Purification and characterisation of Escherichia coli alkaline phosphatase. A biochemical experiment

Munson, Lianna M.; Fall, Ray

doi:10.1016/0307-4412(78)90059-6

Cited by 9 publications

(6 citation statements)

References 21 publications

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“…ALP activity is a marker of VSMC transformation (Lomashvilli et al, 2008). Samples of cell lysates, collected at different time points of calcification, were analyzed for ALP activity (Munson and Fall, 1978) with cell lysates. Protein content was analyzed using the Bradford assay.…”

Section: Methodsmentioning

confidence: 99%

Eotaxin Augments Calcification in Vascular Smooth Muscle Cells

et al. 2016

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Calcification of atherosclerotic plaques in elderly patients represents a potent risk marker of cardiovascular events. Plasma analyses of patients with or without calcified plaques reveal significant differences in chemokines, particularly eotaxin, which escalates with increased calcification. We therefore, hypothesize that eotaxin in circulation augments calcification of vascular smooth muscle cells (VSMCs) possibly via oxidative stress in the vasculature. We observe that eotaxin increases the rate of calcification significantly in VSMCs as evidenced by increased alkaline phosphatase activity, calcium deposition, and osteogenic marker expression. In addition, eotaxin promotes proliferation in VSMCs and triggers oxidative stress in a NADPH oxidase dependent manner. These primary novel observations support our proposition that in the vasculature eotaxin augments mineralization. Our findings suggest that eotaxin may represent a potential therapeutic target for prevention of cardiovascular complications in the elderly. J. Cell. Biochem. 118: 647-654, 2017. © 2016 Wiley Periodicals, Inc.

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Section: Methodsmentioning

confidence: 99%

Eotaxin Augments Calcification in Vascular Smooth Muscle Cells

et al. 2016

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“…These two bands may correspond to the different isozyme of APase on nondenaturing gel. However, three to five bands were also reported to be observed in the native PAGE of APase (Munson and Fall 1978). This analysis revealed that the butanol and acetone treatment removed most of the unwanted protein from the cream solution, and purified the enzyme, alkaline phosphatase, up to homogeneity.…”

Section: Resultsmentioning

confidence: 90%

A three step approach for the purification of alkaline phosphatase from non-pasteurized milk

Upadhyay

Verma

2014

J Food Sci Technol

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In this study, a three step purification of alkaline phosphatase from non-pasteurized milk has been described. It included cream extraction, n-butanol treatment and acetone precipitation. Different parameters such as buffer concentration, temperature, pH, substrate concentration, acetone and n-butanol treatment were optimized to maximize the enzyme activity. The enzyme was fruitfully purified up to homogeneity from the milk, with percentage recovery and fold purification of 56.17 and 17.67 respectively. The kinetic parameters were determined to be 0.927 mM (Km) and 55.86 μM/min (Vmax), with specific activity of 11.31 U/mg. Other optimized parameters were estimated as a buffer concentration of 0.5 M with pH 9.0, temperature optima at 37 °C, with n-butanol and acetone concentration of 20 % (v/v) and 50 % (v/v) respectively. This approach provides a simple and effective method for the purification of alkaline phosphatase from non-pasteurized milk.

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“…Alkaline phosphatase (EC 3.1.3.1) has been the subject of numerous biochemistry lab exercises due to its relative inexpensive cost and the simple, rapid, colorimetric assay used to measure its activity . The enzyme exists as a dimer and requires the presence of two distinct cofactors for its activity in hydrolyzing phosphoric acid monoesters, releasing free phosphate.…”

Section: Introductionmentioning

confidence: 99%

A ten‐week biochemistry lab project studying wild‐type and mutant bacterial alkaline phosphatase

Witherow

2016

Biochem Molecular Bio Educ

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This work describes a 10-week laboratory project studying wild-type and mutant bacterial alkaline phosphatase, in which students purify, quantitate, and perform kinetic assays on wild-type and selected mutants of the enzyme. Students also perform plasmid DNA purification, digestion, and gel analysis. In addition to simply learning important techniques, students acquire novel biochemical data in their kinetic analysis of mutant enzymes. The experiments are designed to build on students' work from week to week in a way that requires them to apply quantitative analysis and reasoning skills, reinforcing traditional textbook biochemical concepts. Students are assessed through lab reports focused on journal style writing, quantitative and conceptual question sheets, and traditional exams. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(6):555-564, 2016.

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Purification and characterisation of Escherichia coli alkaline phosphatase. A biochemical experiment

Cited by 9 publications

References 21 publications

Eotaxin Augments Calcification in Vascular Smooth Muscle Cells

Eotaxin Augments Calcification in Vascular Smooth Muscle Cells

A three step approach for the purification of alkaline phosphatase from non-pasteurized milk

A ten‐week biochemistry lab project studying wild‐type and mutant bacterial alkaline phosphatase

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