Transmembrane movement of heme

Evidence for CO-heme partitioning into and across lipid bilayers was obtained by kinetic and chromatographic studies. Biphasic time courses were observed when CO-heme was rapidly mixed with unilamellar lipid vesicles in a stopped-flow spectrometer. The initial rapid phase depended linearly on lipid concentration and was assigned to heme partitioning between the external solvent phase and the outer lipid layer of the membranes. The rate of the second, much slower phase was independent of both heme and lipid concentration. The fraction of absorbance change associated with this slower phase increased with increasing heme to lipid ratios and reached a maximum of approximately 45%. A similar slow phase was observed when membrane-bound heme was reacted with apomyoglobin. In the presence of excess globin, all of the CO-heme was extracted from the membranes to form native CO myoglobin. Under these conditions, the fractional amount of absorbance change associated with the slow dissociation phase was approximately 45%, regardless of the heme to lipid ratio. These results suggest strongly that the slow phases represent transmembrane movement of heme, from the outer to the inner lipid layer in the association reactions and from the inner to the outer layer in dissociation reactions. The temperature dependence of the rate of CO-heme binding to the outer lipid layer was markedly different from that of transmembrane movement. The rate of the latter, slower process decreased greatly with increasing acyl chain length, whereas the rate of the initial binding process varied little with vesicle composition, as long as the membranes were examined above their melting temperatures. Finally, the two kinetically distinct bound heme fractions could be isolated directly by column chromatography.     

Redistribution of a major cell surface glycoprotein during cell movement

The distribution in living cells of an 80,000-dalton major cell surface glycoprotein of murine fibroblasts has been studied by use of monoclonal antibodies. The presence of the molecule throughout the plasma membrane and on the substrate attached surface of the cell was demonstrated by immunofluorescence. Cell growth kinetics were not altered and the cells remained motile in the presence of the antibody. The uniform distribution of the direct immunofluorescence stain persisted for long periods (greater than 100 h), which indicates that the fluorescent monoclonal antibodies may be used to trace antigen surface distribution during cell functions. In motile cells, but not G0 or confluent cells, the degree of fluorescent staining decreased toward the leading edge; this gradient increased markedly during the time that the antibody was bound to the cells. However, the gradation was not seen with the lipid probe, dihexadecylindocarbocyanine. The antigen was "patched" only by the application of a second antibody directed to the rat monoclonal antibody and the relationships of these patches to the underlying cytoskeleton were characterized.     

Transmembrane movement of dolichol linked carbohydrates during N-glycoprotein biosynthesis in the endoplasmic reticulum

The process of N-linked glycosylation of secretory proteins is characterized by enzymatic reactions occurring on both sides of the endoplasmic reticulum (ER) membrane. On either side multiple glycosyltransferases participate in the stepwise addition of monosaccharides to core oligosaccharide unit that is attached to the lipid carrier dolichyl pyrophosphate. Cytoplasm-oriented glycosyltransferases use nucleotide-activated sugars as substrates, whereas lumen-oriented transferases that act later in the pathway make use of dolichyl phosphate-linked monosaccharides. The completely assembled core oligosaccharide is transferred to proteins on the lumenal side of the ER. The topological organization of this biosynthetic pathway requires the translocation of lipid-linked mono- and oligo-saccharides across the ER membrane. The transfer of the substrates and intermediates depend on specific translocators, i.e. so called flippases.     

Epithelial sheet movement: effects of tunicamycin on migration and glycoprotein synthesis

Corneas with central epithelial wounds, 3 mm in diameter, were organ cultured in the presence of tunicamycin (TM) (1 microgram/ml), an antibiotic that inhibits glycosylation of asparagine-linked glycoproteins. Compared with control corneas, which healed in 22 hr, corneas cultured in the presence of TM for the entire culture time or for only the first 6 hr displayed a progressively slower epithelial healing rate that essentially dropped to zero by 24 hr of culture time. At 24 hr, approximately 75% of the wound was covered. After repeated washings with TM-free culture media (6X, 10 min each), this effect could consistently be reversed in corneas exposed to TM for 6 hr. Incorporation of [3H]glucosamine into trichloroacetic acid-precipitable proteins of migrating epithelial sheets was reduced to 14% that of controls after 12 hr of culture with TM, whereas [14C]leucine incorporation was not significantly affected. The decreased glycosylation was reflected on the cell surface after 12 and 20 hr culture in the presence of TM: apical cell membranes of the first six cells of the leading edge of the migrating sheet bound significantly fewer ferritin-concanavalin A particles per micrometer of membrane than did controls. These results indicate that synthesis of asparagine-linked glycoproteins is required for continued migration of corneal epithelial sheets. The asparagine-linked glycoproteins that are required for migration probably include cell-surface glycoproteins.     

Transmembrane movement of phosphatidylglycerol and diacylglycerol sulfhydryl analogues

Transmembrane movement of phospholipids is a fundamental step in the process of biological membrane assembly and intracellular lipid sorting. To facilitate study of transmembrane movement, we have synthesized analogues of phosphatidylglycerol and diacylglycerol in which a sulfhydryl group replaces a hydroxyl group in the polar head group. A rapid, continuous assay for the movement of phospholipids across single-walled lipid vesicles was developed that exploits the reactivity of these analogues toward 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a nonpenetrating, colorimetric, sulfhydryl reagent. In the reaction of DTNB with vesicles containing phosphatidylthioglycerol, a phosphatidylglycerol analogue, two kinetic phases were seen, which represent the reaction of DTNB with phosphatidylthioglycerol in the outer and inner leaflets of the bilayer. Analysis of the slow second phase indicated that the half-time for phosphatidylthioglycerol transbilayer movement was in excess of 8 days. In a similar experiment using dioleoylthioglycerol, a diacylglycerol analogue, the reaction was complete within 15 s. The large difference in translocation rates between these two lipids indicates that the primary barrier to transmembrane movement is the polar head group and implies that phospholipid translocation events in biological membranes may not be unlike those for molecules similar to the polar head groups alone.     

Control of glycoprotein synthesis

Hen oviduct membranes have been shown to catalyze the transfer of GlcNAc from UDP-GlcNAc to GlcNAc-beta 1-2Man alpha 1-6(GlcNAc beta 1-2 Man alpha 1-3) Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X (GnGn) to form the triantennary structure GlcNAc beta 1-2Man alpha 1-6[GlcNAc beta 1-2(GlcNAc beta 1-4)Man alpha 1-3]Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X. The enzyme has been named UDP-GlcNAc:GnGn (GlcNAc to Man alpha 1-3) beta 4-N-acetylglucosaminyltransferase IV (GlcNAc-transferase IV) to distinguish it from three other hen oviduct GlcNAc-transferases designated I, II, and III. Since GlcNAc-transferases III and IV both act on the same substrate, concanavalin A/Sepharose was used to separate the products of the two enzymes. At pH 7.0 and at a Triton X-100 concentration of 0.125% (v/v), GlcNAc-transferase IV activity in hen oviduct membranes is 7 nmol/mg of protein/h. The product was characterized by high resolution proton NMR spectroscopy at 360 MHz and by methylation analysis. In addition to triantennary oligosaccharide, hen oviduct membranes produced about 20% of bisected triantennary material, GlcNAc beta 1-2Man alpha 1-6[GlcNAc beta 1-2(GlcNAc beta 1-4)Man alpha 1-3] [GlcNAc beta 1-4]Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X. Maximal GlcNAc-transferase IV activity requires the presence of both terminal beta 1-2-linked GlcNAc residues in the substrate. Removal of the GlcNAc residue on the Man alpha 1-6 arm or of both GlcNAc residues reduces activity by at least 80%. A Gal beta 1-4GlcNAc disaccharide on the Man alpha 1-6 arm reduces activity by 68% while the presence of this disaccharide on the Man alpha 1-3 arm reduces activity to negligible levels. A similar substrate specificity was found for GlcNAc-transferase III, the enzyme which adds a bisecting GlcNAc in beta 1-4 linkage to the beta-linked Man residue. Since a bisecting GlcNAc was found to prevent GlcNAc-transferase IV action, the bisected triantennary material found in the incubation must have been formed by the sequential action of GlcNAc-transferase IV followed by GlcNAc-transferase III. Activities similar to GlcNAc-transferase IV were also detected in rat liver Golgi-rich membranes (0.4 nmol/mg/h) and pig thyroid microsomes (0.1 nmol/mg/h).     

Transmembrane movement of diether phospholipids in human erythrocytes and human fibroblasts

We have synthesized spin-labeled (SL) and fluorescently labeled diacyl, 1-alkyl-2-acyl-, and di-alkyl glycerophospholipids. The sn-2 chain was a short chain with either a nitroxide group or a 7-nitro-2, 1,3-benzoxadiazol-4-yl (NBD). After incorporation in the exoplasmic leaflet of human erythrocytes, we found that SL-phosphatidylcholine (PC) redistributed very slowly across the plasma membrane, less than 20% reaching the cytoplasmic leaflet in 3 h at 37 degrees C. In contrast, SL-phosphatidylserine (PS) accumulated on the cytoplasmic leaflet with the same plateau corresponding to 90% of the probes inside. The characteristic times for inward redistribution were different for the three PS analogues: at 37 degrees C, the t(1/2) for the diacyl, alkyl-acyl, and dialkyl compounds were 2.3, 3.5, and 41 min, respectively. ATP depletion or incubation with N-ethylmaleimide inhibited the rapid translocation of the PS derivatives. The diether PS bearing an NBD group translocated very slowly in human erythrocytes and no acceleration by ATP could be measured. On the other hand, in human fibroblasts, the diether NBD-PS and SL-PS were both transported from the exoplasmic to the cytoplasmic monolayer of the plasma membrane as it is the case for the transport of the respective diester PS analogues. These results prove that the ether bonds do not prevent completely PS binding and translocation by the aminophospholipid translocase despite a probable hindrance due to the ether linkage on the sn-2 chain. Because of the high stability of the ether linkage, SL and NBD diether analogues should be useful to investigate lipid traffic in cultured cells.     

Transmembrane location of oligosaccharide-lipid synthesis in microsomal vesicles

The oligosaccharide-lipid which is the precursor of asparagine-linked oligosaccharides of eucaryotic glycoproteins is synthesized from sugar nucleotides in the endoplasmic reticulum. The transmembrane location of the assembly of this oligosaccharide-lipid has been studied in vitro in rat liver microsomes. Protease treatment of these sealed vesicles which are derived from the endoplasmic reticulum resulted in the inactivation of a number of enzymes of oligosaccharide-lipid synthesis. Three early steps, the synthesis of dolichol--phosphate--mannose, of dolichol--phosphate--glucose and of dolichol--pyrophosphoryl--di--N--acetylchitobiose, as well as the final steps, the addition of glucose residues to oligosaccharide-lipid, were inactivated under conditions where only the cytoplasmic side of the membrane was accessible to protease. This finding, and the fact that no activities were latent to protease in intact microsomal vesicles, suggest that oligosaccharide-lipid is assembled on the cytoplasmic side of the microsomal membrane. However, the possibility of enzymes spanning the bilayer with their active sites facing the lumen cannot be ruled out. These results are discussed in relation to the segregation of newly made glycoprotein products within the lumen of the endoplasmic reticulum.     

Transmembrane calcium fluxes during Al stress

The primary Al lesion is suggested to be blockage of the root plasma membrane Ca²⁺ channels. Resulting decrease in net Ca²⁺ uptake into the root tip cells leads to Ca²⁺ deficiency in the cytoplasm and disturbance of the cell Ca²⁺ homeostasis, effects that can deleteriously influence cell structure and function. Contribution of internal Ca²⁺ stores to maintaining cytoplasmic Ca²⁺ concentration at physiological levels is considered to be insufficient in meristematic cells at the root tip. It is suggested that differential blockage of Ca²⁺ channels may be at the core of differential tolerance to Al, opening up the possibility of manipulating Al tolerance at the molecular level.     

A rat serum glycoprotein whose synthesis rate increases greatly during inflammation.

Crossed immunoelectrophoresis of rat serum demonstrated considerably increased serum concentrations of at least ten different proteins during turpentine-induced inflammation. One protein, which moved during electrophoresis like an alpha 1 globulin, showed a particularly large increase. This protein was purified to homogeneity by ammonium sulfate fractionation followed by chromatography on DEAE-cellulose. Sephadex G-100, and concanavalin A-Sepharose, and finally disc electrophoresis in polyacrylamide gel. It has a molecular weight of 56,000 determined by equilibrium ultracentrifugation. An apparent molecular weight of 68,000 was estimated for the reduced protein by electrophoresis in polyacrylamide gel plus sodium dodecyl sulfate, suggesting that the native protein is composed of a single polypeptide chain. It has an E2801%, 1 cm of 5.2, an isoelectric pH of 4.7, and contains 19% carbohydrate. The protein does not inhibit bovine trypsin or chymotrypsin. Its physical properties and amino acid composition distinguish this protein from all other rat serum proteins hitherto characterized. During acute inflammation, induced 25 h previously, rats incorporated 20 times more [14C]leucine into this particular protein than did normal rats. However, incorporation into total serum protein during acute inflammation increased only slightly. Regardless of whether inflammation was induced by surgical injury or by a subcutaneous turpentine injection, within 48 h the serum concentration of this major acute-phase protein rose from the normal value of 0.46 g/liter to a maximum value of 7.2 g/liter, which constituted 10% of the total serum protein.     

Sweetening up yeast glycoprotein synthesis

An engineered yeast strain is capable of carrying out the full range of human protein glycosylation reactions.     

Tunicamycin--an inhibitor of yeast glycoprotein synthesis

Tunicamycin, a glucosamine-containing antibiotic, halted synthesis of the external glycoproteins invertase, acid phosphatase and mannan by yeast protoplasts within 30 min; formation of two intracellular proteins, alpha-glucosidase and alkaline phosphatase, and of glucan continued at the control rate for at least 60–80 min. No accumulation of mannan-free acid phosphatase or invertase was evident in treated cells. Utilization of hexoses and incorporation of ¹⁴C-amino acids into protein were not affected. Incorporation of ³H-glucosamine into trichloroacetic acid-insoluble products was only partially reduced. In yeast tunicamycin acts primarily as an inhibitor of glycoprotein synthesis and not of general glucosamine metabolism.     

Lymphocyte membrane alpha-1-acid glycoprotein: a cellular synthesis during lymphocyte activation

Soluble alpha 1 acid-glycoprotein is considered an "acute phase protein" with an inhibitory effect on lymphocyte activity; it has recently been shown that a lymphocyte modulatory variant of alpha 1 acid-glycoprotein has a positive role on T cell activation. It is not clear whether the presence of this glycoprotein on lymphocyte membranes is due to an endogenous production or to a passive uptake of soluble alpha 1 acid-glycoprotein by its carbohydrate moiety. Our data show an increase of membrane alpha 1 acid-glycoprotein both in peripheral blood lymphocyte and T-enriched lymphocytes after phytohemagglutinin stimulation. Peripheral blood lymphocyte enzymatic treatment by neuraminidase does not affect alpha 1 acid-glycoprotein expression while pronase digestion induces a strong decrease of alpha 1 acid-glycoprotein positive lymphocytes and a resynthesis after phytohemagglutinin stimulation. Furthermore, the presence of alpha 1 acid-glycoprotein was prevalently, found on helper/inducer lymphocytes. These data support the hypothesis of a synthesis of alpha 1 acid-glycoprotein by T lymphocytes during their activation process.     

Radioautographic study of acid glycoprotein synthesis by tracheal and bronchial glands during ontogeny]

The synthesis of acid glycoproteins in the glands of the respiratory pathway has been studied radioautographically by means of sodium sulfate in the cat ontogenesis. 35S-sulfate incorporation into the fetal gland has been stated to increase by the time of delivery. After birth and in young kittens, 35S-sulfate incorporation into the gland decreases. Acid glycoproteins are intensively synthesized by the tracheal and bronchial gland in young and adult animals. In old animals this process is lowered. The synthesis of acid glycoproteins by the tracheal and bronchial glands in ontogenesis changes both quantitatively and qualitatively.     

Transmembrane movement and distribution of cholesterol in the membrane of vesicular stomatitis virus.

The transmembrane movement and distribution of cholesterol in the vesicular stomatitis virus membrane were studied by following the depletion of cholesterol from virions to interacting phospholipid vesicles and by exchange of radiolabeled cholesterol between virions and phospholipid-cholesterol vesicles. The kinetics of the cholesterol exchange or depletion reactions revealed the presence of two exponential rates: a rapid rate, dependent on the vesicle to virus ratio, and a slower rate, independent of the vesicle to virus ratio. The kinetics of cholesterol movement could be best interpreted by a model of the virion membrane considered as a two pool system in which approximately 30% of the cholesterol resides in the outer monolayer and approximately 70% in the inner monolayer. The half-time for equilibration of the two pools was calculated to be 4--6 h and was assumed to represent the time required for transmembrane movement of cholesterol across the bilayer. The initial rate of transfer of cholesterol from virus into vesicles increased when vesicle phospholipids contained more unsaturated and shorter chain fatty acids. Furthermore, the transfer of cholesterol appeared to occur by a collisional mechanism requiring membrane-membrane contact. Interaction with lipid vesicles did not significantly affect the integrity of the virion membrane as assessed by the relative inaccessibility of internal proteins to lactoperoxidase-catalyzed iodination and by the small loss of [3H]amino acid labeled protein from the virus.     

The biosynthesis of oligosaccharide-lipids. Isolation of an oligosaccharide-P-P-lipid acceptor

An oligosaccharide-P-P-lipid has been isolated from porcine liver by extraction with organic solvents and purified by chromatography on silica gel and DEAE-cellulose. The purified oligosaccharide-lipid was shown to contain mannose and N-acetylglucosamine in an approximate ratio of 1:1 and our results suggest that the major oligosaccharide component in the preparation was a tetrasaccharide with the composition (Man)2 (GlcNAc)2. When the oligosaccharide-lipid was incubated with GDP-[14C]mannose and a solubilized enzyme preparation from rabbit liver in the presence of MgCl2, three radioactive products could be isolated. The oligosaccharides in the products could be identified as a penta-, a hexa-, and a heptasaccharide. These products were formed by the stepwise addition of mannose to the growing oligosaccharide chain and GDP-mannose was indicated as the glycosyl donor in each reaction.     

Transmembrane potential responses during HL-60 promyelocyte differentiation

Myeloid cells, including granulocytes and monocyte/macrophages, are important in disease-associated inflammatory reactions. These cells come from a common progenitor, the promyelocyte. The human promyelocytic cell line, HL-60, can be induced to terminally differentiate into granulocytes or monocyte/macrophages in a controlled fashion providing a model to study various aspects of myelomonocytic differentiation. The expression of several ion channels is controlled in HL-60 cells in a differentiation specific pattern. The purpose of this study was to determine if lineage-specific ion channel expression during HL-60 differentiation resulted in differences in functional responses to external stimuli. This was investigated by examining transmembrane potential responses in HL-60 promyelocytes, HL-60-derived polymorphonuclear cells (PMNs), and monocytes to various stimuli using the transmembrane potential sensitive dye, diSBAC₂-(3). Exposure of HL-60 promyelocytes to ionomycin or ATP produced a membrane hyperpolarization. Studies using ion substitutions and ion channel blockers indicate that the hyperpolarization was mediated by K_Ca channels. During HL-60 promyelocyte differentiation to PMNs, the membrane potential response to ionomycin and ATP shifted from a hyperpolarization to a depolarization over 7 days. Conversely, HL-60-derived monocytes exhibited a membrane hyperpolarization in response to ionomycin and ATP. HL-60-derived monocytes also exhibit a Cl⁻ conductance specifically induced by ATP. Lineage-specific expression of ion channels during HL-60 cell differentiation is important in determining the transmembrane potential response of these cells. This may be translated into functional responses of various myelomonocytic cells during disease-associated inflammatory reactions. © 1996 Wiley-Liss, Inc.     

Enzymatic transfer of oligosaccharide from oligosaccharide-lipids to an Asn-Ala-Thr containing heptapeptide

Thyroid rough microsomes catalyzed the transfer of oligosaccharide from previously labeled thyroid [Man-¹⁴C] oligosaccharide-lipids to a synthetic dinitrophenylated heptapeptide containing a sequence Asn-Ala-Thr. The resulting product revealed an N-glycosidic attachment, probably to the asparaginyl residue in this sequence. The reaction which was time-dependent up to 1 h and exhibited an apparent Km of 94 μM for the DNP-heptapeptide was favoured by dimethylsul-foxide and inhibited by EDTA.