39 research outputs found
Mps1Mph1 kinase phosphorylates Mad3 to inhibit Cdc20Slp1-APC/C and maintain spindle checkpoint arrests
<div><p>The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mad3/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1<sup>Mph1</sup> kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1<sup>Mph1</sup> kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We show biochemically that Mad3 phospho-mimics are potent APC/C inhibitors <i>in vitro</i>, demonstrating that Mad3p modification can directly influence Cdc20<sup>Slp1</sup>-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1<sup>Mph1</sup> kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests.</p></div
Development of an Interference-free Biosensor for L-glutamate using a Bienzyme Salicylate Hydroxylase/L-glutamate Dehydrogenase System
An amperometric biosensor was developed for the interference-free determination Of L-glutamate with a bienzyme-based Clark electrode. This sensor is based on the specific dehydrogenation by L-glutamate dehydrogenase (GLDH, EC 1.4.1.3) in combination with salicylate hydroxylase (SHL, EC 1.14.13.1). The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. The principle of the determination scheme is as follows: the specific detecting enzyme, GLDH, catalyses the specific dehydrogenation Of L-glutamate consuming NAD(+). The product, NADH, initiates the irreversible decarboxylation and the hydroxylation of salicylate by SHL in the presence of oxygen. This results in a detectable signal due to the SHL-enzymatic consumptions of dissolved oxygen in the measurement Of L-glutamate. The sensor has a fast steady-state measuring time of 20 s with a quick response (1 s) and a short recovery (1 min). It shows a linear detection range between 10 mu M and 1.5 MM L-glutamate with a detection limit of 3.0 mu M. A Teflon membrane, which is used to fabricate the sensor, makes the determination to avoid interferences from other amino acids and clectroactive substances. (C) 2007 Elsevier Inc. All rights reserved
Development of an l-glutamate biosensor using the coimmobilization of l-glutamate dehydrogenase and p-hydroxybenzoate hydroxylase on a Clark-type electrode
A bienzyme-based Clark electrode was developed for the interference-free determination of L-glutamate. This sensor is based on the specific dehydrogenation by L-glutamate dehydrogenase (GLDH, EC 1.4.1.3) in combination with p-hydroxybenzoate hydroxylase (HBH, EC 1.14.13.2). The enzymes were entrapped by a poly(carbamoyl) sulfonate hydrogel on a Teflon membrane. The principle of the determination scheme is as follows: the specific detecting enzyme, GLDH, catalyses the specific dehydrogenation of L-glutamate consuming NADP(+). The product, NADPH, initiates the irreversible hydroxylation of p-hydroxybenzoate by HBH in the presence of oxygen. This results in a detectable signal due to the HBH-enzymatic consumptions of dissolved oxygen in the measurement Of L-glutamate. The sensor has a fast steady-state measuring time of 20 s with a quick response (2 s) and a short recovery (1 min). It shows a linear detection range between 10 mu M and 1.5 mM L-glutamate and a detection limit of 5 mu M. A Teflon membrane, which is used to fabricate the sensor, makes the determination to avoid interferences from different amino acids and electroactive substances. (c) 2007 Elsevier B.V. All rights reserved
Development of a glucose-6-phosphate biosensor based on coimmobilized p-hydroxybenzoate hydroxylase and glucose-6-phosphate dehydrogenase
This work reports the development of an amperometric glucose-6-phosphate biosensor by coimmobilizing p-hydroxybenzoate hydroxylase (HBH) and glucose-6-phosphate dehydrogenase (G6PDH) on a screen-printed electrode. The principle of the determination scheme is as follows: G6PDH catalyzes the specific dehydrogenation of glucose-6-phosphate by consuming NADP(+). The product, NADPH, initiates the irreversible the hydroxylation of p-hydroxybenzoate by HBH in the presence of oxygen to produce 3,4-dihydroxybenzoate, which results in a detectable signal due to its oxidation at the working electrode. The sensor shows a broad linear detection range between 2 mu M and 1000 mu M with a low detection limit of 1.2 mu M. Also, it has a fast measuring time which can achieve 95\% of the maximum current response in 20 s after the addition of a given concentration of glucose-6-phosphate with a short recovery time (2 min). (C) 2006 Elsevier B.V. All rights reserved
A Disposable, Screen-printed Electrode for the Amperometric Determination of Azide Based on the Immobilization with Catalase or Tyrosinase
A disposable, screen-printed electrode based on the immobilization of catalase or tyrosinase was developed to construct biosensors for the amperometric determination of azide. The determination principles for azide by these two methods are based on inhibiting the enzymatic consumption of an electrode-detectable substance (hydrogen peroxide or catechol) on an enzyme-immobilized electrode. Both of these methods show a sensitive detection range and a short measuring time
Amperometric Trienzyme ATP Biosensors Based on the Coimmobilization of Salicylate Hydroxylase, Glucose-6-phosphate Dehydrogenase, and Hexokinase
Two types of amperometric ATP biosensors were developed by using the coimmobilization of salicylate hydroxylase (SHL, EC 1.14.13.1), glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1. 1.49), and hexokinase (HEX, EC 2.7. 1.1.) on a Clark-type oxygen electrode and on a screen-printed electrode. The principles of the determination schemes are as follows: HEX transfers the phosphate group from ATP to glucose to form glucose-6-phosphate. G6PDH catalyzes the specific dehydrogenation of glucose-6-phosphate by consuming NAD(+). The product, NADH initiates the irreversible decarboxylation and hydroxylation of salicylate by SHL to consume dissolved oxygen and generate catechol. This results in a detectable signal on a Clark-type electrode due to the SHL-enzymatic consumption of oxygen, or a detectable signal on a screen-printed electrode due to the SHL-enzymatic generation of catechol in the measurement of ATP. Both sensors show high performance characteristics with broad detection ranges, short measuring times, and good specificities. (C) 2008 Elsevier B.V All rights reserved
Understanding the practical consequences of metabolic interactions – a software package for teaching and research
METSTOICH, a metabolite balancing software package, was developed for use in teaching metabolic pathways and their interactions. Based on the metabolism of Baker's Yeast, the package has been used to examine the relationship between cell yield, cell composition, P/O ratio, and energy (ATP) utilization during cell growth. After the simulation was developed, a number of problem sets were developed which targeted particular cellular interactions. These had increasing levels of difficulty. The simulation was then trialed in the postgraduate course BIEN502 (Biochemistry for Bioengineering). Initial trials indicated that the package provides a useful supplement to traditional methods in teaching metabolism. Student evaluation of the course indicated that the simulation was considered a very useful supplement to traditional teaching methods, and that it was easy to use and to understand. The simulation was supported by a large help file which included background theory, nomenclature and the problem sets. Some minor operational faults and some suggestions by the students for further improvement were incorporated into a revised simulation. This will be trialed further in CENG565 (Environmental Biotechnology) and CENG361 (Biochemical Engineering). In addition, a supplementary grant will allow it to be trialed in Biochemistry, where the more basic biochemical details will be focused upon
Determination of poly(3-hydroxybntyrate) using a combination of enzyme-based biosensor and alkaline hydrolysis
The combination of an enzyme-based biosensor and alkaline hydrolysis was developed for the measurement of poly(3-hydroxybutyrate) (PHB). The principle of the determination is based on that the alkaline condition converts PHB to produce its monomer, 3-hydroxybutyrate (3-HB), which generates a detectable current signal by an amperometric biosensor through coupled two-enzyme reactions on an electrode. This method takes less than 40 min, and results in a linear detection range of 0.5 - 110 mg L-1 PHB with a detection limit of 0.3 mg L-1 by the saturated production of 3-HB; it can also take less than 15 min and result in a linear detection range of 1.0 - 160 mg L-1 PHB with a detection limit of 0.5 mg L-1 by a part production of 3-HB. The method also shows simple operation and high reproducibility
Amperometric Determination of Tyrosinase Catecholase Activity Using a Screen-printed Electrode
A fast amperometric method for the determination of tyrosinase-catecholase activity has been proposed. The principle of the method is as follows: tyrosinase-catecholase converts catechol to benzoquinone in the presence of oxygen; benzoquinone is subsequently reduced at the working electrode which generates an appropriate current signal. The method has a good analytical characteristics and a broad detection range (0.01-10.00 U mL(-1)). It is easy to handle, and requires short measuring time (1 min)