Rapid purification of cytochrome c oxidase from Paracoccus denitrificans

Two methods are described for the purification of cytochrome c oxidase from Triton X-100 extracts of the periplasma membrane of Paracoccus denitrificans. The first is a large-scale procedure for the preparation of 100-250 nmol of cytochrome c oxidase (10-20 mg) in 1 week. The second is a rapid procedure for isolating up to 25 nmol in 2-3 days. Owing to the high yields given by fast protein liquid chromatography (FPLC) on Mono Q columns, the overall yield is about 20%, whereas the yield in many other previously published procedures does not exceed 10%. The use of FPLC on Mono Q also offers a considerable saving of time.     

Isolation and partial characterization of angiotensinase A and aminopeptidase M from urine and human kidney by lectin affinity chromatography and high-performance liquid chromatography

Angiotensinase A (ATA) and aminopeptidase M (APM) were partially purified from human urine specimens and human kidney particles using wheat germ lectin affinity chromatography, anion-exchange Fast Protein Liquid Chromatography (FPLC) (Mono Q), chromatofocusing (Mono P, FPLC) and Superose 12 gel filtration. APM, a globular 5-nm glycoprotein, is localized in the brush border membrane of the proximal tubule; angiotensin II-degrading ATA is present on glomerular endothelia and podocytes and, to lesser extent, in the brush border. For the first time, both peaks of ATA and APM activity from urine samples were separated by the above-mentioned techniques with only slight overlap; ATP (146,000 dalton: pI4.8) was enriched more than 20-fold and APM (153,000 dalton, pI4.7) more than 50-fold compared with the activity of the starting material. Using similar separation steps, ATA and APM solubilized from kidney particles could not be resolved into two distinct peak fractions, however, except after hydrophobic interaction chromatography. Thus urine is a major source for the preparation of individual ATA and APM fractions, necessary to generate specific anti-enzyme antibodies for diagnostic purposes.     

Purification of the glycoprotein glucose oxidase from Penicillium amagasakiense by high-performance liquid chromatography

Fast protein liquid chromatography (FPLC) in combination with ion-exchange chromatography on a Mono Q column was used to purify glucose oxidase from Penicillium amagasakiense to homogeneity. Purification was performed with a mixed pH and salt gradient, with 20 mM phosphate buffer (pH 8.5) as starting buffer (A) and 50 mM acetate buffer (pH 3.6) with 0.1 M NaCl as elution buffer (B). Elution conditions were optimized to permit the simultaneous purification and separation of the glucose oxidase isoforms. Three peaks, each consisting of 1-2 isoforms and exhibiting a homogeneous titration curve profile, were resolved with a very flat linear gradient of 5.0-5.1% B in 40 ml. Three more peaks, each consisting of several isoforms, were eluted at 10%, 30% and 100% B. Optimization of the elution conditions and separation of the glucose oxidase isoforms was only possible because of the rapidity of each purification step and the high resolution provided by FPLC and Mono Q.     

Reconstitution of neutral amino acid transport from partially purified membrane components from Ehrlich ascites tumor cells

Solubilized protein fractions have been obtained from plasma membranes of Ehrlich ascites cells either by extraction with 0.5% Triton X-100 or by extraction with 2% cholate. Partial purification of the solubilized protein fraction has been obtained by utilizing a combination of ammonium sulfate precipitation and column chromatography. Leucine-binding activity has been detected in the Triton X-100 solubilized membrane fraction. The leucine-binding activity was measured by equilibrium dialysis and was saturable with high levels of leucine or phenylalanine and is not strongly effected by alanine. These properties are similar to those previously identified as System L. In addition, the cholate extracted protein fraction was partially purified and reconstituted into liposomes. Sodium dependent uptake of alanine and leucine could be demonstrated in the reconstituted vesicles. Concentrative uptake was dependent upon a sodium gradient. A membrane potential produced by valinomycin mediated potassium diffusion in the presence of sodium also stimulated amino acid transport in reconstituted liposomes.     

Purification of the membrane-form variant surface glycoprotein of Trypanosoma brucei

The membrane-form variant surface glycoprotein (mfVSG) is anchored in the plasma membrane of African trypanosomes by a diacylglycerol residue. On cell rupture the anchor is rapidly cleaved by an endogenous phospholipase C. A purification procedure is described which results in native mfVSG devoid of lipase activity. A total membrane fraction is prepared in the presence of the SH-inhibitor p-chloromercuribenzenesulphonic acid (pCMBS). Membrane proteins are solubilized in the presence of pCMBS and the detergent Zwittergent 3-12, conditions which inhibit the activity of the phospholipase. mfVSG is then purified by successive chromatography on rabbit anti-VSG affinity and cation-exchange columns (25% yield). The isolated protein is electrophoretically pure and partitions into the detergent phase on Triton X-114 phase separation, proving that it retains the diacylglycerol anchor.     

Efficient high-performance liquid chromatographic system for protein purification

An efficient high-performance liquid chromatographic system, consisting of an affinity column and a high-performance size-exclusion column, was developed and applied to the purification of growth hormone receptors from rabbit livers. When a 6-ml sample of Triton X-100 extracts containing 16 mg of protein was applied to the system, 1200-fold purified receptor with a 10% recovery of binding activity from homogenates was obtained within 3-4 h. The purified receptor exhibited one main band on sodium dodecyl sulphate polyacrylamide gel electrophoresis, and the affinity constant (Ka = 6.0.10(9) M-1) was found to be comparable with that of 1% Triton X-100 extract (4.4.10(9) M-1). The injection of 1 ml of 3 M urea solution prior to receptor elution with 10 ml of 6 M urea solution was effective in removing non-specific binding proteins.     

Purification of cytochromes P-450 derived from liver microsomes of untreated and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated marmoset monkeys

The purification of multiple forms of cytochrome P-450 (P450) from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated marmosets using fast protein liquid chromatography (FPLC) is described. The main aim was to achieve a better separation of certain closely related P450 sub-forms from each other than that previously obtained using conventional chromatography. An 8-aminooctyl-Sepharose fraction of cholate-solubilized microsomes was obtained first and, after fast desalting on Sephadex G-25, loaded on to a preparative Mono Q column. Five of the six gradient peaks contained P450 and were each rechromatographed on an analytical Mono Q column. The pass-through peak was fractionated further using a Mono S column. Other HPLC-quality anion- and cation-exchange gels were compared. For removal of excess of non-ionic detergent, five types of hydroxyapatite gels were compared. Seven purified forms of P450 and cytochrome b5 and P420 were isolated and characterized according to PHAST sodium dodecylsulphate-polyacrylamide gel electrophoretic apparent molecular masses, catalytic, spectral and magnetic properties and also TCDD-binding capacity (molar ratio of [14C]TCDD to P450). There are at least two sub-forms which appear to be TCDD inducible, one showing a substantial ethoxyresorufin-O-deethylase activity and the other having a high TCDD-binding molar ratio. Two other forms appear to be constitutive, as deduced from comparisons with forms purified from untreated animals.     

Angiotensinase A (aminopeptidase A): properties of chromatographically purified isoforms from human kidney

Angiotensin-II-cleaving angiotensinase A (aminopeptidase A, E.C. 3.4.11.7, ATA) plays an important role in glomerular haemodynamics. the pathophysiology of essential arterial hypertension and the induction of vascular disorders. In order to study biochemical and immunological properties of ATA, two isoforms (I and II) of the glycoprotein were isolated for the first time from human kidney cortex. Kidney cortex homogenate, digested with bromelain, was fractionated by ammonium sulphate precipitation and subsequent hydrophobic interaction chromatography, using a fast protein liquid chromatographic (FPLC) system. By anion-exchange FPLC (Mono Q column), the isoforms of ATA were eluted in two distinct peaks and were further purified by size-exclusion FPLC and preparative polyacrylamide gel electrophoresis. Biochemical, immunological and immunohistological characterization disclosed differences in the intrarenal localization, glycosylation Michaelis constant and apparent molecular mass (native and sodium dodecyl sulphate gel electrophoresis) but similar properties in the double-immunodiffusion technique. Polyclonal rabbit antibodies, raised against ATA isoforms I and II, precipitated an analogous antigen in urine from patients with renal tubular damage.     

质粒DNA的阴离子交换色谱法纯化及内毒素去除

采用Fractogel EMD TMAE(M)强阴离子交换介质分离纯化质粒脱氧核糖核酸（DNA）,该介质对质粒DNA的动态载样量达0.62 mg/mL。用Triton X-114或Triton X-100预先处理质粒DNA的裂解澄清液后,经阴离子交换色谱分离纯化获得的质粒DNA中内毒素含量分别为6.42 EU/mg和9.50 EU/mg,显著低于未经Triton处理的裂解澄清液（67.82 EU/mg）。该法实现了阴离子交换色谱一步纯化质粒DNA的目的,具有简便、省时、成本低等特点。     

Isolation of cytochrome P-450 components from marmoset liver microsomes by high-performance liquid chromatography.

A fast protein liquid chromatographic (FPLC) system with pre-packed and laboratory-packed columns was used for the analytical and preparative isolation of marmoset monkey cytochrome P-450 (P450) and NADPH-P450-reductase. Chromatographic separations also allowed the recovery of cytochrome b5, NADH-b5-reductase and epoxide hydratase. Cholate-solubilized liver microsomes from phenobarbital-induced marmosets were crudely purified on 8-aminooctyl-Sepharose or 6-aminohexyl-Sepharose and then fractionated into several isoenzyme groups using hydroxyapatite. Further purification on Mono S or CM-Sepharose and finally on phenyl-Superose, phenyl-Sepharose or octyl-Sepharose yielded a P450 fraction which was apparently homogeneous as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the automated Phast system using silver staining. Removal of excess of non-ionic detergent was effected by hydroxyapatite columns, and this was compared with other methods. For the isolation of P450 isoenzymes from untreated marmosets, Mono Q columns were employed and yielded at least two highly purified forms. NADPH-P450-reductase was recovered from the 8-aminooctyl-Sepharose column or crudely fractionated on DEAE-Sepharose Fast Flow. Subsequent purification via 2',5'-ADP-Sepharose and Superose 12 chromatography resulted in a homogeneous preparation.     

猪肝中单胺氧化酶B的分离纯化

依据单胺氧化酶B(monoamine oxidase B, MAOB)的疏水特性,建立了一种从猪肝中分离纯化MAOB的新方法。用含有1% Triton X-100的膜蛋白裂解液制备粗酶,以饱和度为20%～50%的硫酸铵反抽提进行粗提,再利用自制的配基密度为75.7 μmol/mL的苯基疏水色谱及Sepharose Q High Performance离子交换色谱进一步分离纯化,得到纯化倍数为18.2、酶比活为135 U/mg的MAOB。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析显示为相对分子质量约60 000的单一蛋白质带。采用高效液相色谱-电喷雾串联质谱对该酶进行鉴定,证实为MAOB。本研究所用分离纯化方法可以有效纯化MAOB, 为MAOB的深入研究提供技术支撑。     

Simultaneous purification of the neuroproteins synapsin I and synaptophysin.

A procedure for the simultaneous purification of synapsin I and synaptophysin from calf brain was developed. Demyelinated membranes were extracted with 2% Triton X-100 and 2 M KCl. The extracted proteins were separated by weak cation-exchange chromatography on carboxymethyl-Sepharose Fast Flow. Synaptophysin was finally purified by preparative sodium dodecyl sulphate-polyacrylamine gel electrophoresis and synapsin I by affinity chromatography using a calmodulin-Sepharose column. The recovery obtained was 40 micrograms/g in brain for synaptophysin and 25 micrograms/g in brain for synapsin I.     

Chick 28,000 Mr vitamin D-dependent calcium-binding protein in intestine, kidney and cerebellum. Purification using chromatofocusing

A purification method has been developed for chick 28,000 Mr vitamin D-dependent calcium-binding protein, involving Blue Sepharose CL-6B column chromatography, heat treatment and chromatofocusing with a microparticulate anion exchanger (Mono P). It allowed the rapid and reproducible purification of milligram amounts of homogeneous calcium-binding protein with good yields from chick intestine, kidney and cerebellum. The calcium-binding proteins thus obtained have the same molecular weight of 28,000, heat stability, calcium binding capability and apparent isoelectric point of 4.0. These physico-chemical properties are in good agreement with those of proteins isolated by a previous procedure, which gave a low and variable yield of calcium-binding protein.     

Rapid analysis of membrane glycopeptides by gel permeation high-performance liquid chromatography

A rapid procedure for the analysis of glycopeptides has been developed using gel permeation high-performance liquid chromatography (HPLC). Glycopeptides derived by exhaustive pronase digestion of glycoproteins from radiolabeled human tumor and normal cell lines were chromatographed on DuPont GF-250 and GF-450 gel permeation columns in buffers containing non-ionic detergents. Effective separations of glycopeptides ranging in molecular mass from less than 600 daltons to more than 20,000 daltons, equivalent to the separation range of Sephadex G50 chromatography, were achieved in 7 min. The separations were dependent upon the use of an isocratic mobile phase, that contained a low-ionic-strength Tris buffer and Nonidet P-40 or Triton X-100. The mobilities of protein standards indicated the occurrence of a biphasic elution system, which favored the separation of species with molecular masses below 20,000 daltons. Glycopeptides isolated by this method could be applied directly to lectin or ion-exchange columns or could be digested with neuraminidase, endo H or other enzymes without further treatment. Removal of sialic acid from the glycopeptides caused a dramatic increase in retention time. Using this method, glycopeptides could be isolated rapidly and in high yield. The ease, speed and reproducibility of the separations and compatibility of the solvent systems with affinity or ion-exchange chromatography techniques make this gel permeation HPLC method an ideal initial step in the purification of glycopeptides.     

Recovery of the contact site A glycoprotein of Dictyostelium discoideum from sodium dodecyl sulfate-polyacrylamide gel and characteristics of monoclonal antibodies against the recovered protein

Monoclonal antibodies were produced against a cell-cell adhesion (contact site A) glycoprotein of Dictyostelium discoideum, isolated by preparative gel electrophoresis. The glycoprotein was recovered by electroelution from a polyacrylamide gel strip and used for the production of monoclonal antibodies. Four of the five antibodies obtained bound specifically to the protein moiety of the contact site A glycoprotein. The specificities of the antibodies were in striking contrast to those of antibodies raised against the contact site A glycoprotein purified by Triton X-114 phase separation and DEAE chromatography. The majority of the latter antibodies recognized the carbohydrate moiety of the contact site A glycoprotein and cross-reacted heavily with other membrane glycoproteins.     

Analytical fractionation of microsomal cytochrome P-450 isoenzymes from rat liver by high-performance ion-exchange chromatography

Ion-exchange Fast Protein Liquid Chromatography (FPLC) on Mono Q and Mono S was optimized for the analytical separation of microsomal cytochrome P-450 species from rat liver. The effects of detergent, pH, gradient profile and column load on resolution are demonstrated. Successive application of anion- and cation-exchange chromatography leads to eleven separated P-450 fractions. The altered microsomal P450 pattern after treatment of rats with various inducers is reflected by distinct elution profiles. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and enzymatic analysis imply that several FPLC fractions contain more than one P-450 species. Preliminary results are presented showing the suitability of immobilized metal affinity chromatography (MAC) for general P-450 fractionation and thus for the further resolution of Mono Q and Mono S fractions. Scale-up for preparative P-450 fractionation is easily done by adapting the optimized analytical FPLC procedures to Q- and S-Sepharose Fast Flow.     

Partial purification and characterization of epidermal plasminogen activator and their inhibitor

Plasminogen activator (PA) and PA inhibitor were partially purified from 2-d-old rat epidermis and characterized in order to elucidate the enzyme-inhibitor interaction in epidermis. PA extracted with buffer containing KSCN was first purified by Blue-Sepharose affinity chromatography. Separation of two PAs, with relative molecular mass (Mr) of 66000 and 44000, was accomplished by Con A-Sepharose column chromatography. The Mr 66000 enzyme had the properties of tissue-type PA (t-PA), while the Mr 44000 enzyme showed those of urokinase-type PA (u-PA) as determined by immunological and fibrin-binding studies. PA inhibitor was extracted in 1,4-piperazinediethanesulphonic acid buffer and purified by (NH4)2SO4 precipitation, gel filtration followed by a Mono Q column in an FPLC system. This inhibitor showed Mr 60000 and inhibited human u-PA activity in a dose- and time-dependent manner, but did not inhibit the activity of t-PA from human and murine melanoma cells or plasmin. It inhibited epidermal PA, Mr 44000, more effectively than it did the Mr 66000 epidermal PA. It was stable at 60 degrees C for 60 min or between pH 5 and 11. This study indicates that both u-PA and t-PA function in normal rat epidermis. On the other hand, an inhibitor which preferentially acts against u-PA exists, but inhibitor to t-PA does not appear to operate under normal epidermal functions.     

Analysis, purification and properties of a 50,000-dalton membrane-associated phosphoprotein from human platelets

Recently, the development of a monospecific antiserum against a 46,000/50,000-dalton membrane protein from human platelets which was stoichiometrically and reversibly phosphorylated in intact human platelets in response to vasodilators was reported. Using this antiserum, the subcellular distribution and the purification of this vasodilator-stimulated phosphoprotein (VASP) from human platelets has now been analysed. The VASP of human platelets is primarily a membrane-associated protein and can be purified to apparent homogeneity by salt extraction and sequential ion-exchange and dye-ligand chromatography with a purification factor of 1200 and a yield of 13%. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing and non-reducing conditions indicated that purified monomers of this VASP are linked by interchain disulphide bonding.     

Determination of Triton X-100 in influenza vaccine by high-performance liquid chromatography and capillary electrophoresis

Triton X-100 is applied to influenza vaccines at different stages of the manufacturing process to prevent aggregation and precipitation of biomolecules. Furthermore it is used to disintegrate the virus particles in split vaccine and to guarantee the homogeneity during production and utilisation. The final concentration of Triton X-100 has to be determined because the concentration changes in manufacturing process. The determination of the total amount of Triton X-100 as well as the separation of its ethylene oxide oligomers was possible with high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). In HPLC a change of the column and eluent was necessary, in CE different electrolytes were used for the various separation effects. The HPLC method for the analysis of total Triton was preferred for the quantification of Triton X-100 in influenza vaccine because of better linearity, reproducibility and detection sensitivity compared to CE. In the end products an average concentration of 0.117 mg/mL was found. Received: 19 December 1996 / Revised: 27 February 1997 / Accepted: 6 March 1997     

Visualization of the turkey erythrocyte beta-adrenergic receptor

We have recently described the affinity chromatography purification of the turkey erythrocyte beta-adrenergic receptor. The minute amounts obtained initially precluded extensive biochemical characterization. To improve the yield of the receptor, the erythrocyte membranes have been prepared by a new method. This procedure resulted in a 10-fold higher receptor density in comparison with the membrane preparation used previously. The new membranes also contained a catecholamine-sensitive guanine triphosphatase and an adenylate cyclase sensitive to Gpp(NH)p and l-epinephrine. Solubilization by a double digitonin extraction resulted in a preparation containing 4-6 pmoles of 3H-dihydroalprenolol binding sites per mg of membrane protein. A single step of affinity chromatography on alprenolol-sepharose of the soluble digitonin extract resulted in an additional 1,000-fold purification of the receptor. The overall purification factor was 20,000 relative to the binding activity of the crude membrane preparations. Electrophoresis is SDS-polyacrylamide of iodinated purified beta-receptors revealed, after autoradiography, the presence of four major components. Three of these, corresponding to molecular weights of 170,000, 33,000, and 30,000, respectively, were not affected by reduction with beta-mercaptoethanol and were not observed when the digitonin extracts were loaded on the affinity gel in the presence of an excess of l-propranolol. A fourth 52,000-dalton component (60,000 daltons after reduction with beta-mercaptoethanol) remained apparent even when affinity purification was prevented by addition of l-propranolol. Our results suggest that the beta-adrenergic receptor is composed of at least three subunits that interact by noncovalent bonds.