Partial purification of a recombinase activity from human placenta and assay of recombination using rec-blot

We have designed an assay for the detection of recombination intermediates. The assay can also be used for screening for recombinases. In this assay, the nonradioactive plasmid was immobilized on a nylon membrane and the radioactive plasmid counterpart was allowed to recombine in the presence of a DNA binding protein fraction from human placenta. After incubation for 30 min at 30 degrees C, the membrane was washed extensively and dried. The recombination was visualised by autoradiography and quantitated by counting the corresponding areas of the membrane. Recombination was confirmed by transformation using pRDK plasmids and by gel electrophoresis. These results indicate the presence of recombinase activity in the DNA binding protein fraction of human placenta.     

S-Adenosylhomocysteine hydrolase from human placenta. Affinity purification and characterization.

S-Adenosylhomocysteine hydrolase (EC 3.3.1.1) was purified to homogeneity from human placenta by using S-adenosylhomocysteine-agarose affinity chromatography. The enzyme is a tetramer with a native Mr of 189 000 and subunit Mr of 47 000-48 000; there were nine cysteine residues per subunit and no disulphide bonds. The pI was 5.7. H.p.l.c. analysis revealed that the enzyme contained four molecules of tightly bound cofactor (NAD) per tetramer, of which 10-50% was in the reduced form. The enzyme had four binding sites per tetramer for adenosine, of which 10-35% were found to be occupied. Two types of adenosine-binding sites could be distinguished on the basis of differences in rates of dissociation of the enzyme-adenosine complex, and by examining binding of adenosine at 0 degree C and 37 degrees C. The enzyme catalysed the interconversion of adenosine and 4',5'-dehydroadenosine; the equilibrium constant for this reaction was 2.1 and favoured 4',5'-dehydroadenosine formation. Variability in the specific activity of preparations of S-adenosylhomocysteine hydrolase was related to the NAD+/NADH ratio of the preparation. The capacity to bind radioactively labelled adenosine depended on the adenosine content of the purified enzyme. The rate of adenosine binding and the sensitivity of S-adenosylhomocysteine hydrolase to inactivation by adenosine were both diminished in the absence of dithiothreitol.     

Partial purification and characterization of a copper-induced anionic peroxidase of sunflower roots

Treatment of 14-day-old sunflower seedlings with a toxic amount of copper (50 μM of CuSO₄) during 5 days caused significant increase in peroxidase activity in roots. Qualitative analysis of soluble proteins using native anionic PAGE followed by detection of peroxidase activity with guaïacol as electron donor in the presence of H₂O₂ revealed five stimulated peroxidases, named A1, A2, A3, A4, and A5. These peroxidases had differential behavior during the period of treatment. A1, A2, A3 and A4 were stimulated in the first period of stress, but rapidly suppressed at 72 h. A5 showed a progressive stimulation which was even increased at 120 h. A1 was partially purified, identified using liquid chromatography coupled to mass spectrometry (LC-MS/MS), and characterized. Effects of pH and temperature on its activity were determined with guaïacol as electron donor. Optima were obtained at pH 8 and at 40 °C. Analysis of substrate specificity showed that A1 was active on coniferyl alcohol but not on IAA. Enzymatic activity was inhibited by a high concentration of H₂O₂.     

Immunoaffinity purification and characterization of nucleotide pyrophosphatase from human placenta

Nucleotide pyrophosphatase [EC 3.6.1.9] was purified to homogeneity from human placenta using a monoclonal antibody affinity column. By sodium dodecylsulfate--polyacrylamide gel electrophoresis, the purified enzyme showed a major band at a molecular size of 130 K. The enzyme was a glycoprotein with N-linked oligosaccharides consisting of both complex- and oligomannoside-types. Substrate specificity to hydrolyze phosphodiester and phosphosulfate linkages as well as other properties were similar to those of nucleotide pyrophosphatase and phosphodiesterase from other sources.     

Partial purification and characterization of a human 3-methyladenine-DNA glycosylase.

A DNA glycosylase was purified about 30-fold from cultured human lymphoblasts (CCRF-CEM line) and was found to cleave 3-methyladenine from DNA alkylated with methyl methanesulfonate. The enzyme did not promote the release of 1-methyladenine, 7-methyladenine, or 7-methylguanine from DNA nor did it act on denatured methylated DNA. It produced apurinic sites in DNA alkylated with N-methyl-N-nitrosourea and ethyl methane-sulfonate as well as methyl methanesulfonate but not in untreated DNA or in DNA alkylated with nitrogen mustard or irradiated with ultraviolet light or X-rays. The glycosylase was free of detectable endonuclease activity in experiments with untreated DNA or DNA exposed to ultraviolet light; low levels of endonuclease activity, obtained when X-irradiated, alkylated, or depurinated DNA was the substrate, were attributed to contaminant apurinic endonuclease activity. This 3-methyladenine-DNA glycosylase has an estimated molecular weight of 34,000, is not dependent on divalent metal ions, and shows optimal activity at pH 7.5--8.5.     

Partial purification and characterization of a folatebinding protein from human choroid plexus

A folate-binding protein (binder) from human choroid plexus was solubilized with Triton X-100 and partially purified in three steps: (1) affinity chromatography, (2) Sephadex G-200 column chromatography, and (3) polyacrylamide gel electrophoresis. When the partially purified binder was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the binding activity was located in the region of the gel with a molecular weight between 45,000 and 60,000. The specific activity of the binder after the three purification steps was 1.2 g folic acid/mg protein, a 316-fold purification. Binding activity of the partially purified binder decreased below pH 6.0 and above pH 8.0 was unaffected by treatment with ribonuclease or deoxyribonuclease, but was abolished with trypsin, chymotrypsin, or protease (Streptomyces griesus). The binding of folic acid to the human binder was inhibited by folate > H₄-folate > methyl-H₄-folate dihydrofolate pteroic acid methotrexate aminopterin.     

Partial purification and characterization of gamma-secretase from post-mortem human brain

One characteristic feature of Alzheimer's disease is the deposition of amyloid beta-peptide (Abeta) as amyloid plaques within specific regions of the human brain. Abeta is derived from the amyloid beta-peptide precursor protein (beta-APP) by the intramembranous cleavage activity of gamma-secretase. Studies in cells have revealed that gamma-secretase is a large multimeric membrane-bound protein complex that is functionally dependent on several proteins, including presenilin, nicastrin, Aph-1, and Pen-2. However, the precise biochemical and molecular nature of gamma-secretase is as yet to be fully elucidated, and no investigations have analyzed gamma-secretase in human brain. To address this we have developed a novel in vitro gamma-secretase activity assay using detergent-solubilized cell membranes and a beta-APP-derived fluorescent probe. We report that human brain-derived gamma-secretase activity co-purifies with a high molecular weight protein complex comprising presenilin, nicastrin, Aph-1, and Pen-2. The inhibitor profile and solubility characteristics of brain-derived gamma-secretase are similar to those described in cells, and proteolysis occurs at the Abeta40- and Abeta42-generating cleavage sites. The ability to isolate gamma-secretase from post-mortem human brain may facilitate the identification of brain-specific modulators of beta-APP processing and provide new insights into the biology of this important factor in the pathogenesis of Alzheimer's disease.     

Partial purification and characterization of phosphatidylinositol kinases from human platelets

Most of human platelet phosphatidylinositol (PI) kinase activity (approx. 80%) was associated with the membrane fraction and its majority was released by the extraction with Triton X-100 after KCl treatment. Two major activity peaks (mPIK-I and mPIK-III) were obtained by Mono Q column chromatography. They were distinct from each other with regard to Mr (76,000 and 80,000 as determined by gel-filtration chromatography), apparent Km values for ATP, effect of arachidonic acid and phosphatidylserine and detergent requirement. Triton X-100 inhibited the activity of mPIK-I but rather weakly enhanced the mPIK-III activity, and sodium cholate remarkably inhibited both mPIK-I and mPIK-III activities. Their products were identified to be phosphatidylinositol 4-phosphate. On the other hand, about 20% of PI kinase activity was recovered from the cytosolic fraction and two activity peaks (cPIK-I and cPIK-II) were resolved on Mono Q column chromatography. There were no significant differences in biochemical properties between cPIK-I and cPIK-II. Both of them had Mr approx. 550,000 as determined by gel-filtration chromatography and were activated by sodium cholate to a greater extent than by Triton X-100. The results suggest that the major PI kinases (mPIK-I and mPIK-III) are PI 4-kinase and mPIK-I is distinct from PI 4-kinases in other sources especially with regard to the effect of Triton X-100.     

Partial purification and properties of glutathione peroxidase from carp hepatopancreas.

1. Two glutathione peroxidases (GSH-Px), D1 and D2, were partially purified from the hepatopancreas of common carp Cyprinus carpio by ion-exchange, gel filtration, and hydrophobic chromatography. The enzymatic properties of those enzymes were examined. 2. The D1 and D2 differed in following enzymatic properties. The pH optima of D1 and D2 were 8.0 and 9.0, respectively. 3. When stored at pH 4.0 for 36 hr, D1 kept 70% of maximal activity while only 30% of that was shown in D2. 4. D1 and D2 lost activity by 50% when incubated at 55 degrees C and 65 degrees C for 10 min, respectively.     

DNA polymerase alpha-DNA primase from human placenta. Immunoaffinity purification and preliminary characterization

Highly purified DNA polymerase alpha-DNA primase from normal human tissue (human placenta) has been prepared by immunoaffinity purification on immobilized anti-human DNA polymerase alpha monoclonal antibody SJK 287-38. According to data from SDS electrophoresis this preparation consists of subunits of 180, 160, 145, 140 kDa (a cluster of DNA-polymerizing subunits), 73 kDa (function unknown) and 59, 52 kDa (corresponding to primase). Three active enzyme forms of 270, 460 and 575 kDa have been revealed using native electrophoresis followed by detection of DNA polymerase activity.     

Partial purification and characterization of human sperminogen

A recently recognized non-proacrosin zymogen referred to as sperminogen has been purified from human spermatozoa, and several of its properties have been determined. The purification procedure included acid extraction of washed ejaculated sperm at pH 3.0, followed by gel filtration of the solubilized extract over a Sephadex G-75 superfine column. The sperminogen eluted from the column in a single band that was completely separated from the proacrosin band. This separation was confirmed by a gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) zymograph. This zymograph also demonstrated that the final sperminogen preparation contained four forms of zymogen, with molecular weights between 32,000 and 36,000. At neutral pH, the sperminogen was converted into spermin, its enzymatically active form, yielding a sigmoidal curve typical of zymogen autoactivation. The effects of several factors on the rate of this autoconversion indicate specific differences between sperminogen and proacrosin. Spermin hydrolyzed N-alpha-benzoyl-L-arginine ethyl ester (BzArgOEt), and was inhibited by lima bean trypsin inhibitor, pancreatic trypsin inhibitor, N-acetyl-L-leucyl-L-leucyl-L-argininal (leupeptin), and tosyl-L-lysine chloromethyl ketone, indicating that the enzyme has a trypsin-like specificity and probably belongs to the class of trypsin-like enzymes. Since acrosin is generally believed to be the only trypsin-like enzyme in mammalian sperm, the demonstration of human sperminogen and spermin necessitates further inquiry into the functions and the relationships between sperm proteinase systems.     

Partial purification and characterization of human erythrocyte phosphoglycollate phosphatase.

Human erythrocytes contain a phosphatase that is highly specific for phosphoglycollate. It shows optimum pH of 6.7 and has Km 1 mM for phosphoglycollate. The molecular weight appears to be about 72000. The enzyme is a dimeric molecule having subunits of mol. wt. about 35000. It could be purified approx. 4000-fold up to a specific activity of 5.98 units/mg of protein. The activity of the enzyme is Mg2+-dependent. Co2+, and to a smaller extent Mn2+, may substitute for Mg2+. Half-maximum inhibition of the phosphatase by 5,5'-dithiobis-(2-nitrobenzoate), EDTA and NaF is obtained at 0.5 microM, 1 mM and 4 mM respectively. Moreover, it needs a univalent cation for optimum activity. Phosphoglycollate phosphatase is a cytoplasmic enzyme. Approx. 5% of its total activity is membrane-associated. This part of activity can be approx. 70% solubilized by freezing, thawing and treatment with 0.25% Triton X-100.     

Purification and properties of glutathione peroxidase from human placenta.

Glutathione peroxidase (glutathione--H2O2 oxidoreductase; EC 1.11.1.9) was purified to homogeneity from human placenta by using (NH4)2SO4 precipitation, ion-exchange chromatography, Sephadex gel filtration and preparative polyacrylamide-disc-gel electrophoresis. Glutathione peroxidase from human placenta is a tetramer, having 4g-atoms of selenium/mol of protein. The molecular weight of the enzyme is about 85000 with a subunit size of about 22,000. Kinetic properties of the enzyme are described. On incubation with cyanide, glutathione peroxidase is completely and irreversibly inactivated and selenium is released as a low-molecular-weight fragment. Reduced glutathione, beta-mercaptoethanol and dithiothreitol protect the enzyme from inactivation by cyanide and the release of selenium. Properties of human placental glutathione peroxidase are similar to those of isoenzyme A reported earlier by us from human erythrocytes. The presence of isoenzyme, B, reported earlier by us in human erythrocytes, was not detected in placenta. Also selenium-independent glutathione peroxidase (isoenzyme II), which is specific for cumene hydroperoxide, was not present in human placenta.     

Partial purification and characterization of isopenicillin N epimerase activity from Streptomyces clavuligerus

Epimerase activity, which converts isopenicillin N to penicillin N, has been partially purified from cell-free extracts of Streptomyces clavuligerus. No stimulating cofactors of this activity were found, and neither EDTA nor anaerobic incubation caused significant inhibition of activity. Although pyridoxal phosphate did not stimulate epimerase activity, the presence of this cofactor was necessary for the stabilization of enzymic activity during the purification process. Epimerase activity was purified 35.5-fold by a combination of salt precipitation, gel filtration, and ion exchange chromatography. Gel filtration indicated that the epimerase has a molecular weight of 60 000 and sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the 35.5-fold purified epimerase showed a major protein band running near that location. Pyridoxal phosphate antagonists did not uniformly inhibit epimerase activity, but the inhibitory effect of hydroxylamine could be partially reversed by pyridoxal phosphate.