Controlled Pore Glass Chromatography Research Articles

ADAM10 and BACE1 are localized to synaptic vesicles.

Synaptic degeneration and accumulation of the neurotoxic amyloid β-peptide (Aβ) in the brain are hallmarks of Alzheimer disease. Aβ is produced by sequential cleavage of the amyloid precursor protein (APP), by the β-secretase β-site APP cleaving enzyme 1 (BACE1) and γ-secretase. However, Aβ generation is precluded if APP is cleaved by the α-secretase ADAM10 instead of BACE1. We have previously shown that Aβ can be produced locally at the synapse. To study the synaptic localization of the APP processing enzymes we used western blotting to demonstrate that, compared to total brain homogenate, ADAM10 and BACE1 were greatly enriched in synaptic vesicles isolated from rat brain using controlled-pore glass chromatography, whereas Presenilin1 was the only enriched component of the γ-secretase complex. Moreover, we detected ADAM10 activity in synaptic vesicles and enrichment of the intermediate APP-C-terminal fragments (APP-CTFs). We confirmed the western blotting findings using in situ proximity ligation assay to demonstrate close proximity of ADAM10 and BACE1 with the synaptic vesicle marker synaptophysin in intact mouse primary hippocampal neurons. In contrast, only sparse co-localization of active γ-secretase and synaptophysin was detected. These results indicate that the first step of APP processing occurs in synaptic vesicles whereas the final step is more likely to take place elsewhere.

Synapsin Phosphorylation by Src Tyrosine Kinase Enhances Src Activity in Synaptic Vesicles

Synapsins are synaptic vesicle-associated phosphoproteins implicated in the regulation of neurotransmitter release. Synapsin I is the major binding protein for the SH3 domain of the kinase c-Src in synaptic vesicles. Its binding leads to stimulation of synaptic vesicle-associated c-Src activity. We investigated the mechanism and role of Src activation by synapsins on synaptic vesicles. We found that synapsin is tyrosine phosphorylated by c-Src in vitro and on intact synaptic vesicles independently of its phosphorylation state on serine. Mass spectrometry revealed a single major phosphorylation site at Tyr(301), which is highly conserved in all synapsin isoforms and orthologues. Synapsin tyrosine phosphorylation triggered its binding to the SH2 domains of Src or Fyn. However, synapsin selectively activated and was phosphorylated by Src, consistent with the specific enrichment of c-Src in synaptic vesicles over Fyn or n-Src. The activity of Src on synaptic vesicles was controlled by the amount of vesicle-associated synapsin, which is in turn dependent on synapsin serine phosphorylation. Synaptic vesicles depleted of synapsin in vitro or derived from synapsin null mice exhibited greatly reduced Src activity and tyrosine phosphorylation of other synaptic vesicle proteins. Disruption of the Src-synapsin interaction by internalization of either the Src SH3 or SH2 domains into synaptosomes decreased synapsin tyrosine phosphorylation and concomitantly increased neurotransmitter release in response to Ca(2+)-ionophores. We conclude that synapsin is an endogenous substrate and activator of synaptic vesicle-associated c-Src and that regulation of Src activity on synaptic vesicles participates in the regulation of neurotransmitter release by synapsin.

Effect of size, proteic composition, and heat treatment on the colloidal stability of proteolyzed bovine casein micelles

The casein micelles of reconstituted nonfat milk that have been fractionated by controlled pore glass chromatography showed a relationship between their size and their proteic composition: The fractions containing the smaller particles were richer in κ-casein than the fractions containing the bigger ones, in accordance with the casein micelle model of submicelles. The initial aggregation rate of micelles of different sizes, partially proteolyzed with chymosin (para-casein micelles), was measured in conditions of enzyme excess in which aggregation is the rate-limiting step of enzymatic coagulation, showing higher rates for the smaller micelles with the production of less compact para-casein micelle networks. This behavior could be explained in terms of electrostatic and steric colloidal stabilization due to their lower negative net charge and size and to a higher surface density of hydrophobic “patches” of proteolyzed κ-casein related to a higher probability of effective collisions between particles. Differences in the β-casein content did not seem to affect the initial aggregation rate of the micelles. On the contrary, the modifications of the micelle surface by heating affected the colloidal stability of the hydrolyzed micelles in different ways. The denaturation of the whey proteins and the formation of covalent complexes with κ-casein modify the micelle surface, increasing specially the steric stabilization, and produces a diminution in the number of hydrophobic sites that could be able to give interparticle hydrophobic interactions.

Regulated delivery of AMPA receptor subunits to the presynaptic membrane.

In recent years, a role for AMPA receptors as modulators of presynaptic functions has emerged. We have investigated the presence of AMPA receptor subunits and the possible dynamic control of their surface exposure at the presynaptic membrane. We demonstrate that the AMPA receptor subunits GluR1 and GluR2 are expressed and organized in functional receptors in axonal growth cones of hippocampal neurons. AMPA receptors are actively internalized upon activation and recruited to the surface upon depolarization. Pretreatment of cultures with botulinum toxin E or tetanus toxin prevents the receptor insertion into the plasma membrane, whereas treatment with alpha-latrotoxin enhances the surface exposure of GluR2, both in growth cones of cultured neurons and in brain synaptosomes. Purification of small synaptic vesicles through controlled-pore glass chromatography, revealed that both GluR2 and GluR1, but not the GluR2 interacting protein GRIP, copurify with synaptic vesicles. These data indicate that, at steady state, a major pool of AMPA receptor subunits reside in synaptic vesicle membranes and can be recruited to the presynaptic membrane as functional receptors in response to depolarization.

Chicken-embryo fibroblasts produce two types of interferon upon stimulation with Newcastle disease virus

Controversy has long surrounded the question of whether chickens, like mammals, can produce two types of interferon (IFN). Recently, type-I and type-II chicken IFNs have been cloned. Our study focuses on the further characterization of native fibroblast and spleen IFNs and shows that chicken embryo fibroblasts (CEFs) produce a mixture of type-I and type-II IFNs. IFN was purified by three different methods, controlled pore-glass chromatography, ion-exchange chromatography and preparative SDS-PAGE. Three protein bands showing IFN-like anti-viral activity, from CEFs which had been virus-stimulated for IFN production, were detected at 25, 27 and 29 kDa. Polyclonal antibodies produced against these bands showed partial cross-reaction with purified media from mitogen-stimulated spleen cells in ELISA, western blot analysis and anti-viral activity neutralization assay. Differences between purified conditioned media from CEF and spleen were found with respect to the stimulation of macrophages for nitric oxide production, pH stability and signal transduction pathways; only CEF IFN activated the IFN-stimulated gene factor-3 complex, whereas both CEF and spleen IFNs activated the IFN regulatory factor-1 gene. These findings concur with the differences that are known to exist between mammalian type-I and type-II IFNs. Attempts at sequencing the 25 and 27 kDa proteins by Edman degradation yielded evidence of N-terminal blockage.

Amphiphysin, a novel protein associated with synaptic vesicles.

To obtain access to novel proteins of the neuronal synapse, we have raised antisera against proteins of synaptic plasma membranes and used them for immunoscreening brain cDNA expression libraries. One of the newly isolated cDNAs encodes an acidic protein of 75 kDa with a distinct architecture of structural domains and multiple potential phosphorylation sites. Light and electron microscopy employing monospecific antisera raised against the expression product indicate a synapse-specific, presynaptic localization of this protein in many synapses of the chicken and rat nervous system. Its overall distribution in brain is very similar to that of synaptophysin, a ubiquitous protein of synaptic vesicles. In addition to brain, the protein or its mRNA is expressed in adrenal gland and anterior and posterior pituitary, but was not detected in a variety of other tissues. In controlled pore glass chromatography the native protein copurifies with synaptic vesicles and largely remains associated with them under various washing conditions. However, its amino acid sequence is very hydrophilic and it segregates into the aqueous phase in detergent phase partition. An earlier step of synaptic vesicle purification, sucrose cushion centrifugation, separates a vesicle-bound fraction of this protein from an unbound fraction. This seems to be a new, perhaps peripheral, protein of synaptic vesicles for which we propose the name, amphiphysin.

Purification of bursh border membrane vesicles from rat renal cortex by size-exclusion chromatography

Size-exclusion chromatography with controlled pore glass (CPG) was used in the further purification of renal brush border membrane vesicles (BBMV) isolated by the Ca precipitation method. The BBMV obtained had an almost spherical shape and their average diameter was about 95 nm in isotonic solution. The specific activities of alkaline phosphate and leucine aminopeptidase in the BBMV preparation were increased 18- and 17-fold, respectively, over those in the crude homogenate. The uptake of d-glucose by the purified BBMV in the presence of a sodium gradient reached 8.53 nmol/mg protein at 20 s. These results indicate that CPG chromatography is a suitable procedure by which to obtain purified renal BBMV of homogenous size and with high specific marker enzyme activity for use in the study of membrane transport.

Characterization of recombinant human lymphotoxin (tumor necrosis factor-β) produced by a mammalian cell line

Lymphotoxin (LT) was purified from serum-free conditioned media of a recombinant mammalian cell line transfected with human lymphotoxin cDNA. The purification scheme consisted of controlled pore glass chromatography, Q-Sepharose ion-exchange chromatography, and concanavalin A-Sepharose chromatography. The purified protein was found to be homogeneous by reverse-phase high-performance liquid chromatography and had an approximate specific activity of 130 × 10 6 units per milligram protein as determined by the L-929 cytotoxicity assay. Purified LT had an isoelectric point of approximately 6.85 and an apparent molecular weight of 50,000 by gel permeation high-pressure liquid chromatography. However, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two distinct bands at approximate molecular sizes of 25 and 20 kDa were observed. Both the bands were immunoreactive by Western blot analysis and found to be associated with biological activity. The two forms of lymphotoxin differed from each other with respect to protein structure. Amino-terminal amino acid sequence analysis revealed that the 25-kDa LT sequence starts with LeuProGly-residues whereas that of the 20-kDa LT begins with HisLeuAla; thus the latter form is truncated by 20 amino acid residues from the amino terminal. Two species of LT also differed from each other with respect to carbohydrate structure. Enzymatic removal of sialic acid reduced the molecular weight of 25 kDa by approximately 5 kDa whereas that of the 20-kDa LT was unchanged. A reduction in an apparent molecular size by ~4 kDa of both species of LT was observed on removal of N-linked oligosaccharides. Treatment with O-Glycanase had minimal effect on either form of LT. The recombinant lymphotoxin described here was found superior in its solubility behavior as compared to bacterial cell derived LT. Overall, mammalian cell line derived recombinant LT appears closer in its properties to natural LT than does bacterial cell derived recombinant LT.

Effect of heat treatment on casein micelle profiles obtained by controlled pore-glass chromatography

SummaryThe effect of heat treatment on the profiles of casein micelles obtained at 340 and 280 nm from controlled pore-glass chromatography of milk was studied in a series of unhomogenized whole milk samples which had been heated in a pilot plant at 75, 90, 105, 120, 135 and 150 °C for holding times of 4, 16, 32, 72, 150 and 300 s. A second experiment was carried out in which whole milk was preheated at temperatures between 70 and 110 °C for 13, 60 and 180 s before being treated at 120 °C for 4 s or 150 °C for 16 s. The average micelle size in some of the samples was determined by photon correlation spectroscopy. Profiles were divided into four fractions in which micelle size decreased from fraction 1 to fraction 4. The dimensions of these fractions were expressed in terms of size relative to that of the total profile. Fraction 4 was not affected by any of the heat treatments. Fractions 1–3 were significantly affected only by temperatures above 120 °C. Between 120 and 150 °C fraction 1 increased significantly while fractions 2 and 3 showed a corresponding decrease. Holding time affected fraction 1 only above 120 °C. For fraction 2 there was no effect in < 32 s at temperatures less than 150 °C. Fraction 3 was the most sensitive to changes in holding time, but no effect was observed below 105 °C. Average micelle size measurements indicated that a substantial size increase only occurred in those milks heated at 135 °C for longer than 16 s and in all the samples at 150 °C. To assess whether this increase in micelle size was reversible, samples were dialysed against a 70 mm-NaCl solution containing 10 mm-EDTA and then separated by column chromatography on Sephacryl S-300. The proportion of materials which was undissociated by dialysis remained unchanged for temperatures below 120 °C, but progressively increased with treatments more severe than 120 °C for 16 s. Column profiles were not influenced by variations in the conditions of preheating

Characterization of synapsin I binding to small synaptic vesicles.

The binding of synapsin I, a synaptic vesicle-associated phosphoprotein, to small synaptic vesicles has been examined. For this study, synapsin I was purified under nondenaturing conditions from rat brain, using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and characterized. Small synaptic vesicles were purified from rat neocortex by controlled pore glass chromatography as the last purification step, and binding was characterized at an ionic strength equivalent to 40 mM NaCl. After removal of endogenous synapsin I, exogenous dephospho-synapsin I bound with high affinity (Kd, 10 +/- 6 nM) to synaptic vesicles. The binding saturated at 76 +/- 40 micrograms synapsin I/mg of vesicle protein, which corresponded to the amount found endogenously in purified vesicles. Synapsin I binding exhibited a broad pH optimum around pH 7. Other basic proteins, specifically myelin basic protein and histone H2b, did not compete with synapsin I for binding to vesicles. Other membranes purified from rat brain and membranes derived from human erythrocytes did not show the high affinity binding site for synapsin I found in vesicles. The binding of three different forms of phosphosynapsin I to vesicles was investigated. Synapsin I, phosphorylated at sites 2 and 3 by purified calcium/calmodulin-dependent protein kinase II, bound with a 5-fold lower affinity to the vesicles than did dephospho-synapsin I. In contrast, synapsin I, phosphorylated at site 1 by purified catalytic subunit of cAMP-dependent protein kinase, bound with an affinity close to that of dephospho-synapsin I. Synapsin I phosphorylated on all three sites bound to the vesicles with an affinity comparable to that of synapsin I phosphorylated on sites 2 and 3. Under conditions of higher ionic strength (150 mM NaCl equivalent), synapsin I bound with a 5-fold lower affinity to vesicles, and no effect of phosphorylation on binding was observed under these conditions.

Characterisation of cerebroside 3-sulphate dispersions

Galactosyl ceramide 3-sulphate (cerebroside 3-sulphate) was tritiated using [3H]NaBH4 with PdCl2 as catalyst. Quantitative purification of the three components of the product was achieved by chromatography on Florisil. Dispersions, prepared by either low energy sonication or by dilution from organic solvent, were compared by controlled pore glass chromatography, ultra-centrifugation and electron microscopy. Both dispersion techniques were shown to form unilamellar vesicles, the average size of vesicles produced by sonication being far larger than those produced by solvent dilution. The diameter of isolated vesicles produced by solvent dilution was in the range 10–80 nm.

Small-pool high-yield factor VIII production.

Hemophilia care depends on several factors for the production of purified FVIII: plasma procurement, plasma logistics, production method, and efficacy. The latter two are restrictive factors, both for the supply and the safety of FVIII preparations. Conventional production methodology unavoidably recovers only 10 to 20% of usable protein, therefore requiring large pools of source plasma. Related to pool size is the transmission of diseases, which poses unnecessary risks for patients. The development of new technologies to better recover FVIII allows reduction of the pool size: crush-thaw, controlled pore-glass chromatography, and heparin double-cold precipitation techniques. This review will reflect on current production methods, pool size concept, small-pool approaches in FVIII production, and future developments.

老鼠干擾素(Mouse Interferon)之快速純化方法

本文提供一種快速的純化老鼠干擾素方法，用此法所得到的干擾素很穩定，比活性（Specific activity）為2.1×10^(6)單位／毫克蛋白質，而尖峯部份（peak fraction）的比活性則為5.7×10^(6)單位／毫克蛋白質，為粗干擾素（crude interferon）的438倍，整個純化過程需時才二天，因此我們相信應用毛孔玻璃色譜（Controlled Pore Glass Chromatography）和高效能液相層析分析色譜（High Performance Liquid Chromatography）將是一種快速且有效的純化方法。

Aggregation states of alcohol-soluble storage proteins of barley, rye, wheat and maize.

AbstractTwo approaches were used to study the aggregation states of prolamins extracted from milled whole grain of wheat, barley, rye and maize with 500 ml litre−1 aqueous propan‐1‐ol. In the first, a comparison was made of the electrophoretic patterns of the samples separated by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS‐PAGE) under reducing and non‐reducing conditions. In the second, they were chromatographed on a column of controlled pore glass (CPG) using a modified acetic acid, urea, cetyltrimethyl ammonium bromide (AUC) solvent and aliquots of the major peaks were reduced and analysed by SDS‐PAGE. When the CPG chromatography was repeated under reducing conditions some of the peaks were eliminated indicating that they were composed of disulphide‐linked aggregates. The results from both approaches showed that in barley, rye and wheat the HMW prolamins and some of the S‐rich prolamins (B hordein, some γ‐secalins and subunits of high molecular weight gliadins) are present predominantly or partially in high molecular weight (above 1 × 106 daltons) aggregates which appear to be stabilised by disulphide bonds. Other S‐rich prolamins (including some γ‐secalins, α, β and γ‐gliadins) and all the S‐poor prolamins (‘C’ hordein, ω‐secalin, ω‐gliadin) are present predominantly or only as monomers. These results are discussed in relation to the structural and genetic homology of the prolamins in the three species. Although prolamin aggregates are also present in maize, these are of lower molecular weight.

PREPARATION OF HIGH-PURITY FACTOR VIII BY CONTROLLED PORE GLASS CHROMATOGRAPHY

PREPARATION OF HIGH-PURITY FACTOR VIII BY CONTROLLED PORE GLASS CHROMATOGRAPHY

Preparation of stable intermediate-purity factor VIII concentrate with a note on high-purity factor VIII.

Cryoprecipitate prepared by a rapid thawing technique was pooled in batches of 600--720 donor units and washed with ice-cold Tris-citrate-NaCl solution. After dissolving at 37 degrees C, it was adsorbed with Al(OH)3 and kaolin, and cleared by centrifugation. The supernatant, diluted with 5% dextrose was passed repeatedly through a bed of Celite, filtered through a 293 mm X 0.3 micrometer membrane disc and lyophilized. Typical composition was 15 U . ml-1 factor VII, and 40 mg . ml-1 protein with a yield of 300 U/l of starting plasma. The crude factor VIII concentrate was also a suitable material for preparation of high-purity factor VIII by controlled pore glass chromatography.

Preparation of Stable Intermediate-Purity Factor VIII Concentrate with a Note on High-Purity Factor VIII

Cryoprecipitate prepared by a rapid thawing technique was pooled in batches of 600-720 donor units and washed with ice-cold Tris-citrate-NaCl solution. After dissolving at 37 °C, it was adsorbed with Al(OH)(3) and kaolin, and cleared by centrifugation. The supernatant, diluted with 5% dextrose was passed repeatedly through a bed of Celite, filtered through a 293 mm X 0.3 μm membrane disc and lyophilized. Typical composition was 15 U · ml^-1 factor VIII, and 40 mg · ml^-1 protein with a yield of 300 U/l of starting plasma. The crude factor VIII concentrate was also a suitable material for preparation of high-purity factor VIII by controlled pore glass chromatography.

A Comparison of Controlled Pore Glass Chromatography and Ultra Centrifugation for Detecting Igm Antibody in Congenital Rubella Infection

Screening programmes for inapparent congenital rubella infection have created a need for accurate methods of laboratory diagnosis of this disease in the newborn. Demonstration of rubella-specific immunoglobulin M (IgM) antibody in either cord blood or neonatal serum is accepted as the most convenient method available. This study reports a chromatographic method with Controlled Pore Glass for demonstrating rubella-specific IgM antibody. The technique is more practical for use in the diagnostic laboratory than other currently advocated methods of serum separation and is at least as sensitive.

Controlled pore glass chromatography of protein-sodium dodecyl sulphate complexes

The inclusion of urea has been found to eliminate adsorption of proteinsodium dodecyl sulphate (SDS) complexes to controlled pore glass. Using buffer containing 6 M urea, 0.5% SDS and glass with pore diameter 12.3 nm, it is possible to determine protein molecular weights in the range 3500–12,000. Results with glass of larger pore diameter (25.5 nm) are similar to those reported in the absence of urea in the molecular-weight range 12,000–140,000. Controlled pore glass chromatography also permits the study of the relative importance of conformation free of charge effects for those proteins which deviate from the normal calibration curve for SDS-polyacrylamide gels.

Lymphocytic Choriomeningitis virus. I. Concentration and purification of the infectious virus.

Two procedures for the purification of infectious lymphocytic choriomeningitis virus from cell culture fluid have been developed. If large quantities of very pure virus are to be prepared, infected L cells are maintained with a medium supplemented with calf serum, the proteins of which have been largely removed by pretreatment with polyethylene glycol. Two days after infection of the cultures, the media are collected and the virus is concentrated by treatment with polyethylene glycol 40,000. Purification with a 10,000-fold increase of specific infectivity is achieved with steric chromatography on controlled-pore glass beads with pore sizes of 42 to 44 nm and centrifugation in density gradients prepared with amido trizoate. An alternative method begins with precipitation of the virus from infected cell cuture medium with zinc acetate, followed by controlled-pore glass chromatography and density centrifugation in a discontinuous sucrose gradient. Purification thus obtained is 200-fold in terms of specific infectivity.