Partial purification of an intercalated disc-containing cardiac plasma membrane fraction

The sarcolemma of cardiac muscle cells contains a specialised junctional region, the intercalated disc which includes three types of intercellular junction, the macula and fascia adherens and the nexus or gap junction. To facilitate the isolation of these junctions a procedure for the partial purification from mouse hearts of a subcellular fraction containing the intercalated disc region of the sarcolemma was developed. This involved investigating methods of tissue disruption that preserve the integrity of the intercalated disc and minimise myofibrillar entrapment of organelles. Examination of the distribution of marker enzymes showed that relative to the homogenate the intercalated disc fraction prepared by sucrose density centrifugation was only enriched 1.5- to 3-fold in 5'-nucleotidase and (Na+ + K+)-ATPase activities, whereas mitochondrial and sarcoplasmic reticulum marker enzymes were low. The properties of the intercalated disc-containing fraction were compared with the vesicular sarcolemmal fractions devoid of junctional complexes prepared by other methods.     

Partial purification of a plasma membrane flavoprotein and NAD(P)H-oxidoreductase activity

Plasma membrane flavins and pterins are considered to mediate important physiological functions such as blue light photoperception and redox activity. Therefore, the presence of flavins and pterins in the plasma membrane of higher plants was studied together with NAD(P)H-dependent redox activities. Plasma membranes were isolated from the apical hooks of etiolated bean seedlings (Phaseolus vulgaris L. cv. Limburgse Vroege) by aqueous two-phase partitioning. Fluorescence spectroscopy revealed the presence of two chromophores. The first showed excitation maxima at 370 and 460 nm and an emission peak at 520 nm and was identified as a flavin. The second chromophore was probably a pterin molecule with excitation peaks at 290 and 350 nm and emission at 440 nm. Both pigments are considered intrinsic to the plasma membrane since they could not be removed by treatment with hypotonic media containing high salt and low detergent concentrations. The flavin concentration was estimated at about 500 pmol mg^?1 protein. However difficulties were encountered in quantifying the pterin concentrations. Protease treatments indicated that the flavins were non-covalently bound to the proteins. Separation of the plasma membrane proteins after solubilisation by octylglucoside, on an ion exchange system (HPLC, Mono Q), resulted in a distinct protein fraction showing flavin and pterin fluorescence and NADH oxidoreductase activity. The flavin of this fraction was identified as flavin mononucleotide (FMN) by HPLC analysis. Other minor peaks of NADH:acceptor reductase activity were resolved on the column. The presence of distinct NAD(P)H oxidases at the plasma membrane was supported by nucleotide specificity and latency studies using intact vesicles. Our work demonstrates the presence of plasma membrane flavins as intrinsic chromophores, that may function in NAD(P)H-oxidoreductase activity and suggests the presence of plasma membrane bound pterins.     

A domain-specific marker for the hepatocyte plasma membrane. III. Isolation of bile canalicular membrane by immunoadsorption

Previous immunolabeling studies (Roman, L.M., and A.L. Hubbard, 1983, J. Cell Biol., 96:1548-1558; Roman, L.M., and A.L. Hubbard, 1984, J. Cell Biol., 98:1488-1496, companion paper) established leucine aminopeptidase (LAP) as a specific marker for the bile canalicular (BC) domain of the rat hepatocyte plasma membrane (PM). In this study, we have isolated membrane from a sonicated PM vesicle fraction using anti-LAP-coated Staphylococcus aureus cells as a solid-phase immunoadsorbent. The extent and specificity of the immunoadsorption were assessed by following the behavior of LAP (the BC marker) and 32P-labeled membrane phospholipids (a uniform membrane marker). The BC fraction obtained was significantly enriched in LAP (yield: greater than 70% of PM-LAP). Alkaline phosphatase, 5'-nucleotidase, and a 110,000-dalton glycoprotein, HA-4, were enriched in the BC fraction to the same extent as LAP (enzyme or antigen/LAP = 1.0). However, alkaline phosphodiesterase I was not enriched to the same degree (enzyme/LAP = 0.5). Contamination of this BC fraction by membrane derived from the sinusoidal domain and endoplasmic reticulum, as determined from the distribution of the asialoglycoprotein receptor and NADH cytochrome c reductase, respectively, was small (less than 13%).     

Large-scale purification of presynaptic plasma membranes from Torpedo marmorata electric organ

The presynaptic plasma membrane (PSPM) of cholinergic nerve terminals was purified from Torpedo electric organ using a large-scale procedure. Up to 500 g of frozen electric organ were fractioned in a single run, leading to the isolation of greater than 100 mg of PSPM proteins. The purity of the fraction is similar to that of the synaptosomal plasma membrane obtained after subfractionation of Torpedo synaptosomes as judged by its membrane-bound acetylcholinesterase activity, the number of Glycera convoluta neurotoxin binding sites, and the binding of two monoclonal antibodies directed against PSPM. The specificity of these antibodies for the PSPM is demonstrated by immunofluorescence microscopy.     

Partial purification of somatomedin from human plasma.

Fractional precipitation of human plasma using ethanol, followed by chromatography on S.P. Sephadex, yielded a somatomedin-enriched fraction freed from substantial amounts of inhibitory substances. Heat coagulation of the proteins present in this fraction allowed the recovery of appreciable amounts of active components which were then chromatographed on Sephadex G-75 and S.P. Sephadex. A major part of the activity was associated with components less than 4,000 daltons, suggesting that somatomedin, or an active fragment thereof, had been dissociated from a carrier protein by the heat treatment. The range of pH employed throughout was 5.3-9.8. Recoveries of about 30% of biological activity with fold-purification up to 38, as measured by radioactive sulphate uptake in the chick pelvic cartilage assay, were higher than those obtained using acid-ethanol extraction.     

Partial purification and characterization of the Ca2(+)-pumping ATPase of the liver plasma membrane

A Ca2(+)-pumping ATPase has been characterized in rat hepatocyte plasma membranes. The enzyme has high Ca2+ affinity, and properties typical of a P-type ion pump. At variance with the Ca2+ pumps of other eukaryotic plasma membranes, it is not stimulated by calmodulin. The steady state concentration of the phosphoenzyme formed in the presence of ATP is increased by La3+. The enzyme cross-reacts with a monoclonal antibody (mAb-5F10) raised against the human erythrocyte Ca2+ pump. The enzyme has been purified using a mAb-5F10 antibody affinity column. CNBr digestion of the isolated protein has yielded two peptides which have been sequenced. One of them matches perfectly a sequence contained in the erythrocyte Ca2+ pump, the other is very homologous to another domain in the erythrocyte pump. In spite of the absence of calmodulin stimulation, 125I-calmodulin overlay experiments on the purified liver ATPase under denaturing conditions have revealed that the enzyme binds calmodulin even more strongly than the erythrocyte pump. Immunocytochemical experiments on liver slices using the mAb-5F10 antibody have shown that the enzyme is located predominantly in the blood sinusoidal domain of the hepatocyte plasma membrane.     

Partial purification and characterization of an ascorbate-reducible b-type cytochrome from the plasma membrane of Arabidopsis thaliana leaves

Summary.  The plant plasma membrane (PM) contains more than one b-type cytochrome. One of these proteins has a rather high redox potential (can be fully reduced by ascorbate) and is capable of transporting electrons through the PM. Four genes encoding proteins with considerable homology to the sequences of cytochrome b ₅₆₁ proteins in the animal chromaffin granule membrane have recently been identified in the genome of Arabidopsis thaliana. In order to characterize the cytochrome b ₅₆₁ located in the Arabidopsis PM, first PM vesicles were purified by aqueous polymer two-phase partitioning from the leaves of 9-week-old A. thaliana. PM proteins were solubilized by nonionic detergent, and the fully ascorbate-reducible b-type cytochrome was partially purified by anion-exchange chromatography steps. Potentiometric redox titration of the fraction, containing the fully ascorbate-reducible b-type cytochrome after the first anion-exchange chromatography step, revealed the presence of two hemes with redox potentials of 135 mV and 180 mV, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions containing the fully ascorbate-reducible b-type cytochrome after the second anion-exchange chromatography step revealed the presence of a single polypeptide band at about 120 kDa. However, heat treatment (15 min, 90 °C) before electrophoresis was able to split the 120 kDa band into two bands with molecular masses of about 23 and 28 kDa. These values are lower than the apparent molecular mass for the fully ascorbate-reducible b-type cytochrome purified from Phaseolus vulgaris hypocotyls (about 52 kDa) but are in good agreement with those characteristic for the cytochrome b ₅₆₁ proteins purified from chromaffin granule membranes (about 28 kDa) and the four polypeptides predicted from the Arabidopsis genome (24–31 kDa). Received May 4, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Institute of Biophysics, BRC, Hungarian Academy of Sciences, POB 521, 6701 Szeged, Hungary.     

Partial purification and characterization of (Na++K+)-ATPase from garfish olfactory nerve axon plasma membrane

Summary The (Na⁺+K⁺)-ATPase of garfish olfactory nerve axon plasma membrane was purified about sixfold by treatment of the membrane with sodium dodecyl sulfate followed by sucrose density gradient centrifugation. The estimated molecular weights of the two major polypeptide components of the enzyme preparation on sodium dodecyl sulfate gels were 110,000 and 42,000 daltons, which were different from those of the corresponding peptides of rabbit kidney (Na⁺+K⁺)-ATPase. No carbohydrate was detected in the 42,000-dalton component either by the periodic acid-Schiff reagent or by the more sensitive concanavalin A-peroxidase staining procedure. The molecular properties of the garfish (Na⁺+K⁺)-ATPase, such as theK _m for ATP, pH optimum, energies of activation, Na and K ion dependence and vanadium inhibition, were, however, similar to those of the kidney enzyme.The partially purified garfish (Na⁺+K⁺)-ATPase was reconstituted into phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted enzyme was found to catalyze a time and ATP dependent²²Na⁺ transport. The ratio of²²Na⁺ pumped to ATP hydrolyzed was about 1; under the same reconstitution and assay conditions, eel electroplax (Na⁺+K⁺)-ATPase, however, gave a²²Na⁺ pumped to ATP hydrolyzed ratio of nearly 3.     

Partial purification of the apolipoprotein E receptor of rat liver membrane

The liver has two distinct lipoprotein receptors; one is the apolipoprotein (apo) B, E receptor and the other the apo-E receptor. In this study, the protein to which apo-E HDLc (cholesterol-induced high density lipoprotein containing apo-E as the predominant protein species) bound specifically was partially purified from the rat liver membrane by ion exchange chromatography and preparative gel electrophoresis. The molecular weight of the protein was estimated to be about 36K daltons and the protein bound to 125I-apo-E HDLc in a specific and saturable manner, suggesting that the protein is the apo-E receptor.     

The mechanism of action of beta-bungarotoxin at the presynaptic plasma membrane.

The beta-bungarotoxin-induced depolarization of the synaptosomal plasma membrane monitored by the efflux of 86Rb+ is potentiated by raising the albumin in the incubation, is Ca2+-dependent and is due neither to inhibition of the (Na+ + K+)-dependent ATPase nor to activation of the voltage-dependent Na+ channel. Occupancy of the beta-bungarotoxin-binding site by dendrotoxin inhibits partially the action of beta-bungarotoxin. The efflux of 86Rb+ is parallelled by a release of lactate dehydrogenase from the synaptosome, and the two processes are maximal with 2 nM-toxin. Digitonin induces a release of 86Rb+ and lactate dehydrogenase closely similar to that seen with beta-bungarotoxin. It is concluded that the toxicity of beta-bungarotoxin for mammalian nerve terminals can be largely accounted for by specific site-directed phospholipase A2-induced permeabilization of the plasma membrane.     

Octadecyl silica: a solid phase for protein purification by immunoadsorption.

Immunoaffinity chromatography involves binding of an antigen or antibody to a solid matrix, usually agarose, frequently using the cyanogen bromide method. These methods are laborious, rather expensive, and their use has been mostly restricted to immunopurifications on the microscale. We propose here the use of octadecyl silica (SiCl8) beads, a matrix for HPLC, as an alternative solid phase for protein immunopurification and immunoadsorption. Antibodies or antigens are strongly bound to SiCl8 by a simple incubation; radiolabeled antibodies can only be eluted from SiCl8 by detergent-containing solutions. After the remaining free binding sites have been saturated with bovine serum albumin, SiCl8 is incubated with the antigen- or antibody-containing crude preparations and is then poured into a minicolumn. The nonspecifically bound proteins are removed by washing; specific proteins are eluted by disruption of the antigen-antibody complexes with a low pH buffer. With this methodology, we have obtained high purity preparations of proteins in single steps, even when these proteins are present in trace amounts (picograms) in a complex mixture such as human serum. Similarly, specific antibodies against an intracellular parasite (Trypanosoma cruzi) were completely absorbed from human serum with SiCl8 coated with parasite antigens.     

Partial purification and characterization of cysteine proteinase inhibitor from chicken plasma

A high-molecular-weight cysteine proteinase inhibitor (CPI) was purified from chicken (Gallus gallus) plasma using polyethylene glycol (PEG) fractionation and affinity chromatography on carboxymethyl–papain–Sepharose-4B. The CPI was purified 96.8-fold with a yield of 28.9%. Based on inhibitory activity staining for papain, CPI was shown to have an apparent molecular mass of 122 kDa. No inhibitory activity was obtained under reducing condition, indicating that CPI from chicken plasma was stabilized by disulfide bonds. CPI was stable in temperature ranges from 40 to 70 °C for 10 min; however, more than 50% of the inhibitory activity towards papain was lost within 30 min of heating at 90 °C. CPI was stable in the presence of salt up to 3%. The purified CPI exhibited the inhibitory activity toward autolysis of arrowtooth flounder (Atheresthes stomias) and Pacific whiting (Merluccius productus) natural actomyosin (NAM) in a concentration-dependent manner.     

alpha-latrotoxin triggers transmitter release via direct insertion into the presynaptic plasma membrane

alpha-latrotoxin, a component of black widow spider venom, binds to presynaptic nerve terminals and stimulates massive neurotransmitter release. Previous studies have demonstrated that alpha-latrotoxin first binds to two high-affinity receptors on nerve terminals, neurexins and CLs (CIRLs and latrophilins), and then executes a critical, second step of unknown nature that stimulates neurotransmitter release. We now demonstrate that incubation of alpha-latrotoxin with synaptosomes at 0 degrees C results in its peripheral membrane association. Incubation at 37 degrees C, however, converts the toxin into an operationally integral membrane protein, and induces generation of a protease-resistant fragment that consists of the entire N-terminal domain of alpha-latrotoxin and becomes protease sensitive after lysis of synaptosomes. Our data suggest that alpha-latrotoxin inserts into the presynaptic plasma membrane after receptor binding, resulting in an intracellular location of the N-terminal sequences. Membrane insertion of the N-terminal domain of alpha-latrotoxin occurs spontaneously, independently of membrane recycling or transmembrane ion gradients. We postulate that alpha-latrotoxin acts intracellularly in triggering release, and propose that non-selective cation channels induced by alpha-latrotoxin may be a by-product of membrane insertion.     

Partial purification of a boar sperm membrane protein inducing sperm agglutinating antibody

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.