Cloning, sequencing, and cDNA-directed expression of the rat renal CYP4A2: Arachidonic acid ω-hydroxylation and 11,12-epoxidation by CYP4A2 protein

Mong-Heng Wang, David E. Stec, Michael Balazy, Vladimir Mastyugin, Chung S. Yang, Richard J. Roman, Michal Laniado Schwartzman

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Abstract

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), the ω- hydroxylation product of arachidonic acid, is the major metabolite produced in the kidney. It has potent biological effects on renal tubular and vascular functions and on the long-term control of arterial pressure. The synthesis of 20-HETE is catalyzed by enzymes of the CYP4A family, among which CYP4A2 is the most abundant isozyme expressed in the kidneys of rats. We have cloned and sequenced the CYP4A2 cDNA from the kidney of Lewis-Wistar rats and directed its expression using baculovirus and Sf9 insect cells. A high level of expression of CYP4A2 was evident by Northern, Western, and spectral analyses revealing a P450 content of 0.3 nmol/mg microsomal protein. To study CYP4A2-catalyzed arachidonic acid ω-hydroxylation, SP9 cells were coinfected with CYP4A2 and NADPH cytochrome P450 oxidoreductase (OR) recombinant viruses. CYP4A2/OR membranes metabolized lauric acid at a high rate (7 and 5.5 nmol/min/nmol P450 in the presence and absence of b5, respectively). However, arachidonic acid ω-hydroxylase activity was barely detectable. When purified OR was added to the membranes expressing CYP4A2 protein, a concentration-dependent production of 20-HETE was observed. Maximal synthesis of 20-HETE of 0.89 nmol/min/nmol P450 was achieved at OR:CYP4A2 ratio of 14:1. The ω-hydroxylation of arachidonic acid was dependent on the presence of b5. Furthermore, increasing OR concentrations yielded additional arachidonic acid metabolite identified by GC/MS as 11,12-EET. Microsomes prepared from isolated renal microvessels selectively expressed CYP4A2 protein and readily metabolized arachidonic acid to two major metabolites, 20-HETE and 11,12-DHET, the hydrolytic metabolite of 11,12-EET. It is suggested that CYP4A2 functions as the renal microvessel arachidonate ω- hydroxylase and that it can also catalyze the 11,12-epoxidation of arachidonic acid.

Original languageEnglish (US)
Pages (from-to)240-250
Number of pages11
JournalArchives of Biochemistry and Biophysics
Volume336
Issue number2
DOIs
StatePublished - Dec 15 1996

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Hydroxylation
Epoxidation
Cloning
Arachidonic Acid
Rats
Organism Cloning
Complementary DNA
Kidney
Metabolites
Proteins
Oxidoreductases
lauric acid
Mixed Function Oxygenases
Microvessels
Cytochrome P-450 CYP4A
cytochrome P-450 CYP4A2 (rat)
Membranes
Sf9 Cells
NADPH-Ferrihemoprotein Reductase
Baculoviridae

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Cloning, sequencing, and cDNA-directed expression of the rat renal CYP4A2 : Arachidonic acid ω-hydroxylation and 11,12-epoxidation by CYP4A2 protein. / Wang, Mong-Heng; Stec, David E.; Balazy, Michael; Mastyugin, Vladimir; Yang, Chung S.; Roman, Richard J.; Laniado Schwartzman, Michal.

In: Archives of Biochemistry and Biophysics, Vol. 336, No. 2, 15.12.1996, p. 240-250.

Research output: Contribution to journalArticle

Wang, Mong-Heng ; Stec, David E. ; Balazy, Michael ; Mastyugin, Vladimir ; Yang, Chung S. ; Roman, Richard J. ; Laniado Schwartzman, Michal. / Cloning, sequencing, and cDNA-directed expression of the rat renal CYP4A2 : Arachidonic acid ω-hydroxylation and 11,12-epoxidation by CYP4A2 protein. In: Archives of Biochemistry and Biophysics. 1996 ; Vol. 336, No. 2. pp. 240-250.
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abstract = "20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), the ω- hydroxylation product of arachidonic acid, is the major metabolite produced in the kidney. It has potent biological effects on renal tubular and vascular functions and on the long-term control of arterial pressure. The synthesis of 20-HETE is catalyzed by enzymes of the CYP4A family, among which CYP4A2 is the most abundant isozyme expressed in the kidneys of rats. We have cloned and sequenced the CYP4A2 cDNA from the kidney of Lewis-Wistar rats and directed its expression using baculovirus and Sf9 insect cells. A high level of expression of CYP4A2 was evident by Northern, Western, and spectral analyses revealing a P450 content of 0.3 nmol/mg microsomal protein. To study CYP4A2-catalyzed arachidonic acid ω-hydroxylation, SP9 cells were coinfected with CYP4A2 and NADPH cytochrome P450 oxidoreductase (OR) recombinant viruses. CYP4A2/OR membranes metabolized lauric acid at a high rate (7 and 5.5 nmol/min/nmol P450 in the presence and absence of b5, respectively). However, arachidonic acid ω-hydroxylase activity was barely detectable. When purified OR was added to the membranes expressing CYP4A2 protein, a concentration-dependent production of 20-HETE was observed. Maximal synthesis of 20-HETE of 0.89 nmol/min/nmol P450 was achieved at OR:CYP4A2 ratio of 14:1. The ω-hydroxylation of arachidonic acid was dependent on the presence of b5. Furthermore, increasing OR concentrations yielded additional arachidonic acid metabolite identified by GC/MS as 11,12-EET. Microsomes prepared from isolated renal microvessels selectively expressed CYP4A2 protein and readily metabolized arachidonic acid to two major metabolites, 20-HETE and 11,12-DHET, the hydrolytic metabolite of 11,12-EET. It is suggested that CYP4A2 functions as the renal microvessel arachidonate ω- hydroxylase and that it can also catalyze the 11,12-epoxidation of arachidonic acid.",
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AU - Balazy, Michael

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AU - Roman, Richard J.

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N2 - 20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), the ω- hydroxylation product of arachidonic acid, is the major metabolite produced in the kidney. It has potent biological effects on renal tubular and vascular functions and on the long-term control of arterial pressure. The synthesis of 20-HETE is catalyzed by enzymes of the CYP4A family, among which CYP4A2 is the most abundant isozyme expressed in the kidneys of rats. We have cloned and sequenced the CYP4A2 cDNA from the kidney of Lewis-Wistar rats and directed its expression using baculovirus and Sf9 insect cells. A high level of expression of CYP4A2 was evident by Northern, Western, and spectral analyses revealing a P450 content of 0.3 nmol/mg microsomal protein. To study CYP4A2-catalyzed arachidonic acid ω-hydroxylation, SP9 cells were coinfected with CYP4A2 and NADPH cytochrome P450 oxidoreductase (OR) recombinant viruses. CYP4A2/OR membranes metabolized lauric acid at a high rate (7 and 5.5 nmol/min/nmol P450 in the presence and absence of b5, respectively). However, arachidonic acid ω-hydroxylase activity was barely detectable. When purified OR was added to the membranes expressing CYP4A2 protein, a concentration-dependent production of 20-HETE was observed. Maximal synthesis of 20-HETE of 0.89 nmol/min/nmol P450 was achieved at OR:CYP4A2 ratio of 14:1. The ω-hydroxylation of arachidonic acid was dependent on the presence of b5. Furthermore, increasing OR concentrations yielded additional arachidonic acid metabolite identified by GC/MS as 11,12-EET. Microsomes prepared from isolated renal microvessels selectively expressed CYP4A2 protein and readily metabolized arachidonic acid to two major metabolites, 20-HETE and 11,12-DHET, the hydrolytic metabolite of 11,12-EET. It is suggested that CYP4A2 functions as the renal microvessel arachidonate ω- hydroxylase and that it can also catalyze the 11,12-epoxidation of arachidonic acid.

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