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J A Fox

Publications and source records attributed to J A Fox.

At least 55 records · Page 3Linked to original sources
PubMed1987

8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 1. Purification and characterization.

The 8-hydroxy-5-deazaflavin (coenzyme F420) reducing hydrogenase from the obligate anaerobe Methanobacterium thermoautotrophicum delta H has been purified 41-fold to apparent homogeneity. The major active enzyme form is a high molecular weight aggregate of Mr ca. 800,000, composed of three subunits, alpha (Mr 47K), beta (Mr 31K), and gamma (Mr 26K). The hydrogenase is purified aerobically in reversibly inhibited form, and conditions for anaerobic reductive activation with H2, high salt, thiols, and electron acceptors have been defined. The minimal species transferring electrons from H2 to coenzyme F420 appears to be an alpha beta delta (Mr 115K) complex. The tightly associated redox cofactors per 115K species are 0.6-0.7 nickel atom, 0.8-0.9 flavin adenine dinucleotide (FAD), and 13-14 iron atoms in iron-sulfur centers. The subunits have been separated by denaturing gel electrophoresis, which has permitted determination of amino acid composition, subunit N-terminal sequencing, and preparation of subunit-directed antibodies. There is iron associated with the alpha-subunit, but placement of the nickel and FAD has not been established.

Amino Acid Sequence↗
PubMed1987

8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 2. Kinetic and hydrogen-transfer studies.

Steady-state kinetic parameters have been obtained for the pure 8-hydroxy-5-deazaflavin-reducing hydrogenase. With H2 and 8-hydroxy-5-deazariboflavin (F0) as substrates, Km (H2) = 12 microM, Km (F0) = 26 microM, and Kcat = 225 s-1. In the back-direction, F0H2 is reoxidized (anaerobically) at 225 s-1. Initial velocity patterns, product inhibition patterns, dead-end inhibition by carbon monoxide, and transhydrogenation to Procion Red HE-3B suggest a two-site hybrid ping-pong mechanism. A kinetic derivation for the rate equation is provided in the Appendix. Studies with D2 and with D2O reveal that no steps involving D transfer are substantially rate determining. Further, D2 yields F0H2 with no deuterium at C5 while in D2O a 5-monodeuterio F0H2 product is formed, indicating complete exchange of hydrogens from H2 with solvent before final transfer of a hydride ion out from reduced enzyme to C5 of F0.

Aerobiosis↗
PubMed1987

Purification of phosphatidylinositol kinase from bovine brain myelin.

A membrane-bound phosphatidylinositol (PI) kinase (EC 2.7.1.67) was purified by affinity chromatography from bovine brain myelin. This enzyme activity was solubilized with non-ionic detergent and chromatographed on an anion-exchange column. Further purification was achieved by affinity chromatography on PI covalently coupled to epoxy-activated Sepharose, which was eluted with a combination of PI and detergent. The final step in the purification was by gel filtration on an Ultrogel AcA44 column. This procedure afforded greater than 5500-fold purification of the enzyme from whole brain myelin. The resulting activity exhibited a major silver-stained band on SDS/polyacrylamide-gel electrophoresis with an apparent Mr 45,000. The identity of this band as PI kinase was corroborated by demonstration of enzyme activity in the gel region corresponding to that of the stained protein. The purified enzyme exhibited a non-linear dependence on PI as substrate, with two apparent kinetic components. The lower-affinity component exhibited a Km similar to that observed for the phosphorylation of phosphatidylinositol 4-phosphate by the enzyme.

1-Phosphatidylinositol 4-Kinase↗
PubMed1987

Insulin-stimulated diacylglycerol production results from the hydrolysis of a novel phosphatidylinositol glycan.

We recently described the insulin-dependent release of a carbohydrate substance from plasma membranes which regulated certain intracellular enzymes (Saltiel, A. R., and Cuatrecasas, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5793-5797). This enzyme-modulating substance appeared to arise from the phosphodiesterase hydrolysis of a novel inositol-containing glycolipid. This is supported by observations that insulin stimulated the rapid generation of [3H]myristate-labeled diacylglycerol in cultured BC3Hl myocytes. Myristoyl diacylglycerol production in these cells was unaffected by epinephrine, although arachidonate-labeled diacylglycerol was rapidly produced in response to stimulation by this alpha-1 adrenergic agent. The production of distinct species of diacylglycerol was apparently due to hormonally specific hydrolysis of different precursors. A novel glycolipid was identified on silica TLC or high pressure liquid chromatography which served as a substrate for the insulin-stimulated phosphodiesterase reaction. This glycolipid was metabolically labeled with radioactive inositol, glucosamine, and myristic acid, suggesting a phosphatidylinositol (PI)-glycan structure. Treatment of this glycolipid with a PI-specific phospholipase C resulted in the generation of two products: an inositol phosphate-glycan which modulated the activity of the low Km cAMP phosphodiesterase and myristoyl diacylglycerol. Insulin caused the rapid hydrolysis of the PI-glycan, which was then apparently resynthesized. These data further suggest that insulin stimulates the activity of a phospholipase C which selectively hydrolyzes a novel PI-glycan, releasing a carbohydrate enzyme modulator as well as a unique species of diacylglycerol.

Cell Line↗
PubMed1987

Glycosyl-sn-1,2-dimyristylphosphatidylinositol is the membrane anchor for Trypanosoma equiperdum and T. (Nannomonas) congolense variant surface glycoproteins.

We have analysed the structures of the Trypanosoma (Nannomonas) congolense and T. equiperdum variant surface glycoprotein (VSG) membrane anchors. Myristic acid uptake, phospholipase treatment, and nitrous acid deamination showed that, for each species, the anchor is glycosyl-sn-1,2-dimyristylphosphatidylinositol, as has been previously described for T. brucei. Osmotic lysis of these trypanosomes resulted in the release of soluble VSG, lacking fatty acid. In both species and in T. evansi, an endogenous phospholipase C, which cleaved diacylglycerol from membrane form VSG, was identified.

Animals↗
PubMed1987

Purification of a phosphatidylinositol-glycan-specific phospholipase C from liver plasma membranes: a possible target of insulin action.

Insulin stimulates the hydrolysis of a phosphatidylinositol-glycan, resulting in the generation of two related inositol phosphate-glycan enzyme modulators and diacylglycerol. A phosphatidylinositol-glycan-specific phospholipase C that catalyzed this reaction was found in the plasma-membrane fraction of rat liver. The enzymatic activity was measured by the release of diacylglycerol from the glycosylated-phosphatidylinositol-membrane-anchored variant surface glycoproteins of African trypanosomes. The enzyme also catalyzed the production of an inositol phosphate-glycan from an insulin-sensitive phosphatidylinositol-glycan precursor. The enzyme was solubilized with neutral nonionic detergent and purified to near homogeneity by anion-exchange chromatography on DEAE-cellulose, followed by hydrophobic chromatography on butyl-agarose. The resulting enzyme preparation was purified approximately 20,000-fold from whole liver and exhibited one major silver-stained band of Mr 52,000 on NaDodSO4/PAGE. Gel permeation chromatography of the purified activity revealed a Stokes radius of 35 A and an apparent molecular weight of 62,000, suggesting that the enzyme was tightly associated with lipid or detergent but existed as a monomer in its active form. The enzyme was specific for glycosylated phosphatidylinositol; no hydrolysis of phosphatidylinositol, phosphatidylinositol 4,5-bisphosphate, or phosphatidylcholine was observed. The enzyme was calcium independent and thiol activated. These data suggest a role for the phosphatidylinositol-glycan-specific phospholipase C as an effector for some of the metabolic actions of insulin.

Animals↗
PubMed1986

Purification and characterization of a novel glycan-phosphatidylinositol-specific phospholipase C from Trypanosoma brucei.

A novel membrane-bound glycan-phosphatidylinositol-specific phospholipase C, which catalyzes the conversion of membrane form variant surface glycoproteins to soluble variant surface glycoproteins, with the release of sn-1,2-dimyristylglycerol, has been isolated from Trypanosoma brucei. The activity was solubilized from trypanosome membrane fractions in non-ionic detergent and purified by anion exchange chromatography on DEAE-cellulose followed by chromatography on phosphatidylinositol-Sepharose. The enzyme constitutes about 0.1% of the total cellular protein and has an apparent molecular weight of 39,800. The enzyme shows a head group specificity for molecules containing carbohydrate covalently linked to glycan-phosphatidylinositol, but can also act on the monoacyl derivative of membrane form variant surface glycoprotein. It shows no specific ion requirements but is stimulated by thiol-reducing agents and inhibited by ions that thiols chelate.

Animals↗
PubMed1986

Insulin-stimulated hydrolysis of a novel glycolipid generates modulators of cAMP phosphodiesterase.

Insulin action may involve the intracellular generation of low molecular weight substances that modulate certain key enzymes. The production of two substances that regulate the activity of adenosine 3',5'-monophosphate phosphodiesterase was evaluated in cultured myocytes by incorporation of radiolabeled precursors. Insulin caused the rapid hydrolysis of a chemically undefined membrane glycolipid, resulting in the production of two related complex carbohydrates as well as diacylglycerol. Both the glycolipid precursor and the aqueous products were monitored by labeling with radioactive inositol and glucosamine. Depletion of the labeled precursor and the appearance of labeled water-soluble products and diacylglycerol occurred within 30 seconds after hormone treatment and was followed by rapid resynthesis of the precursor. The aqueous products that were radioactively labeled appeared chromatographically and electrophoretically identical to phosphodiesterase modulating activities produced by insulin from the same cells. The purified radiolabeled and bioactive substances had similar chemical properties. Hydrolysis of the glycolipid precursor and subsequent generation of products could be reproduced by incubation of extracted lipids with a phosphatidylinositol-specific phospholipase C. These studies suggest that insulin stimulates an endogenous, selective phospholipase C activity that hydrolyzes a novel glycolipid, resulting in the generation of a complex carbohydrate-phosphate substance containing inositol and glucosamine that may mediate some of the actions of the hormone.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Preliminary experience with defibrotide in severe lower limb ischaemia.

Eight patients with severe lower limb ischaemia, aged 65-80, received defibrotide intravenously for periods from 5 to 21 days (mean 13 days). All patients had intractable rest pain. Five had ischaemic ulcers and 3 had minor gangrene. Five had previous arterial surgery and 6 lumbar sympathetic ganglion injections. Pretreatment ankle pressure indices ranged from 0 to 0.5 (mean 0.19). Rest pain, sleep disturbance and analgesic requirement were assessed on a nominal scale. Rest pain improved in 4 and sleeping pattern in 2 patients. One patient showed a diminution in analgesic requirement. Pressure indices improved in 5 patients. Amputation was performed in 4 patients. Adverse reactions included vomiting and diarrhoea (2), thrombophlebitis at infusion site (3) and generalized skin reaction (1). All patients had 'end-stage' peripheral vascular disease but some showed symptomatic benefit. Further evaluation of defibrotide is indicated.

Aged↗
PubMed1985

The mechanism of action of ethanolamine ammonia-lyase, an adenosylcobalamin-dependent enzyme. Evidence that the hydrogen transfer mechanism involves a second intermediate hydrogen carrier in addition to the cofactor.

Ethanolamine ammonia-lyase catalyzes the adenosylcobalamin (AdoCbl)-dependent conversion of ethanolamine to acetaldehyde and ammonia. During this reaction, a hydrogen atom migrates from the carbinol carbon of ethanolamine to the methyl carbon of acetaldehyde. Previous studies have shown that this migrating hydrogen equilibrates with the hydrogens on the 5'-(cobalt-linked) carbon of the cofactor. On the basis of those studies, a two-step mechanism for hydrogen transfer has been postulated in which the migrating hydrogen is first transferred from the substrate to the cofactor, then in a subsequent step is returned from the cofactor to the product. We now show that this migrating hydrogen is transferred not only to the cofactor, but also to a second acceptor at the active site. Hydrogens on this acceptor do not exchange with water during the course of the reaction, but are released to water when the enzyme is denatured. The catalytic significance of this second hydrogen acceptor was demonstrated by the findings that the transfer of hydrogen to this acceptor required both AdoCbl and active enzyme and that hydrogen at the second acceptor site could be washed out by unlabeled ethanolamine. On the basis of these results, we propose an expanded hydrogen transfer mechanism in which AdoCbl and the second acceptor site serve as alternative intermediate hydrogen carriers during the course of ethanolamine deamination.

Ammonia-Lyases↗
PubMed1985

Controlled trial of Iodosorb in chronic venous ulcers.

Cadexomer iodine (Iodosorb) is a hydrophilic starch powder containing iodine, which is a suitable dressing for granulating wounds such as venous ulcers. A total of 61 outpatients with chronic venous ulcers participated in a randomised optional crossover trial using cadexomer iodine or a standard dressing for their ulcers. The trial lasted for 24 weeks or until the ulcer had healed. Two patients withdrew during the course of the trial. Both treatments were highly effective, but the epithelium of ulcers dressed with cadexomer iodine grew again significantly faster (p less than 0.001). At the midpoint of the trial (12th week) 13 of 30 patients receiving standard treatment were changed to cadexomer iodine, while only three of 29 receiving cadexomer iodine changed to the standard dressing (p less than 0.02). In most cases ulcers were dressed and rebandaged daily by the patients themselves after instruction and supervision. This may be better than having dressings and bandages applied by professionals less regularly.

Adult↗
PubMed1985

Conductance and selectivity properties of a substate of the rabbit sarcoplasmic reticulum channel.

In this paper I describe the occurrence and properties of a subconductance state of the cation-selective channel of the sarcoplasmic reticulum. The substate conductance is 60% of the major state conductance in every salt solution examined. When single channel conductance is plotted vs. ion concentration (for potassium or thallium salt solutions) the Michaelis-Menten constant is nearly the same for both conductance states, while the maximum conductance is reduced for the subconductance state. Both conductance states show anomalous conductance behavior in mixed potassium-thallium solutions that may be modeled in the same way. These results indicate that the ionic selectivity of the channel is the same for both open states.

Animals↗