A procedure for the purification of ferritin from human liver by heating a methanol-treated homogenate

A simple, rapid technique for purification of ferritin from human liver tissue is described. Methanol, at a final concentration of 40% (v/v) in liver homogenate, precipitates the majority of proteins but does not affect ferritin. Subsequent heating of this homogenate at 75 degrees C for 10 min results in a purified ferritin preparation as judged by immunoelectrophoresis and polyacrylamide gel electrophoresis. The resultant purified ferritin contained the same amount of iron as the original endogenous ferritin. There were no significant differences (paired t tests) in the amount of protein in the purified ferritin preparation when measured by rocket immunoelectrophoresis and by the Lowry procedure, suggesting that the antigenecity of ferritin was unaffected by the methanol and heat treatment. Both endogenous liver ferritin and radiolabeled human liver ferritin added to liver homogenates were recovered after methanol and heat treatment with similar yields (77 +/- 7% and 70 +/- 2%, respectively) when compared with the standard treatment of heating a homogenate at 75 degrees C. The overall ferritin yield with this rapid procedure was 40%.     

Partial purification and characterization of a triacyglycerol lipase from rat liver cytosol.

A triacyglycerol lipase (EC 3.1.1.3) was purifiec about 60-fold from rat liver cytosol by delipidation with acetone and ethyl ether, hydroxyapatitie and Sephadex G-100 column chromatographies and isoelectrofocusing electrophoresis. The partially purified enzyme had a molecular weight of approximately 42 000 and an isolectric point of 7.2. The Km for trioleylglycerol was 0.33 mM and the pH optimum was around 8.0. The activity of the enzyme was not dependent on serum lipoproteins, but was stimulated about 2-fold by several proteins such as serum albumin, lipoproteins, gamma-globulin and ovalbumin. The lipase hydrolyzed trioleyglycerol to oleic acid and glycerol. NaCl had no effect on the enzymatic activity. Some physical and kinetic properties of the partially purified lipid-free lipase were different from those of crude non-delipidated lipase and also from those of a neutral triacylglycerol lipase which was recently purified partially from pig liver cytosol (Ledford, J.H. and Alaupovic, P. (1975) Biochim. Biophys. Acta 398, 132-148).     

A simplified procedure for the purification of rat liver tyrosine aminotransferase.

Rat liver tyrosine aminotransferase was purified by chromatography on CM-Sephadex C-50 and DEAE-cellulose, (NH4)2SO4 fractionation and gel filtration on Sephadex G-200. Livers from 400 rats can be easily worked up by this procedure. Furthermore, this purification method has the advantage that hepatic tryptophan 2,3-dioxygenase, which, like tyrosine aminotransferase, is induced by glucocorticosteroids, can be purified from the same homogenate. Tyrosine aminotransferase purified by this method was shown to be specific for 2-oxoglutarate. Its subunits have a molecular weight of 45 000. The following "apparent" Michaelis constants were determined: L-tyrosine, 1.7 X 10(-3) M; 2-oxoglutarate, 5.9 X 10(-4) M; and pyridoxal 5'-phosphate, 2.1 X 10(-6) M. Tyrosine aminotransferase, depleted of its cofactors, binds 4 molecules of pyridoxal 5'-phosphate per 90 000 daltons with a KA of 2.2 X 10(5) M-1.     

An improved purification procedure for rat liver lysosomal phospholipase A1

A purification procedure is presented for the isolation of lysosomal acid phospholipase A1 (PLA1) from livers of non-pretreated rats, in a high yield and purity. The purification starts from a crude mitochondrial-lysosomal fraction. PLA1 is solubilised and subsequently purified by chromatography on concanavalin A-Sepharose, by chromatofocusing, and by gel filtration. After chromatofocusing, the enzyme is already purified 50200-fold with a yield of 50%, and after gel filtration 56600-fold with a yield of 7%. Purified PLA1 exhibits a specific activity of approx. 8.2 mumol phosphatidylethanolamine (preferred substrate) hydrolysed per min per mg protein, and upon chromatofocusing an apparent isoelectric point of 5.3 Gel filtration of purified PLA1 suggests a molecular mass of about 29 kDa, whereas in SDS-PAGE two proteins of 27 kDa and 55 kDa (mass ratio about 1/2) were visualised.     

Xanthine oxidase and aldehyde oxidase: a simple procedure for the simultaneous purification from rat liver

Aldehyde oxidase (AO) and xanthine oxidase (XO) are cytosolic enzymes that have been involved in some pathological conditions and play an important role in the biotransformation of drugs and xenobiotics. The increasing interest in these enzymes demands for a simple and rapid procedure for their purification. This paper describes for the first time a method that allows simultaneous purification of both enzymes from the same batch of rat livers. It involves few steps, is reproducible and offers high enzyme yields with high specific activities. The rat liver homogenate was fractionated by heat denaturation and by ammonium sulphate precipitation to give a crude extract containing both enzymes. This extract was chromatographed on an Hydroxyapatite column that completely separated AO from XO. Further purification of XO by anion exchange chromatography on a Q-Sepharose Fast Flow column resulted in a highly purified (1200-fold) preparation, with a specific activity of 3.64 U/mg and with a 20% yield. AO was purified about 1000-fold at a yield of 15%, with a specific activity of 3.48 U/mg, by affinity chromatography on Benzamidine-Sepharose 6B. The purified enzymes gave single bands of approximately 300 kDa on a polyacrylamide gel gradient electrophoresis and displayed the characteristic absorption spectra of highly purified enzymes.     

An improved large scale procedure for the purification of porcine pancreatic lipase

A modified procedure for purifying porcine pancreatic lipase (triacyglycerol acyl-hydrodrolase, EC 3.1.1.3) is described. In comparison to the previous procedure reported by Verger, R., de Hass, G.H., Sarda, L and Desnuelle, P. (1969) Biochim. Biophys. Acta 188, 272--282) it is more rapid, more reproducible and results in a purer enzyme preparation. No colipase could be detected in the mixture of isoenzymes and, naturally, in the different separated lipases. In this process, butanol treatment is omitted. After pancreas powder extraction, a batch procedure was used for adsorption of DEAE-cellulose. Sephadex filtration (pH 8.0) was made in a largeer size column. Finally the isoenzymes were separated on CM-cellulose as in the Verger procedure, but under slightly modified conditions. Lipase LB was fully homogeneous as judged by end group determination, gel electrophoresis (in the presence of absence of sodium dodecyl sulfate) and sedimentation equilibrium.     

A simple and rapid procedure for the purification of phenobarbital-inducible cytochrome P-450 from rat liver microsomes.

A rapid and simple procedure has been developed for the purification of a phenobarbital-inducible form of cytochrome P-450 from the liver microsomes of phenobarbitalpretreated rats. Within 2 days approximately 1000–1500 nmol of highly purified cytochrome P-450 with a specific content of 16 nmol/mg protein can be recovered from 4 g of microsomal protein. The procedure consists of solubilization of microsomal protein with sodium cholate, fractionation with polyethylene glycol, and column chromatography at room temperature on DEAE-cellulose. The resulting DEAE-cellulose fraction electrophoreses on polyacrylamide gels in the presence of sodium dodecyl sulfate as a major protein band with a minimum molecular weight of 52,000 and a few faint bands. Further chromatography on QAE Sephadex A-25 essentially removes these faint bands and increases the specific content slightly to 17 nmol/mg protein. Relatively low amounts of this form of cytochrome P-450 appear to be present in microsomes of untreated rats since less than 1% can be recovered as the DEAE-cellulose fraction by this procedure. An identical form is inducible by phenobarbital in rats of different ages and sex. In a reconstituted system under optimal assay conditions, this form of cytochrome P-450 catalyses the N-demethylation of benzphetamine with a turnover number greater than 100 and hydroxylates testosterone at the 16α position but not at the 6β or 7α position.     

Cell-density-dependent release of hepatic lipase from cultured rat hepatocytes

Cultured rat hepatocytes release the enzyme hepatic lipase. In this study we investigated the effect of cell density on this metabolic function under a variety of experimental conditions. The release of hepatic lipase from cultured rat hepatocytes exhibits a cell-density dependence, the secretion per mg cell protein being increased with increasing cell density. When cell density dependence was taken into consideration no significant effect of insulin on the release of hepatic lipase from cultured hepatocytes was observed, whereas glucagon suppressed the release. Glucose stimulating the release of the enzyme, especially in cultures with high cell density.     

An improved purification procedure for sulfite oxidase from bovine liver.

Sulfite oxidase (Sulfite:O2 oxidoreductase, EC 1.8.3.1) has been purified 2,440-fold from bovine liver. The procedure developed was used to isolate the enzyme from 1,000 g of liver and permitted the rapid isolation of enzyme with a very high specific activity (40,405 mU/mg). The enzyme preparations obtained have been characterized by electrophoretic and spectrophotometric analysis and the molecular mass and the Stokes radius of the enzyme have been determined.     

A simple procedure for the purification of rat alpha-fetoprotein

A simple purification procedure for obtaining a high yield of electrophoretically and immunologically pure rat α-fetoprotein from amniotic fluid is described. Rat amniotic fluid is passed through an anti-rat albumin immunoabsorbent column to remove albumin. The albumin-free eluate is then chromatographed on DEAE-Sephacel to separate α-fetoprotein from transferrin and other minor protein contaminants. This two-step purification procedure results in a recovery of approximately 70% of the rat α-fetoprotein originally present in the amniotic fluid.     

Resolution of components from rat liver homogenate in reorienting density gradients

The subcellular components of rat liver homogenate were separated into seven distinct components when discontinuous gradients were used. The studies reported here demonstrate the similarity of the separation when compared with the work of previous investigators (4).     

High-yield purification of glucokinase from rat liver

A rapid and reliable method for the purification of rat liver glucokinase was developed. The procedure consists of DEAE-cellulose ion-exchange chromatography, Phenyl-Sepharose hydrophobic interaction chromatography, DEAE-Affi Gel Blue dye-ligand chromatography, and duplicate steps of glucosamine-Sepharose affinity chromatography. Glucokinase was purified to a specific activity of 290 units/mg protein in a yield of 55% in 6 days. The final enzyme preparations were completely homogeneous in most experiments as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The estimated molecular weight (51,000) and sigmoidal saturation function for glucose of purified glucokinase were in good agreement with published data.     

Improved procedure of purification of inorganic pyrophosphatase from Bacillus stearothermophilus.

An improved method for isolation of inorganic pyrophosphatase (EC 3.6.1.1) from Bacillus stearothermophilus is described. The enzyme was purified to more than 90% after two chromatographic steps. A molecular weight of 140,000 daltons was estimated by density gradient centrifugation. The isoelectric point was found to be 4.0.     

Enzyme-linked immunosorbent assay for rat hepatic triglyceride lipase

A noncompetitive enzyme-linked immunosorbent assay to measure rat hepatic triglyceride lipase (H-TGL) was developed. Antibodies to rat H-TGL were purified from goat antisera by immunoadsorption on an H-TGL-Sepharose 4B column. Routinely, Immulon 2 Removawell strips were coated with the purified antibody overnight at 4 degrees C. After blocking the wells with bovine serum albumin (BSA) for 2 hr at room temperature, standards (0.85 ng/ml-13.1 ng/ml) or samples were added to the wells and were incubated with the bound anti-rat H-TGL overnight at 4 degrees C. The standards and samples had been pretreated with 5-20 mM SDS for 30 min at room temperature and were then diluted so that the final SDS concentration in the assay was 1 mM or less. The pretreatment with SDS was necessary to achieve maximal immunoreactivity. The sample incubation was followed by an overnight incubation at 4 degrees C with an anti-rat H-TGL-horseradish peroxidase conjugate. Rat H-TGL was detected by the color development after the addition of 0.4 mg/ml of o-phenylenediamine in 0.01% H2O2, 0.1 M citrate phosphate, pH 5.0. A linear relationship was obtained between absorbance at 490 nm and the amount of highly purified rat H-TGL used as a standard. Inclusion of 1 M NaCl in the assay buffer (1% BSA, 0.05% Tween 20, 10 mM phosphate, pH 7.4) during the sample and conjugate incubations minimized non-specific interactions. Recoveries of purified rat H-TGL added to a rat liver perfusate sample ranged from 98.6% to 103%.(ABSTRACT TRUNCATED AT 250 WORDS)