Chicken erythrocyte membranes: Comparison of nuclear and plasma membranes from adults and embryos

Nuclear membranes and plasma membranes of chicken erythrocytes were tested for some enzyme activities and polypeptide content. Both membranes show ATPase and NADH cytochrome c reductase activities, which were higher for nuclear membrane preparations; besides the nuclear membrane ATPase is not stimulated by Na + K + ions. SDS solubilization of these membranes followed by separation using SDS polyacrylamide gel electrophoresis yields about 25 bands. Nuclear membranes and plasma membranes possess specific bands; some differences were seen when adult and embryonic membranes were compared.     

Purification and analysis of mitochondrial membrane proteins on nondenaturing gradient polyacrylamide gels

A nondenaturing gradient polyacrylamide gel electrophoresis method is described for the resolution of membrane proteins. Bovine heart inner mitochondrial membranes were solubilized in Triton X-100 and individual complexes were identified by staining for activity and protein. Succinate dehydrogenase was isolated by band excision and shown by electrophoresis under denaturing conditions to be highly purified. In addition, the electrophoretic transfer of NADH dehydrogenase to nitrocellulose was demonstrated. The enzyme was identified on the resulting blot by activity staining and the binding of monospecific antibodies.     

Auxin-Stimulated NADH Oxidase Purified from Plasma Membrane of Soybean

NADH oxidation by plasma membrane vesicles purified from hypocotyls of etiolated soybean seedlings by two-phase partition was stimulated 2- to 3-fold by auxins, indole-3-acetic acid, 2,4-dichlorophenoxy acetic acid (2,4-D), and α-naphthaleneacetic acid. The stimulation was concentration dependent in the presence or absence of detergent with a maximum for 2,4-D at 1 micromolar. The NADH oxidation activity was solubilized with the zwitterionic detergent CHAPS and purified by ion exchange chromatography and gel filtration approximately 2000-fold over the total homogenate. Both the partially purified fraction and an active band from nondenaturing gel electrophoresis revealed the same three bands when analyzed by denaturing gel electrophoresis. When obtained from plasma membrane vesicles from the region of rapid cell elongation, the NADH oxidase complex retained auxin responsiveness throughout purification (3- to 5-fold stimulation by 1 micromolar 2,4-D).     

Tubulin: An Integral Protein of Mammalian Synaptic Vesicle Membranes

Abstract: The major protein in isolated synaptic vesicles from bovine cerebral cortex has been compared to tubulin by sodium dodecyl sulphate-urea polyacrylamide gel electrophoresis, by two-dimensional gel electrophoresis and by peptide mapping following limited proteolysis of the protein by Staphylococcus aureus protease. The results establish in purified synaptic vesicles the presence of tubulin, which is composed of the α and β subunits. In the presence of ethyleneglycol bis (aminoethyl ether)- N, N' -tetraacetic acid (EGTA) or magnesium in the isolation buffers, the synaptic vesicles contained mainly the α-tubulin whereas the β subunit was less abundant. Similarly, synaptosomal plasma membranes that were prepared in the presence of EGTA also contained more of α-tubulin than of the β subunit. Non-ionic detergents such as Triton X-100 or Nonidet P-40 failed to solubilize the tubulin from the synaptic vesicles. Ionic detergents such as deoxycholate and sodium dodecyl sulphate solubilized all the vesicle proteins, including tubulin. The results indicate that α-tubulin is an integral vesicle membrane protein, whereas most of the β sub-unit is peripherally attached and can be easily dissociated from the vesicle membrane with EGTA.     

Phospholipid transfer protein from maize seedlings is partly membrane-bound

Specific antibodies raised against phospholipid transfer protein from maize seeds, react with mitochondria or microsomes solubilized by sodium deoxycholate. A single precipitin line was observed with both types of solubilized membranes when the double immunodiffusion technique was used. When the solubilized membranes were separated by fast-protein liquid chromatography (FPLC) on a reverse phase column, prior to the immunodiffusion, only fractions co-migrating with the pure phospholipid transfer protein, reacted with the antibody. Alternatively, solubilized membranes were submitted to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting; the detection of antigen-antibody complexes by a peroxydase reagent revealed the presence of a band co-migrating with the pure protein. All these observations strongly suggest that phospholipid transfer proteins are membrane-bound. The physiological significance of this finding will be discussed.     

Purification of NADH-cytochromeb 5 reductase from rat liver plasma membranes

Summary An NADH-cytochromeb ₅ reductase was purified from rat liver plasma membranes. Rat liver plasma membranes were prepared by aqueous two-phase partition. Peripheral proteins were removed by EDTA extraction and integral membrane proteins were solubilized with Triton X-100. The NADH-cytochromeb ₅ reductase was purified by hydroxyapatite, anion exchange, and gel filtration chromatographies. The purified preparation was homogeneous and estimated to have an apparent molecular weight of 32 kDa on SDS-polyacrylamide gel electrophoresis. Two tryptic peptides of the purified enzyme had sequence homologies with rat, human, and bovine NADH-cytochromeb ₅ reductases.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - BCA bicinchonicic acid - EDTA ethylenediamine tetraacetate acid disodium salt - FeCN ferricyanide - HPLC high-performance liquid chromatography - NADH nicotinamide adenine dinucleotide reduced form - PMSF -phenylmethylsulfonyl fluoride     

Yeast mitochondrial dehydrogenases are associated in a supramolecular complex

Separation of yeast mitochondrial complexes by colorless native polyacrylamide gel electrophoresis led to the identification of a supramolecular structure exhibiting NADH-dehydrogenase activity. Components of this complex were identified by N-terminal Edman degradation and matrix-assisted laser desorption ionization mass spectrometry. The complex was found to contain the five known intermembrane space-facing dehydrogenases, namely two external NADH-dehydrogenases Nde1p and Nde2p, glycerol-3-phosphate dehydrogenase Gut2p, D- and L-lactate-dehydrogenases Dld1p and Cyb2p, the matrix-facing NADH-dehydrogenase Ndi1p, two probable flavoproteins YOR356Wp and YPR004Cp, four tricarboxylic acids cycle enzymes (malate dehydrogenase Mdh1p, citrate synthase Cit1p, succinate dehydrogenase Sdh1p, and fumarate hydratase Fum1p), and the acetaldehyde dehydrogenase Ald4p. The association of these proteins is discussed in terms of NADH-channeling.     

Bovine dihydropteridine reductase

Dihydropteridine reductase has been purified to homogeneity from bovine liver and bovine adrenal medulla by precipitation with polyethylene glycol, ion exchange chromatography, gel filtration, and affinity chromatography on 5-AMP-Sepharose 4B. The enzymes from the two tissues seem identical by the criteria of gel filtration chromatography, affinity chromatography, polyacrylamide gel electrophoresis in the presence and absence of dodecyl sulfate, isoelectric focusing, amino acid analysis, and binding of NADH. Fluorescence studies show two independent binding sites for NADH and a dissociation constant of 10 nM at pH 6.8. Isoelectric focusing of the enzyme as purified in the presence of NADH revealed three different bands, which by removal of this coenzyme were converted into a single band, corresponding to pI 5.7. The enzyme contains no carbohydrate or zinc.     

Purification and characterization of microsomal and lysosomal beta-glucuronidase from rat liver by use of immunoaffinity chromatography.

Rat liver beta-glucuronidase (EC 3.2.1.31), both from microsomal and lysosomal fractions, were purified about 9500-fold over the homogenate with high yield using affinity chromatography prepared by coupling purified specific immunoglobulin G against rat preputial gland beta-glucuronidase to Sepharose 2B and isoelectric focusing. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of approximately 310000. In dodecylsulfate polyacrylamide gel electrophoresis, the microsomal beta-glucuronidase showed a single band corresponding to a molecular weight of 79000, while the lysosomal beta-glucuronidase had three distinct bands which consisted of one major and two minor bands corresponding to molecular weight of 79000, 74000, and 70000, respectively. A broad pH activity curve with a single optimum at pH 4.4 was observed in both the microsomal and the lysosomal beta-glucuronidases. Immunological gel diffusion technique with rabbit antiserum against rat liver lysosomal beta-glucuronidase revealed that both enzymes had the same or quite similar antigenic determinants.     

NADPH: nitroblue tetrazolium reductase found in plasma membrane of human neutrophil

After phorbol 12-myristate 13-acetate (PMA) stimulation the increase of NADPH:nitroblue tetrazolium reductase activity in the plasma membrane almost corresponded with the stimulated activity of respiratory burst oxidase. Solubilization of plasma membranes from PMA-activated neutrophils with n-octyl glucoside resulted in high recoveries of the two enzymatic activities. When solubilized plasma membrane was subjected to non-denaturing polyacrylamide gel electrophoresis in the presence of 35 mM n-octyl glucoside, we could see three major bands stained with NADPH-dependent nitroblue reductase activity giving molecular masses of approx. 95, 45 and 40 kDa, respectively. Activity was specific for NADPH but not for NADH. These bands also stained weakly in the plasma membranes obtained from resting cells. The activities for NADPH oxidase and nitroblue tetrazolium reductase were found to elute as a very similar protein peak on an anion-exchange HPLC, at about 0.32 M KCl. This elution peak also contains 45 and 40 kDa proteins showing NADPH:nitroblue tetrazolium reductase activity.     

Comparison of plasma membranes and endoplasmic reticulum fractions obtained from whole white adipose tissue and isolated adipocytes.

The influence of the mode of preparation upon some of the characteristics of white adipose tissue plasma membranes and microsomes has been reported. Plasma membrane fractions prepared from mitochondrial pellet were shown to have higher specific activities of (Mg2+ + Na+ + K+)-ATPase than plasma membranes originating in crude microsomes. Isolation of fat cells by collagenase treatment was found to result in a decrease in specific activity of the plasma membrane enzymes; in plasma membranes prepared from isolated fat cells, the specific activity values obtained for (Mg2+ + Na+ +k+)-ATPase and 5'-nucleotidase were only 42% and 6.3% respectively of those obtained in plasma membranes prepared from whole adipose tissue. Purification of whole adipose tissue crude microsomes by hypotonic treatment caused extensive solubilization of the endoplasmic reticulum marker enzymes, NADH oxidase and NADPH cytochrome c reductase. The lability of endoplasmic reticulum marker enzymes, however, was found to be greatly diminished in the preparations from isolated fat cells. The possibility that NADH oxidase and NADPH cytochrome c reductase activities found in the plasma membranes are microsomal enzymes adsorbed by the plasma membranes is discussed. The peptide patterns as well as the NADH oxidase and NADPH cytochrome c reductase activity patterns of plasma membranes and purified microsomes were compared by means of sodium dodecyl sulfate or Triton X-100 polyacrylamide gel electrophoresis.     

Isolation ofSpiroplasma citri membranes and characterization of membrane proteins by two-dimensional gel electrophoresis

This paper describes a method for isolating plasma membranes fromSpiroplasma citri and for comparing membrane and cytoplasmic proteins by two-dimensional gel electrophoresis. Plasma membranes ofS. citri were stabilized against fragmentation by coating cell with Concanavalin A just prior to lysis. After lysis of the cells by ultrasonic irradiation, membranes were purified by differential centrifugation and step gradients. The purified fraction, which consisted essentially of extended sheets of membranes, exhibited membrane-boundpara-nitrophenylphosphatase specific activity 1.5-fold over that of the whole-cell lysate. Only traces of soluble NADH oxidase were present in the membrane preparation. The latter fraction appeared homogeneous upon sorbitol density gradient centrifugation and banded at an equilibrium density of 1.107 g/ml. The plasma membrane proteins were then analyzed by two-dimensional polyacrylamide gel electrophoresis. Approximately 40 different proteins were detected in the membrane preparations. By comparison with the patterns obtained for whole-cell extracts and cytoplasmic fractions, a protein map ofS. citri could be established in which membrane and cytoplasmic proteins were identified.     

Purification and comparative properties of the glycoprotein nicotinamide adenine dinucleotide glycohydrolase from rat liver microsomal and plasma membranes.

NAD glycohydrolase, or NADase (NAD+ glycohydrolase, EC 3.2.2.5) was solubilized with porcine pancreatic lipase from isolated fractions of microsomes and plasma membranes obtained from rat livers. The enzyme from each organelle was further purified by DEAE-cellulose chromatography, gel filtration and isoelectric focusing. The solubilized, partially purified enzymes had similar molecular weights, pH-activity profiles and Km values. Marked charge heterogeneity was observed for the microsomal enzyme on isoelectric focusing between pH 6 and 8 with maximum activity focusing at pH 8.0. Plasma membrane NADase displayed a single peak at pH 6.7. Treatment of the partially purified microsomal or plasma membrane enzyme with neuraminidase resulted in a single peak of activity on isoelectric focusing (pH 3.5--10) with a pI of 9.2. Polyacrylamide gel electrophoresis of either NADase revealed a periodate-Schiff positive band which was coincident with enzyme activity. Compositional analyses of the microsomal enzyme focusing at pH 8.0 confirmed the presence of hexoses, hexosamines and sialic acid. Differences in carbohydrate composition might be important in determining the subcellular distribution of this enzyme.     

Antenna chlorophyll a complexes in mutant and developing barley

The chlorophyll a antenna of photosystems I and II were each isolated after detergent treatment by gel electrophoresis or sucrose gradient centrifugation from a b-less mutant of barley grown in daylight and from wildtype barley developed in intermittent light. We identified each fraction by both its electrophoretic position and PS I activity (P700 content) in the case of the mutant, and by both PS I and PS II activity (DCIP reduction from DPC) in the light-limited plants. The proportion of Chl a in each photosystem was estimated from the amount in each gel or sucrose gradient band, and from addition of the areas under the absorption spectra (650–710 nm) of each fraction to match the spectrum of the solubilized thylakoids. The latter method was possible because the spectrum (77 K) of each fraction was unique; in the mutant about 70% of chlorophyll is associated with PS I and 30% with PS II. In the light-limited plants, the reverse is true with nearly 70% associated with PS II. RESOL analyses of both absorption and fluorescence emission spectra of all isolated fractions indicated an abnormal arrangement of antenna chlorophyll molecules in the light-limited, developing membranes even though their reaction centers are fully functional.Abbreviations DCIP dichlorophenolindophenol - DOC deoxycholate - DPC diphenylcarbazide - DL daylight - ImL intermittent light - LHC light-harvesting Chl a/b protein complex - PAGE polyacrylamide gel electrophoresis DPB-CIW No. 778     

Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes

The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.     

Some molecular properties of NAD(P)H dehydrogenase from rat liver

NAD(P)H dehydrogenase (EC 1.6.99.2) purified from rat liver cytosol revealed three discrete bands, of mol.wts. about 27000, 18000 and 9000, when subjected to polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate. Elution of the bands from the gel and individual re-electrophoresis on separate gels showed that the 27000-mol.wt. band yielded three bands similar to those obtained with the intact enzyme, whereas the 18000-mol.wt. band retained its characteristic mobility. Amino acid analysis of native enzyme and protein extracted from each of the three bands from sodium dodecyl sulphate/polyacrylamide gels suggests that the native enzyme is composed of two subunits and that each subunit consists of two dissimilar non-covalently bound polypeptides, so that altogether the enzyme is composed of four polypeptides, two of mol.wt. 18000 and two of mol.wt. 9000. NAD(P)H dehydrogenase was active over a wide pH range with no sharp optimum. The same K(m) value for NADH but different values for V(max.) were obtained for the enzyme purified from Sprague-Dawley and Wistar rats. In immunodiffusion, however, the enzymes from the two rat strains showed a reaction of complete identity. NAD(P)H dehydrogenase was effectively inhibited by thiol-blocking reagents, indicating that the activity is dependent on free thiol group(s). By amino acid analysis six cysteine residues were found per mol of enzyme. Guanidino-group- and amino-group-selective reagents had only moderate inactivating effects on the enzyme activity.     

Purification and Some Properties of Exo-type Fucoidanases from Vibrio sp. N-5

Three fucoidanases were purified from Vibrio sp. N-5 by ammonium sulfate fractionation and chromatography with DEAE-Toyopearl 650 M, Sephacryl S-300 HR, and chromatofocusing. The purified enzymes gave a single band on disc polyacrylamide gel electrophoresis. The molecular weights of the enzymes, E-1, E-2, and E-3 were 39,500, 68,000, and 68,000, respectively, by SDS polyacrylamide gel electrophoresis and 158,000, 68,500, and 67,500 by gel filtration. The enzymes hydrolyzed gagome-fucoidan to give small oligosaccharides containing sulfate as main product.     

Isolation and Characterization of Tubules and Plasma Membranes from Cytophaga columnaris

Tubular structures are released from cells of Cytophaga columnaris after lysis of the cells. To determine the nature of these tubules, they were purified and their composition was determined. Tubules were isolated after treating cell lysates with 1.0% sodium dodecyl sulfate at pH 8.1, which solubilizes all structural components except tubules. Plasma membranes from the same organism were isolated by discontinuous sucrose gradient centrifugation of lysed cells. Both tubules and membranes are composed of lipids and proteins. Lipids extracted from tubules and plasma membranes produced similar patterns when examined by thin-layer chromatography. Proteins solubilized from membranes were separated into 14 bands by polyacrylamide gel electrophoresis, whereas those solubilized from tubules separated into only 5 bands. The presence of lipids in tubules from C. columnaris supports the idea that they are derived from membranes of intact cells. In this respect they are similar to tubules produced by cells of Clostridium botulinum and different from other tubular structures ("rhapidosomes") found in cells of Saprospira grandis.     

Association of Ferredoxin-NADP Oxidoreductase with the Chloroplastic Pyridine Nucleotide Dehydrogenase Complex in Barley Leaves

Barley (Hordeum vulgare L.) leaves were used to isolate and characterize the chloroplast NAD(P)H dehydrogenase complex. The stroma fraction and the thylakoid fraction solubilized with sodium deoxycholate were analyzed by native polyacrylamide gel electrophoresis, and the enzymes detected with NADH and nitroblue tetrazolium were electroeluted. The enzymes electroeluted from band S from the stroma fraction and from bands T1 (ET1) and T2 from the thylakoid fraction solubilized with sodium deoxycholate had ferredoxin-NADP oxidoreductase (FNR; EC 1.18.1.2) and NAD(P)H-FeCN oxidoreductase (NAD[P]H-FeCNR) activities. Their NADPH-FeCNR activities were inhibited by 2′-monophosphoadenosine-5′-diphosphoribose and by enzyme incubation with p-chloromercuriphenylsulfonic acid (p-CMPS), NADPH, and p-CMPS plus NADPH. They presented Michaelis constant NADPH values that were similar to those of FNRs from several sources. Their NADH-FeCNR activities, however, were not inhibited by 2′-monophosphoadenosine-5′-diphosphoribose but were weakly inhibited by enzyme incubation with NADH, p-CMPS, and p-CMPS plus NADH. We found that only ET1 contained two polypeptides of 29 and 35 kD, which reacted with the antibodies raised against the mitochondrial complex I TYKY subunit and the chloroplast ndhA gene product, respectively. However, all three enzymes contained two polypeptides of 35 and 53 kD, which reacted with the antibodies raised against barley FNR and the NADH-binding 51-kD polypeptide of the mitochondrial complex I, respectively. The results suggest that ET1 is the FNR-containing thylakoidal NAD(P)H dehydrogenase complex.     

Characterization of cholecystokinin receptor subunits on pancreatic plasma membranes

To determine the size and subunit structure of the pancreatic cholecystokinin (CCK) receptor, 125I-CCK33 was covalently cross-linked to its receptor on mouse pancreatic acinar plasma membranes utilizing the bifunctional cross-linker disuccinimidyl suberate. When CCK was cross-linked at pH 7.4 to either purified plasma membranes or to isolated pancreatic acini and then followed by preparation of plasma membranes, the major labeled protein band revealed by polyacrylamide gel electrophoresis was Mr = 120,000 in the absence of reducing agent and Mr = 80,000 in the presence of reducing agent. A similar banding pattern was also observed when different cross-linkers, ethylene glycol bis(succinimidyl succinate) or dithiobis (succinimidyl propionate), were employed. At pH 6.0, where CCK binding to its receptors is optimal, the labeling pattern was similar to that seen at 7.4, although the two bands were more heavily labeled. Both the binding of CCK to its receptors on plasma membranes and the appearance of the two cross-linked proteins on gels were inhibited in a parallel manner by increasing concentrations of unlabeled CCK8; similar results were observed with dibutyryl cyclic GMP, a competitive inhibitor of CCK binding and action. The data indicate, therefore, that the CCK receptor possesses subunit structure whereby an Mr = 76,000 binding subunit is linked to an Mr = 40,000 nonbinding subunit by a disulfide bond.