Biomedical subjects

R H Elder

Publications and source records attributed to R H Elder.

At least 19 recordsLinked to original sources

PubMed2002

Changes in the expression of cytochrome P450 isozymes and related carcinogen metabolizing enzyme activities in Schistosoma mansoni-infected mice.

Mixed-function oxidase enzymes metabolize most xenobiotic agents. Western blotting was used to investigate the effect of Schistosoma mansoni infection on the expression of various cytochrome P450 (CYP) isozymes and specific enzyme assays to study related metabolic functions in mouse liver microsomes. Male BK-TO mice were infected with 200 cercariae per mouse and their livers were assayed at 6, 15, 30 and 45 days post-infection (p.i.) and compared with appropriately matched controls. The expression of each of the CYP isozymes (1A1, 2B1/2, 2C6, and 4A) was either unaffected or transiently increased up to 30 days post-infection. By 45 days, a significant loss of signal was observed, particularly for CYP 1A1 and 2B1 /2 where no signal could be detected. Evidence supporting these findings was obtained from enzyme assays specific for particular CYP isozymes. The activity of ethoxyresorufin O-deethylase (CYP 1A1) was reduced by 97% and that of pentoxyresorufin O-depentylase (CYP 2B1 /2) by 96% at 45 days p.i. Similarly, the activity of ethoxycoumarin hydroxylase was progressively reduced over the period under study. It is believed that N-nitrosamines are activated principally by N-nitrosodimethylamine N-demethylase I which was significantly increased at both 30 and 45 days p.i. To further investigate metabolic competency following S. mansoni infection, the in vitro binding of benzo(a)pyrene metabolites to DNA was measured, using isolated liver microsomes to activate benzo(a)pyrene. Benzo(a)-pyrene-DNA adduct formation was markedly increased at 6,15 and 30 days with a maximum at 15 days, but decreased at 45 days p.i. It was concluded that S. mansoni infection changes the expression of different CYP isozymes and also the activity of phase I drug-metabolizing enzymes at different periods of infection and may thus change the liver's capacity to activate or detoxify many endogenous and exogenous compounds. Such alterations may also change the therapeutic actions of drugs that are primarily metabolized by the P450 system, when administered to patients with schistosomiasis.

Animals↗

PubMed2001

Increased susceptibility to streptozotocin-induced beta-cell apoptosis and delayed autoimmune diabetes in alkylpurine-DNA-N-glycosylase-deficient mice.

Type 1 diabetes is thought to occur as a result of the loss of insulin-producing pancreatic beta cells by an environmentally triggered autoimmune reaction. In rodent models of diabetes, streptozotocin (STZ), a genotoxic methylating agent that is targeted to the beta cells, is used to trigger the initial cell death. High single doses of STZ cause extensive beta-cell necrosis, while multiple low doses induce limited apoptosis, which elicits an autoimmune reaction that eliminates the remaining cells. We now show that in mice lacking the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG), beta-cell necrosis was markedly attenuated after a single dose of STZ. This is most probably due to the reduction in the frequency of base excision repair-induced strand breaks and the consequent activation of poly(ADP-ribose) polymerase (PARP), which results in catastrophic ATP depletion and cell necrosis. Indeed, PARP activity was not induced in APNG(-/-) islet cells following treatment with STZ in vitro. However, 48 h after STZ treatment, there was a peak of apoptosis in the beta cells of APNG(-/-) mice. Apoptosis was not observed in PARP-inhibited APNG(+/+) mice, suggesting that apoptotic pathways are activated in the absence of significant numbers of DNA strand breaks. Interestingly, STZ-treated APNG(-/-) mice succumbed to diabetes 8 months after treatment, in contrast to previous work with PARP inhibitors, where a high incidence of beta-cell tumors was observed. In the multiple-low-dose model, STZ induced diabetes in both APNG(-/-) and APNG(+/+) mice; however, the initial peak of apoptosis was 2.5-fold greater in the APNG(-/-) mice. We conclude that APNG substrates are diabetogenic but by different mechanisms according to the status of APNG activity.

Animals↗

PubMed2000

Dietary fish oil reduces O6-methylguanine DNA adduct levels in rat colon in part by increasing apoptosis during tumor initiation.

There is epidemiological, clinical, and experimental evidence that dietary fish oil, containing n-3 polyunsaturated fatty acids, protects against colon tumor development. However, its effects on colonocytes in vivo remain poorly understood. Therefore, we investigated the ability of fish oil to modulate colonic methylation-induced DNA damage, repair, and deletion. Sprague Dawley rats were provided with complete diets containing either corn oil or fish oil (15% by weight). Animals were injected with azoxymethane, and the distal colon was removed 3, 6, 9, or 12 h later. Targeted apoptosis and DNA damage were assessed by cell position within the crypt using the terminal deoxynucleotidyl transferase-mediated nick end labeling assay and quantitative immunohistochemical analysis of O6-methylguanine adducts, respectively. Localization and expression of the alkyl group acceptor, O6-methylguanine-DNA-methyltransferase, was also determined. Lower levels of adducts were detected at 6, 9, and 12 h in fish oil- versus corn oil-fed animals (P < 0.05). In addition, fish oil supplementation had the greatest effect on apoptosis in the top one-third of the crypt, increasing the apoptotic index compared with corn oil-fed rats (P < 0.05). In the top one-third of the crypt, fish oil feeding caused an incremental stimulation of apoptosis as adduct level increased. In contrast, a negative correlation between apoptosis and adduct incidence occurred with corn oil feeding (P < 0.05). Diet had no main effect (all tertiles combined) on O6-methylguanine-DNA-methyltransferase expression over the time frame of the experiment. The enhancement of targeted apoptosis combined with the reduced formation of O6-methylguanine adducts may account, in part, for the observed protective effect of n-3 polyunsaturated fatty acids against experimentally induced colon cancer.

Analysis of Variance↗

PubMed1999

Expression levels of the DNA repair enzyme HAP1 do not correlate with the radiosensitivities of human or HAP1-transfected rat cell lines.

Apurinic/apyrimidinic (AP) sites in DNA are potentially lethal and mutagenic. They can arise spontaneously or following DNA damage from reactive oxygen species or alkylating agents, and they constitute a significant product of DNA damage following cellular exposure to ionizing radiation. The major AP endonuclease responsible for initiating the repair of these and other DNA lesions in human cells is HAP1, which also possesses a redox function. We have determined the cellular levels of this enzyme in 11 human tumour and fibroblast cell lines in relation to clonogenic survival following ionizing radiation. Cellular HAP1 levels and surviving fraction at 2 Gy (SF2) varied five- and tenfold respectively. However, no correlation was found between these two parameters following exposure to gamma-irradiation at low (1.1 cGy per min) or high (108 cGy per min) dose rates. To examine this further, wild-type and mutant versions of HAP1 were overexpressed, using an inducible HAP1 cDNA expression vector system, in the rat C6 glioma cell line which has low endogenous AP endonuclease activity. Induction of wild-type HAP1 expression caused a > fivefold increase in the capacity of cellular extracts to cleave an oligonucleotide substrate containing a single abasic site, but increased expression did not confer increased resistance to gamma-irradiation at high- or low-dose rates, or to the methylating agent methyl methanesulphonate (MMS). Expression in C6 cell lines of mutant forms of HAP1 deleted for either the redox activator or DNA repair functions displayed no apparent titrational or dominant negative effects. These studies suggest that the levels of endogenous AP endonuclease activities in the various cell lines examined are not limiting for efficient repair in cells following exposure to ionizing radiation or MMS. This contrasts with the correlation we have found between HAP1 levels and radiosensitivity in cervix carcinomas (Herring et al (1998) Br J Cancer 78: 1128-1133), indicating that HAP1 levels in this case assume a critical survival role and hence that established cell lines might not be a suitable model for such studies.

Animals↗

PubMed1998

Inactivation of O6-alkylguanine-DNA alkyltransferase. 1. Novel O6-(hetarylmethyl)guanines having basic rings in the side chain.

A number of novel guanine derivatives containing heterocyclic moieties at the O6-position have been synthesized using a purine quaternary salt which reacts with alkoxides under mild conditions. Initially O6-substituents were investigated in which the benzene ring of the known agent, O6-benzylguanine, was replaced by unsubstituted heterocyclic rings. The ability of these agents to inactivate the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase), both as pure recombinant protein and in the human lymphoblastoid cell line Raji, has been compared with that of O6-benzylguanine. The present paper focuses on O6-substituents with basic rings, and under standard conditions several of them proved more effective than benzyl for inactivation of both recombinant and Raji ATase. Among the pyridine derivatives, the 2-picolyl compound 7 is not very active in contrast to the 3- and 4-picolyl compounds, and this influenced our choice of isomers of other basic ring systems for study. Since halogen substitution in the thiophene ring considerably increased the activity (17 versus 6), similar modifications in the pyridine series were examined. The more polar O6-substituents in this study are on the whole compatible with the stereochemical requirements of the ATase protein, and their pharmacological properties may be valuable in subsequent in vivo investigations, particularly the thenyl (6), 5-thiazolylmethyl (12), 5-bromothenyl (17), and 2-chloro-4-picolyl (21) derivatives.

Enzyme Inhibitors↗

PubMed1998

Levels of the DNA repair enzyme human apurinic/apyrimidinic endonuclease (APE1, APEX, Ref-1) are associated with the intrinsic radiosensitivity of cervical cancers.

A study was made of the relationship between the intrinsic radiosensitivity of human cervical tumours and the expression of the DNA repair enzyme human apurinic/apyrimidinic endonuclease (HAP1). The radiosensitivity of clonogenic cells in tumour biopsies was measured as surviving fraction at 2 Gy (SF2) using a soft agar assay. HAP1 expression levels were determined after staining of formalin-fixed paraffin-embedded tumour sections with a rabbit antiserum raised against recombinant HAP1. Both measurements were obtained on pretreatment biopsy material. All 25 tumours examined showed positive staining for HAP1, but there was heterogeneity in the level of expression both within and between tumours. The average coefficients of variation for intra- and intertumour heterogeneity were 62% and 82% respectively. There was a moderate but significant positive correlation between the levels of HAP1 expression and SF2 (r = 0.60, P = 0.002). Hence, this study shows that there is some relationship between intrinsic radiosensitivity and expression of a DNA repair enzyme in cervical carcinomas. The results suggest that this type of approach may be useful in the development of rapid predictive tests of tumour radiosensitivity.

Animals↗

PubMed1998

Alkylpurine-DNA-N-glycosylase knockout mice show increased susceptibility to induction of mutations by methyl methanesulfonate.

Alkylpurine-DNA-N-glycosylase (APNG) null mice have been generated by homologous recombination in embryonic stem cells. The null status of the animals was confirmed at the mRNA level by reverse transcription-PCR and by the inability of cell extracts of tissues from the knockout (ko) animals to release 3-methyladenine (3-meA) or 7-methylguanine (7-meG) from 3H-methylated calf thymus DNA in vitro. Following treatment with DNA-methylating agents, increased persistence of 7-meG was found in liver sections of APNG ko mice in comparison with wild-type (wt) mice, demonstrating an in vivo phenotype for the APNG null animals. Unlike other null mutants of the base excision repair pathway, the APNG ko mice exhibit a very mild phenotype, show no outward abnormalities, are fertile, and have an apparently normal life span. Neither a difference in the number of leukocytes in peripheral blood nor a difference in the number of bone marrow polychromatic erythrocytes was found when ko and wt mice were exposed to methylating or chloroethylating agents. These agents also showed similar growth-inhibitory effects in primary embryonic fibroblasts isolated from ko and wt mice. However, treatment with methyl methanesulfonate resulted in three- to fourfold more hprt mutations in splenic T lymphocytes from APNG ko mice than in those from wt mice. These mutations were predominantly single-base-pair changes; in the ko mice, they consisted primarily of AT-->TA and GC-->TA transversions, which most likely are caused by 3-meA and 3- or 7-meG, respectively. These results clearly show an important role for APNG in attenuating the mutagenic effects of N-alkylpurines in vivo.

Animals↗

PubMed1997

Targeted deletion of alkylpurine-DNA-N-glycosylase in mice eliminates repair of 1,N6-ethenoadenine and hypoxanthine but not of 3,N4-ethenocytosine or 8-oxoguanine.

It has previously been reported that 1,N6-ethenoadenine (epsilonA), deaminated adenine (hypoxanthine, Hx), and 7,8-dihydro-8-oxoguanine (8-oxoG), but not 3,N4-ethenocytosine (epsilonC), are released from DNA in vitro by the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG). To assess the potential contribution of APNG to the repair of each of these mutagenic lesions in vivo, we have used cell-free extracts of tissues from APNG-null mutant mice and wild-type controls. The ability of these extracts to cleave defined oligomers containing a single modified base was determined. The results showed that both testes and liver cells of these knockout mice completely lacked activity toward oligonucleotides containing epsilonA and Hx, but retained wild-type levels of activity for epsilonC and 8-oxoG. These findings indicate that (i) the previously identified epsilonA-DNA glycosylase and Hx-DNA glycosylase activities are functions of APNG; (ii) the two structurally closely related mutagenic adducts epsilonA and epsilonC are repaired by separate gene products; and (iii) APNG does not contribute detectably to the repair of 8-oxoG.

Adenine↗

PubMed1997

Induction of O6-alkylguanine-DNA-alkyltransferase in the hepatocytes of rats following treatment with 2-acetylaminofluorene.

Molecular and immunohistological techniques have been used to study the induction in rat liver of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (ATase), following an acute dose (60 mg/kg) of the hepatocarcinogen, 2-acetylaminofluorene (2-AAF). An increase in ATase activity was specific to the liver, with a five- to six-fold induction being observed 72 hr after administration of 2-AAF. A similar temporal increase of both activity and ATase protein (detected by immunoblotting) was observed up to 1 week following treatment, but after 2 weeks the activity had returned to control levels. Although maximal induction of hepatic ATase mRNA was observed as early as 24 hr, the levels remained elevated at least 1 week after 2-AAF treatment. Using a rabbit antiserum raised against purified recombinant rat ATase, ATase-specific staining was observed in the nuclei of both nonhepatocytes and hepatocytes in control liver sections. There was, however, a significant differential staining of hepatocytes across the liver lobule, with ATase staining being most intense in the periportal region. In the livers of 2-AAF-treated rats, an increased intensity of staining was observed in hepatocytes throughout the liver lobule, whereas the nonparenchymal cells showed much less, or no, increase in staining. The increased expression of ATase in hepatocytes and its differential distribution across the lobule were confirmed by image analysis. Thus, ATase induction in response to 2-AAF treatment was an hepatocyte-specific response and not confined to any particular region of the liver lobule.

2-Acetylaminofluorene↗

PubMed1996

Inter- and intracellular heterogeneity of O6-alkylguanine-DNA alkyltransferase expression in human brain tumors: possible significance in nitrosourea therapy.

The inter- and intracellular distribution of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase) may be an important factor in the sensitivity or resistance of tumours to treatment with certain alkylating agents, including the methyltriazenes and nitrosoureas. In order to examine this issue 26 human brain tumour sections (23 high grade gliomas and three low grade gliomas) were examined for ATase expression by immunohistochemistry using a rabbit anti-human ATase polyclonal antibody. Positive staining, seen as fine black granules mainly confined to the nucleus, was observed in all the glioma sections examined. There was marked cellular heterogeneity, ranging from cells completely devoid of staining to cells with very intense staining. Semi-quantitatively, in the 23 high grade gliomas examined six had 1+ staining, seven had 2+ staining and 10 had 3+ staining, whereas all three low grade gliomas had 1+ staining. These results are in contrast to published reports showing that approximately 35% of human brain tumour-derived cell lines and xenografts had very low levels of ATase activity and suggest that the complete lack of ATase is not a common occurrence in high grade glioma.

Antineoplastic Agents, Alkylating↗

PubMed1995

Subunit structure of mitochondrial DNA polymerase from Drosophila embryos. Physical and immunological studies.

The subunit structure of mitochondrial DNA polymerase from Drosophila embryos has been examined by a combination of physical and immunological methods. A highly specific rabbit antiserum directed against the native enzyme was developed and found to recognize specifically its two subunits in immunoblot and immunoprecipitation analyses. That and the potent inhibition by the rabbit antiserum of the DNA polymerase and 3'-->5' exonuclease activities of the nearly homogeneous mitochondrial DNA polymerase provide strong evidence for the physical association of the 3'-->5' exonuclease with the two subunit enzyme. An immunoprecipitation analysis of crude enzyme fractions showed that the two subunits of Drosophila mitochondrial DNA polymerase are intact, and an in situ gel proteolysis analysis showed that they are structurally distinct. Template-primer DNA binding studies demonstrated formation of a stable and discrete enzyme-DNA complex in the absence of accessory proteins. Photochemical cross-linking of the complexes by UV light indicated that the alpha but not the beta subunit of mitochondrial DNA polymerase makes close contact with DNA, and limited digestion of the native enzyme with trypsin showed that an approximately 65-kDa proteolytic fragment of the alpha subunit retains the DNA binding function.

Animals↗

PubMed1995

O6-benzylguanine increases the sensitivity of human primary bone marrow cells to the cytotoxic effects of temozolomide.

The sensitivity of human primary bone marrow granulocyte/macrophage precursor cells to the cytotoxic effects of the methylating antitumor agent temozolomide (8-carbamoyl-3- methylimidazo[5,1-d]-1,2,3,5-tetrazin-4-[3H]-1) was investigated using an in vitro colony-forming assay. In the eight samples examined, there was a range of sensitivities with D37 values from 18.2 to > 55 microM. When cells were simultaneously exposed to the O6-alkylguanine-DNA alkyltransferase (ATase) inactivating agent, O6-benzylguanine (O6BeG; 10 microM), the cytotoxicity of temozolomide was substantially increased with D37 values between 5 and 38.5 microM. O6BeG also increased temozolomide sensitivity in the human colon carcinoma cell line, WiDr, and this was shown to correlate with the O6BeG-mediated depletion of ATase activity. Where the extent of sensitization produced by O6BeG could be calculated, there was a correlation between this and the D37 value in the absence of O6BeG (R = 0.996); thus, sensitization was more extensive in the cells that were inherently more resistant to temozolomide. These data have implications for possible increased hematological toxicity in clinical protocols designed to exploit O6BeG or other agents to deplete ATase activity in tumors cells prior to treatment of patients with temozolomide or related agents.

Alkylating Agents↗

PubMed1994

Site-directed mutagenesis of glutamic acid 172 to glutamine completely inactivated human O6-alkylguanine-DNA-alkyltransferase.

DNA repair by O6-alkylguanine-DNA-alkyltransferase involves the stoichiometric transfer of the O6-alkyl group from the guanine lesion to the active-site cysteine residues of the protein. Site-directed mutagenesis of glutamic acid 172 of human O6-alkylguanine-DNA-alkyltransferase (EC 2.1.1.63) to glutamine totally abolished the alkyltransferase activity of the protein. This suggests that glutamic acid 172 is crucial to the alkyl transfer. It may act as a general acid (as CO2H) or base (as CO2-), or have a role as a component of a salt-link (-CO2-.....+N-), vital for the structural integrity of the active site. This is the first mutational inactivation of a protein in this family of DNA repair molecules by means of a residue change outside the highly conserved pentet (PCHRV) which includes the active-site cysteine.

Amino Acid Sequence↗

PubMed1994

Differential inactivation of mammalian and Escherichia coli O6-alkylguanine-DNA alkyltransferases by O6-benzylguanine.

The action of O6-benzylguanine (O6-BzlG) on recombinant mammalian and Escherichia coli O6-alkylguanine-DNA alkyltransferases (ATase; EC 2.1.1.63; methylated-DNA-protein-cysteine methyltransferase) was compared by preincubation of these proteins with the base, followed by measurement of residual ATase activity using [3H]methylated substrate DNA. All of the mammalian proteins examined were inactivated by O6-BzlG (Chinese hamster: I40, 0.04 microM; human and rat: I40, 0.06 microM); however, the murine ATase was substantially more resistant requiring 4-5 fold higher concentrations of O6-BzlG to achieve the same levels of inactivation (I40, 0.28 microM). A similar differential inactivation was seen with human and murine ATases when extracts of 3T6 (murine) cells and Raji (human) cells were compared. Of the two E. coli ATase proteins, only the ogt-encoded protein was inactivated, but approximately 400 times more O6-BzlG was required to achieve a level of inactivation similar to that seen with the human protein (I40, 24.8 microM). When O6-BzlG was present in an oligonucleotide, the differential effect on the murine, human and ogt-encoded ATases was not seen and only the ada-encoded ATase remained refractory under the conditions used.

Animals↗

PubMed1993

Lack of discrimination between DNA ligases I and III by two classes of inhibitors, anthracyclines and distamycins.

We have measured the effects of eight distamycin and two anthracycline derivatives on polynucleotide joining and self-adenylating activities of human DNA ligase I and rat DNA ligases I and III. All test drugs show good inhibitory activity against the three enzymes in the poly[d(A-T)] joining assay. Several distamycins also inhibit the DNA-independent self-adenylation reaction catalysed by the human enzyme and, to a lesser extent, by rat DNA ligases. These results confirm that anthracyclines and distamycins express their inhibitory action against DNA joining activities mainly via specific interactions with the substrate, and suggest that the three test DNA ligases utilize similar, if not identical, mechanisms of recognition and interaction with DNA-drug complexes. Our findings also indicate that distamycins have a greater affinity for human DNA ligase I than for rat enzymes, suggesting that, in this respect, rat DNA ligase I is more similar to rat DNA ligase III than to human DNA ligase I.

Animals↗

PubMed1993

Expression of O6-alkylguanine-DNA-alkyltransferase in situ in ovarian and Hodgkin's tumours.

The cellular expression of O6-alkylguanine-DNA-alkyltransferase (ATase) may be an important factor in determining tumour sensitivity to certain alkylating agents. In a comparative study, we have examined the inter- and intracellular distribution of ATase in tumour biopsies of a series of patients with Hodgkin's disease and ovarian cancer using a rabbit antihuman ATase antiserum. The antibody recognises the ATase protein on western blots of cell-free extracts of a number of ovarian tumours with ATase activities varying from 20 to 420 fmol/mg protein as determined by in vitro assay and there was a linear correlation between ATase activity and the intensity of the band on western blots (r = 0.993). Immunohistochemical staining was seen in all of the ovarian tumours examined and was confined to the nucleus. This is in contrast to the Hodgkin's tissue, where staining was much reduced and present in both nuclei and cytoplasm. The results suggest that in ovarian tumours the general resistance to nitrosourea chemotherapy may be related to the high cellular expression of ATase protein: this is in contrast to the more chemosensitive Hodgkin's disease. This raises the possibility that it might be feasible to predict sensitivity or resistance to these alkylating agents by immunohistochemical staining of tumour or tissue specimens.

Adult↗

PubMed1992

C-terminally truncated human O6-alkylguanine-DNA alkyltransferase retains activity.

A cDNA encoding the human O6-alkylguanine-DNA alkyltransferase (ATase; EC 2.1.1.63; methylated-DNA: protein-cysteine methyltransferase) has been manipulated to generate a C-terminally deleted protein which retains full methyl-transfer activity. The elimination of 22 amino-acid residues from the C-terminus was achieved by endonuclease-SacI digestion of the 623 bp cDNA coding sequence and ligation of a SacI/HindIII linker containing an in-frame stop codon. The truncated protein was characterized by its reduced molecular mass in immunoblots probed with an antiserum against the full-length protein and by fluorography after incubation with [3H]methylated calf thymus DNA. The rate of methyl transfer was virtually identical for the full-length and truncated ATases. The construction of such a truncated, yet still functional, ATase, with a molecular mass of 19.7 kDa should facilitate a detailed n.m.r. structural study and help to determine the functional significance of the C-terminal domain of mammalian ATases.

Animals↗

PubMed1992

Isolation and partial characterisation of a Chinese hamster O6-alkylguanine-DNA alkyltransferase cDNA.

The cDNA encoding Chinese hamster O6-alkylguanine-DNA-alkyltransferase (ATase) has been isolated from a library prepared from RNA isolated from V79 lung fibroblasts which had an upregulated level of this repair activity following stepwise selection with a chloroethylating agent (1, 2). Expression of the cDNA in E. coli produced functionally active ATase at levels of 2.5% of total cellular protein as determined by in vitro assay. The recombinant hamster protein has a molecular weight of 28 kDa as estimated by SDS-PAGE and fluorography and this was identical to that in the upregulated cells. The characteristic PCHRV pentapeptide of the alkyl acceptor site has been identified and there is a 68 amino acid residue region which is 90% conserved across all the mammalian proteins so far analysed: in contrast, the N- and C-terminal domains diverge by as much as 50% between species. Polyclonal antibodies to the human and rat ATases hybridised to the hamster protein on western analysis suggesting at least one common epitope shared across species. However, in antibody inhibition experiments neither of the antisera cross reacted with the hamster ATase in a way which interfered with functional activity whereas the anti-human antibodies inhibited the human ATase and the anti-rat antibodies inhibited the rat and mouse ATases. There may therefore be significant tertiary structural differences between the hamster protein and the other mammalian ATases.

Alkyl and Aryl Transferases↗