Method for Detecting NADPH-Cytochrome P450 Reductase in Liver Microsomal Fractions by Using Native Polyacrylamide Gel Electrophoresis and NADPH-Diaphorase Staining

By combining native polyacrylamide gel electrophoresis (PAGE) and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining, a simple method for detecting NADPH-cytochrome P450 reductase in tissue samples was established. When rat liver microsomal fractions were examined by this method, several bands with different mobility were visualized. Western blot analysis indicated that the band which appeared in the most anodal position among them represented NADPH-cytochrome P450 reductase. SDS-PAGE/Western blot analysis revealed that the molecular weight of the protein forming the band was around 80 kDa, which was identical to that of rat NADPH-cytochrome P450 reductase. The intensity level of NADPH-diaphorase staining assigned to this enzyme estimated by this method increased four times in microsomal fractions prepared from rat fed ethanol chronically compared to that from controls. When a dilution series of a rat liver microsomal fraction was examined by this method and SDS-PAGE/Western blot analysis, their staining intensities representing this enzyme were significantly correlated with each other. Using the naive PAGE/NADPH-diaphorase staining method, NADPH-cytochrome P450 reductase is detected in rat liver microsomes. This method is beneficial because compared with the conventional SDS-PAGE/Western blot analysis, the quantification of NADPH-cytochrome P450 reductase in tissue samples is allowed to be more easily done.

Downregulation of NADPH-cytochrome P450 reductase via RNA interference increases the susceptibility of Acyrthosiphon pisum to desiccation and insecticides

Nicotinamide adenine dinucleotide phosphate(NADPH)-cytochrome P450 reductase(CPR)is involved in the metabolism of endogenous and exogenous substances,and detoxification of insecticides.RNA interference(RNAi)of CPR in certain insects causes developmental defects and enhanced susceptibility to insecticides.However,the CPR of Acyrthosiphon pisum has not been characterized,and its function is still not understood.In this study,we investigated the biochemical functions of A.pisum CPR(ApCPR).ApCPR was found to be transcribed in all developmental stages and was abundant in the embryo stage,and in the gut,head,and abdominal cuticle.After optimizing the dose and silencing duration of RNAi for downregulating ApCPR,we found that ApCPR suppression resulted in a significant decrease in the production of cuticular and internal hydrocarbon contents,and of cuticular waxy coatings.Deficiency in cuticular hydrocarbons(CHCs)decreased the survival rate of A.pisum under desiccation stress and increased its susceptibility to contact insecticides.Moreover,desiccation stress induced a significant increase in ApCPR mRNA levels.We further confirmed that ApCPR participates in CHC production.These results indicate that ApCPR modulates CHC production,desiccation tolerance,and insecticide susceptibility in A.pisum,and presents a novel target for pest control.

Bioinformatics analysis for structure and function of CPR of Plasmodium falciparum

Objective:To analyse the structure and function of NADPH-cytochrome p450 reductase(CYPOR or CPR) from Plasmodium falciparum(Pf),and to predict its’ drug target and vaccine target. Methods:The structure,function,drug target and vaccine target of CPR from Plasmodium falciparum were analyzed and predicted by bioinformatics methods.Results:PfCPR,which was older CPR,had close relationship with the CPR from other Plasmodium species,but it was distant from its hosts,such as Homo sapiens and Anopheles.PfCPR was located in the cellular nucleus of Plasmodium falciparum.335aa-352aa and 591aa - 608aa were inserted the interior side of the nuclear membrane,while 151aa-265aa was located in the nucleolus organizer regions.PfCPR had 40 function sites and 44 protein-protein binding sites in amino acid sequence.The teriary structure of laa-700aa was forcep-shaped with wings.15 segments of PfCPR had no homology with Homo sapien CPR and most were exposed on the surface of the protein.These segments had 25 protein-protein binding sites.While 13 other segments all possessed function sites. Conclusions:The evolution or genesis of Plasmodium falciparum is earlier than those of Homo sapiens.PfCPR is a possible resistance site of antimalarial drug and may involve immune evasion, which is associated with parasite of sporozoite in hepatocytes.PfCPR is unsuitable as vaccine target,but it has at least 13 ideal drug targets.

为从变形体 berghei 的氮的氧化物 synthase 和类似的蛋白质的结构和功能的生物信息学分析和预言

【正】Objective:To search and analyze nitric oxide synthase(NOS) and similar proteins from Plasmodium berghei(Pb).Methods:The structure and function of nitric oxide synthase and similar proteins from Plasmodium berghei were analyzed and predicted by bioinformatics. Results:P6NOS were not available,but nicotinamide adenine dinucleotide 2’-phosphate reduced tetrasodium(NADPH)-cytochrome p450 reductase(CPR) were gained.PiCPR was in the nucleus of Plasmodium berghei,while 134aa-229aa domain was localize in nucleolar organizer. The amino acids sequence of P6CPR had the closest genetic relationship with Plasmodium vivax showing a 73%homology.The tertiary structure of PbCPR displayed the forcep-shape with wings,but no wings existed in the tertiary structure of its’ host,Mus musculus(Mm).137aa-200aa, 201aa-218aa,220aa-230aa,232aa-248,269aa-323aa,478aa-501aa and 592aa-606aa domains of P6CPR showed no homology with MmCPRs’,and all domains were exposed on the surface of the protein.Conclusions:NOS can’t be found in Plasmodium berghei and other Plasmodium species.PbCPR may be a possible resistance site of antimalarial drug,and the targets of antimalarial drug and vaccine.It may be also one of the mechanisms of immune evasion.This study on Plasmodium berghei may be more suitable to Plasmodium vivax.And137aa-200aa, 201aa-218aa,220aa-230aa,232aa-248,269aa-323aa,478aa-501aa and 592aa-606aa domains of PbCPR are more ideal targets of antimalarial drug and vaccine.