A soluble ATP-dependent system for protein degradation from murine erythroleukemia cells. Evidence for a protease which requires ATP hydrolysis but not ubiquitin

A soluble ATP-dependent system for protein degradation has been demonstrated in reticulocyte lysates, but not in extracts of nucleated cells. We report that extracts of undifferentiated murine erythroleukemia (MEL) cells contain a labile ATP-stimulated proteolytic system. The addition of ATP to MEL cell extracts at alkaline pH enhances degradation of endogenous cell proteins and various radiolabeled exogenous polypeptides from 2-15-fold. Nonhydrolyzable ATP analogs had no effect. In reticulocytes, one role of ATP in proteolysis is for ubiquitin conjugation to protein substrates. MEL cells also contain ubiquitin and extracts can conjugate 125I-ubiquitin to cell proteins; however, this process in MEL cells seems unrelated to protein breakdown. After removal of ubiquitin from these extracts by DEAE- or gel chromatography, the stimulation of proteolysis by ATP was maintained and readdition of purified ubiquitin had no further effect. In addition, these extracts degraded in an ATP-dependent fashion casein whose amino groups were blocked and could not be conjugated to ubiquitin. After gel filtration or DEAE-chromatography of the MEL cell extracts (unlike those from reticulocytes), we isolated a high molecular weight (600,000) ATP-dependent proteolytic activity, which exhibits many of the properties of energy-dependent proteolysis seen in crude cell extracts. For example, both the protease and crude extracts are inhibited by hemin and N-ethylmaleimide and both hydrolyze casein, globin, and lysozyme rapidly and denatured albumin relatively slowly. The protease, like the crude extracts, is also stimulated by UTP, CTP, and GTP, although not as effectively as ATP. Also, nonhydrolyzable ATP analogs and pyrophosphate do not stimulate the protease. Thus, some mammalian cells contain a cytosolic proteolytic pathway that appears independent of ubiquitin and involves and ATP-dependent protease, probably similar to that found in Escherichia coli or mitochondria.     

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.     

Trypsin inhibitor in mung bean cotyledons: purification, characteristics, subcellular localization, and metabolism

Trypsin inhibitor was purified to homogeneity from seeds of the mung bean (Vigna radiata [L.] Wilczek). The protease inhibitor has the following properties: inhibitory activity toward trypsin, but not toward chymotrypsin; isoelectric point at pH 5.05; molecular weight of 11,000 to 12,000 (sodium dodecyl sulfate gel electrophoresis) or 14,000 (gel filtration); immunological cross-reactivity against extracts of black gram and black-eyed pea, but not against soybean; no inhibitory activity against vicilin peptidohydrolase, the principal endopeptidase in the cotyledons of mung bean seedlings.

The trypsin inhibitor content of the cotyledons declines in the course of seedling growth and the presence of an inactivating factor can be demonstrated by incubating crude extracts in the presence of β-mercaptoethanol. This inactivating factor may be a protease as vicilin peptidohydrolase rapidly inactivates the trypsin inhibitor. Removal of trypsin inhibitory activity from crude extracts by means of a trypsin affinity column does not result in an enhancement of protease activity in the extracts.

The intracellular localization of trypsin inhibitor was determined by fractionation of crude extracts on isopycnic sucrose gradients and by cytochemistry with fluorescent antibodies. Both methods indicate that trypsin inhibitor is associated with the cytoplasm and not with the protein bodies where reserve protein hydrolysis occurs. No convincing evidence was obtained which indicates that the catabolism of trypsin inhibitor during germination and seedling growth is causally related to the onset of reserve protein breakdown.

     

Identification of nuclear factors that enhance binding of the thyroid hormone receptor to a thyroid hormone response element

Using a gel shift assay, we analyzed the binding of in vitro translated alpha- and beta-thyroid hormone (T3) receptors to a T3-response element (TRE) derived from the rat GH gene. No receptor-TRE complexes were observed when translated receptor alone was incubated with the TRE. However, addition of a nuclear extract from liver to the translational products resulted in the formation of two receptor-DNA complexes for both the alpha- and beta-receptors. These complexes were shown to contain translated receptor by comigration of 32P-labeled TRE and 35S-labeled receptor in the gel shift assay. A competition experiment demonstrated that formation of the complexes was sequence specific. Preincubation of the liver nuclear extract at 60 C abolished formation of both complexes indicating that receptor-TRE binding required a heat-labile nuclear factor. Phosphocellulose chromatography of the nuclear extract resulted in separation of the activities required for formation of the two complexes. Analysis of nuclear extracts from different tissues revealed that one complex formed in the presence of all extracts, whereas the second complex appeared predominantly with a nuclear extract from liver. Addition of T3 to the binding reaction had no effect on receptor-TRE complex formation. We suggest that nuclear factors interact with the T3 receptor to enhance hormone-independent binding to a TRE.     

Rapid assay for nitric oxide synthase using thin-layer chromatography

A simple, sensitive, and rapid method to determine the nitric oxide synthase (NOS) activity in crude cell extracts has been developed. The method takes advantage of differential migration of arginine and citrulline on silica gel thin-layer chromatography (TLC) with the specified buffer system. We have shown that products obtained by treating [14C]arginine with crude mouse hippocampal homogenate can be separated by methanol precipitation followed by TLC. The separated products of the enzyme reaction can be quantitated by radiometric scanning of the TLC plate or by counting in a scintillation counter. Inhibition of conversion of l-arginine to l-citrulline by NG-monomethyl-l-arginine acetate, a specific inhibitor of NOS, confirmed the NOS assay described in this investigation. This method is versatile and allows rapid simultaneous assay of several samples in a short period of time. Therefore, this assay is very useful for both qualitative and quantitative estimation of NOS activity.     

Scintillation Proximity Assay for Human DNA Topoisomerase I Using Recombinant Biotinyl-Fusion Protein Produced in Baculovirus-Infected Insect Cells

DNA topoisomerases are well-established targets of important therapeutic agents which include the antibacterial quinolones and anticancer camptothecins. Screens for new classes of topoisomerase inhibitors generally employ methods, such as gel electrophoresis, which are not readily amenable to a rapid high-throughput format. We describe here a high-throughput assay to screen for inhibitors of human DNA topoisomerase I based on the scintillation proximity assay. The assay employs recombinant biotinyl-topoisomerase I fusion protein, a hybrid protein which contains a domain that is biotinylated duringin vivoexpression. The hybrid topoisomerase I fusion protein is found to be biotinylated, active, and nuclear-localized when produced in insect cells using a baculovirus expression system. The biotinyl-topoisomerase I fusion protein can be captured from crude nuclear extracts by immobilization on streptavidin-coated scintillation proximity assay beads. The assay detects binding of³H-labeled DNA to the bead-immobilized enzyme by scintillation counting. The method is also able to detect stabilization of covalent protein–DNA complexes by camptothecin, an inhibitor previously shown to stabilize covalent intermediates that form during catalysis.     

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,quantification, and characterization of proteases in recombinant Escherichia coli

Electrophoresis of crude cell extracts on PAGE gels in the presence of SDS copolymerized with a nonspecific protease substrate has been used to detect, characterize, and quantify intracellular proteases in recombinant Escherichia coli. After electrophoresis, the gels are incubated, SDS is removed, and protease activity is revealed by clear zones on the stained gel due to proteolysis of the nonspecific protease substrate (gelatin or casein). The method differentiates proteases based on activity and molecular weight.     

Purification and partial characterization of poliovirus protease 2A by means of a functional assay.

The purification of poliovirus protease 2A from infected cells by a functional assay is described. A small synthetic peptide was cleaved specifically by an esterase present in poliovirus-infected cells. Since the enzyme proved extremely unstable in crude extracts a rapid and efficient purification procedure had to be developed. By treatment with different detergents followed by high-speed centrifugation, the esterase activity was separated from inactivating cellular enzymes and was solubilized. Purification to more than 90% homogeneity could be achieved by a single chromatography step, namely, by gel filtration through Superose 12 under fast-protein liquid chromatography conditions. The esterase activity was associated with a protein of 17,000 daltons and copurified with poliovirus protein 2A. Furthermore, antibodies to 2A specifically precipitated the esterase activity. Thus, the esterase was identified as poliovirus protease 2A. Inhibition studies with known protease inhibitors revealed that 2A is probably a sulfhydryl protease. Of the metal ions tested, only zinc exerted significant inhibitory effects. The esterase activity was optimal near neutral pH and had an extremely short half-life at physiological temperatures.     

Translational activity of mouse protamine 1 messenger ribonucleoprotein particles in the reticulocyte and wheat germ cell-free translation systems

Protamine 1 mRNAs are inactivated by a block to the initiation of translation in early spermatids and are translationally active in late spermatids in mice. To determine whether translation of protamine 1 mRNAs is inhibited by a protein repressor, the translational activity of ribonucleoprotein particles and deproteinized RNAs were compared in the reticulocyte and wheat germ cell-free translation lysates. To isolate RNPs, cytoplasmic extracts of total testes were fractionated by large-pore gel filtration chromatography. Ribonucleoprotein particles in the excluded fractions stimulated synthesis of radiolabeled translation products for protamine 1 about twofold less effectively than deproteinized RNAs in the reticulocyte lysate, but were inactive in the wheat germ lysate. The ability of translationally repressed protamine 1 ribonucleoprotein particles to form initiation complexes with 80S ribosomes in the reticulocyte lysate was also measured. Protamine 1 ribonucleoprotein particles isolated by gel filtration and in unfractionated cytoplasmic extracts of early spermatids were nearly as active in forming initiation complexes as deproteinized mRNAs. The isolation of ribonucleoprotein particles in buffers of varying ionic strength, protease inhibitors, and several other variables had no major effect on the ability of protamine 1 ribonucleoprotein particles to form initiation complexes in the reticulocyte lysate. These results can be explained by artifacts in the isolation or assay of ribonucleoprotein particles or by postulating that protamine 1 mRNAs are inactivated by a mechanism that does not involve protein repressors, such as sequestration. © 1994 Wiley-Liss, Inc.     

Purification and initial characterization of ubiquitin from the higher plant, Avena sativa

Ubiquitin is a highly conserved, 76-amino acid polypeptide recently demonstrated to be involved in ATP-dependent protein degradation in mammalian cells. From immunoblot analyses with anti-human-ubiquitin antibodies we have detected the presence of free ubiquitin in green leaves, etiolated shoots, and dry seeds of the higher plant, oats (Avena sativa L.). We also find that crude oat extracts contain protease(s) that rapidly degrade both oat and human ubiquitin (t1/2 approximately 10 min at 27 degrees C). This proteolysis apparently cleaves ubiquitin at the carboxyl-terminal glycine dipeptide and results in inactivation of the molecule with respect to ligation but does not affect its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using homogenization conditions that preclude this proteolysis (low pH and the addition of the protease inhibitor p-chloromercuribenzoate) and immunoblotting as an assay for the protein, a procedure for the purification of ubiquitin from etiolated oat shoots was developed. Characterization of purified oat ubiquitin by absorption spectra, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, radioimmunoassay with anti-human-ubiquitin antibodies, and kinetic analyses using the ubiquitin activating enzyme isolated from rabbit liver indicates that this protein is remarkably similar to the mammalian form. Small differences between the oat and human proteins have been observed by amino acid compositional analyses indicating that the two forms are not totally homologous. Immunoblotting of crude oat extracts has revealed the presence of high molecular weight proteins recognized by anti-ubiquitin antibodies that represent ubiquitin-protein conjugates formed in vivo. Taken together, these data provide evidence that higher plants contain a ubiquitin-dependent proteolytic pathway that is mechanistically identical to that present in animals.     

In-gel precipitation of enzymatically released phosphate

The phosphate precipitation reaction using ammonium molybdate and triethylamine under low pH has been applied to gel-based assays for detecting phosphate-releasing enzymes. The sensitivity of the assay is 10 pmol Pi/mm2 of 1.5-mm-thick gel. The assay is applicable to enzymes with a wide range of optimal pH, from acid (pH 4.5) to alkaline phosphatase (pH 9.7), and to enzymes that use acid-labile substrates such as apyrase and glutamine synthetase. Using a negative staining approach, maltose phosphorylase, a phosphate-consuming enzyme, can also be detected. The assay was used to detect glutamine synthetase isoforms, separated by nondenaturing polyacrylamide gel electrophoresis from crude maize extracts. For downstream applications such as staining gels for proteins, the gels with precipitate should be incubated in 10 mM dithiothreitol or beta-mercaptoethanol until the precipitate is dissolved and then thoroughly washed in water. In comparison to calcium phosphate precipitation or the phosphomolybdate-malachite green method, this method is more sensitive. It is a very simple, rapid, versatile, reproducible, and inexpensive method that could be a useful tool in enzymological studies.     

Chemical synthesis and expression of the HIV-1 protease gene in E. coli

The 297bp HIV-1 protease gene was constructed from five discrete synthetic fragments and expressed in E. coli. A soluble protein product of 11.5 Kd was detected by immunoblotting using protease specific antisera. A quantitative assay system, utilizing a synthetic nonapeptide spanning the cleavage site between p17-p24 in the gag polyprotein, was used to measure the specific protease activity in crude extracts. The protease hydrolyzed tyrosyl-proline bonds with an approximate specific activity of 43 pmoles/min/micrograms of total protein. The chemical synthesis of the protease gene and it's expression provides a feasible method for rapid mutant analysis, important for structure-function studies and rational design of potential inhibitors.     

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.     

Evidence for cAMP-dependent protein kinase in the dinoflagellate,Amphidinium operculatum

A cAMP dependent protein kinase (PKA) was identified in the dinoflagellate Amphidinium operculum. In vitro kinase activity towards kemptide, a PKA-specific substrate, was not detectable in crude lysates. However, fractionation of dinoflagellate extracts by gel filtration chromatography showed PKA-like activity toward kemptide at approximately 66 kDa. These findings suggest that possible low molecular mass inhibitors in crude lysates were removed by the gel filtration chromatography. Pre-incubation of extracts with cAMP prior to chromatography resulted in an apparent molecular mass shift in the in vitro kinase assay to 40 kDa. An in-gel kinase assay reflected activity of the free catalytic subunit at approximately 40 kDa. Furthermore, western blotting with an antibody to the human PKA catalytic subunit confirmed a catalytic subunit with a mass of approximately 40 kDa. Results from this study indicate that the PKA in A. operculatum has a catalytic subunit of similar size to that in higher eukaryotes, but with a holoenzyme of a size suggesting a dimeric, rather than tetrameric structure.     

Analysis of multiple forms of nuclear factor I in human and murine cell lines.

Nuclear factor I (NFI) is a group of related site-specific DNA-binding proteins that function in adenovirus DNA replication and cellular RNA metabolism. We have measured both the levels and forms of NFI that interact with a well-characterized 26-base-pair NFI-binding site. Five different NFI-DNA complexes were seen in HeLa nuclear extracts by using a gel mobility shift (GMS) assay. In addition, at least six forms of NFI were shown to cross-link directly to DNA by using a UV cross-linking assay. The distinct GMS complexes detected were composed of different subspecies of NFI polypeptides as assayed by UV cross-linking. Different murine cell lines possessed varying levels and forms of NFI binding activity, as judged by nitrocellulose filter binding and GMS assays. The growth state of NIH 3T3 cells affected both the types of NFI-DNA complexes seen in a GMS assay and the forms of the protein detected by UV cross-linking.     

Detection of peptide nucleic acids in tissue extracts of treated animals by gel mobility shift assay

We have developed a sensitive and reproducible gel mobility shift assay to detect PNA oligomers in tissue of treated animals. PNA present in purified tissue extracts of treated animals is hybridized to a 33P-labelled DNA oligomer probe, and analyzed by polyacrylamide gel electrophoresis. The PNA-DNA hybrid migrates more slowly than the DNA probe alone and can be quantified relative to a standard curve. This detection method is useful for detecting PNAs in many different tissues, including brain, heart, kidney, liver, spleen, and serum, as well as cells in culture.     

AchR亚基基因启动子与分化/未分化肌细胞核因子的相互作用分析

采用凝胶阻滞实验比较分析了鸡烟碱样乙酰胆碱受体（ＡＣｈＲ）亚基基因－２７５／＋３６片段与分化／未分化肌细胞核抽提物的相互作用．发现未分化肌细胞核内存在两种直接识别ＡＣｈＲ启动子的结合活性,其结合反应不能被ＡＣｈＲα亚基基因增强子（含Ｅ盒子）竞争阻断,揭示此结合活性与ＭｙｏＤ家族无关,并表现为基因特异性结合;两种结合活性中一种结合活性既存在于未分化细胞,也存在于分化肌细胞,另一种结合活性只存在于未分化肌细胞．存在于未分化肌细胞、特异识别基因的结合活性不同于ＭｙｏＤ家族,也不同于已发现的Ｉｄ和Ｉ－ｍｆ（两者不能直接结合ＤＮＡ）,可能与基因在未分化肌细胞中表达的负调控有关．     

A fluorescence-resonance-energy-transfer-based protease activity assay and its use to monitor paralog-specific small ubiquitin-like modifier processing

Dynamic modification of proteins with the small ubiquitin-like modifier (SUMO) affects the stability, cellular localization, enzymatic activity, and molecular interactions of a wide spectrum of protein targets. We have developed an in vitro fluorescence-resonance-energy-transfer-based assay that uses bacterially expressed substrates for the rapid and quantitative analysis of SUMO paralog-specific C-terminal hydrolase activity. This assay has applications in SUMO protease characterization, enzyme kinetic analysis, determination of SUMO protease activity in eukaryotic cell extracts, and high-throughput inhibitor screening. In addition, while demonstrating such uses, we show that the SUMO-1 processing activity in crude HeLa cell extracts is far greater than that of SUMO-2, implying that differential maturation rates of SUMO paralogs in vivo may be functionally significant. The high degree of structural conservation across the ubiquitin-like protein superfamily suggests that the general principle of this assay should be applicable to other post-translational protein modification systems.