Studies on the assembly of apo B-100-containing lipoproteins in HepG2 cells

The relationship between apoB-100 and the membrane of the endoplasmic reticulum (ER) has been studied by a combination of pulse-chase methodology and subcellular fractionation. HepG2 cells were pulse-labeled with [35S]methionine for 3 min and chased with cold methionine for periods between 0 and 20 min. ApoB-100 and albumin, present in the membrane as well as in the luminal content of the ER vesicles, were isolated after each chase period. The results indicated that apoB-100 was cotranslationally bound to the membrane of the ER, and from this membrane-bound form, was transferred to the lumen after a delay of 10-15 min. Albumin was, as could be expected for a typical secretory protein, cotranslationally sequestered in the lumen of the ER. Apo-B-100-containing lipoproteins present in the microsomal lumen were analyzed by ultracentrifugation in a sucrose gradient. ApoB-100 occurred on rounded particles in three density regions: (i) d 1.1065-1.170 g/ml (Fraction I), (ii) d 1.011-1.045 g/ml (Fraction II), and (iii) d less than 1.011 g/ml (Fraction III). Fraction I, isolated from cells cultured in the absence of oleic acid, contained a homogenous population of particles with a mean diameter of approximately 200 A. Fraction I isolated from cells cultured in the presence of oleic acid was slightly more heterogeneous and had a mean diameter of approximately 250 A. Fractions II and III had mean diameters of 300 and 500 A, respectively. Cholesterol esters and triacylglycerol were the quantitatively dominating lipid constituents of all three fractions. Pulse-chase experiments indicated that Fraction I contained the newly assembled lipoproteins. With increasing chase time, the apoB-100 radioactivity was redistributed from Fraction I to Fractions II and III, indicating that Fraction I is converted into Fractions II and III during the intracellular transfer. Particles corresponding to Fractions II and III were by far the most abundant lipoproteins found in the medium. The results presented support the possibility of a sequential assembly of apoB-100-containing lipoproteins.     

Function of neutral endopeptidase on the cell membrane of human neutrophils

Intact human neutrophils hydrolyzed N-formyl-Met-Leu-[3H]Phe (fMLP) and released Leu-[3H]Phe, cleaving 45-50% of the peptide within 20 min at 37 degrees C. The dipeptide after its release was then hydrolyzed to free amino acids by a dipeptidase (EC 3.4.13.11). This activity, present in plasma membrane-enriched fractions of neutrophil lysates, was also inhibited over 90% by phosphoramidon, an inhibitor of neutral endopeptidase (NEP, EC 3.4.24.11). Dithiothreitol and EDTA inhibited the activity to a comparable degree, suggesting the requirement for a heavy metal cofactor. Bestatin and amastatin, inhibitors of aminopeptidases (but not human kidney NEP), did not inhibit the rate of fMLP degradation but prevented the production of free phenylalanine and enhanced the accumulation of Leu-Phe. Of other inhibitors, alpha 1-antitrypsin and alpha 2-macroglobulin slightly enhanced the rate of fMLP hydrolysis by neutrophils, and others tested were ineffective. Rabbit antiserum to homogeneous human kidney NEP reacted specifically with a 100-kDa protein present in sodium dodecyl sulfate-solubilized neutrophils. The Mr of this protein was slightly larger than that of the kidney enzyme in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antiserum incubated with intact cells specifically inhibited the degradation of fMLP over 70%. First, we confirm that NEP present on the plasma membrane cleaves fMLP at the Met-Leu bond; then the dipeptide Leu-Phe is cleaved by a dipeptidase. Finally, inhibition of NEP completely blocks fMLP-mediated chemotaxis. Thus, the enzyme may play an important role in modulating chemotactic responses.     

Phosphoenzymes formed from Mg.ATP and Ca.ATP during pre-steady state kinetics of sarcoplasmic reticulum ATPase

We have investigated here the pre-steady state kinetics of sarcoplasmic reticulum ATPase incubated under conditions where significant amounts of Mg.ATP and Ca.ATP coexist, both of them being substrates for the ATPase. We confirmed that these two substrates are independently hydrolyzed by the ATPase, which thus apparently catalyzes Pi production by two simultaneous and separate pathways. External calcium (or the Ca2+/Mg2+ ratio) determines the extent to which Ca2+ or Mg2+ is bound at the phosphorylation site, while internal calcium controls the rate of processing of both the slow, calcium-containing and the fast, magnesium-containing phosphoenzyme. Time-dependent binding of calcium at the catalytic site is correlated with the observed burst of Pi liberation, which therefore results from reequilibration during pre-steady state of magnesium- and calcium-containing phosphoenzyme pools. Independently of direct exchange of metal at the catalytic site, ADP produced by the hydrolysis reaction contributes to reequilibration of these pools through reversal of phosphorylation by the ATP-ADP exchange pathway.     

A polarized epithelial cell mutant deficient in translocation of UDP-galactose into the Golgi complex

Two lectin-resistant mutants derived from a polarized epithelial cell line have been described (Meiss, H.K., Green, R.F., and Rodriguez-Boulan, E.J. (1982) Mol. Cell. Biol. 2, 1287-1294). One of these mutants, the Madin-Darby canine kidney strain II cell line resistant to Ricinus communis agglutinin (MDCKII-RCAr), has been further characterized, and the biochemical defect leading to its altered phenotype has been determined. MDCKII-RCAr cells are shown to be enriched in cell-surface glycoconjugates bearing terminal N-acetylglucosamine residues by in vitro exogalactosylation and by labeling with fluorescent lectins. Binding assays with a sialic acid-specific lectin reveal a 70-75% reduction in sialylation of cell-surface glycoconjugates. The defect is pleiotropic in nature, affecting glycoproteins as well as glycosphingolipids. Analysis of glycosphingolipids shows a strong reduction of galactose-containing glycosphingolipids. Almost 90% of the glycosphingolipids are identified as glucosyl-ceramide. The mutant is not deficient in galactosyl- and sialytransferase activities. However, Golgi vesicles isolated from MDCKII-RCAr cells translocate UDP-galactose at only 2% of the rate observed for vesicles from wild-type MDCKII cells. The deficiency is specific, because translocation rates of UDP-N-acetylglucosamine and CMP-sialic acid are comparable for vesicles isolated from MDCKII-RCAr cells and wild-type cells. Despite the inability to translocate UDP-galactose into the lumen of the Golgi apparatus, MDCKII-RCAr cells are able to form monolayers with normal apical and basolateral polarity as shown by plasma membrane domain-restricted exogalactosylation.     

Solubilization of the active vasoactive intestinal peptide receptor from human colonic adenocarcinoma cells

The non-ionic detergent n-octyl-beta-D-glucopyranoside was used to solubilize the VIP (vasoactive intestinal peptide) receptor from human colonic adenocarcinoma cell line HT29-D4. The binding of monoiodinated 125I-VIP to the solubilized receptor was specific, time-dependent, and reversible. Scatchard analysis of data obtained from competitive displacement of monoiodinated 125I-VIP by native VIP suggested the presence of two classes of VIP binding sites with Kd values of 0.32 and 46.7 nM. The binding capacities of these two classes were 1.7 x 10(10) and 30.2 x 10(10) sites/mg of proteins, respectively. The solubilized receptor retained the specificity of the human VIP receptor towards the peptides of the VIP/secretin/glucagon family. The order of potency in inhibiting monoiodinated 125I-VIP binding was VIP (IC50 = 1.0 x 10(-9) M) much greater than peptide histidine methionine amide (IC50 = 10(-7) M) greater than growth hormone-releasing factor (IC50 = 3 x 10(-7) M) greater than secretin (IC50 greater than 10(-6) M); glucagon had no effect on VIP binding. The reducing agent dithiothreitol inhibited in a dose-dependent manner the binding of 125I-VIP. Covalent cross-linking experiments between the solubilized receptor and 125I-VIP showed that after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography two major and one minor polypeptides of Mr 67,000, 72,000, and 83,000 were specifically labeled. When analyzed by gel filtration, the n-octyl-beta-D-glucopyranoside-solubilized 125I-VIP-receptor complex was resolved into two major peaks with molecular mass in the range of 60-70 and 270-300 kDa. Thus, the soluble form of the VIP receptor was probably a multimeric complex in which disulfide bonds may play an important role to hold the receptor in an active configuration.     

Purification and characterization of the antisecretory factor: a protein in the central nervous system and in the gut which inhibits intestinal hypersecretion induced by cholera toxin

The antisecretory factors (ASF) are hormone-like proteins which inhibit cholera toxin-induced intestinal hypersecretion. Although ASF concentrations in young control rats were low, those in old control rats and toxin-treated rats were high. Toxin-treated rats had 200 ED50 units/g wet weight of ASF in the pituitary gland, while their intestinal mucosa, bile and milk contained 3, 0.5 and 0.5 units/g. In adult man and in 8-9-month-old pig the pituitary level was about 20 units/g. The isoelectric points of ASF from pig and rat were 4.8 and 5.0, respectively, while the molecular size as determined by gel filtration on Bio-Gel P-150 was the same in both cases (Kav 0.43). The molecular weight as determined by SDS-polyacrylamide gel electrophoresis was 60,000 for ASF from porcine pituitary gland. One ED50 unit of the purified porcine ASF corresponded to about 10(-13) mol (1-5 ng) of protein. There were two different ASF from human pituitary gland: pI 5.2, Kav 0.43; and pI 4.5, Kav 0.6. Since antibodies against porcine ASF failed to neutralize the latter protein, it may be unrelated to porcine ASF; the human pI 5.2-protein and rat ASF were both neutralized, but less effectively than was porcine ASF. All the ASF molecules attached to agarose gel, from which they dissociated again in methyl alpha-D-glucose: porcine and rat ASF were eluted at 0.3-0.9 M methyl alpha-D-glucose, human pI 5.2-ASF at 0.1-0.9 M, and human pI 4.5-ASF at 0.1-1.5 M methyl alpha-D-glucose.     

Use of antibodies specific to defined regions of scorpion alpha-toxin to study its interaction with its receptor site on the sodium channel

Five antibody populations selected by immunoaffinity chromatography for their specificity toward various regions of toxin II of the scorpion Androctonus australis Hector were used to probe the interaction of this protein with its receptor site on the sodium channel. These studies indicate that two antigenic sites, one located around the disulfide bridge 12-63 and one encompassing residues 50-59, are involved in the molecular mechanisms of toxicity neutralization. Fab fragments specific to the region around disulfide bridge 12-63 inhibit binding of the 125I-labeled toxin to its receptor site. Also, these two antigenic regions are inaccessible to their antibodies when the toxin is bound to its receptor site. In contrast, the two other antigenic sites encompassing the only alpha-helix region (residues 23-32) and a beta-turn structure (residues 32-35) are accessible to their respective antibodies when the toxin is bound to its receptor. Together, these data support the recent proposal that a region made of residues that are conserved in the scorpion toxin family is involved in the binding of the toxin to the receptor.     

Permeability alterations and antihaemolysis induced by amphiphiles in human erythrocytes

In an attempt to define the parameters in amphiphilic molecules important for their interaction with the erythrocyte membrane, the effects of cationic, anionic, zwitterionic and nonionic amphiphilic agents (C10-C16) on osmotic fragility and transport of potassium and phosphate in human erythrocytes were studied. All the amphiphiles protected the erythrocytes against hypotonic haemolysis. Half-maximum protection occurred at a concentration which was about 15% of that inducing 50% haemolysis. The concentrations of amphiphiles required to induce protection or haemolysis were related to the length of the alkyl chain in a way indicating that a membrane/aqueous phase partition is the mechanism whereby the amphiphile monomers intercalate into the membrane. At antihaemolytic concentrations all the amphiphiles increased potassium efflux and passive potassium influx. The increase in the fluxes was about the same in both directions through the membrane and there were no clear differences in the effects of the different amphiphilic derivatives at equi-protecting concentrations. Active potassium influx was decreased by cationic, zwitterionic and non-ionic amphiphiles. The ability of the amphiphiles to inhibit the influx was not related to the length of the alkyl chain. Anionic amphiphiles had no or only a weak stimulatory effect on the influx. Phosphate efflux was reduced by all the amphiphiles. The inhibitory potency of the different amphiphiles decreased in the following order; anionic greater than zwitterionic, non-ionic greater than cationic. Short-chained amphiphiles were more potent inhibitors than long-chained. The possible participation of non-bilayer phases (mixed inverted micelles) in the intercalation of amphiphiles into the membrane is discussed.     

Endogenous suppression of neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes

Phospholipase A2 activity was measured in homogenized and acid-extracted human polymorphonuclear leukocytes using [1-14C]oleate-labelled autoclaved Escherichia coli as substrate. In whole homogenate and in the supernatant and particular fractions separated by centrifugation at 150,000 X g, phospholipase activity was barely detectable (1-4 pmol/h per 10(6) cell equivalents). By contrast, acid extracts of these fractions contained over 10-times as much phospholipase activity in the dialyzed supernatants (20-300 pmol/h per 10(6) cell equivalents), whereas phospholipase inhibitor(s) were found in the sediment. The acid-solubilized phospholipase A2 activity was absolutely Ca2+-dependent and optimal at pH 7.0-7.5 with 1.0 mM added Ca2+. Addition of the resuspended sediment of the acid extract dose-dependently suppressed phospholipase activity in the supernatant; less than equivalent amounts were sufficient to inhibit 95%. Suppressor activity was lipid-extractable. After thin layer chromatography of lipid extracts, the bulk of inhibitory activity was recovered from the free fatty acid region. Analysis of the fatty acids by gas liquid chromatography showed that 63% were unsaturated. All unsaturated fatty acids tested were potent inhibitors of phospholipase A2 activity (IC50 3-10 microM). Oleoyl-CoA, hydroxyeicosatetraenoic acids and leukotriene D4 were also inhibitory, while methyl oleate, saturated fatty acids and the prostaglandins E2 and F2 alpha had no effect. These in vitro data indicate that neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes is largely suppressed by endogenous inhibitors and suggest that unsaturated fatty acids and some of their metabolites may partly account for this suppressor activity.     

Effect of decreased ferrochelatase activity on iron and porphyrin content in mitochondria of mice with porphyria induced by griseofulvin

The content of iron and protoporphyrin in liver mitochondria from mice with porphyria induced by griseofulvin was measured. The amount of porphyrin was 0.0076 +/- 0.0043, 4.11 +/- 0.58 and 22.2 +/- 6.8 nmol/mg protein (n = 5) in mitochondria from control animals and animals treated with griseofulvin for 3 days and 4-5 weeks, respectively. The energy coupling of the mitochondria was greatly diminished after 4-5 weeks of treatment, and the ferrochelatase activity was inhibited 80-90%, compared to that of control animals. Mitochondrial preparations isolated by differential centrifugation were contaminated with iron-containing lysosomes which could be removed by Percoll density-gradient centrifugation. In purified mitochondrial preparations no change in the amount of non-heme iron was found after griseofulvin feeding, representing 3.36 +/- 0.15, 3.97 +/- 0.40 and 3.59 +/- 0.23 nmol/mg protein for control animals, 3 days- and 4-5 weeks-treated animals, respectively (n = 4). A mitochondrial iron pool previously identified in rat liver mitochondria and shown to be available for heme synthesis in vitro (Tanger?s, A. (1985) Biochim. Biophys. Acta 843, 199-207) was also present in mitochondria from mice. The magnitude of this iron pool, as well as its availability for heme synthesis, was not changed after treatment of the animals with griseofulvin. The fact that porphyrin, but not iron, accumulated in the mitochondria when ferrochelatase was inhibited is discussed with regard to our understanding of the process of heme synthesis and its regulation.