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Synapses
Published in Nassir H. Sabah, Neuromuscular Fundamentals, 2020
The phosphate group is hydrolyzed back to an OH– group by enzymes referred to as phosphatases, and the process is known as dephosphorylation. Protein phosphatase 1 (PP1) dephosphorylates a variety of proteins as well as K+ and Ca2+ channels, NMDA, and AMPA glutamate receptors. Protein phosphatase 2A (PP2A) also dephosphorylates a range of proteins that overlap with those of PP1, in addition to tau protein that stabilizes microtubules of the cytoskeleton. Excessive phosphorylation of tau protein is associated with Alzheimer’s disease. Protein phosphatase 2B (PP2B), also known as calcineurin, is abundant in neurons and is activated by Ca2+. It activates T cells of the immune system and dephosphorylates AMPA receptors. Protein phosphorylation and dephosphorylation are of fundamental importance in cell functioning as it is the major molecular mechanism through which protein activity in a cell is regulated both in and outside the nervous system.
Toward Understanding the Intelligent Properties of Biological Macromolecules
Published in George K. Knopf, Amarjeet S. Bassi, Smart Biosensor Technology, 2018
Chemiluminescent molecules emit visible light, typically accompanying the breakage of an unstable high-energy bond(s). Because of this property, chemiluminescent molecules have a wide range of applications, including those in the field of biotechnology. In our studies, we have utilized a particularly interesting example: chloro 3-(4-methoxy spiro [1,2-dioxetane-3-29-tricyclo-[3.3.1.1.]-decan]-4-yl) phenyl phosphate (CSPD). This phenyl phosphate species is capable of being dephosphorylated by members of a class of phosphatase enzymes. We have used alkaline phosphatase in our biosensor studies and it carries out the initial dephosphorylation step enzymatically, as shown in Figure 2.13. The dephosphorylated reaction product contains an unstable highly strained four-atom dioxetane ring that undergoes subsequent ring cleavage at the weak -O-O- bond in a second step to form two separate product molecules. The unstable phenolate species then emits a chemiluminescent photon over a broad wavelength range. Chemileuminescence, as an optical process for biosensor use, has distinct advantages over absorption or fluorescence. To begin with, no light source is needed because the chemiluminescent molecule already contains a photon energy equivalent to the free energy stored in its internal electronic structure. There is a concomitant advantage that no optical background source noise can interfere with the chemiluminescent signal being detected, as is often the case with widely used fluorescence detection schemes.
FRET Reporter Molecules for Identification of Enzyme Functions
Published in Grunwald Peter, Biocatalysis and Nanotechnology, 2017
Jing Mu, Hao Lun Cheong, Bengang Xing
Apart from β-Lactamase used in the FRET-based imaging, other types of enzymes have also been widely investigated to build up FRET systems for bioanalysis and medical imaging studies, such as protein kinase and alkaline phosphate. Protein kinases modulate the activities of proteins though the phosphorylation process, a very critical step in the intercellular communication and functioning of the nervous and immune systems. Mutation and dysregulation of protein kinase will cause several human diseases like Alzheimer’s disease and cancer as well. Phosphatases, the other class of enzymes that is opposite to the action of protein kinases, can remove a phosphate group from its substrate, called dephosphorylation. A representative phosphatase in many organisms is alkaline phosphatase, ALP. In humans, alkaline phosphatases are present in all tissues, but particularly concentrate on liver, kidney and bone. Either elevated levels or lowered levels of ALP in some specific tissues will be responsible for many human liver or bone diseases. Different methods have been developed to monitor the activities of protein kinases or phosphatases. Recently, Freeman et al. (Freeman et al., 2010) developed two FRET-based sensing configurations for analyzing casein kinase (CK2), a serine/threonine selective protein kinase (Fig. 13.4). One approach was semiconductor quantum dots (QDs) was linked with serine-containing peptide sequence that can be recognized by CK2. Subsequently, the CK2 catalyzed reaction would result in the phosphorylation of the serine unit with the fluorophore-labeled g-phosphate, g-ATP-Atto-590, leading to the formation the FRET between the QDs and Atto-590 acceptor. The second approach is serine-containing peptide functionalized-QDs first react with CK2 and ATP to yield the phosphorylated peptide. Then interaction with Atto-590-modified antiphosphoserine-antibody would form FRET from QDs to the fluorophore acceptor. Such formed FRET process would provide direct and sensitive readout for the phosphorylation process. Meanwhile, these sensitive methods also provide great possibility to allow the multiplexed analysis of several kinases or several phosphatases in vitro and in vivo.
Sludge: next paradigm for enzyme extraction and energy generation
Published in Preparative Biochemistry and Biotechnology, 2019
Santosh Kumar Karn, Awanish Kumar
Alkaline phosphatase has widespread use in research and industry, specifically in protein labeling and dephosphorylation of nucleic acid. Alkaline phosphatase is a useful tool in the molecular biology lab since DNA possesses a phosphate group at 5′ end. Eliminating this phosphate prevents the ligation and circularization of DNA molecules, easy for radiolabelling, etc. Other important applications of alkaline phosphatase enzyme for immune assays are blotting, sequencing, enzyme-linked immunoabsorbent assays (ELISA), and nonisotopic probing. Alkaline phosphatase is apparently ubiquitous in nature. These properties and their tendency of structure and regulation in multiple forms, often make the isolation of alkaline phosphatases complicated. Studies on pure alkaline phosphatase focused mainly on the clinical significance[17,18] from animal sources. Among possible role of alkaline phosphatases might be providing inorganic phosphate for metabolic excretory and some secretory purpose in plants. Alkaline phosphatases probably have the role in the reproductive system of the mammal; possible human prostatic alkaline phosphatase may be in the dephosphorylation of esters to liberate fructose.[19]