Biomedical subjects

B Kemper

Publications and source records attributed to B Kemper.

At least 55 records · Page 3Linked to original sources

PubMed1997

Efficient assembly of functional cytochrome P450 2C2 requires a spacer sequence between the N-terminal signal anchor and catalytic domains.

Cytochromes P450 (P450) are anchored to the endoplasmic reticulum membrane by an N-terminal transmembrane sequence with the catalytic domain facing the cytoplasmic side. Within the peptide sequence linking these two domains in P450 2C2 is a glycine-rich region from residues 22 to 28. To examine the role of this region, deletion and substitution mutations were constructed, and the activities and spectral properties were determined for the mutant proteins expressed in COS-1 cells, insect cells, and bacteria. Deletion of residues 22 to 28 or substitution of 7 valines for this region inactivated the proteins in COS-1 cells, and no P450 species was detected for these mutations in bacteria or insect cells. Substitution of the three glycine residues with alanine or proline or the entire sequence from 22 to 28 with 7 alanines did not reduce lauric acid hydroxylase activity of the proteins expressed in COS-1 cells. Reducing the number of alanines substituted to 4, 3, and 2 progressively decreased activity in COS-1 cells to undetectable levels when 2 alanines were substituted. The loss of activity in COS-1 cells correlated with decreased expression of hemoprotein with a reduced difference spectrum of 450 nm (P450 species) and a corresponding increase in the inactive P420 species in insect cells and bacteria. The activities expressed per nanomole of P450 in insect microsomes were similar for P450 2C2 and the alanine substitution mutants, including the mutant with 2 alanines which was inactive in COS-1 cells. The rates of conversion of P450 to P420 resulting from incubation at 48 degrees C in vitro were not changed sufficiently to explain the increase in expressed P420 observed for the mutants with 3 or 7 alanines substituted. These data are consistent with a role for the residue 22-28 region as a linker that facilitates the folding of P450; however, once the protein is properly folded into the functional P450 species, this region has little influence on the stability and activity of the enzyme.

Animals↗

PubMed1997

Identification of amino acids of endonuclease VII essential for binding and cleavage of cruciform DNA.

Endonuclease VII is a Holliday-structure-resolving enzyme of bacteriophage T4. The active protein is a homodimer with 157 amino acids/monomer. An amber mutation (amE727 in codon 151) inactivates the nuclease completely, indicating the importance of the seven C-terminal amino acids for nucleolytic activity. The influence of these amino acids on cruciform-DNA binding and cleavage was investigated through functional analysis of C-terminal-truncated proteins derived from deletion constructs. It was found that the three C-terminal amino acids are not necessary for binding and cleavage. A transition from active to inactive protein occurs gradually with truncations of the next four amino acids. Reduction of DNA-binding ability, as measured by electrophoretic mobility shift assays, was determined to be the primary defect in the cleavage-deficient proteins. This was further concluded by the finding that EVII-(1-150)-peptide(amber), a protein with fairly low affinity to cruciform DNA, contributes cleavage activity to reactions of wild-type EVII with cruciform DNA. [Asp62]EVII-(1-156)-peptide lacking one C-terminal amino acid, contains a point mutation in codon 62 that eliminates the nucleolytic activity of the protein while retaining its DNA-binding proficiency. By mixing binding-deficient and cleavage-deficient mutants in the same assay, cleavage of cruciform DNA resumed. Evidence is presented that complementation occurs by heterodimer formation. Our results show that the zinc-binding motif of EVII is not sufficient for cruciform-DNA binding.

Amino Acids↗

PubMed1997

Inhibition of Holliday structure resolving endonuclease VII of bacteriophage T4 by recombination enzymes UvsX and UvsY.

Proteins UvsX, UvsY and Endonuclease VII (Endo VII) of bacteriophage T4 are required for DNA recombination, replication and repair. Endo VII is the product of gene 49 (gp49) and essential for resolution of branches from newly made DNA, prior to packaging into preformed heads. The ability of Endo VII to resolve Holliday structures in vitro suggested an in vivo function for the resolution of recombination intermediates, generated by UvsX and UvsY during the early infection cycle. Here we report results which contrast with this hypothesis. It is shown that the potent endonucleolytic activity of Endo VII with branched DNAs is inhibited in strand transfer reactions by the strand transferase UvsX, and more strongly by the accessory protein UvsY in vitro. The inhibitory effect of UvsX or UvsY is also seen in reactions with Endo VII using two synthetic cruciform DNAs and a C/C-mismatch containing substrate. Low concentrations of UvsY protein (12 ng or 0,76 pmol) were sufficient to reduce the cleavage efficiency of 30 units of Endo VII (about 16 fmol) to 50%. The inhibition is due to a direct protein-protein interaction between Endo VII, UvsX and UvsY as suggested by electrophoretic mobility shift assays (EMSAs). These results were confirmed through affinity chromatography, where UvsX and UvsY bound to Endo VII, immobilized on a NHS-activated Sepharose matrix. This is the first identification of phage-encoded proteins which modulate the potent endonucleolytic activity of gp49 in vitro.

Bacteriophage T4↗

PubMed1996

Different structural requirements at specific proline residue positions in the conserved proline-rich region of cytochrome P450 2C2.

Cytochrome P450 is anchored to the endoplasmic reticulum membrane by an N-terminal transmembrane sequence with the catalytic domain facing the cytoplasmic side. Within the peptide sequence linking these two domains is a highly conserved proline-rich region. In cytochrome P450 2C2, this region has the sequence 30PPGPTPFP37. To examine the structural requirements at these proline residues, each proline was replaced with alanine, glycine, valine, or an acidic amino acid, and the activities of the mutated proteins were determined in transfected COS-1 cells. Lauric acid 1omega-hydroxylase activities of Pro30 and Pro33 mutants were less than 10% of wild type for each substitution except for alanine, which was 25-30%. In striking contrast, substitutions at Pro31, including an acidic residue, did not substantially alter activity. At positions 35 and 37, acidic amino acid substitutions reduced activity to less than 10% of wild type while substitution of the other three amino acids had little effect. The tolerance of substitutions of charged residues at Pro31 suggests that the side chain at this position is exposed to a polar environment; conversely, the reduced activity with charged substitutions, but not with uncharged substitutions at positions 35 and 37, suggests that these residues are exposed to a hydrophobic environment, presumably within the folded protein. The loss of activity with substitutions at Pro30 and Pro33 implies that the motif PXXP is important for the formation of a functional cytochrome P450 and that this sequence might have a helical structure with a repeat of three, as in the left-handed poly-L-proline II helix. Insertion of alanine between positions 29 and 30 did not substantially affect activity, but insertions between either 33 and 34 or 37 and 38 resulted in activity less than 25% of wild type. These data indicate that the position of PXXP, relative to the sequence flanking it on the C-terminal side, may be important for its function.

Amino Acid Sequence↗

PubMed1996

Phenobarbital induction mediated by a distal CYP2B2 sequence in rat liver transiently transfected in situ.

The promoter activities of the genes for cytochrome P450 2B1 (CYP2B1) and cytochrome P450 2C1 (CYP2C1) have been assayed by direct injection of promoter-luciferase chimeric genes into rat liver. Activities of minimal promoters for CYP2C1 and CYP2B1 were detectable in untreated animals but were not increased by treatment of the animals with phenobarbital. After insertion to the 5' side of the minimal promoters of one to three copies of the CYP2B2 sequence from -2318 to -2155, a phenobarbital-responsive element in primary hepatocyte cultures (Trottier, E., Belzil, A., Stoltz, C., and Anderson, A. (1995) Gene (Amst.) 158, 263-268), phenobarbital treatment induced the activity of the CYP2C1 promoter by 5-15-fold and the CYP2B1 promoter by 2.5-5-fold. Mutation of a basal transcription element-like motif and a CCAAT/enhancer binding protein element in the CYP2B1 proximal promoter region reduced expression, but 3-4-fold induction by phenobarbital was retained. Mutation of the "Barbie box," a putative phenobarbital-responsive element (He, J.-S., and Fulco, A. J. (1991) J. Biol. Chem. 266, 7864-7869) in the CYP2B1 proximal promoter did not reduce the relative response to phenobarbital. These results demonstrate that direct injection of DNA into rat liver may be used to assay phenobarbital responsiveness of cytochrome P450 genes. In this system, a distal CYP2B2 element mediates a response to phenobarbital, and proximal elements, including the Barbie box, are not required for the induction.

Animals↗

PubMed1996

Bacteriophage T4 strand transfer protein UvsX tolerates symmetric and asymmetric heterologies in short double-stranded oligonucleotides.

The UvsX protein of bacteriophage T4 catalyzes strand transfer from double-stranded DNA to homologous single-stranded DNA to generate both paranemic and plectonemic joints. We demonstrate here that UvsX mediates strand transfer efficiently from synthetic double-stranded donor oligonucleotides of 30 to 117 bp in length to circular single-stranded recipient M13mp19 DNA. Recovery of a diagnostic BamHI-restriction site, activated in the recipient after strand transfer, demonstrates that recipient and donated strands are perfectly base-paired after the exchange reaction has taken place. The transfer reaction progresses with greatest efficiency using donor DNA with a 3' overhang. Use of donor DNA having recessed 3' ends or blunt ends reduces the transfer efficiency by half. Single-stranded heterologies, centrally located in either strand of the donor DNA and forming either heteroduplex loops or a bulge in the donor are transferred with 80 to 100% efficiency. Also, a centrally located C/C-mismatch in the donor does not affect the transfer efficiency. Double-stranded heterologies are tolerated by the UvsX-catalyzed reaction but have different effects on the transfer efficiencies, depending on length and location in the molecule. A heterology of 24 bp located at the proximal end (start of transfer), the distal end (termination of transfer) and at each end of the donor molecule results in transfer efficiencies of 100%, 50% and 50 to 60%, respectively. Strand transfer efficiency is markedly reduced to about 15% if the 24 bp heterology is at a central location. However, insertion of a 4 bp heterology at this position yields a transfer efficiency of about 30%. Also, large double-stranded heterologies of 187 bp at the proximal end or 590 bp at the distal end of control-donor DNAs derived from plasmid digests did not impair the transfer activity of UvsX. This result differs from published results obtained with strand transfer reactions with large distally located heterologies catalyzed by RecA of Escherichia coli in vitro.

Bacteriophage T4↗

PubMed1996

Sex- and tissue-specific expression of a cytochrome P450 2C2-luciferase transgene.

To study the transcriptional regulation of the rabbit cytochrome P450 2C2 gene (CYP2C2), transgenic mice were generated using a DNA fragment containing the CYP2C2 5'-flanking region fused to a luciferase reporter gene. In one of three transgenic lines, this transgene was expressed in the liver and at a lower level in the kidney, similar to the tissue-specific pattern of this gene in rabbits. Unlike expression in rabbits, the transgene was expressed in a male-specific pattern in liver and kidney, and in the brain of both sexes. Expression of the transgene in male mice was repressed after castration, while expression in females was stimulated after testosterone treatment and periodic injection of rat growth hormone (GH). The expression of this transgene was also regulated developmentally in the liver. These results suggest that this 3500 bp of CYP2C2 5'-flanking region contains the basal transcriptional regulation elements for tissue-specific and developmental expression. Although a position effect can not be ruled out, this transgene may also include the cis-regulatory element for sexual dimorphic expression.

Animals↗

PubMed1996

Reactions of mitochondrial cruciform cutting endonuclease 1 (CCE1) of yeast Saccharomyces cerevisiae with branched DNAs in vitro.

Cruciform-cutting endonuclease 1 (CCE1) is an X-solvase from yeast Saccharomyces cerevisiae [Kleff, S., Kemper, B. & Sternglanz, R. (1992) EMBO J. 11, 699-704]. We report here the purification of the cloned enzyme CCE1 to near homogeneity from over-expressing Escherichia coli cells. The purified protein has a globular shape and an apparent molecular mass of 38 kDa. CCE1 reacts specifically with branched DNAs, preferably with four-armed cruciforms. The enzyme linearizes native supercoiled DNA by cutting at the base of cruciform structures as they occur in derivatives of phage M13. Supercoiling was not required for cleavage per se and a relaxed circular DNA hybrid with a stable cruciform was linearized with the same relative cleavage efficiency. Fully synthetic cruciforms (four-armed X-junctions) were also good substrates for CCE1, provided a symmetric 6-bp sequence (in our case an EcoRI restriction site) was maintained at the junction. Consequently, a synthetic cruciform made from fully randomized oligonucleotide sequences was not a substrate for CCE1. In general, cleavage sites were found clustered in a characteristic pattern in each arm of a cruciform structure. A synthetic three-armed Y-junction was also cleaved by CCE1, but with a lower efficiency than the related four-armed construct. CCE1 resolves efficiently branched synthetic DNAs in vitro. The function is consistent with the idea that CCE1 is responsible for a timely reversal of branched recombination intermediates preceding petite formation in mitochondrial DNA.

Bacteriophage T4↗

PubMed1996

Detection of 81 of 81 known mouse beta-globin promoter mutations with T4 endonuclease VII--the EMC method.

The enzyme mismatch cleavage (EMC) method relies on the use of the resolvase T4 Endonuclease VII to cleave and thus detect mismatches in heteroduplex DNA formed by annealing normal DNA with mutant DNA. Detection is based on cleavage 3' to the mismatch within a few nucleotides. We report the detection of all 81 different homozygous single-basepair changes tested and present in the mouse beta-globin promoter by using the EMC method with a single set of conditions. Efficiency of cleavage was rated as strong, medium, or weak based on the intensity of the cleavage product(s) compared with background bands on autoradiography. We expect this method to detect near 100% of mutations.

Animals↗

PubMed1996

Analysis of the mammalian recombination protein complex RC-1.

Based on a novel cell-free assay for DNA recombination, we previously reported the purification and initial characterization of RC-1, a protein complex catalyzing the recombinational repair of deletions and gaps. RC-1 was isolated from calf thymus nuclear extracts and shown to copurify with several enzymatic activities, among them a DNA polymerase. Here, additional evidence is reported identifying the polymerase as DNA polymerase epsilon. Furthermore, a novel DNA structure-dependent endonuclease associated with RC-1 was observed, which recognizes and cleaves branched DNA substrates at specific sites. Implications of this endonuclease activity for the recombination reaction are discussed.

Base Sequence↗

PubMed1996

Neuropsychological aspects of frontal lobe epilepsy.

While most neuropsychological studies in focal epilepsies are concerned with temporal lobe epilepsy (TLE), only few investigations aim cognitive functioning in unresected patients with frontal lobe epilepsy (FLE). Following functional models arising from lesional studies, we evaluated patients with TLE (21 left, 17 right) and FLE (6 left, 17 right) with respect to attention and speed, motor coordination, verbal/nonverbal fluency, concept formation, response inhibition, anticipatory behavior and memory span. The following results were obtained. When compared to TLE, FLE was associated with significantly poorer results in almost all tests, fluency tests being the exception. No group differences were found with respect to the lateralization of the epileptic focus or the presence or absence of cerebral lesions. Factor analysis of the tests indicated that different subfunctions (speed, STM, motor coordination, response maintenance and inhibition) were assessed. A particular cognitive pattern of impaired motor coordination or response inhibition appeared to be characteristic for patients with FLE.

Adult↗

PubMed1996

The CYP2B1 proximal promoter contains a functional C/EBP regulatory element.

Cytochromes P450 2B1 and 2B2 (CYP2B1 and CYP2B2) are well-known phenobarbital-inducible genes in rat liver. Potential transcriptional regulatory elements in the proximal promoter regions of rat CYP2B genes were analyzed by transfection in HepG2 hepatoma cells and by binding of nuclear proteins. Deletion of sequences from -1,400 to -110 had modest effects on promoter activity, but further deletion to -57 decreased the transcriptional activity by more than 90%, suggesting the presence of strong cis-acting elements in this region. Sequences similar to a basal transcription element (BTE) in CYP1A1 and a proposed phenobarbital responsive element (Barbie box) are present from -89 to -67. However, no protection was detected in these regions by DNase I footprinting assay. Instead, a region (FP1) from -64 to -45 was protected by liver nuclear extracts. Mutation of either the BTE or FP1 sequences of CYP2B1, or both, reduced transcriptional activity by 70-80% in HepG2 cells. FP1 was identified as a functional C/EBP site by co-transfection of C/EBP expression vectors and supershift assays with C/EBP antisera. Binding of liver nuclear proteins to sequences within the -110 to +1 region was not detectably altered by pretreatment of rats with phenobarbital.

Animals↗

PubMed1996

Rapid diagnosis of germline p53 mutation using the enzyme mismatch cleavage method.

The p53 tumor suppressor gene is the most commonly altered gene in human cancers. Germline mutations in p53 are the genetic alteration underlying predisposition to multiple cancers in Li-Fraumeni syndrome and Li-Fraumeni-like syndrome. We describe a patient who presented with developed adrenocortical carcinoma at age 19 months and a cerebral primitive neuroectodermal tumor at age 5 years. The patient did not have a family history of cancer. We used the enzyme mismatch cleavage (EMC) method to screen for mutations in the p53 gene and found a germline mutation in exon 7 (codon 248). Loss of heterozygosity analysis in one tumor revealed loss of the wild-type p53 allele. In our report we demonstrate the EMC method to be a rapid and sensitive method for mutation detection.

Adrenocortical Carcinoma↗

PubMed1995

Improved large-scale preparation of phage T4 endonuclease VII overexpressed in E. coli.

Using PCR, we cloned T4 gene 49, which encodes the endonuclease VII, and the inactive mutant gene 49 amE727 into vector pET-11a. In combination with Escherichia coli host strain BL21 (DE3), this system provided excellent repression of the expression of the highly toxic protein before induction with IPTG. After induction, the proteins were made in high quantities while remaining soluble. Dilution of the crude lysate at 1:10,000 continued to show a highly specific activity in the case of the wild-type enzyme. The protein was purified to homogeneity with a recovery of 33% using two chromatography steps. The yield was 20 times higher and the specific activity 500 times higher than that obtained by using the previously published protocol.

Bacteriophage T4↗

PubMed1995

A universal approach to the expression of human and rabbit cytochrome P450s of the 2C subfamily in Escherichia coli.

Human cytochrome P450s 2C8, 2C9, 2C18, and 2C19 and rabbit cytochrome P450s 2C1, 2C2, 2C4, 2C5, and 2C16 were expressed from their respective cDNAs in Escherichia coli as chimeric enzymes in which a portion of the N-terminal membrane anchor sequence was replaced with a modified sequence derived from P450 17A. For 2C1 and 2C2 removal of the extraneous 3'-untranslated sequence allowed the successful expression of constructs that were unproductive in its presence. The levels of expression varied from 180 to 1500 nmol/liter of culture and the addition of delta-aminolevulinic acid to the culture media increased the amount of spectrally detectable P450 for several of these enzymes 2- to 10-fold. The catalytic properties of the modified human 2C P450s expressed in E. coli were concordant with previously published data for several marker substrates including (S)-mephenytoin for P450 2C19, tolbutamide and tetrahydrocannabinol (THC) for P450 2C9, and taxol for P450 2C8. Interestingly, P450 2C19 catalyzed the 21-hydroxylation of progesterone and, to a lesser extent, catalyzed the formation of 16 alpha-hydroxyprogesterone. The rabbit enzyme P450 2C16 catalyzed the formation of 17 alpha- and 16 alpha-hydroxyprogesterone in addition to 21-hydroxylation. P450 2C19 also catalyzed the methylhydroxylation of tolbutamide and the 7-hydroxylation of THC at rates that were similar to or greater than that of P450 2C9. This work has identified important factors required for the high-level expression of 2C subfamily P450s in E. coli. The availability of these enzymes will facilitate detailed kinetic measurements for known and yet to be identified substrates.

Alleles↗

PubMed1995

The cytoplasmic and N-terminal transmembrane domains of cytochrome P450 contain independent signals for retention in the endoplasmic reticulum.

Microsomal cytochrome P450 is inserted into the membrane of the endoplasmic reticulum (ER) by its N-terminal signal/anchor sequence which also functions as an ER retention signal. To analyze further potential retention signals of cytochrome P450, topological domains of cytochrome P450 2C1 or 2C2, epidermal growth factor receptor, a plasma membrane protein, and bacterial alkaline phosphatase, a secreted protein were exchanged. The N-terminal signal/anchor of cytochrome P450 2C1 functioned as an ER retention signal when placed at the N terminus of several reporter proteins but not when fused at the C terminus of the extracellular domain of epidermal growth factor receptor, with or without a heterologous cytoplasmic domain. Chimeric proteins in which the cytoplasmic domain of cytochrome P450 2C2 was substituted for that of epidermal growth factor receptor were retained in the ER indicating that an independent retention signal is present in the cytoplasmic part of cytochrome P450 2C2. These chimeras were enzymatically active which argues against misfolding as the primary cause of retention. The ER retention signal of the cytoplasmic domain could not be localized to a single amino acid segment by deletion analysis. These results show that cytochrome P450 2C2 contains redundant, complex ER retention signals in its cytoplasmic and N-terminal hydrophobic domains and that the function of the N-terminal signal is context-dependent.

Alkaline Phosphatase↗

PubMed1995

Detection and partial purification of a cruciform-resolving activity (X-solvase) from nuclear extracts of mouse B-cells.

We have identified a cruciform-resolving enzyme (X-solvase) in nuclear extracts from mouse B-cells, called EMX1, by using an exonuclease-resistant cruciform DNA as a substrate. The cruciform was a 104-nt oligonucleotide that spontaneously adopted a branched conformation with four arms, each arm protected by a terminal loop of five T residues. A ligatable nick was left in one arm. After ligation, the covalently closed substrate was used to follow an 1800-fold purification of the mouse X-solvase (EMX1) from crude nuclear extracts by chromatography on DEAE-cellulose, MonoQ and heparin-Sepharose. The purest fractions containing EMX1 show high specificity for cruciform DNA. The cleavage pattern is indistinguishable from that found in the same substrates after treatment with endonuclease VII from phage T4 or endonuclease X3 from the yeast Saccharomyces cerevisiae. EMX1 and yeast endonuclease X3 were also found to be sensitive to anti-(endonuclease VII) antibodies which inhibited their reactions with cruciform DNAs in vitro.

Animals↗

PubMed1995

Identification by in vitro mutagenesis of the interaction of two segments of C2MstC1, a chimera of cytochromes P450 2C2 and P450 2C1.

A hybrid cytochrome P450, C2MstC1, with 306 N-terminal amino acids derived from cytochrome P450 2C2 sequence and 184 C-terminal amino acids from cytochrome P450 2C1 acquires a novel progesterone 21-hydroxylase activity which is absent in the parent enzymes. Extension of the cytochrome P450 2C2 sequence to residue 382 reduced progesterone hydroxylase activity to 5% of that of C2MstC1, while further extension to residue 411 or 462 increased activity back to about 30 or 40%, respectively. In the chimera with cytochrome P450 2C2 sequence to residue 382, substitution of cytochrome P450 2C1 amino acids at positions 368, 369, and 374 increased progesterone hydroxylase activity to a level equivalent to that of C2MstC1. In the chimera with cytochrome P450 2C2 sequence extending to residue 411, substitutions of P450 2C1 amino acids at positions 386 and 388, in addition those at 368, 369, and 374, were required to obtain activities equivalent to that of C2MstC1, which suggests an interaction between these two regions. The lauric acid hydroxylase activities of all chimeras and mutant cytochromes P450 differed by 2-fold or less, demonstrating that the changes in progesterone hydroxylase activity reflected altered interactions with the substrate. Alignment of cytochrome P450 2C1 sequence with cytochromes P450cam, P450BM-3, and P450terp predicts that residues 368/369 and 386/388 are in adjacent antiparallel strands of the same beta-sheet, in agreement with the experimental data suggesting an interaction between these two regions.

Amino Acid Sequence↗