Blue Column Chromatography Research Articles

The Effects of Exercise on DHEA, DHEAS and LDL Protein Carbonyl Concentrations in Healthy Adults

PURPOSE: Dehydroepiandrosterone (DHEA) and its sulphate are naturally occurring steroids that decrease in concentration with increasing age. Decreased levels have been associated with immune system dysfunction and activation, increased oxidative stress, and atherogenesis. This study aimed to: 1) determine the response of DHEA and DHEAS to steady state exercise undertaken in a controlled setting by young healthy adults; 2) assess the effect of a steady state exercise bout on low density lipoprotein carbonyl concentration. METHODS: Plasma was collected from healthy male (n=9) (age 23±3years; VO2max 44.5±7ml/kg/min) and female (n=12) (age 23.2±7years; VO2max 32±8ml/ kg/min) subjects a) at rest (baseline); b) post exercise; and c) 60 minutes post exercise. The exercise task comprised a steady state cycling test at 60% Wmax for 40 minutes. Habitual physical activity was assessed using the IPAQ questionnaire. The concentration of circulating DHEA and DHEAS were assessed by ELISA. LDL was prepared by sepharose blue column chromatography and LDL protein carbonyl content was assessed by ELISA. RESULTS: DHEA levels increased significantly following exercise in men (a: 47.0±5.2ng/ml; b: 52.6±6.8ng/ml) and women (a: 19.3±4.1ng/ml; b: 25.9±6.7ng/ml) (p < 0.01). However, DHEAS levels were seen to decrease significantly following exercise in men (a: 2.1±0.8μg/ml; b: 1.9±0.5μg/ml) and women (a: 2.0 ±1.1 μg/ml; b: 1.8±0.8μg/ml) (p < 0.05). No correlation was observed between hormonal response and physical activity level. LDL protein carbonyl content was reduced following an acute bout of exercise in minimally active subjects (38% decrease) (p < 0.01) and highly active subjects (3% decrease) (P=0.11), however LDL protein carbonyl content was slightly increased in physically inactive subjects (2% increase) (P=0.057). CONCLUSION: DHEA increases and DHEAS decreases following an acute bout of moderate intensity exercise. LDL protein oxidation was reduced in physically active subjects following this type of exercise.

Simultaneous separation and purification of pyruvate kinase and lactate dehydrogenase by dye-ligand chromatography

This work describes an improved downstream processing protocol for the simultaneous separation and purification of two diagnostic enzymes, lactate dehydrogenase (LDH) and pyruvate kinase (PK), from rabbit muscles. The purification scheme was specifically designed so that both purified enzymes were comparable, in terms of specific activity and impurity content, to commercial high-purity analytical grade. The proposed protocol comprises two dye-columns (dye-sorbents), Cibacron blue 3GA and Procion yellow MX-4G both immobilised on CL-Sepharose 6B, which were integrated in the following six-step scheme: (i) muscle extract preparation, (ii) 0–45% ammonium sulphate fractionation, (iii) blue-column chromatography where the two enzymes were separated and LDH was purified, (iv) blue-column chromatography, (v) thermal treatment, and (vi) yellow-column chromatography where PK was purified. This protocol affords LDH of specific activity 470 units/mg (no detectable PK) at 65% overall yield, and PK of specific activity 251 units/mg (no detectable LDH and enolase) at 40% overall yield.

Purification and N-terminal amino acid sequences of two polypeptides encoded by the mcrB gene from Escherichia coli K-12

This report provides a purification method for the two proteins, 51 kDa and 33 kDa, both encoded by the same mcrB gene of the McrBC restriction system in Escherichia coli K-12. The two proteins were produced in large quantity using a T7 expression system and copurified to near homogeneity by DEAE-Sepharose and Affi-Gel blue column chromatography. The N-terminal amino acid sequences of these purified McrB proteins were the same as those predicted from the mcrB DNA sequence by Ross et al. [J. Bacteriol. 171 (1989b) 1974–1981]. The 33-kDa protein totally overlaps the C-terminal part of the 51-kDa protein.

Renaturation of cobra venom phospholipase A2 expressed from a synthetic gene in Escherichia coli

Cobra venom (Naja naja naja) phospholipase A2 (PLA2) contains 14 cysteines in the form of 7 disulfide bonds amongst its 119 amino acids. A gene encoding the PLA2 was synthesized and inserted into a bacterial expression vector containing the phage lambda pL promoter. In order to obtain protein without the initiating methionine at the N-terminus, a Factor Xa site was engineered upstream from the PLA2 gene. Upon heat-induction of the cells transformed with the expression plasmid, the protein is produced as insoluble inclusion bodies. The enzyme was partially purified by washing the inclusion bodies with Triton X-100 and urea. The expressed protein was first denatured with 8 M guanidine-HCl and 10 mM DTT. After digestion with Factor Xa, formation of disulfide bonds and refolding into the fully active form was carried out in the presence of cysteine and Ca2+. The renatured recombinant protein was purified by Affi-gel blue column chromatography. The purified recombinant enzyme had the same specific activity as the native enzyme when assayed on a variety of substrates and cross-reacted with antisera prepared against the native enzyme. This is the first report of the expression of a recombinant PLA2 from any venom.

Partial purification and characterization of CA19-9 antigen from the ascitic fluid of a patient with pancreatic cancer

CA19-9 immunoreactive protein was partially purified from the ascitic fluid of a patient with pancreatic cancer by perchloric acid fractionation, gel chromatography and Affi-gel Blue column chromatography, resulting in a purified sample of 5.0 x 10(6) CA19-9 units per milligram of protein (3700-fold purification). Western blotting analysis of this purified sample revealed a single band of molecular weight 210 kDa. Although the original ascitic fluid showed a high CA125 immunoreactivity, this purified sample had no CA125 immunoreactivity. The elution pattern for CA19-9 activity on Affi-gel Blue column is quite distinct from that for CA125. These results suggest that CA19-9 antigen in carcinoma patients may be identical or very similar to that recently purified from the culture media of the colorectal cell line SW1116 and is distinct from CA125 antigen.

Partial purification and characterization of 7α-hydroxysteroid dehydrogenase from rat liver microsomes

An NADPH-dependent 7α-hydroxysteroid dehydrogenase acting on 3α-hydroxy-7-keto-5β-cholanoic acid was partially purified 160-fold with a yield of 13% from rat liver microsomes using DEAE-cellulose, hydroxyapatite and Affi-Gel ® Blue column chromatography. The specific activity of the purified enzyme was 91.3 nmol chenodeoxycholic acid formed /min per mg of protein. The reaction was reversible, and the optimum pH of the enzyme for the oxidation was about 8.5, whereas that for the reduction was about 5.0. A molecular weight of the enzyme was estimated to be about 130000 by Superose 6 TM gel filtration chromatography. The apparent K m value for 3α-hydroxy-7-keto-5β-cholanoic acid was 35.7 μM and that for NADPH was 90.9 μM. The preferred substrate for the enzyme was 3α-hydroxy-7-keto-5β-cholanoic acid rather than 3α,12α-dihydroxy-7-keto-5β-cholanoic acid, a 7-keto-bile acid analogue. The enzyme also preferred the unconjugated form to the conjugated forms. The enzyme activity was inhibited by Pifp-chloromercuribenzoate; however, the inhibition was prevented by addition of reduced form of glutathione to the reaction mixture, indicating that the enzyme requires a sulfhydryl group for activity.

The Partial Purification and Characterization of Nuclear and Mitochondrial Uracil-DNA Glycosylase Activities from Zea mays Seedlings

Uracil-DNA glycosylase activities from etiolated Zea mays seedling nuclei and mitochondria were partially purified and characterized. Nuclei and mitochondria were separated using sucrose differential and step gradient centrifugation. Experiments with osmotically shocked organelles indicated that enzyme activity from mitochondria was soluble, whereas nuclear enzyme activity was only partially soluble under the conditions tested. Purification using DEAE-cellulose and Affigel Blue column chromatography yielded distinct elution profiles from both columns for each of the organellar enzyme activities. Final purification was 490- and 850- fold for the nuclear and mitochondrial uracil-DNA glycosylase, respectively. Characterization studies demonstrated significant differences between the nuclear and mitochondrial uracil-DNA glycosylase with respect to K(m), temperature, and pH activity optimum, the effect of salts, and substrate preference. Molecular weight as determined by gel filtration was 18,000 for enzymes from both sources. Both were also sensitive to the sulfhydryl group-blocking agent N-ethylmaleimide. A number of uracil analogs were tested for their ability to inhibit nuclear and mitochondrial uracil-DNA glycosylase activities. 5-Azauracil, uracil, 6-aminouracil, 6-azauracil, 5-aminouracil, and 5-fluorouracil all inhibited both activities to variable degrees.

Purification and characterization of N1-methylnicotinamide oxidases I and II separated from rat liver

N 1-Methylnicotinamide (1-CH 3Nmd) oxidases I and II of the livers of Wistar strain male rats were separated by DEAE-cellulose column chromatography and purified by AcA 34 gel filtration, Affi-Gel blue column chromatography, and sucrose density gradient centrifugation. These two enzymes were purified 568- and 82-fold with yields of 1.4 and 0.1% of the total activity in the liver homogenate, respectively. They showed the same molecular weight of 300,000 by AcA 34 gel filtration and a single protein band with a molecular weight of 150,000 in sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The difference in antigenicities of these two enzymes was observed with double immunodiffusion and immunoprecipitation using antibody produced in rabbits against each enzyme. Furthermore, these two enzymes were found to be different from each other in the heat stability, optimum pH values, K m values for 1-CH 3Nmd, and response to various inhibitors.

Partial purification and characterization of myosin light chain kinase from bovine thyroid gland.

Myosin light chain kinase has been purified approximately 1700-fold from bovine thyroid gland, using Affigel blue column chromatography, ammonium sulfate fractionation (0-60% saturation), Sepharose 6B gel filtration, and calmodulin-Sepharose affinity column chromatography. This partially purified kinase was Ca2+ and calmodulin dependent for its activity and specifically phosphorylated the 20,000-dalton light chain of chicken gizzard myosin. At higher CaCl2 concentration, thyroid myosin light chain kinase demonstrated a novel inhibition in the presence of calmodulin. The mechanism of this Ca2+-dependent inhibition is not clear at present. The existence of myosin light chain kinase in thyroid gland provides additional support for the role of the contractile system in the secretion of thyroid hormones.