scholarly journals Characterization of the microtubule-activated ATPase of brain cytoplasmic dynein (MAP 1C).

1988 ◽  
Vol 107 (3) ◽  
pp. 1001-1009 ◽  
Author(s):  
H S Shpetner ◽  
B M Paschal ◽  
R B Vallee
Keyword(s):  
Ionic Strength ◽  
Divalent Cations ◽  
High Sensitivity ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Maximum Activity ◽  
Scanning Transmission ◽  
Sulfhydryl Oxidation ◽  
The Brain

We recently found that the brain cytosolic microtubule-associated protein 1C (MAP 1C) is a microtubule-activated ATPase, capable of translocating microtubules in vitro in the direction corresponding to retrograde transport. (Paschal, B. M., H. S. Shpetner, and R. B. Vallee. 1987b. J. Cell Biol. 105:1273-1282; Paschal, B. M., and R. B. Vallee. 1987. Nature [Lond.]. 330:181-183.). Biochemical analysis of this protein (op. cit.) as well as scanning transmission electron microscopy revealed that MAP 1C is a brain cytoplasmic form of the ciliary and flagellar ATPase dynein (Vallee, R. B., J. S. Wall, B. M. Paschal, and H. S. Shpetner. 1988. Nature [Lond.]. 332:561-563). We have now characterized the ATPase activity of the brain enzyme in detail. We found that microtubule activation required polymeric tubulin and saturated with increasing tubulin concentration. The maximum activity at saturating tubulin (Vmax) varied from 186 to 239 nmol/min per mg. At low ionic strength, the Km for microtubules was 0.16 mg/ml tubulin, substantially lower than that previously reported for axonemal dynein. The microtubule-stimulated activity was extremely sensitive to changes in ionic strength and sulfhydryl oxidation state, both of which primarily affected the microtubule concentrations required for half-maximal activation. In a number of respects the brain dynein was enzymatically similar to both axonemal and egg dyneins. Thus, the ATPase required divalent cations, calcium stimulating activity less effectively than magnesium. The MgATPase was inhibited by metavandate (Ki = 5-10 microM for the microtubule-stimulated activity), 1 mM NEM, and 1 mM EHNA. In contrast to other dyneins, the brain enzyme hydrolyzed CTP, TTP, and GTP at higher rates than ATP. Thus, the enzymological properties of the brain cytoplasmic dynein are clearly related to those of other dyneins, though the brain enzyme is unique in its substrate specificity and in its high sensitivity to stimulation by microtubules.

scholarly journals DNA interference by a mesophilic Argonaute protein, CbcAgo

F1000Research ◽  
2020 ◽  
Vol 8 ◽  
pp. 321
Author(s):  
Nieves García-Quintans ◽  
Laurie Bowden ◽  
José Berenguer ◽  
Mario Mencía
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Pyrococcus Furiosus ◽  
Divalent Cations ◽  
Thermus Thermophilus ◽  
Maximum Activity ◽  
Argonaute Protein ◽  
Starting Point ◽  
Mesophilic Bacterium

Background: The search for putative enzymes that can facilitate gene editing has recently focused its attention on Argonaute proteins from prokaryotes (pAgos). Though they are structural homologues of human Argonaute protein, which uses RNA guides to interfere with RNA targets, pAgos use ssDNA guides to identify and, in many cases, cut a complementary DNA target. Thermophilic pAgos from Thermus thermophilus, Pyrococcus furiosus and Methanocaldococcus jasmanii have been identified and thoroughly studied, but their thermoactivity makes them of little use in mesophilic systems such as mammalian cells. Methods: Here we search for and identify CbcAgo, a prokaryotic Argonaute protein from a mesophilic bacterium, and characterize in vitro its DNA interference activity. Results: CbcAgo efficiently uses 5’P-ssDNA guides as small as 11-mers to cut ssDNA targets, requires divalent cations (preferentially, Mn2+) and has a maximum activity between 37 and 42 °C, remaining active up to 55 °C. Nicking activity on supercoiled dsDNA was shown. However, no efficient double-strand breaking activity could be demonstrated. Conclusions: CbcAgo can use gDNA guides as small as 11 nucleotides long to cut complementary ssDNA targets at 37ºC, making it a promising starting point for the development of new gene editing tools  for mammalian cells.

Divalent cation stimulation of in vitro fibre assembly from epidermal keratin protein

1978 ◽  
Vol 33 (1) ◽  
pp. 255-263
Author(s):  
K. Fukuyama ◽  
T. Murozuka ◽  
R. Caldwell ◽  
W.L. Epstein
Keyword(s):  
Ionic Strength ◽  
Divalent Cation ◽  
Divalent Cations ◽  
Urea Concentration ◽  
Newborn Rats ◽  
Filament Formation ◽  
Stimulation Of ◽  
Millimolar Concentration

Keratin was extracted from purified cornified cells of newborn rats in Tris-HCl-buffered 8 M urea containing beta-mercaptoethanol. Microfilaments were assembled in vitro by reducing the ionic strength of buffer and the urea concentration. One millimolar concentration of KCl and NaCl did not affect filament formation, but the same concentration of divalent cations greatly altered this process. CaCl2 and MgCl2 induced gelation of keratin by formation of bundles of birefringent macrofilaments. ZnCl2, CuSO4 and HgCl2 formed greater numbers of macrofilaments and the protein aggregated.

Regulatory effects of potassium and inorganic anions on the NADP-specific malic enzyme of Escherichia coli

1985 ◽  
Vol 63 (2) ◽  
pp. 128-136
Author(s):  
Deborah A. Brown ◽  
Robert A. Cook
Keyword(s):  
Escherichia Coli ◽  
Ionic Strength ◽  
Binding Sites ◽  
Malic Enzyme ◽  
Divalent Cations ◽  
Kinetic Studies ◽  
Regulatory Effects ◽  
Metal Cofactors

The effects of K+ and various anions on the catalytic and regulatory properties of the NADP-specific malic enzyme of Escherichia coli are reported. Studies on the susceptibility of the enzyme to proteolysis indicate that K+ binds directly to the enzyme with a resultant change in enzyme conformation. Kinetic studies indicate that the binding of optimal concentrations of K+ results in activation of the enzyme, increasing both the Vmax and the affinity of the enzyme for divalent cations. The inhibition of enzyme activity observed at KCl concentrations greater than 50 mM is shown to be nonspecific, resulting from increasing ionic strength. The mixed cooperativity between malate-binding sites previously reported at optimal K+ concentration is more pronounced at nonoptimal K+ concentrations (0 and 150 mM). The regulatory effect of metal cofactors and the mixed cooperativity between malate-binding sites is abolished when kinetic studies are conducted at low ionic strength or in the presence of acetate. Acetate appears to act as an activator, increasing the affinity of the enzyme for malate and protecting the enzyme against the inhibition caused by high ionic strength. It is postulated that the enzyme is operating in vivo in a partially inhibited state owing to the ionic strength of the cytoplasm. The kinetic studies conducted at higher ionic strength in vitro are therefore more applicable to the in vivo situation.

scholarly journals rnrGene from the Antarctic Bacterium Pseudomonas syringae Lz4W, Encoding a Psychrophilic RNase R

2011 ◽  
Vol 77 (22) ◽  
pp. 7896-7904 ◽  
Author(s):  
Shaheen Sulthana ◽  
Purusharth I. Rajyaguru ◽  
Pragya Mittal ◽  
Malay K. Ray
Keyword(s):  
Pseudomonas Syringae ◽  
Divalent Cations ◽  
Biochemical Property ◽  
Biochemical Properties ◽  
Maximum Activity ◽  
Rnase R ◽  
Content Type ◽  
Antarctic Bacterium ◽  
The Antarctic

ABSTRACTRNase R is a highly processive, hydrolytic 3′-5′ exoribonuclease belonging to the RNB/RNR superfamily which plays significant roles in RNA metabolism in bacteria. The enzyme was observed to be essential for growth of the psychrophilic Antarctic bacteriumPseudomonas syringaeLz4W at a low temperature. We present results here pertaining to the biochemical properties of RNase R and the RNase R-encoding gene (rnr) locus from this bacterium. By cloning and expressing a His6-tagged form of theP. syringaeRNase R (RNase RPs), we show that the enzyme is active at 0 to 4°C but exhibits optimum activity at ∼25°C. The enzyme is heat labile in nature, losing activity upon incubation at 37°C and above, a hallmark of many psychrophilic enzymes. The enzyme requires divalent cations (Mg2+and Mn2+) for activity, and the activity is higher in 50 to 150 mM KCl when it largely remains as a monomer. On synthetic substrates, RNase RPsexhibited maximum activity on poly(A) and poly(U) in preference over poly(G) and poly(C). The enzyme also degraded structuredmalE-malFRNA substrates. Analysis of the cleavage products shows that the enzyme, apart from releasing 5′-nucleotide monophosphates by the processive exoribonuclease activity, produces four-nucleotide end products, as opposed to two-nucleotide products, of RNA chain byEscherichia coliRNase R. Interestingly, three ribonucleotides (ATP, GTP, and CTP) inhibited the activity of RNase RPsin vitro. The ability of the nonhydrolyzable ATP-γS to inhibit RNase RPsactivity suggests that nucleotide hydrolysis is not required for inhibition. This is the first report on the biochemical property of a psychrophilic RNase R from any bacterium.

scholarly journals Features of Borna disease virus reproduction in brain cells and laboratory diagnosis of infection

2019 ◽  
Vol 2019 (2) ◽  
pp. 21-24
Author(s):  
Константин Юров ◽  
Konstantin Yurov ◽  
Светлана Алексеенкова ◽  
Svetlana Alekseenkova
Keyword(s):  
Disease Virus ◽  
Present Report ◽  
Retrograde Transport ◽  
Laboratory Diagnosis ◽  
Experimental Models ◽  
Brain Cells ◽  
Borna Disease Virus ◽  
The Brain ◽  
Borna Disease

The Borna Disease Virus (BDV) is a non-segmented RNA-containing virus belonging to the Bornaviridae family. The pathogen causes progressive meningoencephalitis in animals of various types. Despite numerous studies, some stages of reproduction of Bornavirus remain poorly understood, in particular, receptor-mediated penetration, retrograde transport into the nucleus, assembly and release of the virion, etc. The present report presents the results of demonstrating studies that were aimed at following the immunoenzyme method, the distribution of the main protein BDV ― phosphoprotein P (p24) in the brain cells of naturally susceptible animals, compare nnye data with the results of a number of authors, made mainly in experimental models. Microscopic examination of histological sections of the brain tissue of horses and sheep treated with specific serum against p24 BDV in a light or luminescent microscope observed a specific color in the form of: small granules; diffuse fluorescence of the cytoplasm; larger granules, apparently due to aggregation of endosomes, for axon transportation; formations in the form of beads, demonstrating the transport of viral material along the axon. In vitro results were obtained indicating that it is possible to transport RNPs via a short path through cytoplasmic bridges. The presented results will allow a better understanding of the neuropathogenesis of Born's disease and improve the diagnosis of the disease.

Identification of the phosphomonoesterases that hydrolyze lysopolyphosphoinositides in rat brain and liver

1987 ◽  
Vol 65 (10) ◽  
pp. 890-898 ◽  
Author(s):  
Frederick B. St. C. Palmer
Keyword(s):  
Rat Brain ◽  
Phosphatase Activity ◽  
Divalent Cations ◽  
Liver Microsomes ◽  
Maximum Activity ◽  
Molar Ratio ◽  
Phosphatase Activities ◽  
Rat Brain And Liver ◽  
Ph Range

The phosphatase activities responsible for the sequential dephosphorylation of lysophosphatidylinositol 4,5-bisphosphate (lysoPtdIns(4,5)P2) to lysophosphatidylinositol that precedes reacylation in rat brain and liver microsomes were characterized. LysoPtdIns(4,5)P2 and the intermediate lysophosphatidylinositol 4-phosphate (lysoPtdIns4P) were hydrolyzed by two distinct phosphatase activities which were distinguishable by their substrate and cation requirements. The lysoPtdIns(4,5)P2 phosphatase activity was Mg2+ dependent and partially inhibited by Ca2+, excess Mg2+, and cationic detergent (cetyltrimethylammonium bromide). Activity was maximal at neutral (brain) or slightly alkaline (liver) pH when the Mg2+/lysoPtdIns(4,5)P2 molar ratio was 1.0 in the presence of bovine serum albumin (1 mg∙mL−1). LysoPtdIns4P phosphatase activity did not require divalent cations (not inhibited by EDTA). This activity was inhibited by Ca2+, Mg2+, and substrate concentrations above 0.2 mM. Maximum activity was observed over a broad pH range (6.0–8.5). Both activities were inhibited by lysophosphatidylinositol and lysophosphatidylcholine, but not other lysophospholipids. The lysopolyphosphoinositides are most likely hydrolyzed by the same phosphatases that act on the diacylpolyphosphoinositides, since PtdIns(4,5)P2 and PtdIns4P were also hydrolysed by Mg2+-dependent and cation-independent phosphatases, respectively. Activities with the diacylpolyphosphoinositides differed only in their requirement of detergents for maximum activity in vitro. Specific activities for the diacyl and "lyso" forms of each substrate were very similar when suitably optimized reaction mixtures were used. The subcellular distributions of the two phosphatase activities in both brain and liver were the same when acting on diacyl- or lyso-polyphosphoinositides, as was their response to inhibitors. Alkaline, acid, phosphoprotein, and inositol-1-phosphate phosphatases did not contribute substantially to the hydrolysis of either lysoPtdIns4P or lysoPtdIns(4,5)P2, since the activities were not significantly inhibited by cysteine, dithiothreitol, NaF, or LiCl. Lack of inhibition by 2,3-bisphosphoglycerate and absence of stimulation by cysteine or dithioerythritol, as well as a different subcellular distribution in liver, excluded inositol-1,4,5-trisphosphate and inositol-1,4-bisphosphate phosphatases as sources of the lysoPtdIns(4,5)P2 and lysoPtdIns4P phosphatase activities.

scholarly journals A tunable LIC1-adaptor interaction modulates dynein activity in a cargo-specific manner

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
In-Gyun Lee ◽  
Sydney E. Cason ◽  
Saif S. Alqassim ◽  
Erika L. F. Holzbaur ◽  
Roberto Dominguez
Keyword(s):  
Coiled Coil ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Eukaryotic Cells ◽  
Amphipathic Helix ◽  
Binding Studies ◽  
In Vitro Motility ◽  
Ef Hand ◽  
Specific Manner

Abstract Cytoplasmic dynein-1 (dynein) is the motor responsible for most retrograde transport of cargoes along microtubules in eukaryotic cells, including organelles, mRNA and viruses. Cargo selectivity and activation of processive motility depend on a group of so-called “activating adaptors” that link dynein to its general cofactor, dynactin, and cargoes. The mechanism by which these adaptors regulate dynein transport is poorly understood. Here, based on crystal structures, quantitative binding studies, and in vitro motility assays, we show that BICD2, CRACR2a, and HOOK3, representing three subfamilies of unrelated adaptors, interact with the same amphipathic helix of the dynein light intermediate chain-1 (LIC1). While the hydrophobic character of the interaction is conserved, the three adaptor subfamilies use different folds (coiled-coil, EF-hand, HOOK domain) and different surface contacts to bind the LIC1 helix with affinities ranging from 1.5 to 15.0 μM. We propose that a tunable LIC1-adaptor interaction modulates dynein’s motility in a cargo-specific manner.

scholarly journals DNA interference by a mesophilic Argonaute protein, CbcAgo

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 321
Author(s):  
Nieves García-Quintans ◽  
Laurie Bowden ◽  
José Berenguer ◽  
Mario Mencía
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Pyrococcus Furiosus ◽  
Divalent Cations ◽  
Thermus Thermophilus ◽  
Maximum Activity ◽  
Argonaute Protein ◽  
Starting Point ◽  
Mesophilic Bacterium

Background: The search for putative enzymes that can facilitate gene editing has recently focused its attention on Argonaute proteins from prokaryotes (pAgos). Though they are structural homologues of human Argonaute protein, which uses RNA guides to interfere with RNA targets, pAgos use ssDNA guides to identify and, in many cases, cut a complementary DNA target. Thermophilic pAgos from Thermus thermophilus, Pyrococcus furiosus and Methanocaldococcus jasmanii have been identified and thoroughly studied, but their thermoactivity makes them of little use in mesophilic systems such as mammalian cells. Methods: Here we search for and identify CbcAgo, a prokaryotic Argonaute protein from a mesophilic bacterium, and characterize in vitro its DNA interference activity. Results: CbcAgo efficiently uses 5’P-ssDNA guides as small as 11-mers to cut ssDNA targets, requires divalent cations (preferentially, Mn2+) and has a maximum activity between 37 and 42 °C, remaining active up to 55 °C. Nicking activity on supercoiled dsDNA was shown. However, no efficient double-strand breaking activity could be demonstrated. Conclusions: CbcAgo can use gDNA guides as small as 11 nucleotides long to cut complementary ssDNA targets at 37ºC, making it a promising starting point for the development of new gene editing tools  for mammalian cells.

scholarly journals Intracellular force comparison of pathogenic KIF1A, KIF5, and dynein by fluctuation analysis

2021 ◽  
Author(s):  
Kumiko Hayashi ◽  
Shiori Matsumoto ◽  
Takuma Naoi ◽  
Yuki Idobata
Keyword(s):  
Synaptic Vesicle ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Force Measurement ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Extreme Value Analysis ◽  
Fluctuation Analysis ◽  
Value Analysis

In mammalian cells, there exist approximately 40 types of microtubule motor proteins that are assigned to specific cargo deliveries. For example, the kinesin-1 family motor KIF5 is the major motor responsible for anterograde mitochondrial transport, whereas the kinesin-3 family motor KIF1A is responsible for synaptic vesicle precursor transport. In contrast, cytoplasmic dynein is responsible for retrograde transport of nearly all cargos. The force and velocity of these microtubule motors have been investigated in in-vitro single-molecule experiments. In the present study, we compared the intracellular force and velocity of various types of motors in the mammalian neuronal axon obtained by non-invasive force measurement (fluctuation analysis) and extreme value analysis with those obtained by previous single-molecule experiments. As we found a high correlation between our results and the previous results, we next investigated synaptic vesicle precursor transport by hereditary spastic paraplegia-associated KIF1A variants (V8M, R350G, and A255V). KIF1A-V8M and KIF1A-A255V exhibited force and velocity impairment in mammalian neuronal axons, whereas the physical property of KIF1A-R350G was similar to that of the wild type. We believe that the development of new analytical techniques for investigating intracellular cargo transports is helpful to elucidate the molecular mechanism of KIF1A-associated neurological disorders.

Effect of pH and inhibitors on beta-amyloid fibril assembly

1996 ◽  
Vol 54 ◽  
pp. 752-753
Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel
Keyword(s):  
Amyloid Fibril ◽  
Peptide Fragment ◽  
Hydrophobic Peptides ◽  
Amyloid Fibril Formation ◽  
Effects Of Ph ◽  
Severity Of The Disease ◽  
Kinetics Of ◽  
The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

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