Purification of NADPH-P450 reductase (NPR) from Xenopus laevis and the developmental change in NPR expression

Mori, Tomohiro; Yamazaki, Ayako; Kinoshita, Tsutomu; Imaoka, Shingi

doi:10.1016/j.lfs.2005.12.056

Cited by 2 publications

(2 citation statements)

References 21 publications

(20 reference statements)

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“…It was observed that the specific activity value, which was calculated in the kinetic studies that were conducted for the enzyme, was close to that of the African clawed frog (Xenopus laevis) liver 44 CPR enzyme and a little lower compared to the rainbow trout liver 27 CPR enzyme. K M values were 5.208 µM and 12.82 µ M, respectively, for NADPH and cytochrome c substrates, whereas V max values were 12.76 µ M/min/mL and 42.9 µ M/min/mL, respectively.…”

Section: Resultsmentioning

confidence: 70%

Purification and characterization of NADPH-cytochrome P450 reductase from Lake Van fish liver microsomes and investigation of some chemical and metals' effects on the enzyme activity

Kuzu¹,

Çiftçi²

2015

Turk J Chem

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NADPH-cytochrome P450 reductase was purified from Lake Van fish liver microsomes by primary and secondary DEAE-cellulose column chromatograph with 20.46 µ M/min/mg enzyme specific activities, 54.4% purification yield, and purification of 38-fold. The purity of the enzyme was established, and its monomer molecular weight was determined by SDS-polyacrylamide gel electrophoresis. SDS-PAGE results showed a single band and the molecular weight of NADPH-cytochrome P450 reductase was 70 kDa. In addition, optimum ionic strength, optimum pH, optimum temperature, and stable pH values were determined for the enzyme in the kinetic studies performed. K M and V max were determined for NADPH and cytochrome c. Effects of some metals ions, antibiotics, and some other drugs used in aquarium fisheries on the activity of the enzyme were investigated. IC50 values and K i values of metals showing an inhibitory effect were calculated.

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Section: Resultsmentioning

confidence: 70%

Purification and characterization of NADPH-cytochrome P450 reductase from Lake Van fish liver microsomes and investigation of some chemical and metals' effects on the enzyme activity

Kuzu¹,

Çiftçi²

2015

Turk J Chem

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“…Reciprocally, a 25-kDa band was detected specifically in heart, liver, and kidney. Previous studies have shown that the expression of specific genes, especially those participating in energy metabolism and stress responses, can be restricted to the heart, liver, and kidney (35,36). In addition, the cardiac CCRK presents several interesting molecular characteristics that could lead to diagnostic or therapeutic applications.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Novel Cardiac Isoform of the Cell Cycle-related Kinase That Is Regulated during Heart Failure

Qiu¹,

Dai²,

Jain

et al. 2008

Journal of Biological Chemistry

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Myocardial infarction (MI) is often followed by heart failure (HF), but the mechanisms precipitating the transition to HF remain largely unknown. A genomic profile was performed in a monkey model of MI, from the myocardium adjacent to chronic (2-month) MI followed by 3 weeks of pacing to develop HF. The transcript of the gene encoding the cell cycle-related kinase (CCRK) was down-regulated by 50% in HF heart compared with control (p < 0.05), which was confirmed by quantitative PCR. The CCRK sequence cloned from a heart library showed a conservation of the N-terminal kinase domain when compared with the "generic" isoform cloned previously but a different C-terminal half due to alternative splicing with frameshift. The homology of the cardiac sequence was 100% between mice and humans. Expression of the corresponding protein, measured upon generation of a monoclonal antibody, was limited to heart, liver, and kidney. Upon overexpression in cardiac myocytes, both isoforms promote cell growth and reduce apoptosis by chelerythrine (p < 0.05 versus control). Using a yeast two-hybrid screening, we found an interaction of the generic but not the cardiac CCRK with cyclin H and casein kinase 2. In addition, only the generic CCRK phosphorylates the cyclin-dependent kinase 2, which was accompanied by a doubling of myocytes in the S and G 2 phases of the cell cycle (p < 0.05 versus control). Therefore, the heart expresses a splice variant of CCRK, which promotes cardiac cell growth and survival; differs from the generic isoform in terms of protein-protein interactions, substrate specificity and regulation of the cell cycle; and is downregulated significantly in HF.Because the fully differentiated cardiac myocyte has a limited capacity for regeneration, most forms of heart disease, including ischemic heart disease and heart failure (HF), 2 are characterized by a loss of cardiomyocytes (1-3). Therefore, deciphering the molecular mechanisms balancing cell growth and apoptosis may help develop strategies to improve the prognosis of both ischemic heart disease and HF, particularly with regard to stem cell therapy (4). Our previous studies have shown that genomic profiling in large mammalian models of heart disease allows the detection of novel potential candidates to promote cardiac cell growth and survival in a context of reversible ischemia or pressure overload (5, 6). In the present study, we extended this investigation to a model of postischemic cardiomyopathy in monkeys. We reasoned that a nonhuman primate model would be ideal, since it is phylogenetically and genetically closer to humans. Genomic profile by DNA microarrays in that model revealed the down-regulation of the cell cycle-related kinase (CCRK) after development of HF. Although this protein has not been characterized in the heart before, it is known that CCRK plays a major role in promoting the growth of cancer cells (7). Therefore, the goal of the present study is to characterize the expression of CCRK in the normal and failing heart and to determine whether this k...

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Purification of NADPH-P450 reductase (NPR) from Xenopus laevis and the developmental change in NPR expression

Cited by 2 publications

References 21 publications

Purification and characterization of NADPH-cytochrome P450 reductase from Lake Van fish liver microsomes and investigation of some chemical and metals' effects on the enzyme activity

Purification and characterization of NADPH-cytochrome P450 reductase from Lake Van fish liver microsomes and investigation of some chemical and metals' effects on the enzyme activity

Characterization of a Novel Cardiac Isoform of the Cell Cycle-related Kinase That Is Regulated during Heart Failure

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