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At least 505 records · Page 28Linked to original sources
PubMed1987

Dot assay for neomycin phosphotransferase activity in crude cell extracts.

A dot assay for determining neomycin phosphotransferase (NPT II) activity in crude cell extracts has been developed. The assay provides for the rapid screening of large numbers of cell cultures generated in gene transformation experiments using NPT II as a dominant selectable marker. Currently, the commonly used procedure for NPT II assay employs a time-consuming electrophoretic protein separation step to eliminate a positive interference resulting from putative protein kinase activities present in crude cell extracts. The dot method we have developed is based upon the ability of nitrocellulose membrane to eliminate that positive interference without a prior protein separation step. It provides a sensitive, reproducible, and significantly more convenient and rapid means of screening large numbers of cell extracts in order to distinguish cultures producing high levels of NPT II from those that do not.

Collodion↗
PubMed1987

Nanosecond time-resolved fluorescence kinetic studies of the 5,5'-dithiobis(2-nitrobenzoic acid) reaction with enzyme I of the phosphoenolpyruvate:glycose phosphotransferase system.

Enzyme I of the bacterial phosphotransferase system is a protein component which undergoes a temperature-dependent monomer/dimer equilibrium. Reaction of sulfhydryl residues with SH-specific reagents inhibits both activity and dimerization. There are four cysteine residues available in each subunit, one of which (Cys 502) is proximate to one of the two tryptophan residues (Trp 498). Previous studies revealed two major lifetimes and spectra, suggesting distinct environments for tryptophan. In this paper, we examine the dynamic quenching of tryptophanyl fluorescence that occurs when an energy transfer acceptor, thio-2-nitrobenzoic acid (TNB), is covalently attached to the sulfhydryl groups. More precisely, we have traced the recovery of nativelike fluorescence lifetime components (and the concomitant loss of "reduced lifetime" amplitudes) that accompanies TNB release. The course of lifetime changes seen when a reducing reagent removes the quencher may be sensitive to a variety of effects, including different SH affinities, different proximities to Trp, changing availability for dimerization, or conformational changes. The prospective value of separating each lifetime component from the mixture is illustrated.

Dithionitrobenzoic Acid↗
PubMed1988

Coupled spectrofluorometric assay for aminoglycoside phosphotransferases.

In order to make accurate kinetic measurements for the substrates of aminoglycoside (AG) phosphotransferases (APHs), we have developed an assay which overcomes many of the limitations of currently used assays. We have adapted the coupled spectrophotometric assay (P. R. Goldman and D. B. Northrop (1976) Biochem. Biophys. Res. Commun. 68, 230-236) for use in a spectrofluorometer. At an excitation wavelength of 340 nm, NADH will emit an intensity peak at 450 nm; NAD does not emit under these conditions. Our assay can accurately measure differences of 0.25 microM. For the APH(3')-II encoded on Tn5, we have redetermined the Km's for the AGs, amikacin (AK), kanamycin (KM), and ribostamycin (Rib), and for ATP. Our values for AK (76 microM) were lower than those derived from the spectrophotometric assay; for KM and Rib we obtained Km values of 5.1 and 3.6 microM, respectively. These values were well below the limit of accuracy (10 microM) for the spectrophotometric assay. In addition, we have begun characterization of an APH from a clinical isolate with a low Km for AK. Thus far, we have determined that this enzyme has Km's of approximately 1 microM for both AK and KM. These results show that the assay is well suited for accurate determinations of kinetic constants for low Km substrates of APH enzymes.

Drug Resistance, Microbial↗
PubMed1990

A quantitative assay for neomycin phosphotransferase activity in plants.

A sensitive, simple, and quantitative assay for determining neomycin phosphotransferase (NPT) activity in plant cell extracts is described. The procedure retains the simplicity of previously published methods, yet offers up to a 140-fold increase in sensitivity. This increase is due to (1) the addition of bovine serum albumin (BSA) to the assay mixture, (2) desalting of crude maize extracts to remove a low-molecular-weight inhibitor of the enzyme, and (3) use of a different extraction buffer and an improved extraction procedure to liberate more enzyme from the cells. This method has been used successfully to detect and quantitate both stable and transient expression of NPT in transgenic tobacco and maize tissue.

Animals↗
PubMed1990

Ultrasensitive enzymatic radioimmunoassay using a fusion protein of protein A and neomycin phosphotransferase II in two-chamber-well microtiter plates.

A new sensitive method for antigen detection employing a phosphorylation reaction is described using human serum albumin as a model. The antigen is initially bound to the surface of polystyrene microtiter plates and reacted with an antibody (rabbit). A microbiologically produced bifunctional fusion protein of protein A and neomycin phosphotransferase II (NPT II) serves as a second immunological reagent by virtue of its protein A component. The detection is based on the phosphorylation of an aminoglycoside antibiotic by the NPT II moiety of the fusion protein using [gamma-32P]ATP as a cosubstrate. This reaction is performed in solution and the evaluation is accomplished by dotting aliquots of the reaction mixture onto phosphocellulose paper, washing with water, and autoradiography. Microtiter plates with a specially designed 10 microliter-volume reaction chamber are particularly advantageous for this procedure. The sensitivity of detection is currently 10 fg (1 pg/ml) of antigen.

Animals↗
PubMed1990

High-sensitivity protein detection by a new "contact-copy" method using a protein A-neomycin phosphotransferase II fusion protein.

A new system for high-sensitivity protein detection by an immunoenzymatic "contact-copy" procedure is described. It is based on two components: (i) a microbiologically produced bifunctional fusion protein of protein A and neomycin phosphotransferase II (protein A-NPT II) in which the protein A moiety acts as a second immunological reagent while NPT II catalyzes the detection reaction and (ii) a novel kanamycin-loaded substrate matrix (kanamycin-cyanuric chloride-activated and sulfanilic acid-derivatized paper) brought into direct contact with a protein-carrying matrix after blot or dot application and initial immunoreaction--the NPT II enzyme reaction with [gamma-32P]ATP as cosubstrate leads to phosphorylation of the substrate kanamycin on the substrate matrix, which is used for further analysis. The contact-copy method has at least the same detection sensitivity as procedures employing 125I-protein A, but allows extremely short exposure times and avoids probe prelabeling. Twenty-five picograms of specific protein blotted from sodium dodecyl sulfate-polyacrylamide gels onto nitrocellulose is detected after 15 min of autoradiography. The limit of detection in dot tests was found to be 10 pg per dot (3 mm2). The method is suitable for quantitative determination of antigens in the range down to 100 pg. Several contact copies of the same original protein-carrying matrix can be produced and used for detection or quantitative analysis without destroying the original matrix.

Collodion↗
PubMed1991

Assay of neomycin phosphotransferase activity in cell extracts.

A simple method for the measurement of neomycin phosphotransferase (NPT) activity in crude extracts of eukaryotic cells is described. This method is based on the elimination of interfering phosphorylated proteins by using phenol-chloroform extraction. This solution phase assay allows the detection of greater than or equal to 0.01 ng of NPT in the crude cell extract. Rapid screening of a large number of cell cultures generated in gene-transfer experiments, using NPT as a selective marker, is made possible by this simple technique. Further, the promoter strength of vector constructs used in gene therapy may also be estimated by this procedure.

Cell Extracts↗
PubMed1991

An immunoaffinity immobilized enzyme assay for neomycin phosphotransferase II in crude cell extracts.

An immunoaffinity immobilized enzyme assay for neomycin phosphotransferase II (NPT II) has been developed. This method combines affinity purification with an enzyme-catalyzed reaction. The assay is mechanically simple and can be semiautomatable since all steps are performed in a microtiter plate. An immunoaffinity step separates NPT II from endogenous kinases, which may produce false positive results, and from endogenous phosphatases and inhibitors, which decrease the apparent NPT activity. This method thus exploits two modes of specificity: antigen-antibody specificity and enzyme catalysis specificity. This gives a high degree of specificity and allows quantitation of 0.1 ppm NPT in crude plant protein extracts. The catalytic ability of the NPT is not significantly hampered by its attachment to the gel, in the Km values for ATP and neomycin and the catalytic number for immobilized NPT are comparable to those for the NPT in solution.

Adenosine Triphosphate↗
PubMed1987

Molecular properties of pyrophosphate:fructose-6-phosphate phosphotransferase from potato tuber.

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) from potato tubers has been purified to homogeneity. The enzyme contains two polypeptides with apparent relative molecular mass (Mr) values of 65,000 and 60,000. These polypeptides give different peptide fragments after limited proteolytic digestion. Antibodies raised against each polypeptide separately are specific for that polypeptide, but both antisera are capable of immunoprecipitating native PFP activity. These antibodies also recognize similar pairs of polypeptides in a range of other plant tissues that contain PFP activity. Based on gel filtration, the Mr value of potato tuber PFP is 265,000. This suggests that the enzyme is a heterotetramer composed of two polypeptides with Mr values of 65,000 and 60,000. In the presence of pyrophosphate, potato PFP dissociates into a 130,000 dimer.

Antibody Specificity↗
PubMed1991

Nucleoside phosphotransferase activity of human colon carcinoma cytosolic 5'-nucleotidase.

A cytosolic 5'-nucleotidase, acting preferentially on IMP and GMP, has been isolated from human colon carcinoma extracts. This enzyme activity catalyzes also the transfer of the phosphate group of 5'-nucleoside monophosphates (mainly, 5'-IMP, 5'-GMP, and their deoxycounterparts) to nucleosides (preferentially inosine and deoxyinosine, but also nucleoside analogs, such as 8-azaguanosine and 2',3'-dideoxyinosine). It has been proposed that the enzyme mechanism involves the formation of a phosphorylated enzyme as an intermediate which can transfer the phosphate group either to water or to the nucleoside. The enzyme is activated by some effectors, such as ATP and 2,3-diphosphoglycerate. Results indicate that the effect of these activators is mainly to favor the transfer of the phosphate of the phosphorylated intermediate to the nucleoside (i.e., the nucleoside phosphotransferase activity). This finding is in accordance with previous suggestions that cytosolic 5'-nucleotidase cannot be considered a pure catabolic enzyme.

2,3-Diphosphoglycerate↗
PubMed1991

Redox active sulfhydryls are required for fructose 2,6-bisphosphate activation of plant pyrophosphate fructose-6-phosphate 1-phosphotransferase.

The classical, alpha/beta-subunit form (Q2) of green tomato pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90), a cytosolic enzyme functional in carbohydrate metabolism, was rapidly inactivated on incubation with the oxidant 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Analysis of the DTNB-treated sample by a fluorescence procedure revealed that inactivation was accompanied by oxidation of sulfhydryl groups, primarily on the alpha-subunit. Phosphate metabolites--fructose 2,6-bisphosphate, fructose 1,6-bisphosphate, Pi, and PPi--protected against DTNB inactivation to varying degrees. The Km values for fructose 6-phosphate and PPi were not changed by DTNB treatment, but the capability for activation by fructose 2,6-bisphosphate was severely diminished. The oxidative inactivation of PFP was reversed by dithiothreitol, but not by monothiols (reduced glutathione or beta-mercaptoethanol). Reactivation was accompanied by restoration of the ability to undergo activation by fructose 2,6-bisphosphate. The findings suggest that sulfhydryl groups are essential for the activation of PFP by fructose 2,6-bisphosphate and raise the possibility that a reversible change in their redox status can take place under certain conditions. Evidence that this is the case was obtained with a preparation from wheat flour which, in the absence of an added oxidant, required reduction by a dithiol for activation by fructose 2,6-bisphosphate (dithiothreitol and reduced thioredoxin h).

Dithionitrobenzoic Acid↗
PubMed1984

Evidence that glucose and sucrose uptake in oral streptococcal bacteria involves independent phosphotransferase and proton-motive force-mediated mechanisms.

Sugar transport and glycolysis in Streptococcus sanguis NCTC 7865, Streptococcus mitis ATCC 903, Streptococcus salivarius NCTC 8606 and several strains of Streptococcus mutans were investigated by following the rate of acid production by washed bacteria at a constant pH of 7.0. The phosphoenolpyruvate-phosphotransferase system (PTS) was inhibited by low concentrations of chlorhexidine. When this PTS-inhibitory concentration of chlorhexidine was added to cells washed and re-suspended in KCl, glucose uptake and glycolysis continued at a greatly-reduced rate. Chlorhexidine abolished glucose and sucrose uptake and metabolism in bacteria washed and incubated in saline. The Na+-inhibition was reproduced in KCl-washed bacteria using the cyclic peptide ionophores, valinomycin and gramicidin, to dissipate K+ and H+ gradients across the cell membrane. Glucose metabolism by Strep. mutans B13 was more resistant to chlorhexidine than that of Strep. mutans NCTC 10449 or Strep. sanguis but was more sensitive to the ionophores. Valinomycin had a greater inhibitory effect on strain B13 than the other two. That ion gradients are important in the chlorhexidine-resistant glucose-uptake mechanism was confirmed using the classical uncoupling agents, carbonylcyanide-m-chlorophenylhydrazone, 2,4-dinitrophenol and KSCN. Glucose metabolism was inhibited in the presence of both the uncouplers and the PTS-inhibitory concentration of chlorhexidine and significant inhibition was also observed in the absence of the PTS inhibitor. Lactate or the ATPase inhibitor, dicyclohexyl carbodiimide (DCCD), had similar inhibitory effects on the non-PTS uptake system.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1990

Evolution of permease diversity and energy-coupling mechanisms with special reference to the bacterial phosphotransferase system.

Different classes of apparently unrelated permeases couple different forms of energy to solute transport. While the energy coupling mechanisms utilized by the different permease classes are clearly distinct, it is proposed, based on structural comparisons, that many of these permeases possess transmembrane, hydrophobic domains which are evolutionarily related. Carriers may have arisen from transmembrane pore-forming proteins, and the protein constituents or domains which are specifically responsible for energy coupling may have had distinct origins. Thus, complex permeases may possess mosaic structures. This suggestion is substantiated by recent findings regarding the evolutionary origins of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS). Mechanistic implications of this proposal are presented.

Biological Evolution↗
PubMed1977

The role of enzyme I in the unmasking of an essential thiol of the membrane-bound enzyme II of the phosphoenolpyruvate-glucose phosphotransferase system of Escherichia coli.

The membrane-bound component of the phosphotransferase system of Escherichia coli, responsible for the phosphorylative uptake of methyl-alpha-D-glucoside has an essential thiol group which becomes available to inactivation by thiol reagents in the presence of the phosphate-accepting sugar or when phosphoenolpyruvate synthesis is inhibited. The form resistant to the thiol reagent requires not only the absence of sugar and an intact phosphoenolpyruvate generating system, but also an intact system generating phosphorylated Hpr which is impaired by heating of a thermosensitive enzyme I mutant.

Escherichia coli↗
PubMed1979

Unmasking of an essential thiol during function of the membrane-bound enzyme II of the phosphenolpyruvate beta-glucoside phosphotransferase system of Escherichia coli.

beta-Glucoside transport by phosphoenolpyruvate-hexose phosphotransferase system in Escherichia coli is inactivated in vivo by thiol reagents. This inactivation is strongly enhanced by the presence of transported substrates. In a system reconstituted from soluble and membrane-bound components, only the particulate component, the membrane-bound enzyme IIbgl appeared as the target of N-ethylmaleimide inaction. The same feature was found in the case of methyl-alpha-D-glucoside uptake via enzyme IIglc. It is shown that the sensitizing effect of substrates is specific and not generalized, methyl-alpha-D-glucoside only sensitizes enzyme IIglc and p-nitrophenyl-beta-D-glucoside only sensitizes enzyme IIbgl towards N-ethylmaleimide inactivation. The inactivation of enzyme IIbgl by thiol reagents is also promoted in vivo by fluoride inhibition of phosphoenolpyruvate synthesis. In toluene-treated bacteria, the presence of phosphoenolpyruvate protects against inactivation by thiol reagents of p-nitrophenyl-beta-D-glucoside phosphorylation. Both results suggest that the inactivator resistent form of enzyme IIbgl is an energized form of the enzyme.

Chloromercuribenzoates↗
PubMed1987

Kinetic and topographical studies of the phosphatidylcholine: ceramide choline phosphotransferase in plasma membrane particles from mouse ascites cells.

The content of endogenous phospholipids in plasma membrane preparations from Ehrlich ascites cells was depleted by exposure to phospholipase C. The enzyme catalyzing the phosphatidylcholine: ceramide choline phosphotransferase reaction was inactivated by this treatment. However, the activity could be restored with exogenous phosphatidylcholines, demonstrating the dependence of the reaction upon the presence of this substrate. Phosphatidylcholines containing unsaturated fatty acids were 10-fold more effective substrates than the saturated molecular species. The activation energy of the reaction was determined to be 17.2 kcal/mol. Selective trypsin treatment of the plasma membranes suggests that the cholinephosphotransferase may have an asymmetric orientation. The reaction kinetics followed a rate equation similar to that of the ping-pong reaction mechanism, which suggests the formation of an enzyme-bound intermediate of the phosphocholine group being transferred. These results are discussed in terms of possible biological functions of the enzyme.

Animals↗
PubMed1975

Comparative study of two ATP : L-arginine phosphotransferases of molecular weight 84 000.

Solen ensisensis muscle arginine kinase (ATP : L-arginine phosphotransferase, EC 2.7.3.3) was isolated in an homogeneous state. Its molecular weight was found to be about 80 000. The properties of this enzyme were compared with those of arginine kinase from Sipunculus nudus, an enzyme which also has a molecular weight of about 80 000. Both enzymes have several reactive thiol groups (8 thiol groups in the Solen kinase and 12 in the Sipunculus enzyme were titrateable with 5,5'-dithio-bis-(2-nitrobenzoic) acid and histidine residues (both enzymes have 6 reactive histidine residues). These kinases were, therefore, highly susceptible to oxidation. Both enzymes show the same pH optimum and absolute specificity towards the guanidine substrate, L-arginine. The reaction kinetics of both enzymes are of the sequential type. In the presence of alpha-aminoacids of Mg2+-ADP, similar spectral effects were obtained. The enzymes differ in their enzymic activities and in their rate of recovery following urea denaturation. The most important difference that appeared to be a special feature of the Sipunculus enzyme is that the spectrum of the Mg2+-ADP-enzyme complex is strongly intensified by L-arginine.

Adenosine Diphosphate↗