Kinase, kinases immobilized

Tani H, Morisaki A, Ishida A, Tokeshi M (2012) On-chip bioluminescence assay of ATP and kinases using immobilized firefly luciferase in three dimensional microfluidic chip. In Proceedings of the 16th international conference on miniaturized systems for chemistry and life sciences, Okinawa, 28 Oct-1 Nov... 

Affinity chromatography of mammalian phosphofructo-kinase on immobilized adenine dinucleotides... 

Motor proteins move along MTs in an ATP-dependent manner. Members of the superfamily of kinesin motors move only to the plus ends and dynein motors only to the minus ends. The respective motor domains are linked via adaptor proteins to their cargoes. The binding activity of the motors to MTs is regulated by kinases and phosphatases. When motors are immobilized at their cargo-binding area, they can move MTs. 

There is evidence for immunosuppressive effects of PAHs in rodents (Davila et al. 1997). For example, strong immunosuppressive effects were reported in mice that had been dosed with benzo[fl]pyrene and 3-methyl cholanthrene, effects that persisted for up to 18 months (Environmental Health Criteria 202). Multiple immu-notoxic effects have been reported in rodents, and there is evidence that these result from disturbance of calcium homeostasis (Davila et al. 1997). PAHs can activate protein tyrosine kinases in T cells that initiate the activation of a form of phospholipase C. Consequently, release of inositol triphosphate—a molecule that immobilizes Ca + from storage pools in the endoplasmic reticulum—is enhanced. 

The immobilized immunoprecipitates are washed twice with lysis buffer containing 0.5 MNaCl and twice with buffer A. The beads are resuspended in 20 /il of kinase buffer also containing the appropriate concentration of the specific peptide. Reactions should also be set up without peptide as a negative control for nonspecific or self-incorporation of radiolabel. To start the reactions, 5 /il of ATP is added (final concentration 0.1 mM unlabeled ATP, 1 /iCi [7 -32P]ATP (per assay) in kinase buffer). The assays are allowed to proceed for 15 to 30 min at 30° with constant shaking at 900 rpm, and stopped by spotting 20 /il of the sample (slurry) onto a square (1.5 X 1.5 cm) of phosphocellulose (P81) paper. The P81 papers are immediately immersed in 500 ml of 1% (v/v) orthophosphoric acid, and then washed 3 times with the same solution (to remove the excess ATP). The washes therefore contain almost all of the radiolabel and must be handled carefully and disposed of appropriately. The papers are briefly rinsed in ethanol and air-dried. The incorporation of 32P-label is measured by Cerenkov counting. 

For luciferin, a firefly luciferase cosubstrate, another method of retention has been evaluated which consisted of incorporating the substrate in acrylic microspheres during their formation, these last being then confined in a polymeric matrix31. Using the suitable co-immobilized enzymes (adenylate kinase and creatine kinase), the three adenylic nucleotides (ATP, ADP and AMP) could be assayed continuously and reproducibly with a selfcontainment working time of 3 h. 

Adenosine triphosphate (ATP) is one of the most important cofactors involved in many of the synthetic reactions going on within the cell. Its recent large scale in vitro enzymatic synthesis from adenosine and acetylphosphate is of particular interest. Three enzymes immobilized in polyacrylamide gel were used adenosine kinase, adenylate kinase and acetate kinase (lip. ... 

An approach to multiplexing analysis was presented by Min et al. [23], who de-velopped a SAMDI-based assay scheme to screen for the activity of different kinases. In this assay scheme, peptide substrates were used that are specific for one type of kinase. A mixture of four substrates was immobilized on the SAM. After incubation with an appropriate kinase, the target surface was rinsed, thus stopping the reaction. Matrix was deposited on the surface and MALDI-MS analysis was carried out (Fig. 8.13). By monitoring the signal intensities for the substrates... 

Mannens, G. Siegers, G. Claeys, A. Purification and immobilization of acetate kinase from Desulfovibrio vulgaris. Biotechnol. Lett., 10, 563-568 (1988)... 

Rudge, J. Bickerstaff, G.F. Thermal stability of immobilized creatine kinase. Biochem. Soc. Trans., 12, 311-313 (1984)... 

Enzymes immobilized on PAN gel, unless stated otherwise. The actual phosphorylating agent, ATP, is regenerated either by acetyl phosphate and acetoldnase, or enolpyruvate phosphate and pyruvate kinase. Phosphorylations by a mixture of the common ribonucleotide triphosphates. Not isolated.4 Soluble enzymes. Enzymes immobilized on agarose.1 Enzymes enclosed in a dialysis bag. 

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