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J Foote

Publications and source records attributed to J Foote.

At least 55 records · Page 3Linked to original sources
PubMed1985

Homotropic effects in aspartate transcarbamoylase. What happens when the enzyme binds a single molecule of the bisubstrate analog N-phosphonacetyl-L-aspartate?

The active sites of aspartate transcarbamoylase from Escherichia coli were titrated by measuring the decrease in the enzyme-catalyzed arsenolysis of N-carbamoyl-L-aspartate caused by the addition of the tight-binding inhibitor, N-phosphonacetyl-L-aspartate. Because the enzyme is a poor catalyst for this non-physiological reaction, high concentrations are required for the assays (more than 1000-fold the dissociation constant of the reversibly bound inhibitor) and, therefore, virtually all of the bisubstrate analog is bound. From the endpoint of the titration, 5.7 active sites were calculated, in excellent agreement with the number, six, based on the structure of the enzyme. Simple inhibition was observed only when the molar ratio of inhibitor to enzyme exceeded five; under these conditions, as shown in earlier physical chemical studies, the R-conformational state of the enzyme is the sole or predominant species. At low ratios of inhibitor to enzyme, the addition of inhibitor caused an increase in activity which is attributable to the conversion of the enzyme from the low-activity T-state to the much more active R-state. Comparison of the linear increase in activity as a function of inhibitor concentration at the low molar ratio (0.01, i.e. 1 inhibitor/600 active sites) with the activity lost at the high ratio provided a direct value for the mean number of active sites converted from the T-state to the R-state as a result of the binding of one bisubstrate analog to an enzyme molecule. This number was four with Mg X ATP or carbamoyl phosphate present and 4.7 for the enzyme in the presence of Mg X PPi, values approaching or identical to the theoretical maximum, 4.7, for a concerted transition with all of the active sites of the molecule changing from the T- to R-state upon the formation of a binary complex of hexameric enzyme with a single inhibitor. With the enzyme in the absence of effectors or with Mg X CTP present, the titrations showed that an average of two and one sites, respectively, of 4.7 possible, changed conformation upon ligand binding. These results were interpreted as a manifestation of an equilibrium between a sub-population of T- and R-state enzyme complexes containing one bound inhibitor molecule. The R-state species would represent 40% of the population for aspartate transcarbamoylase in the absence of extraneous ligands.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1985

Substrate specificity of aspartate transcarbamylase. Interaction of the enzyme with analogs of aspartate and succinate.

The ability of aspartate transcarbamylase from Escherichia coli to catalyze carbamylation of amino acids other than the natural substrate, L-aspartate, was examined. Cysteine, cysteate, cysteinesulfinate, and 3-nitroalanine showed kcat values at pH 7 of 0.16, 0.58, 5.2, and 62 s-1, respectively, while kcat with aspartate was 320 s-1. In a parallel study, competitive inhibition constants of 3-nitropropionate, 3-mercaptopropionate, 3-sulfopropionate, and 3-sulfinopropionate were found to be high, about 0.1 M, compared with that of succinate, 0.56 mM. Although cysteinesulfinate had low activity as a substrate, the pH dependences of kcat and kcat/Km in H2O and D2O observed with the compound closely paralleled those of aspartate. The results of these studies suggest that substrate specificity and reactivity are achieved in part by a strong, highly specific interaction of one or more active site residues with the beta-carboxylate of L-aspartate. Unlike the sigmoidal kinetics found with aspartate, saturation of native aspartate transcarbamylase by cysteine sulfinate showed a lack of cooperativity, even under conditions of activation of the reaction by ATP and inhibition by CTP. The cysteinesulfinate reaction was increased 9-fold by the bisubstrate analog N-phosphonacetyl-L-aspartate. These results were interpreted in terms of an inability of cysteinesulfinate to cause the allosteric conformational change promoted by aspartate.

Amino Acids↗
PubMed1985

On conformational changes in the regulatory enzyme aspartate transcarbamoylase.

Our preliminary studies provide clear answers to only some of the basic questions posed above regarding the nature of conformational changes in ATCase. By exploiting the reverse reaction to develop a kinetic active site titration technique we demonstrated that symmetry-related regions of ATCase are affected equally in the allosteric transition even though the bisubstrate analog is bound to only one of the six active sites. This is a vivid illustration of a "global" change in an enzyme, and it provides powerful support for the view that active site ligands promote a concerted transition of the enzyme from the low-activity to the high-activity conformation. Kinetic experiments show that this conformational change is rapid, requiring only tenths of a second. In contrast, the studies with isolated catalytic subunits provide evidence for a much more local conformational change which occurs after the ligand is bound and which is much slower, lasting over a time period of many seconds. Although the experiments on the holoenzyme do not show directly how many atoms are involved in the conformational change or how large are the displacements, they do indicate, especially in conjunction with other studies, that many amino acid residues in both the catalytic and regulatory chains must be implicated in the T----R allosteric transition. Why PALA has such a different local effect on the catalytic subunit than does the combination of carbamoylphosphate and succinate is not clear, but this uncertainty may be resolved by further NMR studies. Determining how the local changes at the active sites are linked to the global transition affecting the quaternary structure of the enzyme remains a formidable problem. Also further work is needed in order to determine whether unliganded ATCase molecules exist in an equilibrium mixture of T and R conformations even prior to the ligand binding event.

Allosteric Regulation↗
PubMed1984

The postnatal development of blood vessels in the optic nerve of normotensive and hypertensive rats.

The blood vessels in the optic nerve of normotensive and hypertensive rats have been examined at 2, 4, 8 and 12 weeks of age. The pattern of development was found to be different in the two strains, with the number of blood vessels in the hypertensive rat optic nerve being lower at 2 weeks, but greater at 12 weeks than the normotensive rat. There appeared to be no correlation between vascularity and either myelination or changes in the fibre diameter spectrum at the ages studied. It is concluded that while the cause of the increased vascularity of the optic nerve in hypertensive rats is not known, it appears to be without effect in the structural development of the optic nerve.

Animals↗
PubMed1983

An ultraviolet spectrophotometric assay for aspartate transcarbamylase.

A procedure for determining the activity of aspartate transcarbamylase, based upon the greater ultraviolet absorbancy of the products of the reaction catalyzed compared to the reactants, was devised. Extinction coefficients were determined at 205, 210, and 215 nm for the compounds carbamoyl aspartate, acetyl aspartate, and aspartate. These values formed the quantitative basis for a spectrophotometric assay in which an enzymatic reaction is monitored at one of these wavelengths. Use of this procedure was illustrated in four kinetic experiments with the allosteric aspartate transcarbamylase from Escherichia coli, and the nonallosteric catalytic subunit of this enzyme: aspartate saturation curve, arsenate saturation curve (reverse reaction), allosteric activation by a transition-state analog employing acetyl phosphate as substrate, and carbamoyl phosphate progress curve (substrate depletion in the presence of excess cosubstrate). Owing to changes in absorbance on the order of 1000 liter mol-1 cm-1 concomitant with the reaction, the sensitivity of the method is comparable to that of many procedures already in the literature.

Aspartate Carbamoyltransferase↗
PubMed1982

Factors involved in the control of RNA synthesis during regeneration of the optic nerve in the frog.

The incorporation of tritiated uridine into retinal ganglion cells was studied following intra- and extracranial section of the optic nerve in the frog. It was found that incorporation increased to above control levels by two days following extracranial section of the optic nerve and by two weeks following intracranial section and was maintained at an elevated level until the regenerating optic nerve reached the diencephalon, at which point the rate of incorporation fell back to control levels. It is suggested that the signal for turning off increased uridine incorporation lies in the diencephalon, and that functional reconnection is not the signal.

Animals↗
PubMed1981

Kinetics of aspartate transcarbamylase from Escherichia coli for the reverse direction of reaction.

The reverse reaction of aspartate transcarbamylase in which phosphate or arsenate is first coupled to carbamyl aspartate, followed by elimination of aspartate, has been studied under conditions in which one product, aspartate, is removed. Aspartate is converted to oxalacetate by glutamate-oxalacetate transaminase, and the resulting oxalacetate is converted to malate by the NADH, NAD+ oxidoreductase enzyme malate dehydrogenase. Phosphate and carbamyl aspartate saturation curves are nonsigmoidal. The transition state analogue, N-phosphonacetyl-L-aspartate, activates this reverse reaction substantially. Reverse kinetic parameters of the Haldane type are characteristic of the T-state and correlated with the parameters of the usual forward reaction of the T-state. Phosphate and carbamyl aspartate do not alter the thiol reactivity or sedimentation coefficient of the enzyme. These five results indicate that, under the conditions of these experiments, the reverse reaction does not cause the allosteric transition. In a new assay for the forward reaction we couple phosphate production with NADP reduction using phosphorylase a, phosphoglucomutase, and glucose-6-phosphate dehydrogenase.

Adenosine Triphosphate↗
PubMed1981

A study of degeneration, scar formation and regeneration after section of the optic nerve in the frog, Rana pipiens.

Degeneration, scar formation and regeneration have been studied after section of the optic nerve in the frog. In the normal optic nerve two types of macroglial cell were identified: astrocytes and oligodendroglia. Degeneration after injury proceeded rapidly in comparison with mammals but did not lead to the production of a dense scar. Before much scarring had developed, regenerating axons penetrated the lesioned area.

Animals↗
PubMed1980

The role of tryptophan in aspartate transcarbamylase.

Replacement of 7-azatryptophan for tryptophan in two positions on the catalytic chain of aspartate transcarbamylase results in changes in the enzyme's homotropic and heterotropic interactions although there is no change in the enzyme's specific activity. The extent of azatryptophan incorporation was quantitated by amino acid analysis which showed that 85% of the tryptophan residues had been replaced. The substituted enzyme is activated by ATP and inhibited by CTP to a greater extent than is the native enzyme. The aspartate saturation curve in the presence of ATP is identical for the two enzymes, but the curve in the presence of CTP and without effectors is shifted toward higher aspartate concentrations for the azatryptophan-substituted enzyme. At low aspartate concentrations, the native enzyme is activated to a greater extent by the substrate analog succinate. These data suggest that the substitution renders the low substrate affinity conformational state of the enzyme less catalytically efficient. This interpretation is in agreement with possible side chain interactions observed in the three-dimensional structure of the enzyme.

Aspartate Carbamoyltransferase↗
PubMed1980

Isolation and preliminary characterization of single amino acid substitution mutants of aspartate carbamoyltransferase.

In order to isolate functional Escherichia coli aspartate carbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC2.1.3.2) with single amino acid replacements, a series of pyrB nonsense mutants has been isolated. These nonsense mutants were induced by 2-aminopurine mutagenesis and selected by a combination of antibiotic treatments, direct enzyme assays, and suppressibility tests. Suppression of the pyrB nonsense mutation with various suppressors, which insert different amino acids, has resulted in the formation of a series of mutant aspartate carbamoyltransferases, each differing in one amino acid from the wild-type enzyme. After partial purification, kinetic studies revealed that some of the mutant enzymes had altered homotropic and heterotropic interactions. The mutants that had a tyrosine insert showed the most pronounced changes, followed by those with a serine insert. The mutants having a glutamine insert, howevr, were indistinguishable from the wild-type enzyme, supporting the conclusion that, because of the specificity of the mutagen, the glutamine insert had regenerated the wild-type enzyme.

2-Aminopurine↗
PubMed1980

Early events in stricture formation in the guinea pig urethra.

The early stages of urethal stricture formation have been examined by light and electron microscopy after injury of the anterior urethra in the guinea pig. It was found that initially an ulcerated surface formed which was subsequently covered by simple then stratified squamous epithelium. The tissue of the stricture contained elongated myofibroblasts and clumps of multinucleated giant cells. It is suggested that the myofibroblasts are responsible for the formation of the stricture and that the giant cells may be involved in continued collagen synthesis.

Animals↗
PubMed1979

Discriminative effectiveness of the Senior Apperception Test with impaired and nonimpaired elderly persons.

A means was devised of assessing elderly individuals' responses to the Senior Apperception Test (SAT) that would discriminate emotionally-cognitively impaired from nonimpaired. With a matched sample of known impaired and nonimpaired nursing home residents, SAT protocols were scored on four dimensions, and the best discriminating cut-off scores established. Protocols from a second similar matched sample were tested for discriminative effectiveness of previously established criterion scores. Significant discrimination at .01 or better was obtained for all four dimensions in the predicted direction. False positives and false negatives ranged from 7 to 33%. The best predictor score was Interpersonal-relationships, the poorest was mood. This procedure with the SAT appears to be an effective screening method for impairment, and it provides additional useful clinical information.

Affective Symptoms↗
PubMed1977

Neurogenic dysfunction of the urinary bladder in Parkinson's disease.

Involvement of the urinary bladder in Parkinson's disease was recognized in the 1950's, when the accidental discovery of a neurosurgical means of treating the disease brought more attention to it, with increasing research and study. In more recent years, the advent of more specific medical treatment has led to closer examination of the disease, and has enlarged our awareness of its complications. This has been paralleled by progress in the field of urology, with more precise methods of measuring dysfunction of the urinary bladder.

Female↗
PubMedDate not supplied

An enhanced positive reinforcement model for the severely impaired cocaine abuser.

This article describes a cognitive-behavioral treatment approach that has been extensively modified to work with inner-city methadone-maintained cocaine users. Modifications were deemed essential to address the problems of engagement and retention in treatment that are typically encountered with this population. While this approach relies on such basic tenets of treatment as relapse prevention, cognitive restructuring, and psychoeducation, an understanding of the particular psychological vulnerabilities of this population has been incorporated into the model. The modified approach utilizes positive reinforcement extensively. This includes use of concrete reinforcers to facilitate initial engagement, and use of interpersonal reinforcers (therapist positive regard, attention, and respect) to increase program retention and sustain posttreatment change. Preliminary results indicate that 63% of patients can complete this intensive 6-month program, with considerable reductions in cocaine use and significant change in drug injection behavior.

Adult↗