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E F Johnson

Publications and source records attributed to E F Johnson.

At least 55 records · Page 3Linked to original sources
PubMed1994

Differential effects of mutations in substrate recognition site 1 of cytochrome P450 2C2 on lauric acid and progesterone hydroxylation.

Mutations at amino acid positions 107-120, which are part of a predicted substrate recognition site [Gotoh, O. (1992) J. Biol. Chem. 267, 83-90], were analyzed in C2MstC1, a chimera of P450 2C2 and P450 2C1. This hybrid protein has a new activity for progesterone C21-hydroxylation in addition to the lauric acid (omega-1)hydroxylase activity present in both parent proteins. Various substitutions for highly conserved glycines at positions 111 and 117 and tryptophan at position 120 strongly decreased the lauric acid hydroxylase activity of P450 2C2 and C2MstC1 and the progesterone hydroxylase activity of C2MstC1. Activities of mutant proteins with substitutions at 107, 108, and 112-115 were also strongly reduced. Modest or no decreases in activity were observed for substitutions at 109, 110, 116, 118, and 119. Lauric acid hydroxylase activity decreased more in most C2MstC1 mutants than in those of P450 2C2, particularly at positions 107 and 108. A substitution of phenylalanine for valine-112 reduced progesterone hydroxylation by 30-fold while only moderately reducing lauric acid hydroxylase by 40%. This differential effect on two dissimilar substrates demonstrates the importance of residue 112 for substrate interactions. The results are consistent with a model in which residues 107-110 align with the B'-helix of the bacterial proteins P450cam and P450BM-3. This helix is followed by a substrate-contacting loop from 111 to 116, and residues 117-120 align with the C-helices of the bacterial proteins In this alignment, Trp-120 is positioned behind the heme such that it could participate in electron transfer from reductase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Large unresponsive zones appear in cat somatosensory cortex immediately after ulnar nerve cut.

The organization of the primary somatosensory cortex innervated by the ulnar nerve was studied before and immediately after ulnar nerve transection in 11 cats electrophysiologically mapped under Nembutal or Ketamine anesthesia. The cortex was reexamined a second time beginning 42 hr after nerve transection in four cats anesthetized with Nembutal. One additional sham-operated control was also mapped. The region of cortex formerly served by the ulnar nerve remained largely unresponsive to somatic stimulation independent of the type of anesthetic used during recording. Nonetheless, animals anesthetized with Ketamine had more new responsive sites in deafferented cortex following nerve cut than cats anesthetized with Nembutal. New responses, when observed, were evoked by stimulation of a region of skin adjacent to the region served by the ulnar nerve. These findings suggest that the immediate response to deafferentation of somatosensory cortex is a limited acquisition of novel responses restricted to a region immediately adjacent to cortex containing normal afferent input.

Anesthesia↗
PubMed1993

Expression of rabbit cytochromes P4504A which catalyze the omega-hydroxylation of arachidonic acid, fatty acids, and prostaglandins.

The omega-hydroxylation product of arachidonic acid is thought to be a potent vasoconstrictor or a precursor thereof in kidney. In this report, we have measured the capacity of four rabbit CYP4A enzymes, each expressed in COS-1 cells, to catalyze the omega-hydroxylation of arachidonic acid. These rates were compared to those obtained for other substrates such as lauric acid, palmitic acid, and prostaglandins PGE1 and PGA1. With the exception of P4504A5, all of the enzymes tested exhibited relatively high rates for the omega-hydroxylation of arachidonic acid. P4504A5 showed very little activity toward arachidonic or palmitic acids as compared to that toward lauric acid (< 10%). In contrast, P4504A6 and P4504A7 catalyzed the omega-hydroxylation of arachidonic acid at rates that were roughly 50% of that observed for lauric acid. P4504A4 was not active toward lauric acid, but it also catalyzed the omega-hydroxylation of arachidonic acid at a rate that was roughly 20% of that exhibited for PGE1. Thus, each enzyme exhibits a distinct substrate specificity profile across this panel of substrates. A sensitive RNase protection assay was used to provide a more quantitative estimate of the relative abundance of mRNAs encoding P4504A5, P4504A6, and P4504A7 in liver and kidney from control, pregnant, and clofibrate-treated animals. CYP4A5 is the most abundant of the mRNAs, but it was not induced in kidney and only moderately (2-fold) in liver by clofibric acid. CYP4A7 exhibits a similar pattern of induction by clofibrate. In contrast, CYP4A6 is induced 12-fold in liver and 6-fold in kidney. The higher induction ratio largely reflects a lower basal level of expression for CYP4A6 than for CYP4A7 and CYP4A5. Following treatment with clofibrate, the amount of CYP4A6 mRNA is similar to those of CYP4A5 and CYP4A7. Pregnancy did not affect the expression of CYP4A5, CYP4A6, or CYP4A7, although it induced the expression of CYP4A4 to detectable levels in the liver and kidney, where it is not normally found in nonpregnant animals. Our results indicate that the enzyme whose mRNA is most highly induced by clofibric acid (P4504A6) and the enzyme selectively elevated during pregnancy (P4504A4) both exhibit relatively high rates for the omega-hydroxylation of arachidonic acid.

Animals↗
PubMed1993

Hydrophobic side chain requirements for lauric acid and progesterone hydroxylation at amino acid 113 in cytochrome P450 2C2, a potential determinant of substrate specificity.

To determine the requirements for hydrophobic amino acids at position 113 in cytochrome P450 2C2, a series of hydrophobic and uncharged polar amino acids was substituted for isoleucine in P450 2C2 and in C2MstC1, a chimera of P450 2C2 and P450 2C1. Lauric acid hydroxylase activity was determined in COS1 cells transfected with P450 2C2 mutants and both lauric acid and progesterone hydroxylase activities were determined for C2MstC1 variants. In P450 2C2, 40 to 120% of the wild type (omega-1) lauric acid hydroxylase activity was retained in all hydrophobic mutants, but activity was reduced to near background by substitutions of the polar amino acids, tyrosine and cysteine. Likewise, in C2MstC1 mutants, hydrophobic substitutions were 20 to 50% as active as wild type for lauric acid hydroxylation, and polar amino acids again resulted in strong reductions of activity. In contrast, a different pattern of activity for progesterone C21-hydroxylase was observed for C2MstC1 mutants. A valine substitution had a modest effect on activity but substitutions of leucine and alanine reduced progesterone C21-hydroxylase activity 5- to 7-fold, respectively, and the large hydrophobic amino acid, phenylalanine, reduced activity about 30-fold. No changes in the regiospecificity of progesterone hydroxylation were observed for any of the mutants. Similar steady-state levels of immunoprecipitated, radiolabeled protein were observed for each mutant except for the glycine substitution which resulted in degradation of the protein. The different patterns of the effects of the mutations on progesterone and lauric acid hydroxylase activity provide additional support for the critical role of residue 113 in substrate recognition. The low activities in mutant proteins with hydrophilic amino acid substitutions indicate that the hydrophobic nature of this residue is important. The hydrophobic requirements are more stringent for a larger, more rigid steroid substrate than for a saturated fatty acid with a flexible hydrocarbon tail.

Amino Acid Sequence↗
PubMed1993

Cassette mutagenesis of a potential substrate recognition region of cytochrome P450 2C2.

Cassette mutagenesis was used to analyze the effects of mutations at amino acid positions 107-120 in cytochrome P450 2C2 which are part of a predicted substrate recognition site, SRS-1 (Gotoh, O. (1992) J. Biol. Chem. 267, 83-90), and terminate with a highly conserved tryptophan. All the mutant enzymes were expressed in COS1 cells at approximately the same level as wild type as determined by immunoprecipitation of radiolabeled products. Substitutions of amino acids from 107 to 110 and from 116 to 119 resulted in mutant enzymes that retained substantial lauric acid hydroxylase activity suggesting that these residues are not critical determinants of substrate specificity. In contrast, amino acids 112-115 were characterized by strong decreases in activity in at least one mutation tested. A substitution of phenylalanine for valine 112 reduced activity about 3-fold. At position 113, substitution of leucine for isoleucine had little effect, but activity was reduced 15-fold by substitution with cysteine. A conservative mutation at position 114 reduced activity 5-fold, a nonconservative mutation at position 115 resulted in a 30-fold reduction of lauric acid hydroxylation. Substitution of glutamic acid for glycine at position 109 had little effect on activity, while more conservative substitutions of valine for glycines at positions 111 and 117 resulted in about 50- and 7-fold reductions of activity, respectively. Substitution of leucine for tryptophan 120 resulted in a 7-fold reduction of activity indicating that tryptophan at this position contributes to, but is not essential for, enzymatic activity in spite of its presence in nearly all eukaryotic cytochromes P450. The results are consistent with a model in which amino acids 112-114, and possibly 115, align with a loop of bacterial P450cam sequence containing substrate contacting amino acids, tyrosine 96 and phenylalanine 98. Glycines at either end of the loop in cytochrome P450 2C2 appear to be critical for activity.

Amino Acid Sequence↗
PubMed1993

A single amino acid substitution confers progesterone 6 beta-hydroxylase activity to rabbit cytochrome P450 2C3.

A cDNA encoding a naturally occurring variant of cytochrome P450 (P450) 2C3 that catalyzes the 6 beta- and 16 alpha-hydroxylation of progesterone exhibits six differences of nucleotide sequence leading to five amino acid substitutions from that encoding 2C3, a progesterone 16 alpha-hydroxylase that does not catalyze 6 beta-hydroxylation. Analysis of chimeric and mutant enzymes indicates that a Ser/Thr difference at position 364 underlies the difference between the two enzymes in 6 beta-hydroxylase activity as well as sensitivity to the inhibitor, 16 alpha-methylprogesterone. In addition, an Ile/Met difference at position 178 influences the apparent Km for progesterone. The two mutations, S364T and 1178M, together convert 2C3 to a form that exhibits kinetic properties which are similar to the 2C3v enzyme, and the reciprocal mutations in 2C3v convert it to an enzyme that resembles 2C3. Interestingly, position 364 of 2C3 maps to a substrate-contacting domain suggested by models for mammalian P450 enzymes based on the structure of P450cam. Ile178 is highly conserved among mammalian microsomal P450s with the exception of CYP4A and CYP19 enzymes which exhibit a Met at this alignment position.

Amino Acids↗
PubMed1993

Characterization of a cDNA encoding a human kidney, cytochrome P-450 4A fatty acid omega-hydroxylase and the cognate enzyme expressed in Escherichia coli.

A cDNA encoding a cytochrome P-450 4A (CYP4AII) was cloned from a human kidney cDNA library. Northern blot analysis and RNase protection assays indicate that related mRNAs occur in kidney and liver with the highest abundance found in kidney. The enzyme was expressed from its cDNA in Escherichia coli. A solubilized preparation of the enzyme reconstituted with cytochrome P-450 reductase catalyzed the omega-hydroxylation of lauric acid, palmitic acid, and arachidonic acid with turnover numbers of 9.8, 2.2 and 0.55 min-1, respectively. Little or no activity was detected toward prostaglandins A1 and E1.

Amino Acid Sequence↗
PubMed1993

Rabbit prostaglandin omega-hydroxylase (CYP4A4): gene structure and expression.

Genomic DNA containing the entire gene encoding P450 4A4, a cytochrome P450 that is elevated during pregnancy, was isolated on two overlapping recombinant phage. The isolated gene, CYP4A4, encodes a protein that differs at one amino acid position from the predicted sequence of a previously isolated cDNA. The intron/exon structure is highly conserved in relation to the clofibrate inducible genes CYP4A1, CYP4A2, and CYP4A6. However, the 5' flanking sequences of these genes exhibit little identity. Two transcriptional start sites of the CYP4A4 gene have been determined by RNase protection analysis and are located 37 and 40 nucleotides upstream of the initiation codon. Like other CYP4A genes the CYP4A4 promoter does not contain a TATA consensus sequence. CYP4A4 mRNAs are not detected in the lungs, liver, and kidneys of untreated rabbits by RNase protection assays. However, CYP4A4 mRNA is present to varying degrees in all three of these tissues during pregnancy with the greatest abundance observed in the lung and the lowest in the kidney. Treatment of male rabbits with dexamethasone also increases the levels of CYP4A4 mRNA in the lung and liver, but the levels are eightfold less than those seen for pregnant rabbits. Consensus recognition sequences for either the glucocorticoid or progesterone receptors were not found in 1086 bp of sequence upstream of the start of transcription.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of recombinant-expressed cytochrome P450 2C3 from Escherichia coli: 2C3 encodes the 6 beta-hydroxylase deficient form of P450 3b.

Rabbit cytochrome P450 2C3 was expressed from its cDNA in Escherichia coli as a chimeric enzyme in which a portion of the N-terminal membrane anchor sequence of 2C3 was replaced with a modified sequence derived from P450 17 alpha. The nucleotide sequence encoding the N-terminus of P450 17 alpha was modified previously to achieve a high level of expression of P450 17 alpha in E. coli by altering the first eight codons of P450 17 alpha to reflect second codon preferences for high expression and to minimize the potential for the formation of a stable secondary structure of the corresponding RNA transcript. The modified P450 2C3 was expressed at > 400 nmol/liter of culture. P450 2C3 was isolated to apparent electrophoretic homogeneity and a specific content > 14 nmol P450/mg protein. When reconstituted with P450 reductase and dilauroyl-L-alpha-lecithin, the purified E. coli-expressed P450 2C3 catalyzed 16 alpha, but not 6 beta-hydroxylation of progesterone. Expression of unmodified 2C3 from its cDNA in COS-1 cells confirmed the absence of detectable 6 beta-hydroxylase activity. In addition, the enzyme expressed in E. coli is activated by the allosteric effector 5 beta-pregnane-3 beta,20 alpha-diol, with a resultant Vmax = 10 min-1 and Km = 20 microM and is not inhibited by 16 alpha-methylprogesterone. These results indicate that the 2C3 cDNA encodes an enzymatic form characteristic of IIIvo/J and B/J inbred rabbits rather than a second enzymatic form expressed in most outbred and some inbred strains that catalyzes both high efficiency 16 alpha- and 6 beta-hydroxylation of progesterone. Our results have identified the enzyme variant encoded by the 2C3 cDNA and have demonstrated the utility of E. coli for the expression of recombinant P450 enzymes.

Amino Acid Sequence↗
PubMed1992

The peroxisome proliferator-activated receptor mediates the induction of CYP4A6, a cytochrome P450 fatty acid omega-hydroxylase, by clofibric acid.

Gene transfer experiments indicate that induction by the peroxisome proliferators, clofibric acid and WY-14,643, of luciferase expression driven by the promoter and 5'-flanking sequences of the rabbit cytochrome P450 4A6 gene (CYP4A6) is dependent on cotransfection of expression plasmids for the peroxisome proliferator-activated receptor, PPAR. Activation by PPAR is observed in the absence of the inducers. However, a mutant, PPAR-G (Glu282----Gly) activated luciferase expression only in the presence of peroxisome proliferators. Deletion analysis has localized a response element to a 34-base pair segment located 677 base pairs upstream of the CYP4A6 transcription start site that is similar to an element that regulates the response of the rat fatty acyl-CoA oxidase gene to peroxisome proliferators and that binds PPAR.

Animals↗
PubMed1992

Analysis of the catalytic specificity of cytochrome P450 enzymes through site-directed mutagenesis.

The way in which structural diversity encodes the capacity of individual P450 enzymes to metabolize multiple, structurally distinct substrates remains largely unknown. The tools of molecular biology provide a means of identifying amino acid residues among closely related P450s that are determinants of their distinct catalytic properties. Work in our laboratory has identified two substrate specificity-determining segments of the amino acid sequences of subfamily 2C P450s. A pattern has emerged from this work, and that of others, which suggests a model for the structural basis of P450 catalytic diversity.

Amino Acid Sequence↗
PubMed1992

Characterization of a rabbit gene encoding a clofibrate-inducible fatty acid omega-hydroxylase: CYP4A6.

CYP4A6 mRNAs are induced in the rabbit liver and kidney following treatment with the antihyperlipidemic drug clofibrate. As a first step toward the elucidation of the mechanism controlling the induction of this and other CYP4A genes by clofibrate and other peroxisome proliferators, we have cloned and characterized the CYP4A6 gene. Genomic DNA containing the first 12 exons encoding CYP4A6 was isolated as three recombinant lambda phage, two of which were overlapping. The sequence of more than 1000 bp of the 5' upstream region as well as of the first 12 exons has been determined. These 12 exons encode all but approximately 80 bp at the 3' terminus of CYP4A6. Intron/exon junctions within the coding region of the gene are conserved relative to the rat CYP4A1 and CYP4A2 genes. Primer extension analysis indicates that transcription is initiated 33 bp upstream of the start codon. The CYP4A6 promoter region, like that of the rat CYP4A1 and CYP4A2 genes, does not contain a consensus TATA box. However, a consensus Sp1 recognition element is apparent at -46 bp upstream of the transcription start site. In addition, a sequence related to one of two regulatory elements that control the induction of the rat acyl-CoA oxidase gene by ciprofibrate is present upstream of the CYP4A6 promoter.

Acyl-CoA Oxidase↗
PubMed1992

Mapping determinants of the substrate selectivities of P450 enzymes by site-directed mutagenesis.

Point-mutation studies in cytochrome P450s by site-directed mutagenesis have identified key residues that can confer the catalytic properties of one cytochrome P450 onto another. Most of these key residues cluster at sites that map to amino acids forming the substrate-binding site of P450cam, a distantly related enzyme. These sites are found on topological elements of P450cam, which by their surface location and lack of extensive secondary structure are likely to permit genetic variation without extensive disruption of the overall topology of the enzyme. If these topological features of P450cam are conserved in the mammalian enzymes, they are likely to accommodate the structural diversity seen for mammalian P450s in a manner that conserves a basic structure for P450 enzymes but that leads to the catalytic diversity seen for the mammalian enzymes.

Animals↗
PubMed1991

A hypervariable region of P450IIC5 confers progesterone 21-hydroxylase activity to P450IIC1.

Cytochrome P450IIC5 is a hepatic progesterone 21-hydroxylase while the 95% identical P450IIC4 has a greater than 10-fold higher Km for progesterone 21-hydroxylation and the 74% identical P450IIC1 does not hydroxylate progesterone at detectable rates. Previous work demonstrated that the apparent Km of P450IIC4 for progesterone 21-hydroxylation can be markedly improved by replacing a valine at position 113 with an alanine which is present at this position in P450IIC5. In the present studies, a single point mutation in cytochrome P450IIC1 that changed valine at position 113 to alanine conferred progesterone 21-hydroxylase activity to this enzyme. Although the catalytic activity was less than that of P450IIC5, these results indicate the residue 113 plays a critical role in the determination of the substrate/product selectivity in subfamily IIC P450s. By alignment with the sequence of P450cam, the segment of the polypeptide, residues 95-123, containing residue 113 corresponds to a substrate-contacting loop in the bacterial enzyme. The region containing residue 113, which is highly variable among family II P450s, may also be a substrate-contacting loop in the mammalian cytochromes P450. The exchange of this hypervariable region of cytochrome P450IIC1, residues 95-123, with that of P450IIC5 enhanced the 21-hydroxylase activity of the cells transfected with this chimera to levels similar to those of cells transfected with the plasmid encoding P450IIC5. Kinetic analysis of microsomes isolated from the transfected cells showed that the apparent Km for progesterone 21-hydroxylation of the chimera was indistinguishable from that of P450IIC5.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

An inhibitory monoclonal antibody binds in close proximity to a determinant for substrate binding in cytochrome P450IIC5.

We used the expression of chimeric proteins and point mutants to identify amino acids of the hepatic progesterone 21-hydroxylase P450IIC5 which are part of an epitope recognized by an inhibitory monoclonal antibody and which affect substrate binding. Three amino acids of P450IIC5 at positions 113, 115, and 118 were introduced into P450IIC4, which is 95% identical to P450IIC5. The resultant chimeric protein acquired binding of the monoclonal antibody 1F11, which is highly specific and inhibitory for P450IIC5. Point mutants in P450IIC4 showed that two of the three changes, T115S and N118K, contribute to the epitope recognized by this antibody. The T115S mutant bound the antibody weakly (Kd greater than 30 nM) whereas the N118K mutant bound the antibody as tightly as P450IIC5 (Kd less than or equal to 0.7 nM). Thus, residues 115 and 118 are located on the surface of these enzymes, and the Lys/Asn difference at amino acid 118 is largely responsible for the high degree of discrimination which this antibody exhibits between P450IIC5 and P450IIC4. The valine to alanine mutation at position 113 conferred to P450IIC4 a lower apparent Km for progesterone 21-hydroxylation. Because antibody binding was not affected by this mutation, it is tempting to speculate that this residue is buried in the protein where it exerts its effect on the catalytic activity by interaction with the substrate or alters the positions of residues of the active site. The close proximity of the epitope at positions 115 and 118 to Ala113 suggests that the inhibitory monoclonal antibody interferes with substrate binding.

Alanine↗
PubMed1991

Identification of human cytochrome P450s as autoantigens.

Antimicrosomal antibodies in inflammatory liver diseases all seem to be directed against members of the cytochrome P450 family of proteins. These autoantigens seem to be genetically polymorphic, the autoantibodies are inhibitory, and the autoepitopes are generally conserved among species. Anti-P450 autoantibodies share these characteristics with other autoantibodies, for example, antinuclear antibodies in systemic lupus erythematosus. The identification of P450s as human autoantigens is clinically important. Diagnostic tests will be developed on the basis of cloned antigen, facilitating a better diagnosis of drug-induced and idiopathic autoimmune hepatitis. It is unknown what triggers autoantibody production against cytochrome P450 proteins. Furthermore, their pathogenetic role and thus their involvement in tissue destruction is unclear. In this context LKM1 autoantibodies may serve as a model. Although LKM1 antibodies are inhibitory, all LKM1 antibody-positive patients tested so far are extensive metabolizers for drug metabolism mediated by P450IID6 and express this protein in their livers. Thus, the inhibitory LKM1 autoantibody does not sufficiently penetrate through the intact liver cell membrane to inhibit enzyme function in vivo. Presumably, tissue destruction in autoimmune hepatitis is mediated by liver-infiltrating T lymphocytes. T lymphocytes have been cloned from liver tissue that specifically proliferate in the presence of recombinant cytochrome P450IID6. The construction of overlapping cDNA subclones is also valuable to identify immunodominant B cell as well as relevant T cell epitopes.

Autoantibodies↗