Navigation Links
Purification of Two Isoforms of Pyruvate Kinase from Nordic Krill Using an UNO Q1 Column, Rev A


Introduction
Pyruvate kinase (PK, E.C. 2.7.1.40) is one of the key regulatory enzymes of the glycolytic pathway in crustaceans. This enzyme catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate and the phosphorylation of ADP to ATP. In addition to the role of PK in aerobic and anaerobic energy metabolism in invertebrates, regulation of PK activity in response to changes in temperature and oxygen regimes was found to occur in marine mollusks. Others reported changes in PK enzyme characteristics with temperature in fish and the intertidal mussels, suggesting that modification of the biochemical properties of PK may also play a role in temperature adaptation. The Nordic krill (Meganyctiphanes norvegica) is a crustacean which thrives in the open sea under aerobic conditions and a wide range of temperatures. Purification of two isoforms of PK (PK I and PK II) from this species was required to facilitate kinetic and chromatographic studies of these enzymes relating to seasonal temperature adaptation.


Fig. 1. Sample: 5 ml extract; Column: UNO Q1 column; Buffer A: 20 mM Tris-HCl pH 8.0; Buffer B: A+1 M NaCl, pH 8.0; Gradient: 0% B in 10 ml, 030% B in 20 ml, 100% B in 5 ml; Flow rate: 4 ml/min.


Purification
Nordic krill were caught in February and July, deepfrozen and stored at -80 C. For a single extract, two individuals (about 0.4 g) were homogenized in 2.7 ml extraction buffer (50 mM Tris-HCl, 60 mM KCl, 4 mM MgSO4, pH 7.0) with an Ultra-Turrax homogenizer. The suspension was centrifuged and the supernatant was buffer-exchanged into column buffer by gel filtration chromatography. The buffer-exchanged extract was filtered and applied to an UNO Q-1 column (Bio-Rad catalog # 720-0001, see Figure 1). PK I and PK II were well-separated by this procedure at pH 8.0. The previous protocol, using a popular high resolution beaded support, required the use of pH 9.0 buffer to prepare the PK I species, at which pH the enzyme is less stable. The addition of PEP as a substrate stabilizing agent was also required in this pH 9.0 system but not for the UNO-based system. The purified enzyme obtained by this purification process was used to determine that the PK I of Nordic krill does, indeed, exhibit altered physicochemical and kinetic properties with the seasons and may contribute to the temperature adaptations of this species.

Reference
Vetter, R-A.H., and Buchholz, F., Comp. Biochem. Physiol., 116A, 110 (1997).

Data courtesy of Dr. Rolph-Achim Vetter, Biologische Anstalt Helgoland, Meeresstation.


back to top
'"/>

Source:


Page: All 1 2

Related biology technology :

1. High-Level Protein Expression, One-Column Purification, and FLAG Epitope Tagging in E. coli
2. Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications and Agarose Gels
3. Yeast Protein Production System Features High Yields and One-Step Purification
4. Expression and Purification of Recombinant Proteins That Have Native Amino Acid Sequence
5. Easy Cosmid DNA Purification with StrataPrep Kits
6. MinElute PCR Purification Kit
7. QIAquick PCR Purification Kit
8. Microarray Target Purification Kit and the Microarray Target Synthesis Kit (T7)
9. Double-Affinity Purification of GST-His Tagged Proteins Using Vivapure 8-to-96 Well Cobalt-Chelate Kit
10. Ultra-Fast Purification of Small Nucleic Acids
11. RNAClean - RNA & cDNA in vitro Reaction Purification
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:10/10/2017)... International research firm Parks Associates announced today that Tom ... 2017 Annual Meeting , October 11 in Scottsdale, Arizona ... and how smart safety and security products impact the competitive landscape. ... Parks Associates: Smart Home Devices: Main Purchase Driver ... "The residential security market has experienced continued growth, and ...
(Date:10/10/2017)... ... October 10, 2017 , ... USDM Life Sciences , ... life sciences and healthcare industries, announces a presentation by Subbu Viswanathan and Jennifer ... “Automating GxP Validation for Agile Cloud Platforms,” will present a revolutionary approach to ...
(Date:10/9/2017)... 2017  BioTech Holdings announced today identification and ... ProCell stem cell therapy prevents limb loss in ... demonstrated that treatment with ProCell resulted in more ... compared to standard bone marrow stem cell administration.  ... reduction of therapeutic effect.  ...
(Date:10/9/2017)... ... October 09, 2017 , ... At its national board meeting in North ... in Harvard University’s Departments of Physics and Astronomy, has been selected for membership in ... winning team for the 2015 Breakthrough Prize in Fundamental physics for the discovery of ...
Breaking Biology Technology:
(Date:7/20/2017)... DAL ) customers now can use fingerprints instead of their ... (DCA). ... launches biometrics to board aircraft at Reagan Washington National Airport ... Delta,s biometric boarding pass experience that launched in May at ... process to allow eligible Delta SkyMiles Members who are enrolled in CLEAR ...
(Date:5/23/2017)... 2017  Hunova, the first robotic gym for the rehabilitation and functional ... in Genoa, Italy . The first 30 robots will ... USA . The technology was developed and patented at ... IIT spin-off Movendo Technology thanks to a 10 million euro investment from ... click: ...
(Date:4/24/2017)... -- Janice Kephart , former 9/11 Commission ... LLP (IdSP) , today issues the following statement: ... 6, 2017 Executive Order: Protecting the Nation ... instilled with greater confidence, enabling the reactivation of ... are suspended by until at least July 2017). ...
Breaking Biology News(10 mins):