidized phosphatidylcholines correlates with a decrease in lipid order: a 2H NMR analysis.

The enzyme CTP:phosphocholine cytidylyltransferase (CT) binds reversibly to membranes and is active only in its membrane-bound form. Membrane lipid composition influences the equilibrium between its soluble and membrane-bound forms. Whereas the enzyme is not activated by phosphatidylcholine (PC) vesicles, it is activated by PC vesicles that have been oxidized with HClO(4) [Drobnies, A. E., et al. (1998) Biochim. Biophys. Acta 1393, 90-98]. Here we explore the mechanism of activation of CT by a PC oxidized with lipoxidase. Multilamellar vesicles (MLVs) containing > or =5 mol % oxidized 1-palmitoyl-2-arachidonoylPC (PAPC) progressively activated the enzyme, which was fully activated by 25 mol % oxidized PC. The effect of oxidized PAPC on lipid order was investigated by (2)H NMR, using MLVs containing PAPC perdeuterated on the palmitoyl chain. Spectral depaking generated order parameter profiles along the sn-1 chain. The average order parameter (S(CD)) in the plateau region at 37 degrees C decreased from 0.18 to 0.15 with increasing percent of oxidized PAPC (0-25%). The change in S(CD) was even greater near the end of the palmitoyl chain. CT activation was inversely related to lipid order. The major component of the lipoxidase-oxidized PAPC was purified and characterized by mass spectrometry and NMR. This component, 1-palmitoyl-2-(11,15-dihydroxy)eicosatrienoylPC (dihydroxyPAPC), incorporated into PAPC MLVs, also stimulated CT activity and reduced the lipid order parameter. Both effects were reversed by egg sphingomyelin. We propose that CT activation by oxidized PAPC is mediated by effects on lipid packing perturbations. This is the first study to report the effects of a purified oxidized PC on the orientational order along the acyl chain and to correlate the lipid disordering of the oxidized PC with the activation of a membrane-associated regulatory enzyme.

Shuttling of CTP:Phosphocholine cytidylyltransferase between the nucleus and endoplasmic reticulum accompanies the wave of phosphatidylcholine synthesis during the G(0) --> G(1) transition.

The transition from quiescence (G(0)) into the cell division cycle is marked by accelerated phospholipid turnover. We examined the rates of phosphatidylcholine (PC) synthesis and the activity, membrane affinity, and intracellular localization of the rate-limiting enzyme in the synthesis of PC, CTP:phosphocholine cytidylyltransferase (CT) during this transition. The addition of serum to quiescent IIC9 fibroblasts resulted in a wave of PC synthesis beginning at approximately 10 min, peaking at approximately 3 h with a >10-fold increase in rate, and declining to near basal rates by 10 h. CT activity, monitored in situ, was elevated approximately 3-fold between 1 and 2 h postserum. Neither CT mass nor its phosphorylation state changed during the surge in PC synthesis and CT activity. On the other hand, the ratio of particulate/soluble CT surged and then receded in concert with the wave of PC synthesis. During quiescence, CT was confined to the nucleus, as assessed by indirect immunofluorescence. Within 10 min after serum stimulation, a portion of the CT fluorescence appeared in the cytoplasm, where it intensified until approximately 4 h postserum. Thereafter, the cytoplasmic CT signal waned, while the nuclear signal increased, and by 8 h CT was once again predominantly nuclear. The dynamics of CT's apparent translocation in and out of the nucleus paralleled the wave of PC synthesis and the solubility changes of CT. Cytoplasmic CT co-localized with BiP, a resident endoplasmic reticulum protein, in a double labeling experiment. These data suggest that the wave of PC synthesis that accompanies the G(0) --> G(1) transition is regulated by the coordinated changes in CT activity, membrane affinity, and intracellular distribution. We describe for the first time a redistribution of CT from the nucleus to the ER that correlates with an activation of the enzyme. We propose that this movement is required for the stimulation of PC synthesis during entry into the cell cycle.

Activation of CTP:phosphocholine cytidylyltransferase by hypochlorite-oxidized phosphatidylcholines.

CTP:phosphocholine cytidylyltransferase (CT) catalyzes a rate-limiting, regulatory step in mammalian biosynthesis of phosphocholine (PC). Anionic phospholipids, fatty acids and diacylglycerol activate CT and promote its intercalation into the lipid bilayer, whereas zwitterionic phospholipids such as phosphatidylcholines do not. We investigated the effectiveness of polyunsaturated phosphatidylcholines as CT activators after hypochlorite oxidation. Detection and quantitation of oxidized PCs were evaluated by thin layer chromatography, high performance liquid chromatography, and conjugated dienes. Purified CT was assayed in the presence of multilamellar vesicles, containing variable concentrations of oxidized and parent PCs. The results demonstrate that particular species of oxidized PCs activate CT as potently as anionic lipids. The greater the number of double bonds available for oxidation in the fatty acid at the sn-2 position of the PC, the more effective was the oxidized PC as an activator of CT. Oxidized phospholipids at 1:1 bleach/lipid activated CT in the following order: PAPC>PL3PC>PL2PC compared to unoxidized controls. Since oxidized phospholipids decrease bilayer order (M.L. Wratten et al., Biochemistry 31 (1992) 10901-10907) these results are consistent with the activation of CT by perturbations of lipid bilayer packing.

Alpha-1 antitrypsin protease inhibitor typing in immobilized pH gradients.

Alpha-1 antitrypsin deficiency is a cause of liver disease in neonates and emphysema in adults. Protein phenotypes are identified by isoelectric focusing using polyacrylamide gels. The Pharmacia Phastsystem was utilized for electrophoresis in miniature gels to identify heterozygotes for the deficiency. Protein phenotypes were identified by isoelectric focusing in a fixed pH gradient from 4.3 to 5 using the Pharmacia Phastsystem for automated electrophoresis and staining of gels. The gradient is formed with Immobilines to create gels of dimensions 50 x 43 x 0.5 mm. The processing time for 16 specimens is one hour and 45 minutes. This method is a rapid, automated method for the analysis of alpha-1 antitrypsin phenotypes and for establishing the diagnosis of a genetic deficiency of this protein.

Cation transport properties of a synthetic Ca2+-selective peptide ionophore in phospholipid and sarcoplasmic reticulum vesicles.

Transport by the synthetic cyclic peptide ionophore CYCLEX-2E (Deber, C.M. Young, M.E.M., and Tom-Kun, J. (1980) Biochemistry 19, 6194-6198), which in contrast to Ca2+ ionophore A23187 contains no ionizable protons, has been studied with respect to Ca2+ and Na+ transport, and the involvement of exchanged, or counter-transported ions during the transport process. CYCLEX-2E was found to equilibrate Na+ and Ca2+ gradients across phospholipid vesicle membranes. Experiments using the indicator dye Arsenazo III established that calcium ions were indeed reaching the aqueous intravesicular compartments. Absence of metal cations in the external buffer slowed, but did not eliminate, the efflux of Ca2+ from phosphatidylcholine vesicles. As an example of its activity in a biological membrane, CYCLEX-2E was shown to be capable of producing Ca2+ efflux from sarcoplasmic reticulum vesicles which has been loaded with Ca2+ in an ATP-dependent manner. The overall results suggest that in transport by synthetic peptide ionophores typified by CYCLEX-2E, electroneutrality is achieved either through (a) peptide-mediated compensating (but not coupled) fluxes of other cations, or where this is not an option, by (b) transmembrane diffusion of permeant ions such as H+, OH-, or Cl-.