Navigation Links
Preparative Nondenaturing Gel Electrophoresis to Purify NADP-Specific Glutamate Dehydrogenase From Chlorella, Rev B

Contributed by Philip W Miller, Waltraud I Dunn, and Robert R Schmidt, University of Florida, Dept. Microbiology and Cell Science, Gainesville, FL 32611 USA
Florida Agricultural Experiment Station Serial Number T-00256


One of the research goals of this laboratory is to elucidate the pre- and posttranslational mechanisms that regulate both carbon and nitrogen metabolism in plants. Chlorella sorokiniana, a unicellular green alga, has been used extensively as a model system to study enzymes involved in higher plant metabolism. Research in this laboratory has shown that Chlorella sorokiniana possesses seven ammonium inducible, chloroplastic, NADP-specific glutamate dehydrogenase (NADP-GDH) isoenzymes that are regulated by both nitrogen and carbon metabolites (Prunkard et al. 1986). Biochemical, immunochemical, and physical characterization of purified GDH homohexamers revealed that the subunits (α, 55.5 kD; , 53 kD) composing these holoenzymes are nearly identical; however, kinetic characterization of the enzymes showed them to have strikingly different Km values, allosteric properties, and turnover rates (Bascomb and Schmidt 1987). Recent molecular genetic analysis indicates that minor modifications in the primary protein sequences may account for these observed properties. In order to further elucidate the regulation of these isoenzymes, it was necessary to purify to homogeneity a large quantity of active enzyme for polyclonal/monoclonal antibody production and additional protein analysis.

We report here the use of the Model 491 prep cell for the purification of native NADP-GDH protein by nondenaturing PAGE with <2% loss of enzyme activity.

Glutamate Dehydrogenase Isolation
Chlorella sorokiniana cells were cultured in 29 mM NH4+ medium as previously described (Baker and Schmidt 1963). Approximately 130 g fresh weight cells were harvested by centrifugation and washed two times in 0.01 M Tris-HCl (pH 8.5, 4C). Pelleted cells were resuspended in an equal ratio of breakage buffer (w/v) and ruptured by two passages through a French pressure cell (American Inst. Co.) at 20,000 psi. The cell homogenate was centrifuged at 27,000 x g for 45 min and the supernatant was stored overnight at 20C. Frozen supernatant was thawed and the resulting precipitate was removed by centrifugation at 27,000 x g. Initial purification of the NADP-GDH was accomplished using a modified procedure of Yeung et al. (1981), which employs sequential ion exchange and size exclusion chromatography to remove the bulk contaminating proteins from the cell lysate. Procedural modifications involved the addition of NADP+, which functioned as a stabilizer, to a final concentration of 0.1 mM to the gel filtration buffer and all subsequent buffers. As a final modification, a preparative nondenaturing PAGE step using the Model 491 prep cell was substituted for an expensive NADP-affinity resin step.

Sample Preparation
Following ammonium sulfate precipitation and ion exchange chromatography, size exclusion column fractions, in 10 mM KPO4, 2 mM dithiothreitol (DTT), 0.1 mM NADP+ (pH 6.2), possessing NADP-GDH activity were pooled and concentrated via Diaflo filtration from 17.5 ml to 5 ml. The soluble enzyme was reduced by the addition of DTT to a final concentration of 10 mM and placed in 14,000 MW cutoff dialysis tubing. The concentrated sample was dialyzed at 4C against 28.8 mM Tris, 192 mM glycine, 2 mM DTT (pH 8.4) for 30 min. The dialyzed enzyme preparation was clarified by centrifugation at 20,000 x g for 10 min and was combined with 3 ml of 40% sucrose and 1 ml of 0.02% Bromophenol Blue.

Preparative Nondenaturing Gel Electrophoresis
For preparative nondenaturing gel electrophoresis, a 3 cm high 7% acrylamide (28 acrylamide:0.735 bis-acrylamide, pH 8.8) resolving gel and a 2 cm high 2% acrylamide (1.6 acrylamide:0.4 bis-acrylamide, pH 6.6) stacking gel were cast in the 28 mm ID gel tube of the Model 491 prep cell. The resolving gel was polymerized in 374 mM Tris (pH 8.8) using 140 g/ml ammonium persulfate and 1.12 l/ml TEMED. The stacking gel was polymerized in 39 mM Tris (pH 6.6), 12.7% sucrose using 0.07% riboflavin-5-phosphate (Bio-Rad) and 0.365 l/ml TEMED. Stacking gel polymerization was achieved by exposure to two 5 W fluorescent lights for 45 min. Both gels were cooled as per the Model 491 prep cell instruction manual during the polymerization process. All acrylamide stock solutions were pretreated with AG 501-X8 mixed bed resin to remove any contaminating acrylic acid to prevent in vitro acylation of proteins during the electrophoresis process. The preparative gel was pre-electrophoresed for 10 min at 15 mA constant power in upper/lower gel electrophoresis buffer containing 28.8 mM Tris, 192 mM glycine (pH 8.4, 4C). The elution buffer reservoir was filled with elution buffer comprised of 28.8 mM Tris, 192 mM glycine, 2 mM DTT, and 0.1 mM NADP+ (pH 8.4, 4C). The entire Model 491 prep cell was cooled to 4C during the electrophoresis process by operating the unit in a 4C coldroom. The protein sample, 68 mg total protein in 9 ml of loading buffer, was loaded on top of the stacking gel and electrophoresed for 20 min at 15 mA, and then for an additional 3.5 hr at a constant power of 30 mA using Bio-Rads Model 3000/300 power supply.

Fraction Collection and Analysis
The elution buffer was pumped at a rate of 2 ml/min to a fraction collector and 6 ml fractions were collected. The first fraction after the Bromophenol Blue marker eluted, fraction 1, was collected after 2 hr of electrophoresis. A spectrophotometric assay was used to quantitate the deaminating activity of the NADP-GDH in each fraction. One unit of GDH activity was defined as the amount of enzyme activity required to reduce 1 mol of NADP+/min at 38.5C. NADP-GDH activity was detected in fractions 3568 (Figure 1). The multiple peaks of enzyme activity detected presumably correspond to the multiple isoenzymes of the NADP-GDH.

Analysis of Model 491 Prep Cell Purified NADP-GDH
NADP-GDH containing fractions were combined and the protein concentration was determined by the method of Bradford (1976) using the Bio-Rad protein assay kit with Bio-Rads protein standard II as the standard. Fractions under the peaks were pooled, concentrated, and rinsed with 10 mM KPO4 (pH 6.2), then resuspended in 10 mM KPO4 (pH 6.2), 0.1 mM NADP+ to a concentration of 1 mg/ml for further analysis and storage at 20C. The purity of the protein was determined by Tris-Tricine SDS-PAGE (Schagger and von Jagow 1987) in a 10% polyacrylamide resolving, 3% stacking gel using the Mini-PROTEAN II slab cell (Figure 2). Gels were stained using the Bio-Rad Silver Stain Plus kit.

Chlorella sorokiniana NADP-specific GDH was purified an additional 8.5-fold for a final 375-fold purification using the Model 491 prep cell (see Table). Nondenaturing preparative electrophoresis of the partially purified NADP-GDH sample was performed under conditions determined to be optimal for analytical native slab gel electrophoresis. A total of 11.5 mg of extremely pure GDH protein was recovered with <2% loss of activity during the procedure. The resolution of the nondenaturing gel in the Model 491 prep cell was sufficient to separate multiple peaks of NADP-GDH activity corresponding to the multiple isoenzymes of the GDH isoenzymes. Analytical SDS-PAGE of the final purified product revealed a single 53 kD band corresponding to the NADP-GDH -subunit and detected no contaminating proteins. The highly purified NADP-GDH protein was recovered in sufficient quantity in its native form to allow NH2- and COOH-terminal sequencing, antigen affinity column production, and the production of hightiter polyclonal and monoclonal antibodies.

These results suggest that the Model 491 prep cell system is amenable to large-scale purification of partially purified proteins in their native, active form. The high resolution of this technique, evidenced by its ability to separate closely migrating isoenzymes, should allow separation of multiple isoenzymes for the purpose of kinetic, biochemical, and immunochemical analyses.

We thank Dr LO Ingram for the use of the Model 491 prep cell.

Baker AL and Schmidt RR, Intracellular distribution of phosphorus during synchronous growth of Chlorella pyrenoidosa, Biochem Biophys Acta 74, 7583 (1963)

Bascomb NF and Schmidt RR, Purification and partial kinetic and physical characterization of two chloroplast-localized NADP-specific glutamate dehydrogenase isoenzymes and their preferential accumulation in Chlorella sorokiniana cells cultured at low or high ammonium levels, Plant Physiol 83, 7584 (1987)

Bradford MM, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem 72, 248254 (1976)

Prunkard DE et al., Effect of different carbon sources on the ammonium induction of different forms of NADP-specific glutamate dehydrogenase in Chlorella-sorokiniana cells cultured in the light and dark, Plant Physiol 81, 413422 (1986)

Schagger H and von Jagow G, Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa, Anal Biochem 166, 368379 (1987)

Yeung A T et al., Purification of an ammonium-inducible glutamate dehydrogenase and the use of its antigen affinity column-purified antibody in specific immunoprecipitation and immunoadsorption procedures, Anal Biochem 110, 216228 (1981)

Diaflo is a trademark of W R Grace & Co.

back to top


Page: All 1 2 3 4 5 6

Related biology technology :

1. A Rapid Method for the Purification of Analytical Grade Proteins From Plants Using Preparative SDS-PAGE and Preparative IEF
2. Detection of Platinum Species in Plant Material by Preparative Isotachophoresis Using the Model 491 Prep Cell, Rev A
3. Preparative Nondenaturing Gel Electrophoresis Used in the Purification of an Esterase Involved in Insecticide Resistance, Rev C
4. Preparative SDS-PAGE Electrophoresis of a Recombinant Epstein-Barr Virus Encoded Protein and Its Application in Serodiagnostic Test Systems, Rev C
5. Purification and Characterization of beta-Lactoglobulin Genetic Variants A and B Using Preparative Electrophoresis and Isoelectric Focusing
6. A Rapid Method for the Purification of Analytical Grade Proteins From Plants Using Preparative SDS-PAGE and Preparative IEF
7. Semi Preparative Purification of Proteins
8. A Versatile Power Supply for All Electrophoresis Applications
9. A Multiple Mutation Model System as a Test Development and Training Tool for Denaturing Gradient Gel Electrophoresis
10. Continuous-Elution Electrophoresis for Purification of the Baculovirus-Expressed Coronavirus Structural Proteins, Rev A
11. Detection of K-ras Point Mutations in the Pancreas by Constant Denaturing Gel Electrophoresis Using the DCode System
Post Your Comments:
(Date:8/1/2014)... Once a decision has been made ... will discuss how to define your tolerance for lowest ... Allen, Senior Director of Facilities Integration at Fluor Industrial ... Industrial Services, and special guest Carrier Li, Director in ... as they provide an examination of the Conceptual Design ...
(Date:7/31/2014)... (PRWEB) July 31, 2014 Food ... are increasingly searching for food alternatives that offer ... benefits. The food ingredient market has numerous players ... such as Vital Force Technology (VFT). VFT ... than 4,000 energetic patterns, all tested to produce ...
(Date:7/31/2014)... British mathematician (1912-1954), is famous for a number of ... In 1936 he published a paper, which laid the ... of a computer algorithm. He next played a pivotal ... which cracked the German military codes, enabling the Allies ... in the late 1940,s he turned his attention to ...
(Date:7/31/2014)... , July 31, 2014 ... Market Research "Quantum Dots Market - Global Industry Analysis, ... the market was valued at USD 88.5 million in ... by 2023, growing at a CAGR of 53.8% from ... Dots Market Report at Increasing ...
Breaking Biology Technology:Defining Low Cost in Your Solution for Future Facilities, New Webinar for Pharma, Biotech, Medical Device and Healthcare Companies, Hosted by Xtalks 2Functional Ingredient Manufacturer – Vital Force Technology Infuses Raw Materials & Ingredients with Subtle Energy Formula 2A mathematical theory proposed by Alan Turing in 1952 can explain the formation of fingers 2A mathematical theory proposed by Alan Turing in 1952 can explain the formation of fingers 3Global Quantum Dots Market is Expected to Reach USD 8,246.8 Million in 2023: Transparency Market Research 2Global Quantum Dots Market is Expected to Reach USD 8,246.8 Million in 2023: Transparency Market Research 3Global Quantum Dots Market is Expected to Reach USD 8,246.8 Million in 2023: Transparency Market Research 4
... China, March 11 /Xinhua-PRNewswire-FirstCall/ -- China,Bionanometer Industries ... the bionanometer industry has formally signed an ... China Guangzhou,LGLY Advertising LTD., Guangzhou LGLY ... It was,founded in December 1997 and is ...
... March 11 ChanTest, the leading provider of,ion ... customers,will feature the latest in its expanding line ... Toxicology conference March 17-20, in Seattle, Washington.,New FDA ... Channel,Panel(TM) and Brain Channel Panel(TM), which ChanTest designed ...
... The Project on Emerging Nanotechnologies (PEN) and National ... premiere event for the television series Nanotechnology: The ... The eventby invitation onlywill include remarks by U.S. ... Congressional Nanotechnology Caucus. , The series three programs ...
Cached Biology Technology:CBIU Signs Agreement with LGLY Advertising LTD 2ChanTest to Highlight Expanded Ion Channel Testing Services for Preclinical Safety at ToxExpo, Including the World's Largest Catalog of Validated Ion Channels, Booth 2504 2New nanotechnology television series does 'sweat the small stuff' 2
(Date:7/31/2014)... who studied 100 twin pairs have identified a gene mutation ... than six hours of sleep per night. The genetic ... of sleep deprivation. , Results show that a participant with ... an average nightly sleep duration of only five hours, which ... who slept for about six hours and five minutes per ...
(Date:7/31/2014)... completed a 20-year study that looks at the ... times of the year. It finds that burning ... has no measurable negative consequences for the prairie ... The study was conducted by Gene Towne, research ... chief, and Joseph Craine, research assistant professor, both ...
(Date:7/31/2014)... of animal models against the highly infectious and virulent ... disease that kills approximately 30,000 Americans annually. The research ... Immunity . , In the study, the vaccine ... toxins produced by C. difficile , as well ... that mimics the human disease, after only two immunizations. ...
Breaking Biology News(10 mins):Study of twins discovers gene mutation linked to short sleep duration 2Study finds benefits to burning Flint Hills prairie in fall and winter 2Study finds benefits to burning Flint Hills prairie in fall and winter 3C. difficile vaccine proves safe, 100 percent effective in animal models 2
... PalmSecure(TM) Biometric Authentication, Self-Service Check In, Credit Card Scanning ... for Physician PracticesCHICAGO and SUNNYVALE, Calif., April 7 ... ) and Fujitsu , a leading ... today unveiled the Allscripts Patient Kiosk ...
... Company,Limited, a global provider of fingerprint recognition software and ... Fingerprint,Scanner. , FS90 uses a ... a very small form factor. Its size is 57(L) ... one half of Futronic FS80 USB2.0 Fingerprint Scanner but,can ...
... -- Which is a better strategy, specializing in ... more profitable than organic farming? Is it less ... of Wisconsin,s College of Agriculture and Life Sciences ... Wisconsin Integrated Cropping Systems Trial (WICST) in 1990. ...
Cached Biology News:Allscripts Integrates Electronic Health Records with Innovative Patient Kiosk 2Allscripts Integrates Electronic Health Records with Innovative Patient Kiosk 3Allscripts Integrates Electronic Health Records with Innovative Patient Kiosk 4Allscripts Integrates Electronic Health Records with Innovative Patient Kiosk 5Allscripts Integrates Electronic Health Records with Innovative Patient Kiosk 6Allscripts Integrates Electronic Health Records with Innovative Patient Kiosk 7Futronic Launches FS90 USB2.0 Mini Fingerprint Scanner 2
For use with SSP products or any application requiring agarose gel separation • Multiple formats available to meet various throughput needs • Includes replacement parts for each un...
Request Info...
Human RUNX2/CBFA1 MAb (Clone 232902)...
Biology Products: