Purification of brush 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 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.     

Purification of canine plasma renin by affinity chromatography.

An affinity column for the purfication of canine plasma renin was prepared using goat anti-renin (dog kidney) gammaG gloublins. The antiserum was prepared against a purified kidney renin preparation. The anti-renin globulins were coupled to cyanogen bromide activated Sepharose. Using the anti-renin globulin-coupled Sepharose as an immuno-adsorbant, a method was devised allowing purification of plasma renin to a 1,000-fold purity.     

Purification of a completely inactive renin from hog kidney and identification as renin zymogen

Hog renal inactive renin was separated from active renin and completely purified to an electrophoretically homogeneous state by using a new procedure which consisted of affinity chromatography on pepstatin-Sepharose, octyl-Sepharose, Affil-Gel blue and Con A-Sepharose columns, ion exchange chromatography and gel filtration. By this method a 3,000,000-fold purification was obtained with a 6% recovery from a crude kidney extract. This pure preparation was totally inactive and underwent marked activation by trypsin. It is a glycoprotein as judged by affinity to concanavalin A and has an apparent molecular weight of 50,000 as determined by gel filtration on Sephadex G-100. Treatment of the inactive renin with guanidine, urea and Triton X-100 did not cause activation indicating that the inactive renin isolated in the present study is not a product of renin-inhibitor complex.     

Purification and characterization of human kidney renin

By means of a new rapid and small scale purification method, human kidney renin has been purified from a single kidney in a homogeneous state, as judged on SDS-PAGE. The kidney which showed unusually high renin activity was from a patient with cardiomyopathy. 8,000-fold purification was attained by means of only pepstatin-aminohexyl-Sepharose chromatography and FPLC on a Mono Q column, and the yield was 34%. The specific activity was 5.63 mg angiotensin I per mg protein per h at 37 degrees C and pH 6.5 with porcine angiotensinogen as the substrate. The molecular weight was estimated to be 37,000 by SDS-PAGE and 38,000 by HPLC on a TSK G-3000 SW column. The preparation showed three bands on isoelectric focusing. The molecular weight and the profile on isoelectric focusing of the purified renin agreed with those found for the extracts of both the patient's kidney and a kidney with the usual low renin activity.     

Purification of plant hormone extracts by gel permeation chromatography

Gel permeation chromatography on porous polystyrene beads has been adapted for the purification of plant extracts prior to analysis for plant hormones. The retention characteristics of gibberellins, indoleacetic acid, cytokinins and abscisic acid are presented along with chromatograms of some typical plant extracts.     

Purification of alcohol dehydrogenase from bovine liver crude extract by dye-ligand affinity counter-current chromatography

Alcohol dehydrogenase (ADH) was extracted from a crude bovine liver homogenate by dye-ligand affinity counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (x-axis CPC). The purification was performed using two types of polymer phase systems composed of 4.4% polyethylene glycol (PEG) 8000-7.0% dextran T500-0.1 M potassium phosphate buffers and 16% PEG 1000-12.5% potassium phosphate buffers, both containing a procion red dye as an affinity ligand at various pH values. The best purification was achieved using the PEG 1000-potassium phosphate system at pH 7.3 containing 0.05% procion red as a ligand. The upper PEG-rich phase containing procion red was used as the stationary phase and a crude bovine liver homogenate was eluted with the potassium phosphate-rich lower phase at 0.5 ml/min. After elution of bovine liver proteins in the homogenate, ADH still retained in the stationary phase was collected from the column by eluting with the PEG 1000-rich upper phase. Collected fractions were analyzed by ADH enzymatic activity and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to detect contaminant proteins in the ADH fractions. The ADH was purified directly from crude bovine liver extract within 6h with minimum loss of its enzymatic activity.     

Purification of virus-like particles from male-sterile vicia faba and detection by elisa in crude leaf extracts

A method purifying virus-like particles (VLPs) to physical homogeneity from male-sterile Vicia faba (“447” cytoplasm, Ad 23 line) is described for the first time. Purified VLPs were used to prepare a specific antiserum. An Enzyme Linked Immunosorbant Assay (ELISA) was developed with antibodies purified from the specific antiserum in order to detect the presence of VLP in very small amounts of crude leaf extracts (approx. 20mg of fresh tissues). This ELISA allows the detection of male-sterile Vicia faba individuals at a very early decvelopmental stage.     

Purification of lactic acid dehydrogenase from crude bovine heart extract by pH-peak focusing counter-current chromatography

pH-peak focusing counter-current chromatography (CCC) was applied to the purification of lactic acid dehydrogenase (LDH) from a crude bovine heart extract using a cross-axis coil planet centrifuge (CPC). The experiment was performed with two sets of polymer phase systems composed of 16% (w/w) polyethylene glycol (PEG) 1000-12.5% (w/w) potassium phosphate buffer and 15% (w/w) PEG 1540-15% (w/w) ammonium sulfate each at various pH values. The best result was achieved from the PEG 1540-ammonium sulfate polymer phase system by adding a retainer (10 mM acetic acid) to the upper stationary phase and an eluter (100 mM sodium hydroxide) to the lower mobile phase. At a flow-rate of 0.5 ml/min, LDH was eluted as a sharp peak which was well resolved from other proteins. Collected fractions were analyzed by the LDH enzymatic activity and by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis to detect contaminated proteins. LDH was purified directly from crude bovine heart extract in a concentrated state.     

Purification of human plasma inactive renin by immunoaffinity chromatography on profragment-specific IgG

Inactive renin was purified to apparent homogeneity from human plasma by ion exchange, gel filtration, Affi-Gel blue, immunoaffinity chromatography on profragment-specific IgG coupled to Sepharose, and preparative HPLC. By this method, a 460000-fold purification was obtained. The purified renin was totally inactive and was activated by trypsin.     

Purification and characterization of rat urinary renin

Rat urinary renin was purified by a procedure involving ammonium sulfate fractionation, pepstatin-aminohexyl-Sepharose 4B chromatography, ion exchange chromatography and gel filtration. The resulting preparation was essentially homogeneous, as assessed by polyacrylamide gel electrophoresis. The molecular weight of the preparation was estimated to be 39000 by SDS-gel electrophoresis and 40000 by gel filtration. The optimum pH determined with rat angiotensinogen was 7.0, and the Km was 3.6 microM. These properties agreed well with those of purified rat renal renin. The activity of urinary renin was specifically inhibited by anti-renin antibody. These results suggest that urinary renin may originate in the kidney.     

Purification of swine kidney alkaline phosphatase by immunoaffinity chromatography

A homogeneous alkaline phosphatase preparation was obtained from swine kidney cortex by a simple purification step of immunoaffinity chromatography. The enzyme was purified 426 times that of the initial acetone powder with a recovery of 69.6% and a specific activity of 1206 units/mg of protein. The sodium dodecyl sulfate-gel electrophoretic pattern showed a single 80,000-M_r protein band as the monomer of the purified enzyme.     

Purification of dipeptidyl-aminopeptidase IV from human kidney by anti dipeptidyl-aminopeptidase IV affinity chromatography

Summary Dipeptidyl-aminopeptidase IV (DAP-IV) was purified 850 fold with a yield of 16.5% from human kidney cortex. Only two chromatographic procedures of DEAF-cellulose and anti DAP-IV Sepharose were used. The purified enzyme was shown to be homogeneous by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Among various substrates with the structure of X-Y-p-nitroanilide, Lys-Pro-, Gly-Pro- and Arg-Pro-p-nitroanilides were hydrolyzed very fast by DAP-IV. Optimum pH of DAP-IV in human kidney was pH 8.7, Km value for Gly-Pro-pNA was 2.51 ± 0.01 × 10⁴ M and Vmax was 66.6 ± 0.7 mol/min/mg protein.Diisopropylfluorophosphate completely inhibited DAP-IV at 0.1 mM. However, the enzyme was almost unaffected by N-ethylmaleimide, p-chloromercuribenzoate, iodoacetate, phenylmethylsulfonylfluoride, and several metals. The amino acid composition of DAP-IV purified from human kidney was similar to that of DAP-IV purified from pig kidney, liver and intestine. These results indicate that the properties of DAP-IV purified from human kidney are almost same as those from pig kidney.     

Purification and characterization of renin binding protein (RnBP) from porcine kidney

Renin binding protein (RnBP) was purified from porcine kidney using pepstatin affinity column chromatography, DEAE-Sepharose column chromatography, gel filtration on Ultrogel-AcA 34, aminohexyl-Sepharose 4B column chromatography, and high performance liquid chromatography (HPLC) on TSK-gel G-3000 SW. The purified preparation was homogeneous as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, polyacrylamide disc gel electrophoresis and isoelectric focusing on polyacrylamide gel. The isoelectric point was at pH 4.85, and the apparent molecular weight of RnBP was estimated to be 42,000 by SDS-polyacrylamide gel electrophoresis. The preparation did not show any renin activity and was stable for 30 min at 37 degrees C between pH 5.0 and 9.0 or on storage for 4 weeks at 4 degrees C or -80 degrees C. The activity of renin was greatly inhibited by RnBP. From the kinetic analysis of the inhibition we roughly estimated the dissociation constant between renin and RnBP to be about 0.2 nM, assuming that the stoichiometry in the complex, i.e., high molecular weight (HMW) renin, is one to one, and that the complex is inactive. The inhibitory activity of RnBP was lost by acidification at pH 3.0 and the activity of renin was restored. The purified RnBP formed a single precipitin line with the antiserum prepared with the purified HMW renin as antigen, which is RnBP-renin complex (Takahashi, S., et al. (1983) J. Biochem. 93, 265-274), and this line fused with one of the two precipitin lines formed between HMW renin and anti-HMW renin antiserum. The other of the two lines was between renin and anti-HMW renin antiserum. The purified preparation was thus identified as RnBP. The HMW renin was reconstituted with the purified RnBP and renin, and the apparent molecular weight of the reconstituted specimen was estimated to be 60,000 by gel filtration on Ultrogel AcA 44.     

Purification of cholesterol-esterifying enzymes from rat liver cytosol by high-performance liquid chromatography

Three enzymes esterifying cholesterol with long-chain fatty acids were purified approximately 31 000-fold to apparent homogeneity from the cytosol of normal rat liver. The enzymatic activity was tested by incubation of active fractions with tritiated cholesterol and separation of newly formed esters from non-reacted cholesterol by a passage through silica gel cartridges with subsequent assay for radioactivity by liquid scintillation. For the purification of enzymes, active proteins were precipitated by (NH₄)₂SO₄ to 35% saturation. The bulk of inactive proteins was removed by size-exclusion chromatography on TSK G3000 SW. The active fraction was subsequently separated on Separon HEMA BIO 1000 DEAE in gradients of 0–500 mM KCl into three enzymatic activities differing in their retention and these proteins were finally purified by affinity HPLC on columns of cholesterol immobilized on HEMA BIO 1000 E-H. Final purified enzymes showed the same single band in polyacrylamide gel electrophoresis corresponding to 16.5 kDa. Combination of individual enzymes did not increase the overall yield of cholesteryl esters but the reaction-rate was significantly accelerated. These proteins are apparently subunits of a larger complex (M_r 65 000) that can be demonstrated by electrophoresis in the absence of 2-mercaptoethanol. Results presented in this paper indicate that because of good and rapid separation of active proteins, HPLC may be a method of choice for enzyme purifications.     

Purification of alpha 2,6-sialyltransferase from rat liver by dye chromatography.

We describe a simple three-step purification for Gal-beta 1,4-GlcNAc-alpha 2,6-sialyltransferase (EC 2.4.99.1) from rat liver which uses chromatography on Cibacron Blue F3GA and f.p.l.c. It gives a highly purified (11,000-fold) enzyme in 19% yield, which is free of other sialyltransferases, CMP-NeuAc hydrolase, sialidases and proteinases.