Photoactivated cross-linking of proteins within the erythrocyte membrane core.

We describe the reactions of three lipophilic, photoactivated cross-linking reagents, 1,5-diazidonapthalene, 4,4'-diazidobiphenyl, and the reversible 4,4'-dithiobisphenylazide, with erythrocyte membranes. Cross-linking occurs only upon photoactivation. At pH 7 to 8, only spectrin components are cross-linked by these reagents. At pH 5.0 to 5.5 several additional membrane proteins including the major "integral" membrane proteins are also cross-linked, despite equivalent binding of the cross-linkers at neutral and acid pH. The cross-linking rates of various membrane proteins at pH 5.0 to 5.5 depend distinctly upon duration of photoactivation. Bidimensional electrophoresis of membrane proteins after cross-linking with the reversible cross-linker, 4,4'-dithiobisphenylazide, has allowed for the identification of homopolymeric products of cross-linking (e.g. dimers and tetramers of Band 3) and heterocomplexes (spectrin plus other membrane proteins). The data suggest that at reduced pH, cross-linking can proceed not only at the membrane surface but also in the membrane core.     

Protein-protein contacts in solubilized membrane proteins, as detected by cross-linking

The amount of detergent required for the solubilization of membrane proteins needs to be optimised as an excess may cause loss of activity and insufficiency may result in poor solubilization or heterogeneous samples. With sarcoplasmic reticulum Ca2+ -ATPase as an example we show by cross-linking that it can be misleading to choose the proper amount of detergent based on clarification of membrane suspensions, because clarification -as detected by turbidity measurements, for instance- precedes full protein solubilization as monomers. We demonstrate that to assess the extent of sample homogeneity at a given detergent/protein ratio, cross-linking followed by HPLC gel filtration in detergent usefully complements cross-linking followed by SDS-PAGE.     

Periodate induced cross-linking of concanavalin A-reactive membrane proteins of rabbit thymocytes.

Purified plasma membranes of rabbit thymocytes are exposed to sodium periodate and galactose oxidase at conditions similar to those used to induce mitogenic transformation of lymphocytes. The membrane proteins are then fractionated by dodecyl sulfate poly-acrylamide gel electrophoresis. At concentrations of 0.005 M, Na IO4 cross-links 55,000 D and 110,000 D glycoproteins which are known to specifically bind concanavalin A. Galactose oxidase has a similar cross-linking effect, but, at the same time causes proteolytic degradation of membrane proteins. Our data indicate that oxidizing agents, like NaIO4 and galactose oxidase, can indeed cross-link receptors of the thymocyte plasma membrane as has often been proposed as a possible mechanism of their action.     

Effects of the calcium-mediated enzymatic cross-linking of membrane proteins on cellular deformability

Summary Excess calcium binding affects the shape and dynamics of cellular deformation of human erythrocytes. It may be hypothesized that incorporation of calcium may modify cellular deformability by processes which include specific cross-linking of membrane proteins with resultant changes in cell shape and deformability. Since previous studies indicate that accumulation of calcium ions causes development of -glutamyl--lysine bridges in membrane proteins, under control of a membrane transamidating enzyme which specifically requires calcium ions for activation, experiments were devised to examine the relationship between cross-linking and deformability and to determine the effects of specific inhibitor of membrane protein cross-linking on the calcium-dependent modification of erythrocyte to the echinocytic shape. The elastic shear modulus of the membrane was not significantly affected by calcium-induced cross-linking, indicating that induced shape change, not altered elasticity, causes the observed reduction in cellular deformability. These findings support the interpretation that Ca⁺⁺-induced and transamidase-catalyzed cross-linking of membrane proteins contributes to fixation of altered cellular shape and decreased cellular deformability.     

Localization of arabinogalactan proteins in the membrane system of etiolated hypocotyls of Phaseolus vulgaris L.

The subcellular distribution of arabinogalactan protein (AGP) in etiolated bean hypocotyls was studied by isopycnic density centrifugation on sucrose gradients at different Mg²⁺ concentrations. The distribution of hydroxyproline (a major amino acid in AGP) in the membrane-containing fractions indicated that hydroxyproline-containing proteins were associated with rough endoplasmic reticulum, possibly with the Golgi apparatus, and with the plasma membrane. Non-specific binding of hydroxyproline-containing molecules to membranes could be excluded. To detect AGPs, fractions obtained after isopycnic density centrifugation were isoelectrofocused on polyacrylamide gels, and the gels were stained with β-Gal-Yariv reagent. Bands appeared only at low pH values, where also most hydroxyproline was found. In the fractions at low densities (presumably membranefree), several bands were visible supporting the idea of the heterogeneous character of soluble AGP. The distribution of AGP in the membranous fractions strongly indicated that AGP was associated with the plasma membrane. Specific agglutination of protoplasts in the presence of β-Gal-Yariv reagent indicated that AGP was exposed at the outside of the cell membrane.     

Reduced transglutaminase-catalyzed cross-linking of exogenous amines to membrane proteins in sickle erythrocytes

In order to determine the capacity of sickle cells to undergo transglutaminase-catalyzed cross-linking of membrane proteins, human normal and sickle erythrocytes were incubated with [ring-2-14C]histamine in the presence of Ca2+ and ionophore A23187. The [14C]histamine incorporation into membrane components was observed in freshly prepared erythrocytes. Incorporation of radioactivity into spectrin and Band 3 membrane components was significantly (P less than 0.001) less in sickle erythrocytes than in normal cells. Transglutaminase deficiency was excluded by the finding of increased activity of this enzyme in sickle cells from patients with reticulocytosis. The incorporation of [3H]spermine into red cell membranes was also less in sickle erythrocytes than in normal cells under the same conditions of incubation used for [ring-2-14C]histamine. Sickle erythrocytes were more permeable to these amines than normal cells. It is proposed that the gamma-glutamyl sites of membrane proteins in sickle erythrocytes are less accessible for transglutaminase-catalyzed cross-linking to histamine and polyamines in vitro, perhaps due to prior in vivo activation of this enzyme by the increased calcium in sickle cells and/or shielding secondary to altered membrane organization.     

Expression profiling of lymphocyte plasma membrane proteins

The physicochemical properties of plasma membrane proteins of mammalian cells render them refractory to systematic analysis by two-dimensional electrophoresis. We have therefore used in vivo cell surface labeling with a water-soluble biotinylation reagent, followed by cell lysis and membrane purification, prior to affinity capture of biotinylated proteins. Purified membrane proteins were then separated by solution-phase isoelectric focusing and SDS-PAGE and identified by high-pressure liquid chromatography electrospray/tandem mass spectrometry. Using this approach, we identified 42 plasma membrane proteins from a murine T cell hybridoma and 46 from unfractionated primary murine splenocytes. These included three unexpected proteins; nicastrin, osteoclast inhibitory lectin, and a transmembrane domain-containing hypothetical protein of 11.4 kDa. Following stimulation of murine splenocytes with phorbol ester and calcium ionophore, we observed differences in expression of CD69, major histocompatibility complex class II molecules, the glucocorticoid-induced TNF receptor family-related gene product, and surface immunoglobulin M and D that were subsequently confirmed by Western blot or flow cytometric analysis. This approach offers a generic and powerful strategy for investigating differential expression of surface proteins in many cell types under varying environmental and pathophysiological conditions.     

Proteomic mapping of brain plasma membrane proteins

Proteomics is potentially a powerful technology for elucidating brain function and neurodegenerative diseases. So far, the brain proteome has generally been analyzed by two-dimensional gel electrophoresis, which usually leads to the complete absence of membrane proteins. We describe a proteomic approach for profiling of plasma membrane proteins from mouse brain. The procedure consists of a novel method for extraction and fractionation of membranes, on-membrane digestion, diagonal separation of peptides, and high-sensitivity analysis by advanced MS. Breaking with the classical plasma membrane fractionation approach, membranes are isolated without cell compartment isolation, by stepwise depletion of nonmembrane molecules from entire tissue homogenate by high-salt, carbonate, and urea washes followed by treatment of the membranes with sublytic concentrations of digitonin. Plasma membrane is further enriched by of density gradient fractionation and protein digested on-membrane by endoproteinase Lys-C. Released peptides are separated, fractions digested by trypsin, and analyzed by LC-MS/MS. In single experiments, the developed technology enabled identification of 862 proteins from 150 mg of mouse brain cortex. Further development and miniaturization allowed analysis of 15 mg of hippocampus, revealing 1,685 proteins. More that 60% of the identified proteins are membrane proteins, including several classes of ion channels and neurotransmitter receptors. Our work now allows in-depth study of brain membrane proteomes, such as of mouse models of neurological disease.     

Carrot arabinogalactan proteins are interlinked with pectins

Cell wall extracts from a carrot cell culture and tap roots were obtained by sequential extraction with water, EDTA buffer solution and cold sodium hydroxide solution. Arabinogalactan proteins (AGPs) were isolated from the extracts and from the medium of the cell culture and analysed for their molecular weight distribution and carbohydrate composition. Copper ions were used to separate the Yariv positive fractions into AGP fractions with a high and a low level of galacturonic acid (GalA). The GalA rich AGP fractions were incubated with pectin methylesterase and polygalacturonase. This enzyme incubation released GalA fragments from the AGP fractions as monitored by HPAEC and MALDI-TOF MS. At least part of carrot AGPs from the medium and cell walls may be covalently linked to pectin containing a homogalacturonan structural element.     

Phototoxicity of phenothiazine derivatives. II. Photosensitized cross-linking of erythrocyte membrane proteins

Irradiation in the presence of O₂, with near-UV light of five promazine (PZ) derivatives added to erythrocyte ghost membranes, causes covalent cross-linking between proteins as revealed by a progressive decrease in the amounts of proteins separable by electrophoresis after denaturation. The induction of cross-links in the two spectrin subunits is a single-hit process as a function of the irradiation time; relatively the rate constants (in min^?1) of the photoreactions were 0.060 with chlorpromazine (CPZ), 0.039 with methoxypromazine (MTPZ), 0.031 with PZ, 0.029 with triflupromazine (TFPZ) and 0.006 with acepromazine (ACPZ).A main photochemical intermediate implicated in the spectrin aggregation seems to be the cation radical of the PZ derivatives. Indeed, (i) the chemically generated cation radicals can induce the reaction in the dark; (ii) the photoaggregation is regularly reduced upon addition of increasing concentrations of NaN₃; (iii) NaN₃ similarly affects the amount of cross-links induced by the isolated cation radicals. Hydroxyl radicals are also involved in the photocross-linking when the reaction is initiated only by MTPZ and not by the other sensitizers.In the absence of oxygen during irradiation, PZ, MTPZ and ACPZ completely loose their cross-linking activities whereas CPZ and TFPZ remain as efficient as in the presence of oxygen.     

Cerebellar plasma membrane proteins and their antisera

Plasma membranes have been isolated from neonatal through adult cerebella by a sequence of differential centrifugation, aqueous two-phase polymer fractionation and density gradient centrifugation. The protein composition of cerebellar membranes from various aged mice was compared by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Increases in the relative amount of membrane proteins with molecular weights (X 10(-3)) of 400, 340, 270, 220, 54, 44, and 9.5 were most pronounced, while a protein of 66,000 Mr disappeared between birth and Day 25. The relationship of these proteins and others to specific cell types in the cerebellum was examined by preparing membrane fractions from isolated granule and Purkinje cells, as well as from the cerebella of neurological mutant mice: reeler, weaver, staggerer, and nervous. In addition, those membrane proteins on the surface of dissociated cerebellar cells were identified by lactoperoxidase-catalyzed iodination, while glycoproteins were identified by galactose oxidase treatment and NaB3H4 reduction. Rabbit antisera were prepared toward those SDS-PAGE membrane proteins which appeared cell specific or developmentally regulated. Sera from these rabbits were used with indirect immunoperoxidase and immunofluorescence to stain frozen sections of mouse cerebellum and dissociated cerebellar cell cultures. In tissue sections antiserum toward the 400,000 Mr protein (p400) and antiserum p14.7 gave strong reactions with Purkinje cells while anti-p130 reacted preferentially with Purkinje cell somas, anti-p220 stained small cells in the internal granule layer and anti-p30 displayed a coarse, grainy staining of the granule and molecular layers, characteristic of a synaptic localization. Only anti-p220 and anti-p130 bound to freshly dissociated cells or cultured cerebellar cells. Large phase-bright cells in the cultures bound antiserum p130. Anti-p220 reacted specifically with a subpopulation of small round viable cells that bound tetanus toxin and decreased in number from 9% at Day 3 to 0.5% of the cells by Day 11, suggestive of granule neurons.     

Characterization of auxin-binding proteins from zucchini plasma membrane

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948–4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N₃-7-³H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or mutimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may possess transporter or channel function.Abbreviations HPLC high-pressure liquid chromatography - IAA indole-3-acetic acid - azido-IAA 5-N₃-7-³H-IAA - pI isoelectric point - PM plasma membrane - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We thank R. Hopkins and I. Gelford for excellent technical work and our colleagues, especially T. Wolpert and D.L. Rayle, for many helpful discussions. This work was supported by grants to T.L.L. from National Science Foundation (DCB 8904114), National Aeronautics and Space Administration (NAGW 1253) and by a grant to D.L. Rayle and T.L.L. from U.S. Department of Agriculture (90-37261-5779). G.R.H. is supported by a National Aeronautics and Space Administration Predoctoral Fellowship (NGT 50455).     

Developmental regulation of Dictyostelium discoideum plasma membrane proteins

Developmental changes in the plasma membrane proteins of Dictyostelium discoideum have been studied using metabolic labeling with [35S]methionine and two-dimensional electrophoresis. Pulse labeling for 1 h at the early interphase, late interphase, aggregation, and tip formation stages of development showed that the profile of newly synthesized plasma membrane proteins changed dramatically over this interval. Only 14% of the polypeptide species were synthesized at all four stages at detectable levels; 86% of the species changed over this developmental interval according to the criterion that they were synthesized at some but not all of the four stages tested. Long-term labeling during vegetative growth followed by initiation of development showed that the "steady-state" levels of the plasma membrane proteins changed very little over the same period. The only changes were in minor species (33% overall change). Similar analyses of whole cell proteins showed 27 and 20% change, respectively. Cell surface radioiodination revealed 52 external proteins in the plasma membrane. Comparison with the uniform methionine labeling results showed that these proteins were, with one notable exception, minor membrane components. In these external proteins, also, developmental changes were limited and were observed in the less abundant species. These results demonstrate the existence of two general classes of plasma membrane proteins. The first is a population of high-abundance proteins that are present in vegetative cells and are largely conserved through development. These possibly serve "housekeeping" functions common to all stages. The second class consists of low-abundance species that are expressed in a highly stage-specific manner and which presumably participate in developmentally important functions.     

Proteomic analysis of rat hippocampal plasma membrane: characterization of potential neuronal-specific plasma membrane proteins

The hippocampus is a distinct brain structure that is crucial in memory storage and retrieval. To identify comprehensively proteins of hippocampal plasma membrane (PM) and detect the neuronal-specific PM proteins, we performed a proteomic analysis of rat hippocampus PM using the following three technical strategies. First, proteins of the PM were purified by differential and density-gradient centrifugation from hippocampal tissue and separated by one-dimensional electophoresis, digested with trypsin and analyzed by electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) tandem mass spectrometry (MS/MS). Second, the tryptic peptide mixture from PMs purified from hippocampal tissue using the centrifugation method was analyzed by liquid chromatography ion-trap ESI-MS/MS. Finally, the PM proteins from primary hippocampal neurons purified by a biotin-directed affinity technique were separated by one-dimensional electrophoresis, digested with trypsin and analyzed by ESI-Q-TOF-MS/MS. A total of 345, 452 and 336 non-redundant proteins were identified by each technical procedure respectively. There was a total of 867 non-redundant protein entries, of which 64.9% are integral membrane or membrane-associated proteins. One hundred and eighty-one proteins were detected only in the primary neurons and could be regarded as neuronal PM marker candidates. We also found some hypothetical proteins with no functional annotations that were first found in the hippocampal PM. This work will pave the way for further elucidation of the mechanisms of hippocampal function.     

Class III pistil-specific extensin-like proteins from tobacco have characteristics of arabinogalactan proteins

Class III pistil-specific extensin-like proteins (PELPIII) are specifically localized in the intercellular matrix of tobacco (Nicotiana tabacum) styles. After pollination the majority of PELPIII are translocated into the callosic layer and the callose plugs of the pollen tubes, which could suggest a function of PELPIII in pollen tube growth. PELPIII may represent one of the chemical and/or physical factors from the female sporophytic tissue that contributes to the difference between in vivo and in vitro pollen tube growth. PELPIII glycoproteins were purified and biochemically characterized. Because of their high proline (Pro) and hydroxy-Pro (Hyp) content, PELPIII proteins belong to the class of Pro/Hyp-rich glycoproteins. The carbohydrate moiety of PELPIII is attached through O-glycosidic linkages and comprises more than one-half the total glycoprotein. Deglycosylation of PELPIII revealed two backbones, both reacting with PELPIII-specific antibodies. N-terminal amino acid sequencing of these backbones showed that PELPIII is encoded by the MG14 and MG15 genes. Two heterogeneous N-terminal sequences of MG14 and MG15, both starting downstream of the predicted signal peptide cleavage site, seem to be present, which indicates a novel N-terminal processing. Monosaccharide analysis showed that the carbohydrate moiety of PELPIII almost completely consists of arabinose and galactose in an equal molar ratio. Carbohydrate linkage analysis showed terminal and 2-linked arabinofuranosyl residues, as well as terminal and 6-, 3-, and 3,6-linked galactopyranosyl residues to be present, indicating the presence of both extensin-like and Type II arabinogalactan oligosaccharide units. The ability of beta-glucosyl Yariv reagent to bind with PELPIII confirmed the arabinogalactan protein-like characteristics of these proteins.