Porcine Liver Cytosol Research Articles

A microfluidic device containing membrane-immobilized antibodies for successively capturing cytosolic enzymes

A microfluidic device containing membrane-immobilized anti-esterase (ES) antibodies and anti-lactate dehydrogenase (LDH) antibodies was prepared. The membrane was prepared by transferring antibodies that had been separated by non-denaturing two-dimensional electrophoresis to a polyvinylidene difluoride membrane, which was then stained and cut into small pieces (16mm2). In this microfluidic device, >0.014UnitmL−1 of the purified porcine carboxylesterase was specifically captured by membrane-immobilized anti- ES antibodies and >147UnitmL−1 of purified porcine LDH was specifically captured by membrane-immobilized anti-LDH antibodies. Furthermore, ES and LDH in micro-scale aliquots of porcine liver cytosol were successively captured by membrane-immobilized antibodies in the device, and the enzyme activities were quantitatively analyzed by spectrofluorometry. The results indicate that the microfluidic device containing membrane-immobilized antibodies can be used to investigate the activities of several types of intact enzymes.

Micro-Scale Extraction and Analysis of Intact Carboxylesterase after Trapping on an Immunoaffinity Membrane Surface

Porcine liver carboxylesterase was captured using an immunoaffinity membrane, which was prepared by separating an anti-porcine esterase antibody using non-denaturing two-dimensional electrophoresis, followed by transfer to a polyvinylidene difluoride membrane and staining. The activity of this esterase was 0.008 units after it was captured in the tiny spaces (4 mm(2)) of this membrane and eluted by rinsing with 5 μL of aspartic acid solution. The molecular mass of the eluted esterase was m/z 61,885 according to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after the purification of this enzyme from the porcine liver cytosol. The purified enzyme's activity was inhibited by 6,9-diamino-2-ethoxyacridine, and this inhibition was retained even after extracting the enzyme from the immunoaffinity membrane. These results indicate that micro-scale extraction and analysis of a carboxylesterase are possible when the enzyme is trapped using an immunoaffinity membrane and eluted with aspartic acid.

Identification and characterization of a mammalian 14-kDa phosphohistidine phosphatase.

Protein histidine phosphorylation in eukaryotes has been sparsely studied compared to protein serine/threonine and tyrosine phosphorylation. In an attempt to rectify this by probing porcine liver cytosol with the phosphohistidine-containing peptide succinyl-Ala-His(P)-Pro-Phe-p-nitroanilide (phosphopeptide I), we observed a phosphatase activity that was insensitive towards okadaic acid and EDTA. This suggested the existence of a phosphohistidine phosphatase different from protein phosphatase 1, 2A and 2C. A 1000-fold purification to apparent homogeneity gave a 14-kDa phosphatase with a specific activity of 3 micro mol.min-1.mg-1 at pH 7.5 with 7 micro m phosphopeptide I as substrate. Partial amino-acid sequence determination of the purified porcine enzyme by MS revealed similarity with a human sequence representing a human chromosome 9 gene of hitherto unknown function. Molecular cloning from a human embryonic kidney cell cDNA-library followed by expression and purification, yielded a protein with a molecular mass of 13 700 Da, and an EDTA-insensitive phosphohistidine phosphatase activity of 9 micro mol.min-1.mg-1 towards phosphopeptide I. No detectable activity was obtained towards a set of phosphoserine-, phosphothreonine-, and phosphotyrosine peptides. Northern blot analysis indicated that the human phosphohistidine phosphatase mRNA was present preferentially in heart and skeletal muscle. These results provide a new tool for studying eukaryotic histidine phosphorylation/dephosphorylation.

Role of aldehyde oxidase in the hepatic in vitro metabolism of 3-methylindole in pigs.

The metabolism of 3-hydroxy-3-methylindolenine (HMI), a recently discovered metabolite of 3-methylindole (3MI, skatole) produced by porcine liver microsomes, was investigated in vitro using porcine liver cytosol. HMI was rapidly metabolized to a single product, 3-hydroxy-3-methyloxindole (HMOI), by porcine cytosol. By the use of the selective inhibitors menadione and quinacrine, it was shown that the enzyme responsible for the oxidation of HMI into HMOI was aldehyde oxidase (AO; aldehyde:oxygen oxidoreductase, EC 1.2.3.1). The activity of AO in the conversion of HMI to HMOI was measured in a population of pigs (n = 30) with a wide range of 3MI levels in back fat (0.07-0.30 mg/kg). AO activity was found to be negatively correlated (r = -0.70; P < 0.001) with the level of 3MI in fat. The results of the present study suggest that AO plays an important role in the metabolism of 3MI in the pig and that its catalytic activity is related to an adequate 3MI clearance.

Identification and partial purification of GTPase-activating proteins from yeast and mammalian cells that preferentially act on Ypt1/Rab1 proteins

Two GTPase-activating proteins of apparent molecular mass of 100 kDa and 30 kDa have been partially purified from porcine liver cytosol usinig mammalian Ypt1/Rab1 protein as substrate. Both proteins act most efficiently on Ypt1/Rab1 p, but are inactive with H-Ras p21. From the budding yeast Saccharomyces cerevisiae, a cytosolic 40 kDa yptGAP was partially purified. It accelerates the intrinsic GTPase activity of wild-type Yptlp but not of H-Ras p21 or a mutant ypt1p with an animo acid substitution of the effector domain which renders the protein functionally inactive in yeast cells.

Purification and characterization of an NAD +-dependent dehydrogenase that catalyzes the oxidation of thromboxane B 2 at C-11 from porcine liver. Development and application of 11-dehydro-thromboxane B 2 radioimmunoassay to enzyme assay

11-Dehydro-thromboxane B 2 has been identified as a major metabolite of infused as well as endogenous thromboxane B 2 in mammalian plasma and urine. This metabolite is derived from thromboxane B 2 by enzymatic oxidation at C-11 catalyzed by 11-hydroxytrhomboxane B 2 dehydrogenase. A radioimmunoassay for 11-dehydro-thromboxane B 2 has been developed and used for enzyme assay, purification and characterization. Antibodies were generated against 11-dehydrothromboxane B 2 conjugated to bovine thyroglobulin. Labeled marker was prepared by radioiodinating 11-dehydrothromboxane B 2-tyrosine methyl ester conjugate. A sensitive radioimmunoassay capable of detecting 10 pg of 11-dehydro-thromboxane B 2 per assay tube was developed. The antibodies showed minimal crossreaction with thromboxane B 2 (0.03%), prostaglandin D 2 (2.76%) and other eicosanoids (<0.03%). The enzyme activity was determined by assaying NAD +-dependent formation of immunoreactive 11-dehydro-thromboxane B 2 from thromboxane B 2. The enzyme was found to be enriched in liver although significant activity was also detected in gastrointestinal tract and kidney in pig. The enzyme was purified from porcine liver cytosol to apparent homogeneity using conventional and affinity chromatography. The purfied enzyme exhibited coenzyme specificity for NAD + and used thromboxane B 2 as a substrate. The enzyme also catalyzes NADH-dependent reduction of 11-dehydro-thromboxane B 2 to thromboxane B 2 indicating the reversibility of the enzyme catalyzed reaction. The apparent K m values for thromboxane B 2, 11-dehydrothromboxane B 2 and NAD + are 8.1, 8.0 and 23 μM, respectively. Subunit M r was shown to be 55 000, whereas the native enzyme M r was found to be 110 000 indicating that the enzyme is a dimer. The enzyme is sensitive to sulfhydryl inhibitions suggesting cysteine residues are essential to enzyme activity. The availability of a homogeneous enzyme preparation should allow further studies on the substrate specificity and the structure and function of the enzyme.

Purification and characterization of angiotensin-binding protein from porcine liver cytosolic fraction.

A protein that binds angiotensins with high affinity was found in porcine liver cytosol, purified to apparent homogeneity and characterized. The protein was named soluble angiotensin-binding protein (sABP) to distinguish it from angiotensin II receptors present on plasma membranes. Purification of the protein was achieved by a combination of ammonium sulfate fractionation, hydrophobic chromatography, ion-exchange chromatography, hydroxylapatite column chromatography and Mono Q ion-exchange chromatography. Specific angiotensin-binding activity, as measured using 125I-angiotensin II, was enriched more than 3400-fold. SDS/polyacrylamide gel electrophoresis of the purified sABP yielded a single 75-kDa protein band, in good agreement with the molecular mass estimated by affinity labeling. sABP was very similar to the angiotensin II receptor in its sensitivity to reducing agents and in its affinities for angiotensin analogues ([Sar1, Ala8]angiotensin II greater than angiotensin III greater than angiotensin II greater than angiotensin I), suggesting a possible similarity between the ligand-binding sites of sABP and the angiotensin II receptor. To obtain a clue to its physiological role(s), we examined the tissue distribution of sABP and found that this protein is widely distributed not only in the peripheral organs but also in the brain.

Conservation of the DNA binding domain and other properties between porcine and rat glucocorticoid receptors

Activated glucocorticoid receptor protein (GCR) was partially purified from porcine liver cytosol by sequential chromatography on phosphocellulose and DNA-cellulose using a modification of a protocol developed for purification of rat GCR [1–2]. This partially purified preparation, when separated by SDS-polyacrylamide gel electrophoresis and immunoblotted, indicated that a Mr = 94,000 protein band cross-reacts with a monoclonal antibody against rat GCR [3]. A nitrocellulose filter binding assay showed that both the partially purified porcine and rat GCRs interact specifically with a cloned synthetic 24 base pair deoxyoligonucleotide containing the GCR binding sequence in the first intron of the human growth hormone (hGH) gene [4]. This specific protein-DNA interaction is blocked by a single base pair change in the binding site. All three putative domains of the GCR molecule: the steroid binding, immunoreactive, and DNA binding have been conserved between two divergent species.

Sulphoconjugation of steroids in porcine liver: Partial purification of the cytosolic sulphotransferases for pregnenolone and 5α-androst-16-en-3β-ol

Steroid sulphotransferase activities for 5α-androst-16-en-3β-ol and pregnenolone in porcine liver cytosol have been assayed using 3′-phosphoadenosine-5′-phospho[ 35S]sulphate as sulphate donor. 5α-Androst-16-en-3β-ol sulphotransferase activity was obtained from porcine liver cytosol by gel filtration chromatography; activity was linear with time up to about 5 min., the optimum pH was near 8.0 and optimum temperature 37°C. Pregnenolone sulphotransferase activity was partially purified from porcine liver cytosol using DEAE-cellulose chromatography with an ionic gradient of KCl. This enzyme activity was linear with time up to 10 min and had optimum pH and temperature of 8.0 and 37°C, respectively.

Studies on the Enzymatic reduction of C-nitroso compounds. II. Multiple forms of liver C-nitrosoreductase and the identity with alcohol dehydrogenase.

Investigations were made of the multiplicity of the major C-nitrosoreductase in porcine liver cytosol catalyzing NADH-dependent reduction of p-nitrosophenol (p-NSP) to p-aminophenol (P-AmP). A partially purified preparation prepared by precipitation with ammonium sulfate, gel filtration with Sephadex G-100, and ion-exchange chromatography on DEAE-Sephadex A-50 showed two or more apparent Km values for p-NSP and also for NADH. This preparation could be resolved into at least four subfractions having different Km values by affinity chromatography on 5'-AMP-Sepharose, Even a more purified preparation, which was obtained from the Sephadex G-100 gel filtrate by affinity chromatography followed by ion-exchange chromatography, could be resolved into multiple subfractions by isoelectric focusing in polyacrylamide gel. All nitrosoreductase subfractions extracted from the polyacrylamide gel showed NADH-aldehyde reductase (alcohol dehydrogenase [EC 1.1.1.1]) activity and, except for a few minor subfractions, the two activities were in parallel. A commercially supplied, crystalline preparation of equine liver alcohol dehydrogenase also showed a similar multiplicity and the multiple subfractions had the both enzymatic activities, although the pattern of isoelectric focusing was different from those of the porcine liver preparations. Different heat inactivation curves were observed with various preparations, but in each preparation the curve for nitrosoreductase activity always agreed with that for aldehyde reductase activity. The nitrosoreductase preparations and the alcohol dehydrogenase preparation showed very similar pH-activity curves in catalyzing NADH-dependent reduction of p-NSP. Furthermore, ethanol inhibited the NADH-p-NSP reductase reaction competitively and p-AmP inhibited the NADH-aldehyde reductase reaction competitively. These results clearly indicate that the major C-nitrosoreductase in porcine liver was present in multiple forms having different Km values and the enzyme was identical with liver alcohol dehydrogenase.

Studies on the enzymatic reduction of C-nitroso compounds. I. Distribution of c-Nitrosoreductase activity in animal tissues and partial purification of the enzyme from porcine liver.

The subcellular distribution of NADH-p-nitrosophenol (p-NSP) reductase activity at pH 6.0 in porcine liver was studied by spectrophotometric assay. About two-thirds of the activity was found in the cytosol fraction and the pH optimum of this fraction was about 5.5. The activity at pH 5.8 of cytosol fractions from various tissues of rats, quails, frogs, carp, and scallops was also studied. All these fractions showed more or less NADH-p-NSP reductase activity but the activity of NADH-aldehyde reductase (alcohol dehydrogenase [EC 1.1.1.1]) was detected only in these from liver and a few other tissues. Supernatants from sonicated cells of Bacillus subtilis and Escherichia coli also showed C-nitrosoreductase activity but were devoid of aldehyde reductase activity. The major C-nitrosoreductase of porcine liver cytosol was purified 20- to 30-fold by fractionation with ammonium sulfate, gel filtration, and ion-exchange chromatography. The pH optimum of this preparation was 5.5 and activity was strongly inhibited by p-chloromercuribenzoate (p-CMB). The enzyme preparation was stable at 5 degrees C for at least a week in the presence of NADH at pH 8.4. High concentrations of ammonium sulfate also stabilized the enzyme. An equilibrium between monomeric and dimeric forms of the enzyme was found and the molecular weight was estimated to be about 83,000 and 160,000 daltons for the monomeric and dimeric forms, respecively. The enzyme utilized NADH almost specifically and 2 mol of NADH were consumed per mol of p-NSP reduced to p-aminophenol. Nitrosobenzene and aldehydes could also serve as the electron acceptor. The aldehyde reductase activity became concentrated roughly in parallel with the C-nitrosoreductase activity during the course of the purification and these two activities could not be separated even after further purification by 5'-AMP-Sepharose affinity chromatography. N-Nitroso compounds were not affected by this enzyme.

The Preparation of Ligandin with Glutathione‐S‐Transferase Activity from Porcine Liver Cytosol by Affinity Chromatography on Bromosulphophthalein‐Sepharose

A simple and rapid method for the purification of porcine ligandin with glutathione-S-transferase activity is presented. After ion-exchange chromatography on DEAE-Sephadex, ligandin is isolated from porcine liver cytosol by affinity chromatography on bromosulphophthalein-Sepharose and gel filtration of Sephadex G-100. Evidence is presented that the purified ligandin is homogeneous with respect to polyacrylamide-gel electrophoresis (7.5%) and sodium dodecylsulphate-gel electrophoresis. Physico-chemical investigations show that the purified ligandin has properties similar to those of ligandin isolated from other species with respect to molecular weight, amino-acid composition, secondary structure and catalytic activity. As is the case for human and rat ligandin, porcine ligandin binds bilirubin. Evidence is also presented that porcine liver cytosol contains several bromosulphophthalein-binding proteins with basic isoelectric points lacking catalytic activity.