Course Of Purification Procedure Research Articles

25] Carboxylesterases from chicken, sheep, and horse liver

This chapter discusses the determination of carboxylesterases from chicken, sheep, and horse liver. In the course of purification procedure for chicken liver carboxylesterase, chloroform-acetone powder (2 kg) is extracted in 400-g batches, each with 4 liters of 0.1 M phosphate buffer, pH 7.4, at ∼25° for 30 minutes while stirring constantly. The suspensions are centrifuged at 4° for 1 hour at 3000 g to produce clear, reddish brown solutions with a pH of 7.1–7.2. The specific activity of various chloroform–acetone powder extracts has varied from 0.40 to 1.62 (units/ml)/ A 280 , presumably owing to variation in the esterase content of the livers. In the course of purification procedure for sheep liver carboxylesterase, chloroform–acetone powder (400 g) is extracted with 3.6 liters of 0.1 M citrate buffer, pH 4.0, at 4° for 1 hour with constant stirring. The suspension is centrifuged at 2° for 45 minutes at 3000 g. The pH of the clear, reddish brown supernatant is raised to 7.5 by the dropwise addition of 2 N NaOH to the magnetically stirred solution.

19] Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase from chicken liver

This chapter discusses the determination of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase from chicken liver. The thermodynamically favorable condensation of acetyl-CoA and acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA is catalyzed in chicken liver mitochondria by 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). This enzyme is localized in the mitochondrial matrix and occupies a potentially favorable position for the regulation of hepatic ketogenesis. The distinctly different properties of this enzyme from those of the cytoplasmic HMG-CoA synthase are consistent with independent regulation of ketogenesis and cholesterogenesis. 3-Hydroxy-3-methylglutaryl-CoA synthase activity can be conveniently determined by a radiochemical assay, which monitors the incorporation of [1- 14 C]acetyl-CoA (volatile 14 C activity when taken to dryness at 95° in 6 N HCl) into HMG-CoA (nonvolatile 14 C activity when taken to dryness at 95° in 6 N HCl). A spectrophotometric assay can be used to measure the acetyl-CoA–dependent disappearance of acetoacetyl-CoA absorbance at 300 nm. In the course of purification procedure, fresh liver from laying hens is ground in a Universal No. 1 meat chopper and then homogenized in a loose-fitting Potter-Elvehjem tissue homogenizer with a Teflon pestle. One kilogram of liver is homogenized in 2 liters of media containing 0.25 M sucrose, 0.1 m M ethylenediaminetetraacetic acid (EDTA), and 2 m M N -2-hydroxyethylpiperazine- N -2-ethanesulfonic acid (HEPES) brought to pH 7.2 with potassium hydroxide.

22] 3-Hydroxy-3-methylglutaryl-CoA synthase from bakers' yeast

This chapter discusses the determination of 3-hydroxy-3-methylglutaryl-CoA synthase from bakers' yeast. The spectrophotometric assay is based on the absorption of the enol form of acetoacetyl-CoA at 303 nm. The synthase activity is measured by observing the acetyl-CoA stimulated decrease in this absorption, Mg 2+ is routinely included in the assay to increase the apparent extinction coefficient of the acetoacetyl-CoA. Mg 2+ has no direct effect on the enzyme activity. A unit of enzymatic activity is defined as the amount of enzyme necessary to transform 1 μmole of acetoacetyl-CoA into product per minute. Specific activity is expressed in units per milligram of protein. In the course of purification procedure, Fresh bakers' yeast is crumbled into a stainless steel beaker and toluene added to 110 ml/kg of yeast. The mixture is heated in a water bath at 55° until the yeast has warmed to 38°. The yeast is then allowed to autolyze at 38° for 8–12 hours. Within these time limits the yield and specific activity of the HMG-CoA synthase remain approximately constant; further incubation causes losses. At the end of this period, an equal volume of cold deionized water (900 ml/kg of yeast) is added with stirring and the cell debris removed by centrifugation at 15,000 g for 20 minutes.

18] Bovine liver crotonase (enoyl coenzyme A hydratase)

Publisher Summary This chapter discusses the determination of bovine liver crotonase. In the β-oxidation pathway of fatty acids, crotonase catalyzes the hydration of trans-α,β-unsaturated acyl-CoA derivatives to the corresponding L(+)-β-hydroxyacyl-CoA thioesters. Two spectrophotometric methods have been used to monitor this hydration continuously and quantitatively. First, addition of the elements of water across the double bond abolishes the characteristic ultraviolet absorption of the α,β-unsaturated system, which is conjugated with the carbonyl group of the thioester. This change provides the basis of the direct spectrophotometric method. Alternatively, the oxidation of the β-hydroxyacyl-CoA thioester and the accompanying reduction of nicotinamide adenine dinucleotide (NAD) may be followed in the presence of β-hydroxyacyl-CoA dehydrogenase. The spectral changes associated with NAD reduction provide the quantitative basis of this coupled assay method. In the course of purification procedure, ox liver is obtained from a local abattoir shortly after the animal is slaughtered, and is transported on ice to the laboratory. The liver is trimmed of any visible connective tissue, chopped, and wrapped in portions of convenient weight, usually 500 g, then stored in a freezer until use.