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Biomedical subjects

D T Keough

Publications and source records attributed to D T Keough.

At least 19 recordsLinked to original sources
PubMed1999

Purification and characterization of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase and comparison with the human enzyme.

The human malaria parasite Plasmodium falciparum is auxotrophic for purines and relies on the purine salvage pathway for the synthesis of its purine nucleotides. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is a key purine salvage enzyme in P. falciparum, making it a potential target for chemotherapy. Previous attempts to purify this enzyme have been unsuccessful because of the difficulty in obtaining cultured parasite material and because of the inherent instability of the enzyme during purification and storage. Other groups have tried to express recombinant P. falciparum HGXPRT but only small amounts of activity were obtained. The successful expression of recombinant P. falciparum HGXPRT in Escherichia coli has now been achieved and the enzyme purified to homogeneity in mg quantities. The measured molecular mass of 26 229+/-2 Da is in excellent agreement with the calculated value of 26232 Da. A method to stabilise the activity and to reactivate inactive samples has been developed. The subunit structure of P. Jilciparum HGXPRT has been determined by ultracentrifugation in the absence (tetramer) and presence (dimer) of KC1. Kinetic constants were determined for 5-phospho-alpha-D-ribosyl-1-pyrophosphate, for the three naturally-occurring 6-oxopurine bases guanine, hypoxanthine, and xanthine and for the base analogue, allopurinol. Differences in specificity between the purified P. falciparum HGXPRT and human hypoxanthine guanine phosphoribosyltransferase enzymes were detected which may be able to be exploited in rational drug design.

Animals↗
PubMed1991

Localization of the 5-phospho-alpha-D-ribosyl-1-pyrophosphate binding site of human hypoxanthine-guanine phosphoribosyltransferase.

Human erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HPRT) is inactivated by iodoacetate in the absence, but not in the presence, of the substrate, 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRib-PP). Treatment of HPRT with [14C]iodoacetate followed by tryptic digestion, peptide separation and sequencing has shown that Cys-22 reacts with iodoacetate only in the absence of PRib-PP; this strongly suggests that Cys-22 is in or near the PRib-PP binding site. In contrast, Cys-105 reacts with [14C]iodoacetate both in the presence and absence of PRib-PP. Carboxymethylation of Cys-22 resulted in an increase in the Km for PRib-PP, but no change in Vmax. Storage of HPRT also resulted in an increase in the Km for PRib-PP and a decrease in its susceptibility to inactivation by iodoacetate. Dialysis of stored enzyme against 1 mM dithiothreitol resulted in a marked decrease in Km for PRib-PP. The stoichiometry of the reaction of [14C]iodoacetate with Cys-22 in HPRT leading to inactivation (approx. 1 residue modified per tetramer) showed that, in this preparation of HPRT purified from erythrocytes, only about 25% of the Cys-22 side chains were present as free and accessible thiols. Titration of thiol groups [with 5,5'-dithiobis(2-nitrobenzoic acid)] and the effect of dithiothreitol on Km for PRib-PP indicate that oxidation of thiol groups occurs on storage of HPRT, even in the presence of 1 mM beta-mercaptoethanol.

Binding Sites↗
PubMed1990

Expression of active human hypoxanthine-guanine phosphoribosyltransferase in Escherichia coli and characterisation of the recombinant enzyme.

A plasmid, pRG1, has been constructed by incorporating the coding sequence of human hypoxanthine-guanine phosphoribosyltransferase (HPRT) into the expression vector pT7-7. Expression of human HPRT has been achieved in HPRT- Escherichia coli cells transformed with pRG1 and pGP1-2, as shown by: (1) exclusive labelling with [35S]methionine of a polypeptide with the same mobility as purified human HPRT on SDS-PAGE; and (2) measurement of HPRT activity after cell lysis. Although the majority of the recombinant HPRT was present in the particulate fraction after cell lysis and centrifugation, sufficient HPRT activity was present in the supernatant fraction to allow comparison with the HPRT purified from human erythrocytes and the activity in human haemolysates and lymphoblast lysates. Small differences in electrophoretic mobility on native gels were found between HPRT activity from these sources. The Km values of recombinant HPRT for the substrates 5-phospho-alpha-D-ribosyl-1-pyrophosphate and guanine were compared with those of lymphoblast and erythrocyte HPRT.

Chromatography, Gel↗
PubMed1989

Characterization of genomic DNA, mRNA and enzyme protein in cases of HPRT-deficiency.

1. Immunological quantitation of the HPRT proteins, together with DNA and RNA studies have defined further the heterogeneous nature of HPRT-deficiency in our patients. 2. These studies have dictated possible approaches for further characterisation of the HPRT enzyme in our patients. In the two Lesch-Nyhan patients, both the protein and the usual cDNA approach would appear difficult. 3. A BamHI polymorphism has been detected in Patient A. 4. Sequence data confirmed the creation of this BamHI site by a single C----T transition at position 602 in the coding sequence. 5. Sequencing of other patients is proceeding and use is being made of the Polymerase Chain Reaction (PCR)10 for amplification of specific segments of HPRT coding sequence.

Cell Line, Transformed↗
PubMed1988

Biochemical basis of hypoxanthine-guanine phosphoribosyltransferase deficiency in nine families.

The concentration of hypoxanthine-guanine phosphoribosyltransferase (HPRT) cross-reacting material (CRM) was determined in haemolysates and/or lymphoblast lysates from nine patients with complete or partial deficiency of HPRT activity. Two of the patients had the fully developed Lesch-Nyhan syndrome and although they had undetectable HPRT activity, small amounts of CRM were found. HPRT-specific mRNA was not detected in lymphoblast lysates from one of these patients, while lysates from the other had a much reduced concentration. Samples from three patients with less than 0.1% of normal HPRT activity but with minor or no neurological manifestations were also found to contain small amounts of CRM. The other four patients whose HPRT activities ranged from 3 to 10% of normal were found to have CRM concentrations which varied from 26 to 100% of normal. In one patient with a partial deficiency the Km for 5-phospho-alpha-D-ribosyl-1-pyrophosphate was five times normal.

Humans↗
PubMed1987

Human hypoxanthine-guanine phosphoribosyltransferase. Development of a spectrophotometric assay and its use in detection and characterization of mutant forms.

A simple and rapid spectrophotometric assay for the estimation of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity in human tissues is described. It is based on the increase in absorbance at 257.5 nm which occurs when the substrate guanine is converted to its 5'-mononucleotide, GMP. The assay has been developed to measure HGPRT activity in erythrocyte and lymphocyte lysates and in brain homogenates, and has been used in the screening of patients with hyperuricaemia and/or hyperuricosuria for HGPRT deficiency. It has also been used to determine the steady-state kinetic constants of a mutant form of the enzyme. The spectrophotometric assay is compared with the radioactive assay currently used to measure HGPRT activity.

Aged↗
PubMed1987

HPRT-deficiency associated with normal PRPP concentration and APRT activity.

Deficiencies of HPRT are usually associated with increased concentrations of PRPP and increased levels of APRT activity in erythrocytes. We report the case of a male with a partial deficiency of HPRT in whom these two parameters were normal. The clinical features of this patient were those associated with severe hyperuricaemia and gout. Studies of intact erythrocytes showed rates of incorporation of [14C]hypoxanthine and of [14C]adenine into purine nucleotides which were almost indistinguishable from normal. However, HPRT activity in erythrocyte lysates was only 9% of normal. In cell extracts of cultured lymphoblasts, the HPRT activity was 20% of control values and the APRT activity was normal. The PRPP concentration and the rate of de novo purine synthesis in cultured lymphoblasts were both intermediate between controls and lymphoblasts from patients with the Lesch-Nyhan syndrome.

Adenine Phosphoribosyltransferase↗
PubMed1985

The relationship between the carboxylesterase and monoacylglycerol lipase activities of chicken liver microsomes.

The carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) and monoacylglycerol lipase (glycerol-monoester acylhydrolase, EC 3.1.1.23) activities, measured against ethyl butyrate and emulsified monooleoylglycerol respectively, were determined for chicken liver microsomes and highly purified chicken liver carboxylesterase. The activity ratio (ethyl butyrate activity/monooleoylglycerol activity) was approx. 5 for microsomes and approx. 400 for carboxylesterase. Homogenization of microsomes in 0.1 M Tris-HCl buffer (pH 7.92) released all of the ethyl butyrate activity and about half of the monooleoylglycerol activity into a soluble form. Both activities eluted from a Sephadex G-200 column with the same elution volume as that of pure carboxylesterase. This fraction (fraction B) had an activity ratio of approx. 15, an average pI of 5.01 (cf. 4.75 for carboxylesterase), and ran on polyacrylamide gel electrophoresis at pH 8.6 as a number of closely spaced esterase bands with mobilities considerably less than those of the esterase bands present in the carboxylesterase. Fraction B activities against both substrates were completely inhibited by diethyl p-nitrophenyl phosphate and completely precipitated by antibody to carboxylesterase. The remaining half of the monoacylglycerol lipase activity of microsomes was solubilized by treatment with 1.5% (w/v) Triton X-100. This solubilized monoacylglycerol lipase was completely inhibited by diethyl p-nitrophenyl phosphate, showing it to be a serine-dependent enzyme like the carboxylesterases. However, it had no detectable activity against ethyl butyrate, indicating that it is not closely related to the carboxylesterases.

Animals↗
PubMed1984

Enzymatically active zinc, copper and mercury derivatives of the one-iron form of pig allantoic fluid acid phosphatase.

Derivatives of the violet, iron-containing acid phosphatase of pig allantoic fluid have been prepared in which one of the two iron atoms present in the native enzyme has been replaced by zinc, copper or mercury. The derivatives so formed are enzymatically active: the Zn-Fe, Cu-Fe and Hg-Fe enzymes have specific activities of about 80%, 25% and 17% respectively, of the maximum specific activity of the Fe-Fe enzyme in the standard assay at pH 4.9 with p-nitrophenyl phosphate as substrate. In contrast to the Fe-Fe enzyme, the mixed metal derivatives are not rapidly inactivated by H2O2. Visible absorption spectra of the derivatives confirm that all of the visible absorption of the Fe-Fe enzyme is due to one of the iron atoms. Attempts to prepare an active Cu-Cu enzyme were unsuccessful.

Acid Phosphatase↗
PubMed1983

Mössbauer and EPR study of the binuclear iron centre in purple acid phosphatase.

Mössbauer spectra have been determined on 57Fe-enriched samples of both pink (reduced) and purple (oxidized) forms of pig allantoic acid phosphatase (EC 3.1.3.2), and EPR spectra on corresponding unenriched samples. The spectra show unambiguously that both forms of the enzyme contain two distinct, antiferromagnetically coupled, high-spin iron atoms: a ferrous-ferric ion pair in the pink, reduced form, and a pair of ferric ions in the purple, oxidized form.

Acid Phosphatase↗