Cloning and expression of Koala )Phascolarctos cinerus) liver cytochrome P450 reductase

S KONG, Suong Ngo, R MCKINNON, L STUPANS

    Research output: Contribution to journalArticleResearchpeer-review

    Abstract

    The cloning, expression and characterization of hepatic NADPH-cytochrome P450 reductase (CPR) from koala (Phascolarctos cinereus) is described. Two 2059�bp koala liver CPR cDNAs, designated CPR1 and CPR2, were cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CPR cDNAs encode proteins of 678 amino acids and share 85% amino acid sequence identity to human CPR. Transfection of the koala CPR cDNAs into Cos-7 cells resulted in the expression of proteins, which were recognized by a goat-antihuman CPR antibody. The koala CPR1 and 2 cDNA-expressed enzymes catalysed cytochrome c reductase at the rates of 4.9 � 0.5 and 2.6 � 0.4�nmol/min/mg protein (mean � SD, n = 3), respectively which were comparable to that of rat CPR cDNA-expressed enzyme. The apparent Km value for CPR activity in koala liver microsomes was 11.61 � 6.01�?M, which is consistent with that reported for rat CPR enzyme. Northern analysis detected a CPR mRNA band of approximately 2.6�kb. Southern analysis suggested a single PCR gene across species. The present study provides primary molecular data regarding koala CPR1 and CPR2 genes in this unique marsupial species. � 2009 Elsevier Inc. All rights reserved.
    Original languageEnglish
    Pages (from-to)1-9
    Number of pages9
    JournalComparative Biochemistry and Physiology, Part C
    Volume150
    Issue number1
    Publication statusPublished - 2009

    Fingerprint

    Phascolarctidae
    NADPH-Ferrihemoprotein Reductase
    Cloning
    Liver
    Organism Cloning
    Complementary DNA
    Rats
    Enzymes
    Genes
    Cytochrome Reductases
    Amino Acids
    Marsupialia
    Polymerase Chain Reaction
    Proteins
    Polymerase chain reaction
    Liver Microsomes
    Transcription
    Cytochromes c
    Goats
    Cytochrome P-450 Enzyme System

    Cite this

    KONG, S ; Ngo, Suong ; MCKINNON, R ; STUPANS, L. / Cloning and expression of Koala )Phascolarctos cinerus) liver cytochrome P450 reductase. In: Comparative Biochemistry and Physiology, Part C. 2009 ; Vol. 150, No. 1. pp. 1-9.
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    Cloning and expression of Koala )Phascolarctos cinerus) liver cytochrome P450 reductase. / KONG, S; Ngo, Suong; MCKINNON, R; STUPANS, L.

    In: Comparative Biochemistry and Physiology, Part C, Vol. 150, No. 1, 2009, p. 1-9.

    Research output: Contribution to journalArticleResearchpeer-review

    TY - JOUR

    T1 - Cloning and expression of Koala )Phascolarctos cinerus) liver cytochrome P450 reductase

    AU - KONG, S

    AU - Ngo, Suong

    AU - MCKINNON, R

    AU - STUPANS, L

    PY - 2009

    Y1 - 2009

    N2 - The cloning, expression and characterization of hepatic NADPH-cytochrome P450 reductase (CPR) from koala (Phascolarctos cinereus) is described. Two 2059�bp koala liver CPR cDNAs, designated CPR1 and CPR2, were cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CPR cDNAs encode proteins of 678 amino acids and share 85% amino acid sequence identity to human CPR. Transfection of the koala CPR cDNAs into Cos-7 cells resulted in the expression of proteins, which were recognized by a goat-antihuman CPR antibody. The koala CPR1 and 2 cDNA-expressed enzymes catalysed cytochrome c reductase at the rates of 4.9 � 0.5 and 2.6 � 0.4�nmol/min/mg protein (mean � SD, n = 3), respectively which were comparable to that of rat CPR cDNA-expressed enzyme. The apparent Km value for CPR activity in koala liver microsomes was 11.61 � 6.01�?M, which is consistent with that reported for rat CPR enzyme. Northern analysis detected a CPR mRNA band of approximately 2.6�kb. Southern analysis suggested a single PCR gene across species. The present study provides primary molecular data regarding koala CPR1 and CPR2 genes in this unique marsupial species. � 2009 Elsevier Inc. All rights reserved.

    AB - The cloning, expression and characterization of hepatic NADPH-cytochrome P450 reductase (CPR) from koala (Phascolarctos cinereus) is described. Two 2059�bp koala liver CPR cDNAs, designated CPR1 and CPR2, were cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CPR cDNAs encode proteins of 678 amino acids and share 85% amino acid sequence identity to human CPR. Transfection of the koala CPR cDNAs into Cos-7 cells resulted in the expression of proteins, which were recognized by a goat-antihuman CPR antibody. The koala CPR1 and 2 cDNA-expressed enzymes catalysed cytochrome c reductase at the rates of 4.9 � 0.5 and 2.6 � 0.4�nmol/min/mg protein (mean � SD, n = 3), respectively which were comparable to that of rat CPR cDNA-expressed enzyme. The apparent Km value for CPR activity in koala liver microsomes was 11.61 � 6.01�?M, which is consistent with that reported for rat CPR enzyme. Northern analysis detected a CPR mRNA band of approximately 2.6�kb. Southern analysis suggested a single PCR gene across species. The present study provides primary molecular data regarding koala CPR1 and CPR2 genes in this unique marsupial species. � 2009 Elsevier Inc. All rights reserved.

    KW - amino acid

    KW - antibody

    KW - complementary DNA

    KW - messenger RNA

    KW - reduced nicotinamide adenine dinucleotide phosphate ferrihemoprotein reductase

    KW - amino acid sequence

    KW - animal cell

    KW - animal experiment

    KW - animal tissue

    KW - article

    KW - catalysis

    KW - cell strain COS7

    KW - controlled study

    KW - CPR1 gene

    KW - CPR2 gene

    KW - enzyme activity

    KW - gene

    KW - gene amplification

    KW - genetic transfection

    KW - human

    KW - human tissue

    KW - koala

    KW - liver

    KW - liver microsome

    KW - male

    KW - molecular cloning

    KW - nonhuman

    KW - Northern blotting

    KW - nucleotide sequence

    KW - priority journal

    KW - protein expression

    KW - reverse transcription polymerase chain reaction

    KW - sequence homology

    KW - Southern blotting

    KW - Amino Acid Sequence

    KW - Animals

    KW - Base Sequence

    KW - Cloning, Molecular

    KW - Gene Expression Regulation, Enzymologic

    KW - Humans

    KW - Macropodidae

    KW - Male

    KW - Microsomes, Liver

    KW - Molecular Sequence Data

    KW - NADPH-Ferrihemoprotein Reductase

    KW - Phascolarctidae

    KW - Rats

    KW - Rats, Wistar

    KW - Capra hircus

    KW - Metatheria

    KW - Phascolarctos cinereus

    KW - Rattus

    M3 - Article

    VL - 150

    SP - 1

    EP - 9

    JO - Comparative Biochemistry and Physiology, Part C

    JF - Comparative Biochemistry and Physiology, Part C

    SN - 1532-0456

    IS - 1

    ER -