Differences in stability among the human apolipoprotein E isoforms determined by the amino-terminal domain.

Denaturation by guanidine-HCl, urea, or heating was performed on the common isoforms of human apolipoprotein (apo) E (apoE2, apoE3, and apoE4) and their 22-kDa and 10-kDa fragments in order to investigate the effects of the cysteine/arginine interchanges at residues 112 and 158. Previous physical characterization of apoE3 established that apoE contains two domains, the 10-kDa carboxyl-terminal and 22-kDa amino-terminal domains, which unfold independently and exhibit large differences in stability. However, the physical properties of apoE2, apoE3, and apoE4 have not been compared before. Analysis by circular dichroism showed that the different isoforms have identical alpha-helical contents and guanidine-HCl denaturation confirmed that the two domains unfold independently in all three isoforms. However, guanidine-HCl, urea, and thermal denaturation showed differences in stability among the 22-kDa amino-terminal fragments of the apoE isoforms (apoE4 < apoE3 < apoE2). Furthermore, guanidine-HCl denaturation monitored by circular dichroism and fluorescence suggested the presence of a folding intermediate in apoE, most prominently in apoE4. Thus, these studies reveal that the major isoforms of apoE, which are associated with different pathological consequences, exhibit significant differences in stability.

Conformational reorganization of the four-helix bundle of human apolipoprotein E in binding to phospholipid.

Conformational reorganization of the amino-terminal four-helix bundle (22-kDa fragment) of apolipoprotein E (apoE) in binding to the phospholipid dimyristoylphosphatidylcholine (DMPC) to form discoidal particles was investigated by introducing single, double, and triple interhelical disulfide bonds to restrict the opening of the bundle. Interaction of apoE with DMPC was assessed by vesicle disruption, turbidimetric clearing, and gel filtration assays. The results indicate that the formation of apoE.DMPC discoidal particles occurs in a series of steps. A triple disulfide mutant, in which all four helices were tethered, did not form complexes but could release encapsulated 5-(6)-carboxylfluorescein from DMPC vesicles, indicating that the initial interaction does not involve major reorganization of the helical bundle. Initial interaction is followed by the opening of the four-helix bundle to expose the hydrophobic faces of the amphipathic helices. In this step, helices 1 and 2 and helices 3 and 4 preferentially remain paired, since these disulfide-linked mutants bound to DMPC in a manner similar to that of the 22-kDa fragment of apoE4. In contrast, mutants in which helices 2 and 3 and/or helices 1 and 4 paired bound poorly to DMPC. However, all single and double helical pairings resulted in the formation of larger discs than were formed by the 22-kDa fragment, indicating that further reorganization of the helices occurs following the initial opening of the four-helix bundle in which the protein assumes its final lipid-bound conformation. In support of this rearrangement, reducing the disulfide bonds converted the large disulfide mutant discs to normal size.

Effect of arginine 172 on the binding of apolipoprotein E to the low density lipoprotein receptor.

The region of apolipoprotein E (apoE) that interacts directly with the low density lipoprotein (LDL) receptor lies in the vicinity of residues 136-150, where lysine and arginine residues are crucial for full binding activity. However, defective binding of carboxyl-terminal truncations of apoE3 has suggested that residues in the vicinity of 170-183 are also important. To characterize and define the role of this region in LDL receptor binding, we created either mutants of apoE in which this region was deleted or in which arginine residues within this region were sequentially changed to alanine. Deletion of residues 167-185 reduced binding activity (15% of apoE3), and elimination of arginines at positions 167, 172, 178, and 180 revealed that only position 172 affected binding activity (2% of apoE3). Substitution of lysine for Arg(172) reduced binding activity to 6%, indicating a specific requirement for arginine at this position. The higher binding activity of the Delta167-185 mutant relative to the Arg(172) mutant (15% versus 2%) is explained by the fact that arginine residues at positions 189 and 191 are shifted in the deletion mutant into positions equivalent to 170 and 172 in the intact protein. Mutation of these residues and modeling the region around these residues suggested that the influence of Arg(172) on receptor binding activity may be determined by its orientation at a lipid surface. Thus, the association of apoE with phospholipids allows Arg(172) to interact directly with the LDL receptor or with other residues in apoE to promote its receptor-active conformation.

Domains determining agonist selectivity in chimaeric VIP2 (VPAC2)/PACAP (PAC1) receptors.

1 The VPAC2 and PAC1 receptors are closely related members of the Group II G protein-coupled receptor family. At the VPAC2 receptor, VIP is equipotent to PACAP-38 in stimulating cyclic AMP production, whereas at the PAC1 receptor PACAP-38 is many fold more potent than VIP. In this study, domains which confer this selectivity were investigated by constructing four chimaeric receptors in which segments of the VPAC2 receptor were exchanged with the corresponding segment from the PAC1 receptor. 2 When expressed in COS 7 cells all the chimaeric receptors bound the common ligand [125I]PACAP-27 and produced cyclic AMP in response to agonists. 3 Relative selectivity for agonists was determined primarily by the amino terminal extracellular domain of the PAC1 receptor and the VPAC2 receptor. The interchange of other domains had little effect on the potency of PACAP-38 or PACAP-27. 4 For chimaeric constructs with a PAC1 receptor amino terminal domain, the substitution of increasing portions of the VPAC2 receptor decreased the potency of VIP yet increased that of helodermin. 5 This suggests that the interaction of VIP/helodermin but not PACAP with the PAC1 receptor may be influenced (and differentially so) by additional receptor domains.

Functional characterization of apolipoprotein E isoforms overexpressed in Escherichia coli.

Apolipoprotein (apo) E plays an important role in lipid metabolism, and the major isoforms of apoE (apoE2, apoE3, and apoE4) have significantly different metabolic effects. Apolipoprotein E4 is associated with a higher risk of both heart disease and Alzheimer's disease (AD). Patients homozygous for apolipoprotein E2 are predisposed to type III hyperlipoproteinemia, and apoE2 may be protective against AD. Structure/function studies have proved to be a useful tool in understanding how the different apoE isoforms result in different pathological consequences. As these studies continue, it is essential to have a reliable method to produce large quantities of apoE and mutants of apoE. We describe here a method of apoE production in Escherichia coli strain BL21(DE3). The cDNA from apoE isoforms was inserted into a pET32a vector with a T7 promoter and a fusion partner (thioredoxin). The T7 promoter results in high expression of an easily purified His-tagged fusion protein. A thrombin recognition site was positioned in the expression vector so that only two novel amino acids (Gly-Ser) are added to the amino terminus of apoE following the removal of thioredoxin. Approximately 20 mg of apoE is obtained from a 1-liter culture. The major isoforms of apoE produced with this system were extensively characterized for their ability to bind the low-density lipoprotein (LDL) receptor, for their characteristic lipid association preferences, and for their stability as measured by guanidine denaturation. The recombinant proteins behaved identically to plasma-derived apoE isoforms.

Molecular cloning and functional expression of the human glycine transporter GlyT2 and chromosomal localisation of the gene in the human genome.

Neurotransmitter transport systems are major targets for therapeutic alterations in synaptic function. We have cloned and sequenced a cDNA encoding the human type 2 glycine transporter GlyT2 from human brain and spinal cord. An open reading frame of 2391 nucleotides encodes a 797 amino acid protein that transports glycine in a Na+/Cl--dependent manner. When stably expressed in CHO cells, human GlyT2 displays a dose-dependent uptake of glycine with an apparent Km of 108 microM. This uptake is not affected by sarcosine at concentrations up to 1 mM. Radiation hybrid analysis mapped the GlyT2 gene to D11S1308 (LOD=8.988) on human chromosome 11p15.1-15.2.

Deconvolution of gel filtration chromatographs of human plasma lipoproteins.

Gel filtration chromatographs of lipoproteins represent a superposition, or convolution, of the intrinsic polydispersity of the solute and the dispersion due to transport phenomena. We describe a deconvolution technique for improving the resolution of gel filtration chromatographs applicable to lipoproteins and other polydisperse solutes. A matrix of spreading functions, characterizing the dispersive properties of the column, was determined by fitting chromatographic data from a series of monodisperse standards with the solution to the transport equations and interpolating between the fit parameters. A successive approximation scheme was used in which a test distribution was incrementally corrected by an amount proportional to the error between the measured chromatograph and that derived from the test distribution. A nonlinear relaxing function was used to constrain the correction term such that the solution remained physically realizable (i.e., nonnegative absorbance) as it evolved. Deconvolved chromatographs of lipoproteins provided resolution of peaks that were obscured by spreading in the original data. The distribution of particle sizes within each fraction was calculated and verified experimentally by further separating the contents of fractions by gradient gel electrophoresis. Our technique, however, provided comparable resolution of the peaks without the additional experimental procedure.

Analysis of sequences important for herpes simplex virus type 1 latency-associated transcript promoter activity during lytic infection of tissue culture cells.

We describe the analysis of the herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) promoter using an HSV-1-based vector system. Sequences under investigation for LAT promoter activity were analysed for their ability to direct chloramphenicol acetyltransferase gene expression, either in transfection assays or following their insertion into an HSV-1 vector from which the endogenous LAT promoter sequences had been removed. The analysis mapped the main determinants of LAT promoter activity during lytic infection of tissue culture cells to a 277 bp region between -279 and -2 relative to the recognized 5' end of the primary 8.3 kb transcript. The LAT promoter constructs behaved similarly in the context of the virus genome and in the plasmid-based transfection assays. Comparison of the relative activities following infection of fibroblast and neuroblastoma cell lines indicates that sequences upstream from -279 are important for LAT promoter activity in neurons.

The rat vasoactive intestinal polypeptide cyclic AMP response element regulates gene transcriptional responses differently in neonatal and adult rat sensory neurons.

We studied the ability of the rat vasoactive intestinal polypeptide (VIP) cyclic AMP responsive element (CRE) to regulate reporter gene expression through a c-fos promoter in rat sensory neurons transfected in culture by plasmid microinjection. The CRE enhanced the synergistic response of the promoter to combined potassium-evoked depolarisation and forskolin treatment in neonatal but not adult rat neurons. This corresponds to endogenous VIP expression which is induced synergistically by the same stimuli in neonatal but not adult rat neurons. We conclude that VIP expression in sensory neurons, which is induced by axotomy in vivo, could be regulated through the CRE.

The VIP2 receptor: molecular characterisation of a cDNA encoding a novel receptor for vasoactive intestinal peptide.

We have cloned and sequenced a cDNA (RPR4) encoding a new member of the secretin/calcitonin/parathyroid hormone (PTH) receptor family. RPR4 was identified by PCR of rat pituitary cDNA, and a full-length clone was isolated from a rat olfactory bulb cDNA library. When RPR4 was functionally expressed in COS 7 cells, cyclic adenosine monophosphate (cAMP) production was stimulated by vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptides (PACAP-38 and PACAP-27) and helodermin, with equal potency. Peptide histidine isoleucine (PHI) and rat growth hormone releasing hormone (rGHRH) also stimulated cAMP production at lower potency. This suggests that RPR4 encodes a novel VIP receptor which we have designated the VIP2 receptor. In situ hybridisation showed that mRNA for this receptor was present mainly in the thalamus, hippocampus and in the suprachiasmatic nucleus.

Molecular cloning and expression of a cDNA encoding a receptor for pituitary adenylate cyclase activating polypeptide (PACAP).

We have cloned and sequenced a novel cDNA (RPR7) encoding a receptor for pituitary adenylate cyclase activating polypeptide (PACAP). RPR7 was identified by PCR of rat pituitary cDNA, and full-length clones were isolated from a rat olfactory bulb cDNA library. When expressed in COS cells, RPR7 was functionally coupled to increases in intracellular cyclic adenosine monophosphate (cAMP) in response to stimulation by PACAP-38, PACAP-27, vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI). The order of potency of these ligands was PACAP-38-PACAP-27 > VIP > PHI, suggesting that the receptor corresponds to the pharmacologically characterised PACAP Type I receptor.

Intravenous adenosine suppresses cardiac release of endothelin after myocardial ischaemia and reperfusion.

OBJECTIVE: Intravenous adenosine decreases infarct size in experimental models of myocardial ischaemia/reperfusion. Ischaemia/reperfusion is associated with a significant increase in cardiac release of endothelin. The effect of cardioprotective doses of adenosine on endothelin release was explored in dogs undergoing 90 min coronary occlusion and 210 min reperfusion. METHODS: Dogs were assigned to intravenous adenosine in a dose of 0.15 mg.kg-1.min-1 (n = 12) or control (n = 11) during the first 150 min reperfusion. Serial endothelin levels were obtained from the coronary sinus and aortic blood and measured by radioimmunoassay. RESULTS: Adenosine significantly reduced infarct size expressed as a percent of the risk region (28.8 6% v 14.4 2%; p = 0.03). A similar increase in aortic and coronary sinus blood endothelin was observed in both groups during temporary occlusion. A significant transcardiac increase in endothelin levels was present in the control group 60 min after reperfusion whereas no increase occurred in the adenosine treated group [control 5.6(SEM 1.9) v adenosine -0.2(1.4) pg.ml-1; p = 0.02]. Similarly, intravenous adenosine tended to prevent the increase in myocardial endothelin production seen in control animals during the early reperfusion period [control 280(146) v adenosine -57(55) pg.min-1; p = 0.05]. Endocardial blood flow in the ischaemic zone 210 min after reperfusion was significantly higher in the adenosine group, at 0.60(0.02) v 0.38(0.02) ml.min-1.g-1; p < 0.05. A significant correlation between endothelin levels, endocardial flow and infarct size was observed in the control group 3 h after reperfusion: r = 0.73, p = 0.02; r = 0.62, p = 0.03 respectively. This relationship was absent in animals treated with adenosine. CONCLUSIONS: Intravenous adenosine suppresses the release of endothelin from the previously ischaemic myocardium during the early reperfusion period. This effect may in part contribute to the improvement by adenosine in postischaemic microcirculatory flow resulting in attenuation of the "no reflow" phenomenon.

Respiratory alkalosis: no effect on blood lactate decline or exercise performance.

It was the purpose of this study to determine the effects of respiratory alkalosis before and after high intensity exercise on recovery blood lactate concentration. Five subjects were studied under three different acid-base conditions before and after 45 s of maximal effort exercise: 1) hyperventilating room air before exercise (Respiratory Alkalosis Before = RALB, 2) hyperventilating room air during recovery (Respiratory Alkalosis After = RALA), and 3) breathing room air normally throughout rest and recovery (Control = C). RALB increased blood pH during rest to 7.65 +/- 0.03 while RALA increased blood pH to 7.57 +/- 0.03 by 40 min of recovery. Neither alkalosis treatment had a significant effect on blood lactate concentration during recovery. The peak lactate values of 12.3 +/- 1.2 mmol.L-1 for C, 11.8 +/- 1.2 mmol.L-1 for RALB, and 10.2 +/- 0.9 mmol.L-1 for RALA were not significantly different, nor were the half-times (t 1/2) for the decline in blood lactate concentration; C = 18.2 min, RALB = 19.3 min, and RALA = 18.2 min. In C, RALB and RALA, the change in base excess from rest to postexercise was greater than the concomitant increase in blood lactate concentration, suggesting the presence of a significant amount of acid in the blood in addition to lactic acid. There was no significant difference in either the total number of cycle revolutions (C = 77 +/- 2, RALB = 77 +/- 1) or power output at 5 s intervals between RALB and C during the 45 s.(ABSTRACT TRUNCATED AT 250 WORDS)