Search PubMed⌕ Search

Biomedical subjects

I H Fox

Publications and source records attributed to I H Fox.

At least 37 records · Page 2Linked to original sources
PubMed1989

AMP and IMP dephosphorylation by soluble high- and low-Km 5'-nucleotidases.

Three distinct 5'-phosphomonoesterase activities were isolated from soluble fractions of human placenta, cultured human T and B lymphoblasts, and rat liver using 5'-AMP-sepharose 4B affinity chromatography. We define these activities as "low-Km" 5'-nucleotidase, "high-Km" 5'-nucleotidase, and nonspecific phosphatase. High-Km 5'-nucleotidase was eluted with 0.5 M NaCl, low-Km 5'-nucleotidase was eluted with 10 mM ADP, and nonspecific phosphatase was not retained on the column. We have found significant variability in the relative content of high- to low-Km activities in the tissues studied with the ratios ranging from 5.5 to 264. The properties were studied after further purification. The molecular mass of the low-Km enzymes ranged from 72.5 to 209 kDa, optimum pH ranged from 7.4 to 9.0, Km for AMP ranged from 7 to 15 microM, and Km for IMP ranged from 10 to 26 microM. The molecular mass of the high-Km enzymes ranged from 182 to 210 kDa, pH optimum was at 6.5, Km for AMP ranged from 3.0 to 9.4 mM, and the Km for IMP ranged from 0.3 to 0.5 mM. The data indicate that the soluble low- and high-Km 5'-nucleotidase coexist in the mammalian cells and tissues studied. These observations suggest a complex system for the regulation of nucleoside 5'-monophosphate dephosphorylation.

5'-Nucleotidase↗
PubMed1989

Adenosine triphosphate turnover in humans. Decreased degradation during relative hyperphosphatemia.

The regulation of ATP metabolism by inorganic phosphate (Pi) was examined in five normal volunteers through measurements of ATP degradation during relative Pi depletion and repletion states. Relative Pi depletion was achieved through dietary restriction and phosphate binders, whereas a Pi-repleted state was produced by oral Pi supplementation. ATP was radioactively labeled by the infusion of [8(14)C]adenine. Fructose infusion was used to produce rapid ATP degradation during Pi depletion and repletion states. Baseline measurements indicated a significant decrease of Pi levels during phosphate depletion and no change in serum or urinary purines. Serum values of Pi declined 20 to 26% within 15 min after fructose infusion in all states. Urine measurements of ATP degradation products showed an eightfold increase within 15 min after fructose infusion in both Pi-depleted and -supplemented states. Urinary radioactive ATP degradation products were fourfold higher and urinary purine specific activity was more than threefold higher during Pi depletion as compared with Pi repletion. Our data indicate that there is decreased ATP degradation to purine end products during a relative phosphate repletion state as compared to a relative phosphate depletion state. These data show that ATP metabolism can be altered through manipulation of the relative Pi state in humans.

Adenosine Triphosphate↗
PubMed1989

The application of affinity chromatography for the separation of "high Km" and "low Km" 5'-nucleotidase and other AMP metabolizing enzymes.

AMP-sepharose 4B has been widely used as a general ligand affinity chromatography for purification of AMP deaminase, 5'-nucleotidase, adenosine kinase and other adenine nucleotide metabolizing enzymes. Since these enzymes generally differ in their kinetic properties related to the values of Km for AMP and analogous compounds, it was assumed that there may be a specific elution pattern of some of the enzymes which would enable sequential elution from the column during a single run. Using 0.5 M NaCl, 10 mM ATP and 5 mM adenosine as eluting agents, it was possible to separate on AMP-sepharose column AMP deaminase "high Km" and "low Km" 5'-nucleotidase and adenosine kinase. Adenylate kinase, adenosine deaminase and nonspecific phosphatase did not bind to the column. Using human placental extract, AMP deaminase, "high Km" and "low Km" 5'-nucleotidase and adenosine kinase were purified 2.8, 2.9, 105 and 1240 fold, respectively. AMP deaminase and "high Km" 5'-nucleotidase were further separated using phosphocellulose column chromatography and the final purification was 227 and 143 fold, respectively. The specific activities of purified enzyme preparations were 9.1, 1.0, 0.4 and 0.5 mumols/min/mg protein of AMP deaminase, "high Km" 5'-nucleotidase and adenosine kinase, respectively. This approach provides a rapid method for initial purification of these enzymes from crude soluble extracts.

5'-Nucleotidase↗
PubMed1989

Evidence for "low Km" and "high Km" soluble 5'-nucleotidases in human tissues and rat liver.

Two kinetically distinct purine 5'-phosphomono-esterase activities were isolated from soluble fractions of human placenta, cultured human T- and B-lymphoblasts and rat liver using AMP-sepharose chromatography. We have defined these activities as "high Km" and "low Km" 5'-nucleotidase. The relative content of "high Km" and "low Km" activities in the tissues studied ranged from 2 to 264. The optimum pH of "low Km" 5'-nucleotidases ranged from 7.4 to 9.0, Km for AMP from 7 to 15 uM and for IMP from 10 to 26 uM. ATP and ADP were inhibitors of "low Km" enzymes with the apparent Ki values of 55 to 20 uM and 8 to 20 uM for ATP and ADP, respectively. "High Km" 5'-nucleotidases had an optimum pH at 6.5, Km for IMP of 0.3 to 0.5 mM and Km for AMP of 1.0 to 9.4 mM. "High Km" enzymes were activated by ATP with A0.5 values, of 1.7 to 2.3 mM at 100 microM IMP. The data indicate that soluble "low Km" and "high Km" 5'-nucleotidases coexist in mammalian cells and fulfill different functions. These observations suggest a complex system for the regulation of AMP and IMP dephosphorylation.

5'-Nucleotidase↗
PubMed1988

High Km soluble 5'-nucleotidase from human placenta. Properties and allosteric regulation by IMP and ATP.

A human placental soluble "high Km" 5'-nucleotidase has been separated from "low Km" 5'-nucleotidase and nonspecific phosphatase by AMP-Sepharose affinity chromatography. The enzyme was purified 8000-fold to a specific activity of 25.6 mumol/min/mg. The subunit molecular mass is 53 kDa, and the native molecular mass is 210 kDa, suggesting a tetrameric structure. Soluble high Km 5'-nucleotidase is most active with IMP and GMP and their deoxy derivatives. IMP is hydrolyzed 15 times faster than AMP. The enzyme has a virtually absolute requirement for magnesium ions and is regulated by them. Purine nucleoside 5'-triphosphates strongly activate the enzyme with the potency order dATP greater than ATP greater than GTP. 2,3-Diphosphoglycerate activates the enzyme as potently as ATP. Three millimolar ATP decreased the Km for IMP from 0.33 to 0.09 mM and increased the Vmax 12-fold. ATP activation was modified by the IMP concentration. At 20 microM IMP the ATP-dependent activation curve was sigmoidal, while at 2 mM IMP it was hyperbolic. The A0.5 values for ATP were 2.26 and 0.70 mM, and the relative maximal velocities were 32.9 and 126.0 nmol/min, respectively. Inorganic phosphate shifts the hyperbolic substrate velocity relationship for IMP to a sigmoidal one. With physiological concentrations of cofactors (3 mM ATP, 1-4 mM Pi, 150 mM KCl) at pH 7.4, the enzyme is 25-35 times more active toward 100 microM IMP than 100 microM AMP. These data show that: (a) soluble human placental high Km 5'-nucleotidase coexists in human placenta with the low Km enzyme; (b) under physiological conditions the enzyme favors the hydrolysis of IMP and is critically regulated by IMP, ATP, and Pi levels; and (c) kinetic properties of ATP and IMP are each modified by the other compound suggesting complex interaction of the associated binding sites.

5'-Nucleotidase↗
PubMed1988

Acromegalic arthropathy. Characteristics and response to therapy.

Arthropathy was assessed in 19 patients with active acromegaly. Axial or peripheral arthropathy was present in 10 patients, was located most often in large joints or the lumbosacral spine, and was osteoarthritic in nature. The mean duration of acromegaly in patients with arthropathy was 21.6 years, while the mean duration in patients without arthropathy was 7.9 years. A mild-to-moderate improvement in symptoms, estimated functional ability, and crepitus occurred in 8 of 9 patients who were prospectively examined during therapy to lower production of growth hormone. We conclude that this therapy did improve the symptoms of acromegalic arthropathy. Whether objective structural improvements occur remains unclear.

Acromegaly↗
PubMed1988

Isocratic separation of ATP and its degradation products from biological fluids by automated liquid chromatography.

Two groups of metabolites (a) IMP, AMP, ADP, ATP, and cAMP in extracts of fibroblasts and erythrocytes and (b) hypoxanthine, xanthine, adenosine, and inosine in plasma and urine have been separated by ion-pairing reversed-phase chromatography on a microBondapak C18 column, with use of the following reagents: 60 mmol/L KH2PO4, 0.45 mmol/L tetrabutylammonium phosphate, and 1.26 mol/L acetonitrile, pH 3.2 (at 23 degrees C) (group a) and 20 mmol/L KH2PO4, 0.45 mmol/L tetrabutylammonium phosphate, and 0.35 mol/L acetonitrile, pH 2.70 (at 24 degrees C) (group b). Under both sets of conditions, the compounds are completely separated in less than 15 min. The separation is isocratic, so the method is easily adaptable to automation.

Adenosine↗
PubMed1988

Regulation of adenosine kinase by adenosine analogs.

The regulation of adenosine phosphorylation by adenosine analogs was studied using highly purified human placental adenosine kinase [ATP: adenosine 5'-phosphotransferase (EC 2.7.1.20)]. Our observations lead us to classify the analogs into three groups as follows: type I, 5'-N-ethylcarboxamidoadenosine and 5'-methylthioadenosine; type II, N6-cyclohexyladenosine, N6-L-phenylisopropyladenosine, and 2-chloroadenosine; and type III, 6-methylmercaptopurine riboside. Type I compounds are inhibitors of adenosine kinase at 0.5 microM adenosine with IC50 values of 25 microM for 5'-N-ethylcarboxamidoadenosine and 250 microM for 5'-methylthioadenosine. These compounds stimulate adenosine kinase at 5.0 microM adenosine up to a maximum of 30 to 50% above basal velocity. They are not substrates for adenosine kinase. Type II compounds are inhibitors of adenosine kinase at 0.5 microM adenosine with an IC50 of 220 microM for N6-cyclohexyladenosine and 200 microM for N6-L-phenylisopropyladenosine. These analogs also stimulate adenosine kinase at 5.0 microM adenosine. 2-Chloroadenosine, N6-cyclohexyladenosine, and N6-L-phenylisopropyladenosine are phosphorylated by adenosine kinase with apparent Km values of 1,330, and 205 microM, respectively. 6-Methylmercaptopurine riboside (type III) inhibited enzyme activity with an IC50 of 10 microM at 0.5 microM adenosine and 215 microM at 5 microM adenosine and is a substrate for adenosine kinase. These data are consistent with the following: (a) 2-chloroadenosine, N6-cyclohexyladenosine, and N6-L-phenylisopropyladenosine may not be good adenosine receptor agonists in vivo because they are phosphorylated into active derivatives by adenosine kinase; (b) 5'-N-ethylcarboxamidoadenosine and 5'-methylthioadenosine are superior candidates for adenosine receptor agonists in vivo because they are not phosphorylated; (c) 5'-N-ethylcarboxamidoadenosine, 5'-cyclohexyladenosine, N6-L-phenylisopropyladenosine, and 2-chloroadenosine may interact with adenosine kinase at two sites on the enzyme, a catalytic site and a regulatory site; and (d) 6-methylmercaptopurine riboside may interact with the enzyme at the catalytic site only.

Adenosine↗
PubMed1988

Excessive ATP degradation during hemodialysis against sodium acetate.

As the initial step in examining the metabolic basis for acetate intolerance, we have tested the hypothesis that excessive adenosine triphosphate (ATP) degradation occurs during hemodialysis against acetate dialysate (compared with the degree of degradation occurring during dialysis against bicarbonate dialysate). Seven patients undergoing long-term dialysis were infused with carbon 14--labeled 8-adenine, and their response to dialysis against acetate was compared with their response to dialysis against bicarbonate. The following changes were observed. During dialysis against acetate, the mean dialysate uric acid--to-creatinine ratio levels were significantly higher than the mean levels observed after dialysis against bicarbonate (p less than 0.001). The mean dialysate uric acid radioactivity--to-creatinine ratio and inosine, hypoxanthine, and xanthine radioactivity--to-creatinine ratio levels were significantly increased during dialysis against acetate (p less than 0.001). There was no significant change in plasma venous hypoxanthine level, but during dialysis against acetate, the arterial hypoxanthine levels (3.7 +/- 1.6 mumol/L) at 60 minutes were significantly higher than the levels observed after dialysis against bicarbonate (1.4 +/- 0.5 mumol/L) (p less than 0.01). These data provide evidence that excessive ATP degradation occurs during hemodialysis against acetate but not during hemodialysis against bicarbonate dialysate.

Acetates↗
PubMed1988

Musculoskeletal symptoms related to arthropathy in patients receiving dialysis.

We evaluated musculoskeletal complaints related to arthropathy in 28 patients with end stage renal failure receiving maintenance dialysis. Twenty-three of 28 patients had arthritic complaints and 14 had an arthropathy. Six of 14 patients with arthropathy had a pattern resembling calcium pyrophosphate dihydrate deposition (CPPD) disease, 4 patients had moderately severe osteoarthritis, 3 had calcific periarthritis, and 1 patient had acute arthritis with intermittent pain and swelling. Factors which predispose to metabolic arthropathies were observed as follows: 29% elevated ferritin; 39% history of hyperparathyroidism; 68% elevated parathormone; 54% hyperphosphatemia; 36% hypercalcemia, 29% HLA haplotypes A3, B7, or B14; and 60% hyperaluminemia. The arthropathy group had more abnormalities per patient (mean 3.6) than the group without arthropathy (mean 2.7) (p less than 0.05). Our data suggest that (1) arthritic complaints occur frequently in patients receiving dialysis; (2) arthropathy accounted for 61% of the complaints; (3) 43% of patients with arthropathy had CPPD-type; (4) renal osteodystrophy caused 17% of arthritic complaints; and (5) in patients receiving dialysis, there is a high incidence of metabolic abnormalities that are known to be associated with arthropathy.

Adult↗
PubMed1988

The arthropathy of Wilson's disease: clinical and pathologic features.

Twenty-two patients with Wilson's disease were evaluated for arthropathy with history, examination, and radiographs. Knee arthroscopy with cartilage and synovial biopsies was performed in 4 patients. Radiologic findings were minimal, including osteophytes, subchondral cysts, and joint space narrowing. Chondrocalcinosis was observed in 3 patients. Cartilage and synovial biopsies studied by energy dispersive elemental analysis showed copper and sulfur diffusely throughout cartilage in 2 of 4 patients. No crystals were found. The finding of copper in cartilage biopsies suggests that copper deposition may be a factor in the etiology of arthropathy, although definitive studies remain to be performed.

Adult↗
PubMed1987

Radiologic features of a pyrophosphate-like arthropathy associated with long-term dialysis.

In a series of 28 long-term dialysis patients with musculoskeletal complaints, the radiologic findings in six cases resembled those occurring in the arthropathy of idiopathic calcium pyrophosphate dihydrate deposition (CPPD) disease. These findings included osteophytes, subchondral cysts, and cartilage loss in the metacarpophalangeal joints, patellofemoral joints, wrists, and shoulders. Chondrocalcinosis was present in three of the six cases. There were no significant differences in renal function or levels of serum calcium, phosphorus, iron, ferritin, aluminum, or parathormone between these patients and a control group matched for sex and age. Long-term dialysis may be associated with a metabolic arthritis similar to the arthritis which occurs in CPPD deposition disease. The etiology may include deposition of CPPD crystals, hydroxyapatite, or other calcium-containing substances in joints, or it may be related to a number of dialysis-induced metabolic abnormalities.

Arthritis↗
PubMed1987

Hereditary xanthinuria. Evidence for enhanced hypoxanthine salvage.

We tested the hypothesis that there is an enhanced rate of hypoxanthine salvage in two siblings with hereditary xanthinuria. We radiolabeled the adenine nucleotide pool with [8-14C]adenine and examined purine nucleotide degradation after intravenous fructose. The cumulative excretion of radioactivity during a 5-d period was 9.7% and 9.1% of infused radioactivity in the enzyme-deficient patients and 6.0 +/- 0.7% (mean +/- SE) in four normal subjects. Fructose infusion increased urinary radioactivity to 7.96 and 9.16 X 10(6) cpm/g creatinine in both patients and to 4.73 +/- 0.69 X 10(6) cpm/g creatinine in controls. The infusion of fructose increased total urinary purine excretion to a mean of 487% from low-normal baseline values in the patients and to 398 +/- 86% in control subjects. In the enzyme-deficient patients, the infusion of fructose elicited an increase of plasma guanosine from undetectable values to 0.7 and 0.9 microM. With adjustments made for intestinal purine loss, these data support the hypothesis that there is enhanced hypoxanthine salvage in hereditary xanthinuria. Degradation of guanine nucleotides to xanthine bypasses the hypoxanthine salvage pathway and may explain the predominance of this urinary purine compound in xanthinuria.

Adenine↗
PubMed1987

Deferoxamine induced decreases of lipid peroxides in rheumatoid arthritis.

Eleven patients with rheumatoid arthritis (RA) were treated with intraarticular deferoxamine or placebo to test the hypothesis that iron chelation decreased hydroxyl radical mediated lipid peroxidation in RA. Intraarticular administration of deferoxamine 100 mg resulted in predominantly systemic effects with decreased serum ferritin and decreased serum levels of lipid peroxidation products. Similar changes were not detected in synovial fluid at this dose. Iron chelation with deferoxamine may provide a novel approach to preventing tissue injury in RA by inhibiting hydroxyl radical production and lipid peroxidation.

Adult↗
PubMed1986

Human placental cytoplasmic 5'-nucleotidase. Kinetic properties and inhibition.

The kinetic properties of highly purified human placental cytoplasmic 5'-nucleotidase were investigated. Initial velocity studies gave Michaelis constants for AMP, IMP, and CMP of 18, 30, and 2.2 microM, respectively. The enzyme shows the following relative Vmax values: CMP greater than UMP greater than dUMP greater than GMP greater than AMP greater than dCMP greater than IMP. The activity was magnesium-dependent, and this cation binds sequentially with a Km of 14 microM for AMP and an apparent Km of 6 mM for magnesium. A large variety of purine, pyrimidine, and pyridine compounds exert an inhibitory effect on enzyme activity. IMP, GMP, and NADH produce almost 100% inhibition at 1.0 mM. Nucleoside di- and triphosphates are potent inhibitors. ATP and ADP are competitive inhibitors with respect to AMP and IMP as substrates with Ki values of 100 and 15 microM, respectively. Inorganic phosphate is a noncompetitive inhibitor with Ki values of 19 and 43 mM. Nucleosides and other compounds studied produce only a modest decrease of enzyme activity at 1 mM. Our findings suggest that the enzyme is regulated under physiological conditions by the concentrations of magnesium, nucleoside 5'-monophosphates, and nucleoside di- and triphosphates. The nucleotide pool concentration regulates the enzyme possibly by a mechanism of heterogeneous metabolic pool inhibition. These properties of human placental cytoplasmic 5'-nucleotidase may be related to the control of nucleotide degradation in vivo.

5'-Nucleotidase↗
PubMed1986

Characteristics of high affinity and low affinity adenosine binding sites in human cerebral cortex.

The binding characteristics of human brain cortical membrane fractions were evaluated to test the hypothesis that there are A1 and A2 adenosine binding sites. The ligands used were 2-chloro[8-3H]adenosine and N6-[adenine-2,8-3H]cyclohexyladenosine. Binding of chloroadenosine to human brain cortical membranes was time dependent, reversible and concentration dependent. The Kd calculated for chloroadenosine by Scatchard analysis of equilibrium data was 280 nM, with a Bmax of 1.6 pmoles/mg protein, suggesting a single class of binding sites. The specificity of chloroadenosine binding was assessed by the ability of adenosine analogs to compete for binding sites. Using this approach, the apparent Kd was estimated to be 0.74 microM for 5'-N-ethyl-carboxamideadenosine, 1 microM cyclohexyladenosine, and 13 microM for N6-(L-2-phenylisopropyl)adenosine. Isobutylmethylxanthine and theophylline, receptor antagonists, had apparent Kd values of 84 microM and 105 microM, respectively. Hill slope factors ranged from 0.3 to 0.6. Chloroadenosine binding to human brain cortical membranes approached equilibrium at 90 minutes, with a T1/2 of 10 minutes. The kob was 0.080 min-1 and the k1 was 7.5 X 10(4) min-1 M-1. Reversibility of chloroadenosine binding at equilibrium was completed at approximately 10 minutes with a k2 value of 0.074 min-1. The Kd calculated from the rate constants was 990 nM. Cyclohexyladenosine binding was concentration dependent. The Kd calculated for cyclohexyladenosine via Scatchard analysis of equilibrium data was 5 nM with a Bmax of 0.35 pmoles/mg protein. Cyclohexyladenosine binding was displaced by 3 known receptor agonists: N6-(L-2-phenyliso propyl)adenosine (Kd 4 nM), 2-chloroadenosine (Kd 10 nM) and 5H-N-ethyl-carboxamideadenosine (Kd 6 nM). The apparent Kd values for the agonists were 1 to 3 orders of magnitude lower with this ligand as compared to radioactive chloroadenosine. Binding was also displaced by 2 known antagonists, isobutylmethylxanthine and theophylline, with apparent Kd values of 4 microM and 8 microM, respectively. Hill slope factors ranged from 0.5 to 0.8. Our data support the existence of two adenosine binding sites in human cortex compatible with the low affinity (A2) and high affinity (A1) adenosine receptors.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Regulation of deoxyadenosine and nucleoside analog phosphorylation by human placental adenosine kinase.

The enzymes responsible for phosphorylation of adenosine and nucleoside analogs are important in the pathogenesis of adenosine deaminase deficiency and for the activation of specific anticancer and antiviral drugs. We examined the role of adenosine kinase in catalyzing these reactions using an enzyme purified 4000-fold (2.1 umol/min/mg) from human placenta. The Km values of adenosine and ATP are 135 uM and 4 uM, respectively. Adenosine kinase phosphorylates adenine arabinoside with an apparent Km value of 1 mM using adenosine kinase assay conditions. The Km values for 6-methylmercaptopurine riboside and 5-iodotubercidin, substrates for adenosine kinase, are estimated to be 4.5 uM and 2.6 nM, respectively. These data indicate that dadenosine phosphorylation by adenosine kinase is primarily regulated by its Km, and the concentrations of Mg2+, ADP and AMP. The high Km values for phosphorylation of dadenosine and adenine arabinoside suggest that adenosine kinase may be less likely to phosphorylate these nucleosides in vivo than other enzymes with lower Km values. Adenosine kinase appears to be important for adenosine analog phosphorylation where the Michaelis constant is in the low micromolar range.

Adenosine↗