Application of a new surface labeling reagent, EDTA derivative, on erythrocytes and platelets.

The modes of binding of a new class of impermeant metal-chelating probe, the complex of 111In3+ to 1-(p-benzenediazonium) ethylenediamine tetraacetic acid (azo-phenyl-EDTA), to human and rabbit erythrocyte membranes and the effect of binding on the function of rabbit platelets have been studied. The metal chelate, azo-phenyl-EDTA.[111In3+] bound covalently to membrane proteins following reaction with intact erythrocytes. The amount and the pattern of labeling was assessed by sodium dodecyl sulfate (SDS)-polyacrylamide disc and slab gels for radioactivity. The pattern of labeling of intact human erythrocytes by azo-phenyl-EDTA.[111In3+], by pyridoxal phosphate-NaB3H7 and by galactose oxidase-NaB3H4 was also compared. The following results were obtained: (a) The pattern of labeling of intact human erythrocyte by azo-phenyl-EDTA.[111In3+] differed from other commonly used probes for labeling external membrane surfaces. Five polypeptides were labeled by the metal chelates. In addition to the known major proteins (protein band III, PAS-1, PAS-2 and PAS-3 of Fairbanks et al. (1972) Biochemistry 10, 2606--2617) a protein (radioactive band 4) which migrated slightly slower than PAS-3 in SDS gel was labeled heavily by the metal chelate. This protein has an apparent molecular weight of 37,500 in 8.4% acrylamide-SDS gel. About 40% of bound radioactivity was found in this protein. The diazo linkage of the metal chelate to this protein was found to be especially unstable to heat. (b) In rabbit erythrocyte membranes, the metal chelate bound to three polypeptides with apparent molecular weights of 96,000, 43,000 and 33,000 in 8.4% acrylamide gel. They are probably glycoproteins in nature. (c) The binding of the probe to platelets did not affect the platelet aggregability induced by adenosine diphoshpate. In vivo studies indicated that the labeled platelets accumulated at the plague of atherosclerotic rabbits. (d) The bifunctional analog of EDTA may permit new applications of metals with useful physical properties for studies of cell membranes.     

Characterization of the cilia and ciliary membrane proteins of wild- type Paramecium tetraurelia and a pawn mutant

Cilia and ciliary membranes were isolated from axenically grown, wild- type Paramecium tetraurelia strain 51s and from the extreme pawn mutant strain, d495, derived from this parental strain. Over 60 protein bands having molecular weights of 15 to greater than 300 kdaltons were detected by Coomassie Blue staining of whole cilia proteins separated by one-dimensional SDS polyacrylamide gel electrophoresis. About 30 of these protein bands were visible in Coomassie Blue-stained membrane separations. About 60 bands were detected by silver staining of one- dimensional gels of membrane proteins. Differences between Coomassie Blue-stained separations of wild-type and pawn mutant strain d495 membrane proteins were seen in the quantity of a band present at 43 kdaltons. Radioiodination of cell surface proteins labeled approximately 15 protein bands in both wild-type and mutant cilia. The major axonemal proteins were unlabeled. Six membrane glycoproteins were identified by staining one-dimensional separations with iodinated concanavalin A and lentil lectin, two lectins that specifically bind both glucose and mannose residues. Two major neutral sugar species present in an acid hydrolysate of the cilia preparation were tentatively identified as glucose and mannose by gas chromatography of the alditol acetate derivatives.     

The lectin binding sites on the membranes of the nuclear envelope, mitochondria and the cell surface of human lymphoblastoid cells

The membranes of the cell surface, the endoplasmic reticulum, outer and inner mitochondrial leaflet and nuclear envelope were isolated from three human lymphoblastoid cell lines. Membrane components were separated by dodecyl sulfate polyacrylamide gel electrophoresis and the gels incubated with the radioiodinated lectins from lentil, castor bean, scarlet runner bean, gorse seed and Roman snail. After gel slicing and counting, the molecular weights of the lectin binding sites were determined. About 20 glycoproteins were identified as constituents of the plasma membrane, a similar glycoprotein distribution was observed in the endoplasmic reticulum. The outer mitochondrial membrane contained some impurities from the plasma membrane, the inner mitochondrial membrane lacked specific lectin receptors. Two prominent glycoproteins with molecular weights of 70 000 and 60 000 were identified with the castor bean lectin in the nuclear envelope.     

Biogenesis of erythrocyte membrane proteins. In vitro studies with rabbit reticulocytes.

The capability of rabbit reticulocytes to synthesize red cell membrane proteins has been tested in vitro. Reticulocyte-rich blood from phenylhydrazine-treated rabbits was incubated in vitro in a complete amino acid medium containing ferrous salts, glucose, rabbit plasma and [3-H]leucine. Red cell ghost membranes were prepared by hypotonic lysis and leucine incorporation into hemoglobin and total membrane proteins determined. The pattern of incorporation into individual peptides was determined by polyacrylamide gel electrophoresis of labeled membranes on large (19 mm) gels which were then sliced into 1 mm sections; radioactivity was compared with densitometric tracings of Coomassie blue stained analytical (6 mm) gels. Incorporation of [3-H]leucine into both hemoglobin and membrane protein was linear over 1 h. Gel analysis of labeled membranes revealed that the amino acid was primarily incorporated into peptides with molecular weights of 90 000 or less; three peptides of molecular weights 90 000, 60 000 and 33 000 showed the highest specific activity. Synthesis of the four largest peptide species was negligible. Removable of ferrous salts inhibited synthesis of both globin and membrane protein equally (approx. 50%). However, puromycin and cycloheximide preferentially inhibited the synthesis of globin as compared to membrane proteins. Reticulocytes remain capable of synthesizing a number of membrane proteins; these results are consistent with studies of red cell membrane synthesis in anemic rabbits in vivo.     

Formation, size, and solubility in chloroform/methanol of products of protein synthesis in isolated mitochondria of rat liver and Zajdela hepatoma.

The number, size, solubility in chloroform/methanol and some aspects of the formation of the components labeled by radioactive amino acids in isolated mitochondria of rat liver and Zajdela hepatoma were studied. Isolated mitochondria were labeled with radioactive amino acids under various conditions, and the distribution of radioactivity in sodium dodecylsulfate-polyacrylamide gels after electrophoresis of mitochondrial membrane fraction was analysed. 1. Isolated mitochondria of rat liver and Zajdela hepatoma incroporated radioactive amino acids almost exclusively into the membrane fraction. Electrophoretic analysis of this fraction revealed the presence of 15 distinct peaks of radioactivity with corresponding apparent molecular weights of 10 000 to 58 000. The electrophoretic mobility of the labeled components was identical and the general pattern of the radioactivity distribution in the gel for the rat liver and the tumour mitochondria was very similar. 2. Components of the membrane fraction of rat liver mitochondria labeled in vitro displayed an unequal solubility in acidic (2 mM HC1) chloroform/methanol (2/1) mixture; as detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis a single labeled component with apparent molecular weight of 10 000 was soluble in neutral chloroform/methanol. 3. Inverse relation was observed between amino acid incorporation activity of isolated mitochondria and the portion of the label incorporated into the component with apparent molecular weight 10 000. The identity of this component with that soluble in neutral chloroform/methanol mixture has been indicated. 4. The rate of incorporation of [3H]leucine by isolated Zajdela hepatoma mitochondria into the components with lower (10 000-25 000) apparent molecular weights decreased with time, whereas that into components with higher (above 25 000) apparent molecular weight remained approximately constant within the time interval tested (30 min). 5. From the total radioactivity incorporated into the membrane fraction during 5-min pulse labeling of isolated Zajdela hepatoma mitochondria by [3H]leucine up to 25% was recovered in the region of the gel corresponding to a component with apparent molecular weight 10 000. After 25 min chase the radioactivity in this region decreased about 3.5 times while the specific radioactivity of the total membrane fraction did not change significantly. The pattern of radioactivity distribution observed after the pulse was preserved by chloramphenicol. 6. Unlabeled sonicated mitochondria or postribosomal supernatant from rat liver regenerating in the presence of chloramphenicol were incubated with neutral chloroform/methanol extract of in vitro with [14C]leucine labeled rat liver mitochondria. After this incubation several labeled components with apparent molecular weights above 10 000 were recovered in the electrophoreograms of the originally unlabeled fractions.     

Optimal conditions for lactoperoxidase catalyzed radioiodination of external proteins on mouse erythrocytes

Optimal conditions were established for specific labelling of the surface proteins of mouse erythrocytes using lactoperoxidase-catalyzed radioiodination. The levels of H2O2 and I-, and cell concentrations required for restriction of haemoglobin labelling to less than 5% of the total 125I-protein, were different for radioiodination employing direct H2O2 addition or generation of H2O2 with glucose oxidase plus glucose. Preparation of mouse erythrocyte ghosts by hypotonic lysis caused loss of some minor labelled proteins present on intact cells and shifts to lower molecular weights of others. It is therefore important to solubilize labelled cells directly in electrophoresis buffer to avoid artifactual degradation of labelled proteins. The extent of labelling internal cell proteins was measured by a procedure suitable for the comparison of a large number of samples: solubilized radioiodinated erythrocytes were electrophoresed on 14% acrylamide gels and the radioactivity determined in the haemoglobin band which migrates separately from other proteins. The major labelled protein on the mouse erythrocytes had an apparent molecular weight of 92,000, and may be analogous to Band 3 of the human erythrocyte.     

Biogenesis of erythrocyte membrane proteins in vitro studies with rabbit reticulocytes

The capability of rabbit reticulocytes to synthesize red cell membrane proteins has been tested in vitro. Reticulocyte-rich blood from phenylhydrazine-treated rabbits was incubated in vitro in a complete amino acid medium containing ferrous salts, glucose, rabbit plasma and [³H]leucine. Red cell ghost membranes were prepared by hypotonic lysis and leucine incorporation into hemoglobin and total membrane proteins determined. The pattern of incorporation into individual peptides was determined by polycrylamide gel electrophoresis of labeled membranes on large (19 mm) gel which were then sliced into 1 mm sections; radioactivity was compared with densitometric tracings of Coomassie blue stained analytical (6 mm) gels. Incorporation of [³H]leucine into both hemoglobin and membrane protein was linear over 1 h. Gel analysis of labeled membranes revealed that the amino acid was primarily incorporated into peptides with molecular weights of 90 000 or less; three peptides of molecular weights 90 000, 60 000 and 33 000 showed the highest specific activity. Synthesis of the four largest peptide species was negligible. Removal of ferrous salts inhibited synthesis of both globin and membrane protein equally (approx. 50%). However, puromycin and cycloheximide preferentially inhibited the synthesis of globin as compared to membrane proteins. Reticulocytes remain capable of synthesizing a number of membrane proteins; these results are consistent with studies of red cell membrane synthesis in anemic rabbits in vivo.     

Further characterization of a novel phospholipase B (phospholipase A2--lysophospholipase) from intestinal brush-border membranes.

The intestinal brush-border membranes of rats and guinea pigs possess a high molecular weight, calcium-independent phospholipase B (phospholipase A2 - lysophospholipase activities) with the characteristics of a digestive ectoenzyme. A combination of subcellular fractionation, Triton X-114 phase partitioning, chromatofocusing, and preparative sodium dodecyl sulphate - polyacrylamide gel electrophoresis was used to purify a full-length, although denatured, form of this enzyme from the rat. Renaturation of the gel-purified fraction confirmed that both enzyme activities were associated with this protein. Gel slices containing the purified phospholipase B were used to generate a polyclonal antiserum in rabbits that could be used for immunoblotting. The relative mobility of the phospholipase B during electrophoresis in sodium dodecyl sulphate gels was dramatically affected by the percentage of acrylamide and the presence or absence of reducing agents in the gels. This was true for both the purified protein visualized by silver-staining and following electrophoresis of the total proteins of the membrane, with the phospholipase visualized by immunoblotting. Estimates for the molecular mass of the enzyme varied from 130 to 170 kDa in 7.5% gels and from 120 to 130 kDa in 5-10% gradient gels (with a best estimate of 120 kDa). Upon solubilization from the brush-border membrane by papain digestion, the major immunoreactive band migrated with an apparent mass of 80 kDa in both the 7.5% and 5-10% gradient gels. A major cross-reactive band was detected at 97 kDa following immunoblotting of the papain-solubilized proteins from guinea pig brush-border membranes, in agreement with the size of the purified fragment reported in the literature and at 140 kDa following immunoblotting of the intact proteins. Similar immunoblotting produced reaction with a 135-kDa protein from the rabbit brush-border membrane, as well as 95-kDa protein following papain solubilization. These results suggest that while there are species-specific apparent molecular weights, the intestinal brush-border membrane phospholipase B is conserved among species.     

Protein Components of the Purified Sodium Channel from Rat Skeletal Muscle Sarcolemma

Abstract: Sensitive detection systems have been used to study the protein components of the sodium channel purified from rat skeletal muscle sarcolemma. This functional, purified sodium channel contains at least three subunits on 7–20% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis: a large glycoprotein which migrates anomalously in the high-molecular-weight range, a 45,000 molecular weight polypeptide, and a third protein often seen as a doublet at 38,000. The large glycoprotein runs as a diffuse band and stains very poorly with Coomassie blue, but is adequately visualized with silver staining or iodination followed by autoradiography. This glycoprotein exhibits anomalous electrophoretic behavior in SDS-polyacrylamide gels. The apparent molecular weight of the center of the band varies from ～230,000 on 13% acrylamide gels to ～130,000 on 5% gels; on 7–20% gradient gels a value of 160,000 is found. Plots of relative migration versus gel concentration suggest an unusually high apparent free solution mobility. Lectin binding to purified channel peptides separated by gel electrophoresis indicates that the large glycoprotein is the only subunit that binds either concanavalin A or wheat germ agglutinin, and this component has high binding capacity for both lectins. The smaller channel components run consistently at 45,000 and 38,000 molecular weight in a variety of gel systems and do not appear to be glycosylated.     

Characterization of a novel plasma membrane protein, expressed in the midgut epithelia of Bombyx mori, that binds to Cry1A toxins

We describe the properties of a novel 252-kDa protein (P252) isolated from brush border membranes of Bombyx mori. P252 was found in a Triton X-100-soluble brush border membrane vesicle fraction, suggesting that it may be a component of the midgut epithelial cell membrane. P252 was purified to homogeneity, and the amino acid sequence of two internal peptides was determined, but neither of the peptides matched protein sequences in the available databases. The apparent molecular mass of the purified protein was estimated by denaturing gel electrophoresis to be 252 kDa, and it migrated as a single band on native gels. However, gel filtration chromatography indicated an apparent mass of 985 kDa, suggesting that P252 may exist as a homo-oligomer. The associations of P252 with Cry1Aa, Cry1Ab, and Cry1Ac were specific, and K(d) constants were determined to be 28.9, 178.5, and 20.0 nM, respectively. A heterologous competition assay was also done. P252 did not exhibit Leu-pNA hydrolysis activity, and binding to the Cry1A toxins was not inhibited by GalNAc. Binding assays of P252 with various lectins indicated the presence of three antennal N-linked high-mannose-type as well as O-linked mucin-type sugar side chains. While the function of P252 is not yet clear, we propose that it may function with Cry1A toxins during the insecticidal response and/or Cry toxin resistance mechanism.     

Transforming growth factors from a human tumor cell: characterization of transforming growth factor beta and identification of high molecular weight transforming growth factor alpha

Intracellular transforming growth factors (TGFs) were extracted from a human rhabdomyosarcoma cell line and purified to apparent homogeneity by using gel filtration, cation-exchange, and high-performance liquid chromatography. Two types of transforming growth factor activities, TGF-alpha and TGF-beta, were detected. The intracellular polypeptides which belonged to the TGF-alpha family required TGF-beta for full activity in inducing nonneoplastic normal rat kidney fibroblasts to grow in soft agar. These peptides also bound to the membrane receptor for epidermal growth factor. As determined by sodium dodecyl sulfate-polyacrylamide gels, the apparent molecular weight of these intracellular TGF-alpha's was 18 000. Intracellular TGF-beta required either epidermal growth factor or TGF-alpha for stimulation of soft agar growth. The intracellular TGF-beta was purified to homogeneity as judged by a single peak after reverse-phase high-performance liquid chromatography and a single band on a sodium dodecyl sulfate-polyacrylamide gel. The intracellular TGF-beta from the human tumor cell line was similar in all respects tested (migration on sodium dodecyl sulfate-polyacrylamide gels, stimulation of soft agar growth, binding to the membrane receptor for TGF-beta, and amino acid composition) to intracellular TGF-beta from normal human placenta.     

Purification and characterization of a beta-galactoside-binding soluble lectin from rat and bovine brain

A beta-galactoside-binding activity has been detected in mammalian brain extracts using a hemagglutination test and a nerve cell aggregation assay. Inhibition studies suggested the involvement of lectin-carbohydrate interactions in these processes. In an attempt to explore further the biological role of brain lectins, the beta-galactoside-binding activity has been purified to apparent homogeneity from bovine and rat brain by salt extraction of the brain tissue and affinity chromatography on asialofetuin-agarose. The molecular weights determined by gel filtration, under native conditions on Ultrogel AcA-34, were 30,000 for the bovine brain lectin and 32,000 for the rat brain lectin; polyacrylamide gel electrophoresis in SDS gave molecular weights of 15,000 and 16,000, respectively, suggesting that the two brain lectins are dimers. Both lectins have an isoelectric point of 3.9. Amino acid composition data indicate that both lectins contain high proportions of glycine and acidic amino acids. The lectins are specific for beta-D-galactosides and related sugars and the configuration of carbon atoms 1, 2 and 4 seems of primary importance. Moreover, the nerve cell aggregation-promoting activity of the purified lectin is 300-fold that of the crude extracts.     

SDS GEL ELECTROPHORESIS OF RAPIDLY TRANSPORTED PROTEINS IN GARFISH OLFACTORY NERVE

Abstract— Proteins undergoing rapid axonal transport in the garfish olfactory nerve were examined by sodium dodecyl sulphate gel electrophoresis. The distribution of polypeptides and the extent of their labeling by transported molecules was determined in several nerve subfractions including: total particulate, total membrane, mitochondrial and two membrane subfractions rich in axolemma. The polypeptide composition of the various fractions was found to be relatively similar, with each showing a major protein with an estimated MW of 58,000. Specific differences in the concentrations of certain proteins were noted between fractions, including differences between the lower and higher density axolemma rich subfractions. Axonally transported radioactivity was predominantly localized among high molecular weight proteins, with all fractions, except mitochondrial pellet, displaying a major peak of radioactivity centered at 126,000-MW. Several major proteins including the 58,000-MW band were labeled by rapid transport to a much smaller extent. Certain labeled peaks were found to be concentrated in individual fractions, particularly a polypeptide (MW 35,000) more predominantly found in the lower density axolemma rich fraction.
Systemic labeling of the nerve is found to give a general distribution of radioactivity on gels, which is clearly different from the pattern obtained after axonal transport labeling.     

Purification and characterization of a β-galactoside-binding soluble lectin from rat and bovine brain

A β-galactoside-binding activity has been detected in mammalian brain extracts using a hemagglutination test and a nerve cell aggragation assay. Inhibition studies suggested the involvement of lectin-carbohydrate interactions in these processes. In an attempt to explore further the biological role of brain lectins, the β-galactoside-binding activity has been purified to apparent homogeneity from bovine and rat brain by salt extraction of the brain tissue and affinity chromatography on asialofetuin-agarose. The molecular weights determined by gel filtration, under native conditions on Ultrogel AcA-34, were 30 000 for the bovine brain lectin and 32 000 for the rat brain lectin; polyacrylamide gel electrophoresis in SDS gave molecular weights of 15 000 and 16 000, respectively, suggesting that the two brain lectins are dimers. Both lectins have an isoelectric point of 3.9. Amino acid composition data indicate that both lectins contain high proportions of glycine and acidic amino acids. The lectins are specific for β-D-galactosides and related sugars and the configuration of carbon atoms 1, 2 and 4 seems of primary importance. Moreover, the nerve cell aggregation-promoting activity of the purified lectin is 300-fold that of the crude extracts.     

Labelling of intestinal brush border membrane proteins in vivo using diazotised [125I]iodosulfanilic acid

Membrane proteins of the intestinal brush border were labelled in vivo by intraluminal injection of diazotised [¹²⁵I]iodosulfanilic acid, a highly polar molecule. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of brush border membranes labelled in this manner showed 20 protein bands, 11 of which contained significant radioactivity. The most heavily labelled proteins had molecular weights greater than 150 000, indicating that they were the most exposed to the intestinal lumen. Little radioactivity was detected in proteins with molecular weights of less than 94 000. The majority of these smaller proteins were likely to have been brush border core proteins. The evidence that diazotised [¹²⁵I]iodosulfanilic acid bound primarily to brush border membrane proteins when administered in this way, was: (a) the specific activity of brush border proteins was up to 3-fold greater than that of total cell particulate proteins (pelleted at 27 000 × $\text{g}$ from mucosal homogenates); (b) principal peaks in the gel radioactivity profile of total cell particulate proteins corresponded to the most heavily labelled proteins of the isolated brush border membrane; and (c) brush border core proteins showed minimal radioactivity in vivo, but considerably higher radioactivity when brush border membranes were labelled in vitro. A small amount of label was absorbed across the intestinal mucosa. However, secondary labelling of brush border proteins by this absorbed label was minimal, since the specific activity of brush border proteins in jejunum adjacent to the labelled loop was only 0.22% of the level for those proteins in the labelled segment. Since this technique did not affect the cellular morphology, enzyme activity or biochemical integrity of the membrane, it should prove useful as a means of accurately studying in vivo turnover rates of brush border membrane proteins.     

Separation and characterization of the outer membrane of Pseudomonas aeruginosa.

A method is described for the preparation of outer and cytoplasmic membranes of Pseudomonas aeruginosa, and the outer membrane proteins characterized. Isolated outer and cytoplasmic membranes differed markedly in the content of 2-keto-3-deoxyoctonate (lipopolysaccharide) and phospholipid as well as in the localization of certain enzymes (NADH oxidase, succinate dehydrogenase, D-lactate dehydrogenase, malate dehydrogenase, and phospholipase), and also in the microscopic morphology. The outer membrane preparation showed activity neutralizing a certain bacteriocin or bacteriophages, whereas the cytoplasmic membrane preparation showed no neutralizing activity. The protein composition of membrane preparations from five different strains of P. aeruginosa [P14, M92 (PAO1), PAC1, P15, and M2008 (PAT)] were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. More than 50 protein bands were detected in the cytoplasmic membrane preparation. The protein compositions of outer membranes from the five different strains were very similar: at least 6 major bands were found (apparent molecular weights: Band D, 50,000; band E, 45,000; band F, 33,000; bands G and H, 21,000; and band I, 8,000). The protein composition of outer membranes was affected by some physiological growth conditions. Some features of major outer membrane proteins were also studied. Band F showed anomalous migration on SDS polyacrylamide gel electrophoresis depending on the solubilizing conditions or pretreatment with TCA. Band I seemed to be a protein analogous to the lipoprotein which had been found in the outer membrane of Escherichia coli.     

Lectin-Reactive Components in White Matter Membranes from Normal and Multiple Sclerosis Brains

Abstract: Polypeptides derived from human white matter membranes reacted with the radioiodinated lectins concanavalin A, Lens culinaris phytohemagglutinin, Ricinus communis agglutinin and wheat germ agglutinin after electrophoresis in polyacrylamide pore gradient gels. The molecular weights of these lectin-reactive bands were estimated by comparison with radioiodinated protein standards by using the linear relationship between log of the molecular weight and log of the gel concentration reached by the protein after electrophoresis in a polyacrylamide gradient gel. The molecular weight estimates for components reactive with concanavalin A were 176,800, 141,200, 72,800, 52,800, 44,700, 40,000, 24,800 and 23,900. The molecular weights of the bands reactive with both wheat germ agglutinin and Lens culinaris phytohemagglutinin were 138,000, 113,500, 92,100, 52,800, 44,700, 24,800 and 23,900. Wheat germ agglutinin was bound also to a band with a molecular weight of 72,800. Ricinus communis agglutinin bound to bands with estimated molecular weights of 138,000, 72,800, 52,800, 44,700, 24,800 and 23,900. The electrophoretic pattern of lectin-reactive polypeptides derived from normal-appearing white matter of multiple sclerosis brains was not qualitatively different from the lectin-binding pattern of control brain membrane polypeptides.     

Isolation of a 32P-labeled polypeptide of low molecular weight from phosphorylated human erythrocyte membranes.

The incorporation of 32P into well washed human erythrocyte membranes was studied in a medium containing [gamma-32P]ATP, Mg2+, and EGTA. Following phosphorylation, the membranes were completely solubilized in 1% sodium dodecyl sulfate and subjected to gel electrophoresis in dodecyl sulfate polyacrylamide. A large incorporation of radioactivity was observed in a band which migrated faster than component 7 (nomenclature of T. L. Steck, (1972), J. Mol. Biol. 66, 295) but slower than the bromophenol blue tracking dye, and did not stain with Coomassie Blue. Isolation of this band by preparative gel electrophoresis revealed that 41% of the radioactivity was associated with a 32P-labeled polypeptide. This polypeptide was further purified by gel chromatography on Sephadex LH-20 in chloroform-methanol-HCl, and Bio-Gel A 1.5m in dodecyl sulfate. Its amino acid composition is characterized by a high content of acidic residues. The calculated minimal molecular weight is 15084. Based upon the recovery of amino acids, the polypeptide fraction comprises at least 1.8% by weight of the total erythrocyte membrane proteins. An apparent molecular weight of 15000 was estimated by gel chromatography in dodecyl sulfate, while a range of 14000-16000 was estimated by electrophoresis in dodecyl sulfate polyacrylamide. The state of phosphorylation of this peptide may reflect a physiological function in the intact red cell.     

Characterization of a Novel Plasma Membrane Protein, Expressed in the Midgut Epithelia of Bombyx mori, That Binds to Cry1A Toxins

We describe the properties of a novel 252-kDa protein (P252) isolated from brush border membranes of Bombyx mori. P252 was found in a Triton X-100-soluble brush border membrane vesicle fraction, suggesting that it may be a component of the midgut epithelial cell membrane. P252 was purified to homogeneity, and the amino acid sequence of two internal peptides was determined, but neither of the peptides matched protein sequences in the available databases. The apparent molecular mass of the purified protein was estimated by denaturing gel electrophoresis to be 252 kDa, and it migrated as a single band on native gels. However, gel filtration chromatography indicated an apparent mass of 985 kDa, suggesting that P252 may exist as a homo-oligomer. The associations of P252 with Cry1Aa, Cry1Ab, and Cry1Ac were specific, and K_d constants were determined to be 28.9, 178.5, and 20.0 nM, respectively. A heterologous competition assay was also done. P252 did not exhibit Leu-pNA hydrolysis activity, and binding to the Cry1A toxins was not inhibited by GalNAc. Binding assays of P252 with various lectins indicated the presence of three antennal N-linked high-mannose-type as well as O-linked mucin-type sugar side chains. While the function of P252 is not yet clear, we propose that it may function with Cry1A toxins during the insecticidal response and/or Cry toxin resistance mechanism.     

Analysis of transmembrane proteins from eukaryotic cells

The topography and properties of plasma membrane proteins from mouse L-929 cells are studied by comparing their availability for enzymatic labeling on the external and internal surfaces of the membrane. In order to study the internal surface, phagolysosomes are prepared from cells after they ingest latex particles. The plasma membrane surrounding these seems to have an “inside-out” orientation. The sugars of the membrane glycoproteins in intact phagolysosomes are not available for interaction with lectins or available for periodate-borotritide labeling. A comparison of the lectin-binding proteins lableled by lactoperoxidase-catalyzed iodination on the external cell surface with those labeled on the internal cell surface suggests that a variety of plasma membrane glycoproteins span the lipid bilayer. Using two-dimensional gel electrophoresis it has been shown that selected proteins are labeled at both the internal and external faces of the plasma membrane. Analysis of the 2-D gel electrophoregrams reveals that there are two distinct prominent proteins at 60,000 and 100,000 daltons which are enzymatically iodinated from both sides of the membrane. The partial hydrolysis of the 100,000 dalton protein reveals that different peptides are iodinated when the iodination is performed on intact cells or on the phagolysosomes. These proteins are extensively phosphorylated in cells incubated with inorganic ³²P. We conclude that the phagolysosome is probably oriented in an “inside-out” configuration and that this membrane preparation can be used to study the topographic organization of membrane proteins. The use of oriented membranes, selective labeling of proteins, and affinity separation of proteins in combination with gel electrophoresis to define the position and properties of proteins is discussed.