An activity gel assay for the detection of DNA helicases and nucleases from cell-free extracts.

An activity gel assay was developed for the detection of DNA helicases in crude extracts. The assay was based on the ability of DNA helicases to unwind radioactive fragments from single-stranded M13 circles that were immobilized in an SDS polyacrylamide gel. The displaced radioactive strands were detected by blotting them to a filter and visualizing the resulting bands by autoradiography. Experiments with purified proteins demonstrated that DNA helicases, endonucleases and exonucleases could produce activity bands. A one-dimensional gel assay was sufficiently sensitive to allow detection of DNA helicase I, DNA helicase II, DNA helicase IV, the RecQ helicase as well as 3 unidentified putative DNA helicases in crude extracts of Escherichia coli. Exonuclease and endonuclease activities from crude extracts could be distinguished from DNA helicase activities by their ATP-independence and from each other by their band morphologies.     

Detection of the catalytic activities of DNA polymerases and their associated exonucleases following SDS-polyacrylamide gel electrophoresis.

A method is described to detect DNA polymerases and nucleases in homogeneous or crude enzyme preparations after electrophoresis in SDS-polyacrylamide gels(2) containing the appropriate template or substrate. DNA polymerases are electrophoresed in a gel containing gapped calf thymus DNA and after a renaturation treatment, the gel is incubated in a reaction mixture in which one deoxyribonucleoside triphosphate is [alpha-32P]-labelled. Incorporation of radioactivity into DNA is detected at the vicinity of the polymerase band by autoradiography. An associated nuclease activity can be measured after electrophoresis in a gel containing 32P-labelled gapped DNA, when nucleolytic digestion is seen as a clear band in the resulting autoradiogram. The gels can subsequently be stained with Coomassie blue to establish identical molecular weights of polymerase, nuclease and protein bands. Applications of this technique are discussed.     

A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts

A general method is described for the detection and quantification of low amounts of neomycin phosphotransferase in crude cell extracts. The assay is based on the electrophoretic separation of the enzyme from other interfering proteins and detection of its enzymatic activity by in situ phosphorylation of the antibiotic kanamycin. Both kanamycin and [γ³²P]ATP acting as substrates are embedded in an agarose gel placed on the polyacrylamide gel containing the separated proteins. After the enzymatic reaction, the phosphorylated kanamycin is transferred to P81 phosphocellulose ion exchange paper and the radiolabeled kanamycin is visualised by autoradiography. With this method 1 ng of active enzyme can easily be detected. Both prokaryotic and eukaryotic cell extracts can be examined, and changes in the size of enzymatically active proteins can be determined.     

Detection of protein kinase activity in sodium dodecyl sulfate-polyacrylamide gels

A procedure is described for identifying protein kinase activity in protein samples following electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Protein kinase activity is detected by renaturation of the enzymes within the gel followed by phosphorylation with [gamma-32P]ATP of either substrates included in the polyacrylamide gel or of the kinase itself. Then, after removal of the unreacted [gamma-32P]ATP by washing the gel in the presence of an anion-exchange resin, the positions (Mr) of the protein kinase activity are visualized by autoradiography. Studies using a purified catalytic subunit of cAMP-dependent protein kinase indicate that enzyme concentrations as low as 0.01 microgram can easily be detected on gels containing 1 mg/ml casein. The technique is also useful for identifying active subunits of multisubunit enzymes. The active subunit of casein kinase II, for example, can readily be determined by renaturing the dissociated enzyme in gels containing casein. Putative protein kinases present in crude mixtures of proteins can also be detected following separation by gel electrophoresis and can be characterized on the basis of molecular weight and identity of the phosphorylated amino acid. Using this technique, at least three major protein kinases were detected in a mixture of proteins prepared by subfraction of red blood cell membranes.     

Non-disruptive detection of DNA polymerases in nondenaturing polyacrylamide gels

A non-disruptive method is described with which DNA polymerases can be detected in homogeneous preparations and unfractionated cell extracts after electrophoresis in nondenaturing polyacrylamide gradient gels. The technique involves diffusion of DNA polymerase activity into an overlay assay agarose gel, the synthesis of radioactive DNA, removal of excess substrates and autoradiography. Cell extracts from a variety of organisms were studied using this method. The activity from Escherichia coli crude extracts migrated in a position corresponding to a higher molecular mass than did purified preparations of DNA polymerase I. DNA polymerases of higher organisms generally migrated in positions corresponding to 400--900 kDa, in some cases, close to 200 kDA.     

Solid-phase tyrosine-specific protein kinase assay in multiwell substrate-immobilized polyacrylamide gel

Since tyrosine-specific protein kinase (TPK) is much less abundant than Ser/Thr-specific kinases in cells, determination of TPK activity in crude cell extracts or column chromatography eluates has been difficult. This is compounded by the absence of a rapid, economical method for the separation of high endogenous protein phosphorylation background from exogenously added tyrosine-containing substrates. We have developed a new solid-phase assay, which provides high sensitivity and efficiency at a low cost for assaying the TPK activity of crude enzyme preparations. This assay utilizes immobilized tyrosine-containing synthetic polymers such as (Glu:Tyr, 4:1)n in polyacrylamide gels. The kinase reaction is started by adding crude enzyme solutions and [tau-32P]ATP-metal ion mixtures into microtiter-size wells made in the gels. After the phosphorylation reaction, the reaction mixtures are removed and the gels are prewashed in water followed by electrophoresis to completely remove free radioactive ATP. 32P incorporation into the immobilized TPK-specific substrate can be detected by autoradiography and quantitated by cutting the gel pieces and counting them with a liquid scintillation counter. The simple, rapid method should facilitate screening of TPK inhibitors and activators as well as examining the substrate specificity of TPKs. Other enzymes, including Ser/Thr-specific protein kinases, can also be analyzed by this technique.     

High-performance liquid affinity chromatography and in situ fluorescent labeling on thin-layer chromatography of glycosphingolipids

A method combining high-performance liquid affinity chromatography and in situ fluorescent labeling on thin-layer chromatography is introduced for determination of glycosphingolipids. Glycolipids in crude extract from rat liver were separated quantitatively from neutral lipids and phospholipids with a phenylboronic acid-derivatized silica gel column. Glycolipids were eluted quantitatively with approximately 98% of crude extract recovered. This column is useful for selective cleanup of glycosphingolipids in crude extract from tissue. Simultaneously, a fluorometric determination of glycosphingolipids with 7-amino-4-methylcoumarin after NaIO4 oxidation on a TLC plate was introduced and its condition was optimized. Glycolipids in amounts ranging from 1 to 100 pmol are easily detectable and give linear responses over the respective ranges. The method is fast and useful for the determination of glycolipids from small amounts of biological samples and requires a minimum amount of about 1 mg of biological specimen for determination of glycolipids.     

Using proteases to avoid false identification of DNA-protein complexes in gel shift assays.

Gel mobility shift assays using crude nuclear extracts may result in the formation of multiple DNA-protein complexes reflected by their discrete gel mobilities. Identification of the multiple complexes can sometimes be complicated by the presence of protease activities in the extract as demonstrated here. We describe a simple protease-mediated partial digestion method that can be coupled with the gel shift assay to overcome the problem. The combined approach enables us to identify gel complexes that arise from protein degradation and therefore is suitable for analyzing those DNA-binding proteins exhibiting prominent protease sensitivity. The method should prove particularly informative in the search for tissue-specific complexes when crude extracts from different sources are compared by the gel shift assay.     

Simultaneous measurements of rates of protein de novo synthesis and its steady-state levels by immunoblotting

Immunoblotting was used for simultaneous measurements of rates of de novo synthesis and steady-state levels of a model protein-cytochrome P450r. This is the major phenobarbital-inducible P450 form in cultured chick-embryo hepatocytes (L. Oron, and S. Bar-Nun, 1984) Biochem. Biophys. Res. Commun. 125, 1096-1102). Cultured hepatocytes were metabolically labeled with [35S]methionine, and cellular proteins were resolved by gel electrophoresis and then quantitatively electroblotted onto nitrocellulose. The blot was directly exposed to autoradiography and the rates of de novo synthesis were estimated from the 35S label of P450r. The same blot was then incubated with specific anti-P450r antibodies, followed by 125I-labeled secondary antibodies, and the blot was reexposed to autoradiography through white paper. The paper masked the 35S-labeled proteins and the steady-state levels were estimated from the 125I-immunodecorated P450r. By this simple method, "true" inducers of cytochrome P450r, such as phenobarbital, can be defined, whereas "false" inducers, which primarily affect the degradation of P450r, can be detected.     

Heterogeneity of mammalian DNA ligase detected on activity and DNA sequencing gels.

A new method to detect DNA ligase activity in situ after NaDodSO4 polyacrylamide gel electrophoresis has been developed. After renaturation of active polypeptides the ligase reaction occurs in situ by incubating the intact gel in the presence of Mg++ and ATP. Further treatment with alkaline phosphatase removes the unligated 5'-32P-end of oligo (dT) used as a substrate and active polypeptides having ligase activity are identified by autoradiography. Analysis on DNA sequencing gels of the oligo (dT) reaction products present in the activity bands ensures that the radioactive material detected in activity gels or in standard in vitro ligase assays corresponds unambiguously to a ligase activity. Using these methods, we have analysed the purified phage T4 DNA ligase, and the activities present in crude extracts and in purified fractions from monkey kidney (CV1-P) cells. The purified T4 enzyme yields one or two active peptides with Mr values of 60,000 and 70,000. Crude extracts from CV1-P cells contain several polypeptides having DNA ligase activity. Partial purification of these extracts shows that DNA ligase I isolated from hydroxylapatite column is enriched in polypeptides with Mr 200,000, 150,000 and 120,000, while DNA ligase II is enriched in those with Mr 60,000 and 70,000.     

Separation of cellular iron containing compounds by electrophoresis

High resolution separation of metalloproteins and other iron compounds based on native gel electrophoresis followed by⁵⁹Fe autoradiography is described. Lysates of mouse spleen erythroid cells metabolically labeled with⁵⁹Fe-transferrin were separated on 3–20% polyacrylamide gradient gels in the presence of Triton X100 and detected by autoradiography. In addition to ferritin and hemoglobin, several compounds characterized by their binding of iron under different conditions were described. Iron chelatable by desferrioxamine migrated in the region where several high-molecular weight compounds were detected by silver staining. The technique is nondissociative, allowing identification of iron compounds with the use of specific antibodies. Cellular iron transport and the action of iron chelators on specific cellular targets can be investigated in many small biological samples in parallel.     

Sensitization of amino acid derivatives obtained from Edman degradation with radioactively-labeled iodohistamine

The minimum amount of proteins and peptides required for sequencing is constantly decreasing as more sensitive microsequencing methods are developed. The sensitization of and Edman degradation product is one such method. We took the 2-anilino 5-thiazolinone amino acid intermediates obtained from Edman degradation by conventional sequencing procedures, and quantitatively reacted them with a primary amine. The amine used was radioactive [125I]iodohistamine, which affords highly sensitive detection. The labeled amino acid derivatives were separated by thin layer chromatography. Ten femtomoles of a labeled derivative can be detected by autoradiography.     

Detection of DNA and RNA polymerase activities in situ following electrophoresis in polyacrylamide gels.

A procedure is described for the detection of DNA dependent DNA and RNA polymerase activities in intact polyacrylamide gels that contain DNA. After electrophoresis under non-denaturing conditions, the intact gels are incubated with DNA or RNA polymerase reaction mixture in which one of the four deoxyribonucleoside or ribonucleoside triphosphates is radioactively labeled. The acid insoluble radioactivity associated with the intact gel is then analyzed by autoradiography of the intact gel or by liquid scintillation spectrometry of the sliced gel. Inhibition of the enzymatic activities by low molecular weight compounds such as N-ethylmaleimide or rifampin can be demonstrated by this procedure.     

A protein-tyrosine kinase in the nuclear matrix from rat liver

Protein kinase activity in isolated nuclei from rat liver was detected in situ after electrophoresis on SDS-polyacrylamide gel containing no exogenous protein substrate. After renaturation of polypeptides, the gel was incubated with [gamma-32P]ATP and divalent cations. Among five major protein kinase activities observed as radioactive bands by autoradiography, a protein kinase autophosphorylating on tyrosine (Mr 30,000) was identified and found to be localized in the nucleus, particularly in the nuclear matrix. The intensity of the activity band representing the level of the protein-tyrosine kinase in rat liver nuclei did not appreciably change during 3-24 h after partial hepatectomy.     

Analyses of genetic variants of l-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster by two-dimensional gel electrophoresis and immunoelectrophoresis

A protein spot corresponding to l-glycerol-3-phosphate dehydrogenase (α-GPDH, E.C. 1.1.1.8, NAD⁺ oxidoreductase) has been identified on a two-dimensional gel (isoelectric focusing-SDS gel) containing up to 150 stained protein spots from a crude Drosophila homogenate. Preliminary identification of the α-GPDH spot was made by including a suitable amount of purified Drosophila α-GPDH in crude fly homogenates prior to electrophoresis and observing an intensity enhancement of the corresponding protein spot on the gels. When three purified electrophoretic variants (slow, fast, and ultrafast) were mixed and analyzed by two-dimensional gel electrophoresis, horizontal displacements of the three protein spots were observed. Immunoprecipitation of the enzyme prior to electrophoresis and gene mapping further confirmed the identity of the α-GPDH protein spot. The α-GPDH spot can also be detected by autoradiography of a two-dimensional gel from a single fly extract, where it has been estimated to constitute 0.5–1% of the total soluble protein. Mutants which express no apparent α-GPDH activity were analyzed by two-dimensional gels and immunoelectrophoresis in an attempt to identify and characterize the inactive proteins. It is suggested that these techniques provide a powerful tool for the analysis of CRM⁺-null activity mutants of a specific gene-enzyme system.     

Simultaneous detection of kinase and phosphatase activities of polynucleotide kinase using molecular beacon probes

Phosphorylation and dephosphorylation of DNA by polynucleotide kinase (PNK) has an important role in DNA damage repair, replication, and recombination. Traditionally, it is assayed by denaturing gel electrophoresis and autoradiography, which are tedious and not sensitive. We report on the development of a sensitive and simple method for PNK assay based on DNA ligation using a molecular beacon. Enzyme activity of PNK is measured down to a limit of 0.002 unit/ml. The method not only provides a universal platform for simultaneous monitoring of kinase and phosphatase activities, but also shows great potential in biological research, drug discovery, and clinical diagnostics.     

Purification and properties of the human placental uracil DNA glycosylase

Human placental uracil DNA glycosylase was purified 3700-fold to apparent homogeneity as defined by SDS gel analysis. Its immunological characteristics were examined using three monoclonal antibodies prepared against partially purified human placental uracil DNA glycosylase. Immunoblot analysis demonstrated that, even in crude isolates, only one glycosylase species of molecular weight 37,000 could be detected. Each of the three monoclonal antibodies quantitatively recognized the highly purified enzyme by ELISA. The glycosylase is a single polypeptide with a molecular weight of 37,000 as defined by both Sephadex gel filtration and by SDS-polyacrylamide gel electrophoresis analysis. The enzyme is heat-stable, with a t 1/2 of greater than 30 min at 42 degrees C or at 45 degrees C. Surprisingly, inhibitor analysis demonstrated that the glycosylase was inhibited by preincubation with either 5-fluorouracil or 5-bromouracil. However, no significant inhibition was observed when either compound was added directly to the enzyme assay.     

A sensitive method for detection of calmodulin-dependent protein kinase II activity in sodium dodecyl sulfate-polyacrylamide gel

A procedure for detecting protein kinase activities of the alpha and beta subunits of calmodulin-dependent protein kinase II separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. After electrophoresis, the gel was immersed in 6 M guanidine HCl for 1 h and then in a buffer containing 0.04% Tween 40 for 16 h at 4 degrees C for renaturation of the resolved polypeptides. The renatured polypeptides in the gel were incubated with [gamma-32P]ATP for phosphorylation of either the substrate included in the polyacrylamide gel or the kinase itself. After removal of the unreacted [gamma-32P]ATP, the protein kinase activities were visualized by autoradiography. Two radioactive protein bands of Mr 50,000 and 60,000, which corresponded to the alpha and beta subunits, were detected only when the phosphorylation was carried out in the presence of Ca2+ and calmodulin. Approximately 0.05 micrograms of the enzyme could be detected on a gel containing no protein substrate. When microtubule-associated protein 2 was included in the gel, the sensitivity of the detection of calmodulin-dependent protein kinase II in the gel was more than one order of magnitude higher than that in the gel containing no protein substrate.     

M13 clones carrying point mutations: identification by solution hybridization

A solution hybridization procedure for the rapid identification of M13 clones carrying a particular sequence is described. The method, which employs a radiolabeled oligonucleotide probe, can discriminate between sequences which differ by only a single base, and can therefore be used for the identification of mutant sequences created by oligonucleotide-directed mutagenesis. Samples of phage-containing supernatant from cultures of M13-infected Escherichia coli are incubated with radiolabeled probe in the presence of sodium dodecyl sulfate. The mixtures are then subjected to agarose gel electrophoresis to separate hybrid molecules from unbound probe and hybridization is detected by autoradiography. This solution hybridization procedure is quicker and more convenient than membrane hybridization and has the added advantage that more than one probe can be used on a given gel.