Rapid isolation of vesicular and micellar carriers of biliary lipids by ultracentrifugation

A simple, rapid, and new method has been developed to isolate and to quantitate the vesicular carrier of biliary lipids by isopycnic ultracentrifugation. The method combines the use of Metrizamide, as an inert centrifugation media to change the density of bile for isopycnic separation of vesicles, and a vertical rotor, to decrease both the time of centrifugation and the pressure of the hydrostatic column in the ultracentrifuge tube. Vesicles harvested from bile-Metrizamide density gradients were identified by negative staining electron microscopy. The buoyant densitites of biliary vesicles varied between 1.010 and 1.030 g/ml. The diameter of vesicles in fractions with d less than 1.020 g/ml was 82 +/- 10 nm and in fraction with d approximately 1.030 g/ml was 57 +/- 8 nm. Gel filtration chromatography with Ultrogel AcA 34 was used to validate the quantitive isolation of vesicles by the ultracentrifugal method. In experiments with bile-Metrizamide continuous preformed density gradients, greater than 93% of vesicular cholesterol was found in fractions with d less than 1.030 g/ml after 285 min of centrifugation at 50,000 rpm in a VTi vertical rotor (Beckman Instruments, Inc.). When 16% Metrizamide was dissolved in bile and centrifuged for 120 min, greater than 96% of total vesicular cholesterol was found in the top 0.4 ml of the 5-ml centrifuge tube, as assessed by gel filtration chromatography. This fraction contained less than 8% of cholesterol carried in micelles, as assessed by gel filtration chromatography. The variation coefficient of this short ultracentrifugal method to isolate biliary vesicles was 4.6%.(ABSTRACT TRUNCATED AT 250 WORDS)     

大丽轮枝菌毒素的分离、提纯及生物测定

试验用Czapek's培养液培养大丽轮枝菌(Verticillium dahliae Kleb.)不同致病力类型的5个菌株的培养滤液,经浓缩、离心和透析制成的粗毒素,再经DEAE-纤维素柱层析得到初提毒素,最后通过琼脂糖凝胶过滤层析获得纯毒素样品。生物活性测定表明初提毒素的最低生物活性浓度为5.0—5.5μg/ml。纯毒素的最低生物活性浓度为4.0—4.5μg/ml。配制相同浓度的不同致病力类型菌株的毒素液,它们的培养滤液、粗毒素或纯毒素对棉苗的致萎能力相同。     

Casting immobilized pH gradients into cylindrical polyacrylamide gels

A novel method is described for casting immobilized pH gradients in polyacrylamide gel rods of small diameter (2 mm), based on the principle of rotational centrifugation. The tubes are filled vertically with equal volumes of dense and light solution (250 microliter each) titrated to the extremes of the desired pH gradient, and then tilted at 2.5 degrees to the level. After 5 min at rest, to allow for sliding of the two menisci to equilibrium position, the glass tubes are rotated for 3 min at 180 rpm, followed by an additional 3 min at 180 rpm by reversing the sense of rotation. A homogeneous linear gradient is thus produced. The rotating platform is then raised to 90 degrees and the gels allowed to polymerize under standard conditions. Formation of linear and reproducible pH gradients is ensured by using stabilizing density gradients of low viscosity (0-5% glycerol, having a maximal ratio viscosity/density of 1.1).     

Simple and rapid purification of pediocin PA-1 from Pediococcus pentosaceous NCDC 273 suitable for industrial application

The use of pediocins as food additives or drugs requires a simple and rapid method by which large quantities of homogeneous pediocin are produced at industrial level. Two centrifugation steps required during initial stages of purification i.e. separation of cells from fermentation broth and collection of precipitates after ammonium sulphate precipitation are the major bottlenecks for their large scale purification. In the present work, pediocin production by a new a dairy strain, Pediococcus pentosaceous NCDC 273 (identical to pediocin PA-1 at nucleotide sequence level), was found to be optimum at initial pH of 6.0 and 7.0 of basal MRS supplemented with 20g/l of glucose or lactose at 20 and 24h, respectively. Immobilization of cells through entrapment in alginate-xanthan gum gel beads with chitosan coating resulted in negligible cell release during fermentation. Thus, the cell free extract was directly collected through decantation, avoiding the need of centrifugation step at this stage. Subsequent ammonium sulphate precipitation at isoelectric point of pediocin PA-1 (8.85), using magnetic stirrer at high speed (approx. 1200rpm), resulted in forceful deposition of precipitates on the wall of precipitation beaker allowing their collection using a spatula, avoiding centrifugation step at this stage also. Further purification using cation-exchange chromatography resulted in yield of 134.4% with more than 320 fold purification with the specific activity of 19×10(5)AU/mg. The collection of single peak of pediocin at 41.9min in RP-HPLC, overlapping with standard pediocin PA-1, resulted in yield of 1.15μg from 20μl of sample applied. The overlapping of RP-HPLC peak and SDS-PAGE band corresponding to 4.6kDa, confirmed the purity and identity of pediocin 273 as pediocin PA-1.     

Characterization and purification of a calcium-sensitive ATP diphosphohydrolase from pig pancreas

An ATP diphosphohydrolase (EC 3.6.1.5) from the pancreas of the pig has been characterized and purified. The enzyme which has an optimum pH between 8 and 9 is specific for diphospho- and triphosphonucleosides. The Km values for ADP and ATP are 7.4 and 7.3 x 10(-4) M, respectively, and the purified enzyme has specific activities of 13 and 15.2 mumol of Pi/min/m of protein, respectively. It requires calcium or magnesium ions and it is insensitive to ATPase inhibitors, namely oligomycin, ouabain, and ruthenium red, and to levamisole, an inhibitor of alkaline phosphatase. Denaturation experiments, by heat and trypsin treatments, indicated that only one enzyme is involved. This is confirmed by the solubilization and purification process and by polyacrylamide gel electrophoresis. A 270-fold purification was obtained by centrifugation and successive column chromatography on Sepharose 4B and Affi-Gel blue. It is a glycoprotein with a molecular weight of 65,000 as estimated by polyacrylamide gel electrophoresis.     

Partial purification and characterization of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas

This study reports the presence of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas, and its partial purification and some properties. Crude enzyme preparation was obtained by extraction from acetone-dried powder of the pancreas at pH 7.6. For solubilization of enzyme, freezing and thawing were carried out. Crude enzyme extract was fractionated with ammonium sulfate precipitation, gel filtration on Sephadex G-200 column and ion-exchange chromatography on DEAE-cellulose. Partially purified enzyme showed 2897-folds purification. The enzyme activity on polyacrylamide gel electrophoresis showed good agreement with a main protein band stained with Coomassie brilliant blue. Molecular weight of this enzyme from the pancreas was estimated to be 300 000 by gel filtration on Sephacryl S-300 column. Optimum pH was between 8.5 and 9.0, and K_m value for glycylproline-p-nitroanilide tosilate was 0.33 mM. This enzyme from the pancreas was a serine enzyme and was relatively stable to heat at 60°C for 10 min.     

A novel low-molecular weight chondroitin sulphate proteoglycan isolated from cartilage.

Proteoglycans were isolated from cartilage by extraction with 4M-guanidinium chloride followed by direct centrifugation in 4M-guanidinium chloride/CsCl at a low starting density, 1.34 g/ml. N-Ethylmaleimide was included in the extraction solvent as a precaution against contamination of proteoglycans with unrelated proteins mediated by disulphide exchange. A novel, discrete, low-buoyant-density proteoglycan (1.40--1.35 g/ml) was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its proteoglycan nature was revealed by the shift in the molecular size observed on gel electrophoresis after treatment with chondroitinase ABC. The core protein was monodisperse. The proteoglycan was further purified by gel chromatography with and without addition of hyaluronate. The proteoglycan constitutes less than 2% (by weight) of the total extracted proteoglycans and is not capable of interacting with hyaluronate. The same proteoglycan was purified in larger quantities by sequential associative and dissociative CsCl-density-gradient centrifugation, zonal rate sedimentation in a sucrose gradient and gel chromatography on Sepharose CL-4B. The pure proteoglycan had a molecular weight of 76 300 determined by sedimentation-equilibrium centrifugation and an apparent partial specific volume of 0.59 ml/g. It contained about 25% protein (of dry weight) and had remarkably high contents of leucine and cysteine as compared with other proteoglycans. The proteoglycan contained two to three large chondroitin sulphate chains and some oligosaccharides.     

Isolation and Characterization of a Novel Antitumor Substance,Renastacarcin

A new antitumor substance designated renastacarcin has been isolated from the cultured broth of Streptomyces violascens by means of DEAE-cellulose and Sephadex G-100 column chromatography. The homogeneity of renastacarcin was confirmed by electrophoreses on a polyacrylamide gel and a cellulose acetate membrane as well as by ultracentrifugal analysis. A sedimentation coefficient at 55,430 rpm was found to be 2.12S and the molecular weight was calculated to be 34,500.     

Purification and properties of a DNA-dependent ATPase induced by bacteriophage T4.

A DNA-dependent ATPase formed after T4 phage infection is purified to apparent homogeneity. The molecular weight of the purified enzyme is 50 000 when determined by glycerol gradient centrifugation and by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The enzyme at an earlier stage in purification (prior to DEAE-cellulose chromatography) exists as a complex with a molecular weight of 100000. However, molecular weight determinations by Sephadex gel chromatography give considerably decreased molecular weights for the complex and for the enzyme after DEAE-cellulose chromatography. The enzyme is stimulated to varying degrees by a variety of single-stranded polydeoxyribonucleotides or by single-stranded DNA, but no chemical change in the polynucleotide has been detected as a result of the enzyme action.     

Membrane proteins of Rhodopseudomonas spheroides III. Isolation,purification, and characterization of cell envelope proteins

Cell envelopes (cell wall and cell membrane) from aerobically grown cells of Rhodopseudomonas spheroides were isolated and purified by a combination of differential centrifugation and centrifugation through 40% sucrose. Cell envelope protein from aerobically grown cells was resolved by dodecyl sulphate-polyacrylamide gel electrophoresis. Biochemical characterization of selected envelope membrane proteins demonstrated heterogeneity between different protein species. Amino acid analyses of individual proteins revealed between 50–60 mole% nonpolar residues.Envelope membranes derived from anaerobically grown cells were also isolated and purified by a combination of differential centrifugation, column chromatography on Sepharose 2B, and centrifugation in 40% sucrose. The dodecyl sulphate-polyacrylamide gel patterns of anaerobic and aerobic envelope membrane proteins were very similar and the results suggest a common protein structure.     

Biochemical studies of potential-dependent sodium channels

The data on interaction of sodium channels of excited membranes with various neurotoxin groups are considered in the paper including the data on using biologically active neurotoxin derivatives as a specific label. These derivatives carry fluorescent or photoactive groups possessing high specific radioactivity, which permits developing the methods for sodium channel solubilization and solubilized preparation stabilization. The high-purified preparations are characterized by the methods of gel chromatography, centrifugation within the gradient density, gel electrophoresis and affinity chromatography. Experiments on reconstruction of solubilized preparations in the bilayer of lipid vesicles are under discussion.     

Purification and structural characterization of a cartilage matrix protein.

The cartilage matrix protein is a major non-collagenous protein in bovine cartilage. It was purified from a 5 M-guanidinium chloride extract of bovine tracheal cartilage by sequential CsCl-density-gradient centrifugation, gel chromatography in guanidinium chloride and differential precipitation. The molecular weight of the intact protein is 148 000, determined by sedimentation-equilibrium centrifugation. It was dissociated to three subunits of molecular weight 52 000 by reduction of disulphide bonds. The cartilage matrix protein was insoluble in low-salt solutions and behaved abnormally on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The content of cysteine was high, whereas the contents of aromatic amino acids were low. The carbohydrate content was 3.9% (w/w). Glycopeptides obtained after papain digestion were heterogenous on gel chromatography. Asparagine/aspartic acid was enriched in the purified glycopeptides, indicating the presence of N-glycosidic linkages to protein.     

Glicentin is present in the pig pancreas

Specimens from porcine pancreas and ileal mucosa were extracted in acid/ethanol, subjected to gel permeation chromatography, ion-exchange chromatography, enzymatic peptide degradation, reverse-phase HPLC, and analysed for glucagon-like and glicentin-like immunoreactivity by region-specific radioimmunoassays. Results obtained with all methods were consistent with the hypothesis that glicentin is present in the pig pancreas in small amounts.     

Competence related proteins in the supernatant of competent cells of Bacillus subtilis

Summary We report that centrifugation at relatively high g-forces reduces the ability of competent cells of Bacillus subtilis to bind and take up DNA, and to be transformed. The centrifugation supernatant from competent cells restores this reduction of competence; the supernatant from non-competent cells is inactive. Phosphocellulose chromatography of centrifugation supernatants from radioactive competent cultures gave rise to six sharp peaks, together, these were shown by subsequent SDS polyacrylamide gel electrophoresis to contain over 60 different polypeptide bands. Peak II, which showed competence restoring activity, produced three polypeptides. When these bands were further examined, one of these exhibited DNA binding activity and the other two each contained a different endonuclease. Competence restoring activity was not recovered from the SDS polyacrylamide gel of peak II. The three peaks from non-competent cultures produced altogether five faint bands in gel electrophoresis. None of these bands were similar to those found in peak II.This work was performed in partial fulfillment of the requirements of Georgetown University for the degree of Doctor of Philosophy     

Natural purified porcine gastric inhibitory polypeptide (GIP) stimulates exocrine pancreas secretion due to contamination with a cholecystokinin-like substance

The effects of purified natural gastric inhibitory polypeptide-enterogastrone III (GIP-EG III) and a fraction which is further purified by high pressure liquid chromatography (GIP-HPLC) were investigated on the endocrine and exocrine isolated perfused pancreas of rats. At the dose of 5 ng/ml used for both GIP preparations, only GIP-EG III significantly stimulated volume and amylase secretion of the exocrine pancreas. The response of insulin release to stimulation by GIP-EG III or GIP-HPLC was not significantly different. In the presence of cholecystokinin-octapeptide (CCK-8) at a concentration which gave half-maximal stimulation of amylase secretion, GIP-EG III almost doubled the response of the exocrine pancreas, whereas GIP-HPLC had no additional effect. CCK-8 alone significantly increased total insulin output under hyperglycemic conditions. We conclude that porcine GIP purified by gel chromatography contains a CCK-like substance which can be removed by further purification on high pressure liquid chromatography without affecting the insulinotropic activity. Some of the reported effects of GIP could be due to contamination.     

Purification and chemical characterization of human hexosaminidases A and B.

N-Acetyl-beta-hexosaminidases A and B were purified to homogeneity from human placenta. In the initial step of purification, the enzymes were adsorbed on concanavalin A-Sepharose 4B and eluted from the column with alpha-methyl D-mannosides. Subsequent purification steps included DEAE-cellulose column chromatography, QAE-Sephadex [diethyl-(2-hydroxypropyl)aminoethyl-Sephadex] column chromatography, Sephadex G-200 gel filtration and preparative disc polyacrylamide-gel electrophoresis, followed by another QAE-Sephadex chromatography for the hexosaminidase A preparation, and DEAE-cellulose column chromatography, calcium phosphate gel chromatography, Sephadex G-200 gel filtration, QAE-Sephadex chromatography and CM-cellulose chromatography for the hexosaminidase B preparation. The purified preparations, particularly hexosaminidase A, had significantly higher specific enzyme activities than previously reported. The preparations moved on polyacrylamide-gel electrophoresis as single protein bands, which also stained for enzyme activity. Sedimentation-equilibrium centrifugation indicated homogenous dispersion of the enzymes, and the molecular weight was estimated as about 110000 for both enzymes. Complete amino acid and carbohydrate compositions of the two isoenzymes were determined, and, in contrast with previous suggestions, no sialic acid was found in the enzymes.     

Pancreatic anionic trypsin: evidence for the existence of a 30 kDa form.

1. An anionic form of trypsin has been isolated from pancreas of various species (rat, pig, dog and cow). 2. The purification procedure included affinity chromatography on STI-Sepharose 4B and ion-exchange chromatography on DEAE-Sephadex A-50. 3. The preparation was homogenous as checked by SDS-polyacrylamide slab gel electrophoresis, resulting in an estimated molecular weight of 30 kilodaltons (kDa) for this anionic form. 4. Antibodies against the anionic form from rat pancreas cross-reacted towards the anionic enzyme from porcine pancreas but not with the dog or bovine enzyme, nor with all the studied cationic forms. 5. Limited proteolysis of tubulin, a cytoskeletal protein, with an anionic or cationic form of trypsin showed striking differences in the size of produced peptides.     

Purification and characterization of porcine heart type 2A protein phosphatases.

Protein phosphatases 2A1 and 2A2 were isolated from porcine heart tissue extracts by precipitation at pH 5.0 and separated by chromatography on DEAE-Sephacel. Phosphatase 2A1 was then purified to apparent homogeneity by chromatography on phenyl-Sepharose, aminohexyl-Sepharose, Sephacryl S-300, and L-tyrosine-agarose. Phosphatase 2A2 was purified to apparent homogeneity by chromatography on phenyl-Sepharose, DEAE-Sephacel, aminohexyl-Sepharose and L-tyrosine-agarose. Purified phosphatases 2A1 and 2A2 had specific activities of 2200 and 2710 nanomoles of phosphate released from phosphorylase a/mg protein, respectively. The apparent molecular weights of phosphatases 2A1 and 2A2 on gel filtration were 155 and 105 kDa, respectively. Both enzymes contain 70 and 37 kDa subunits and 2A1 also contains a 57 kDa subunit. The 37 kDa catalytic subunit (2Ac) was obtained from the purified phosphatases by treatment with room temperature ethanol followed by sucrose density gradient centrifugation or gel filtration chromatography.     

Preparative electrophoresis on agarose submerged gels of two aggregating proteoglycan monomers from articular cartilage

Analytical electrophoresis on polyacrylamide-agarose gels of aggregating proteoglycan monomers from baboon articular cartilage produces two distinct bands, corresponding to two different aggregating monomer populations. A preparative electrophoresis procedure is described for isolating the two monomers. Proteoglycans were extracted from young baboon articular cartilage in 4 M guanidinium chloride containing proteolysis inhibitors and aggregated after hyaluronic acid addition. The aggregates were separated from non-aggregated proteoglycans by isopycnic centrifugation, followed by gel chromatography on Sepharose CL-2B. The monomers of the aggregates were obtained by isopycnic centrifugation under dissociative conditions. Two monomers were separated by preparative electrophoresis on 0.8 % agarose submerged gels. Approximately 60 % of the proteoglycans were recovered from the gel using a freeze-squeeze procedure. Aliquots of the separated monomers gave single bands when submitted to analytical polyacrylamide-agarose gel electrophoresis. Their migration and appearance were similar to that of the two bands present in the non separated preparation of monomers.     

Selective Augmentation of Stem Cell Populations in Structural Fat Grafts for Maxillofacial Surgery

Structural fat grafting utilizes the centrifugation of liposuction aspirates to create a graded density of adipose tissue. This study was performed to qualitatively investigate the effects of centrifugation on stem cells present in adipose tissue. Liposuction aspirates were obtained from healthy donors and either not centrifuged or centrifuged at 1,800 rpm for 3 minutes. The obtained fat volumes were divided into three layers and then analyzed. The results demonstrate that centrifugation induces a different distribution of stem cells in the three layers. The high-density layer displays the highest expression of mesenchymal stem cell and endothelial markers. The low-density layer exhibits an enrichment of multipotent stem cells. We conclude that appropriate centrifugation concentrates stem cells. This finding may influence the clinical practice of liposuction aspirate centrifugation and enhance graft uptake.