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Biomedical subjects

J F Morrison

Publications and source records attributed to J F Morrison.

At least 145 records · Page 8Linked to original sources
PubMed1982

The influence of visceral mechanoreceptors on sympathetic efferent discharge in the cat.

1. Recordings have been made from eighty-three single sympathetic efferent units in the hypogastric nerve in two types of preparation. In all animals the baroreceptors were denervated to exclude changes in sympathetic discharge resulting from any variations in arterial pressure, and the spinal cord was sectioned at the 6th lumbar segment to exclude changes in efferent discharge that might have been due to pelvic nerve afferents from the bladder or other viscera. In some animals the afferent pathways were sectioned from all pelvic and lower abdominal viscera other than the bladder, so that the vesical afferent pathway was the only neural pathway that might mediate reflex events from these viscera. The hypogastric nerve afferent pathway was excited by bladder distension or by bladder contractions induced by electrical stimulation of the sacral cord.2. Approximately half the units gave an increase in spike rate during distension or during contraction of the bladder. Three-quarters of the units tested also gave an increase in spike rate during colonic distension. In no units that exhibited convergence of afferent input did colonic and vesical stimuli cause responses of opposite sign.3. Approximately 10% of units showed a reduction in discharge rate when the bladder was distended or caused to contract.4. The estimated intravesical pressure thresholds for these reflexes were in the range 8-56 mmHg which extends beyond that of the mechanoreceptors which form the afferent limb of the reflex.5. These reflex studies indicate that the hypogastric nerve afferents from the bladder can elicit sympathetic reflexes within the physiological range of intravesical pressures, and that vesico-sympathetic reflexes can be elicited in the absence of pelvic nerve afferent inputs.In 75% of sympathetic efferent units that respond to bladder distension or contraction, there is evidence for convergent inputs with similar actions from the colon.

Action Potentials↗
PubMed1982

An electrophysiological study of somatic and visceral convergence in the reflex control of the external sphincters.

1. Mass wave and single unit discharges have been recorded from pudendal efferents innervating the external anal and urethral sphincters in chloralose anaesthetized or decerebrate cats.2. Reflex discharges in these neurones were elicited by electrical stimulation of the contralateral pudendal nerve, the posterior cutaneous nerve of thigh, or the vesical or colonic branches of the pelvic nerve. The latencies of the evoked responses were 5.5-20 msec. The vesical branches of the pelvic nerve produced discharges less consistently than the other nerves.3. Irrespective of whether afferent stimulation produced an early evoked response there was always a prolonged period of depression of pudendal nerve excitability following the stimulus. Condition-test interactions showed that this depression began within 50 msec of the stimulus and that its duration varied between 150 and 2500 msec in single units, with a modal value of 500 msec.4. No evoked response or depression of excitability was seen when afferents in the hypogastric or lumbar colonic nerves were stimulated.5. Increasing intravesical or intracolonic pressure, within physiological limits, produced a graded reduction in the size of evoked discharges.6. Short trains of stimuli (four shocks in 20 msec) applied to the raphé nucleus, were capable of inhibiting test responses in pudendal efferents for periods of up to 800 msec.7. The possible functional roles of two groups of sphincteric reflex interneurones, with either excitatory or inhibitory receptive fields, are discussed.

Action Potentials↗
PubMed1981

Chemical mechanism of the reaction catalyzed by dihydrofolate reductase from Streptococcus faecium: pH studies and chemical modification.

The variation with pH of the kinetic parameters associated with dihydrofolate reductase from Streptococcus faecium has been used to gain information about the chemical mechanism of the reaction catalyzed by the enzyme. The pH dependence of log V/K for dihydrofolate showed that a group with a pK value of 4.7 must be ionized and that a group with a pK value of 6.6 must be protonated for activity. Temperature and solvent perturbation studies indicate that these groups are probably the carboxyls of the glutamate moiety of dihydrofolate and of an aspartate residue on the enzyme, respectively. The similarity of the pH profile and the magnitude of the pK value for the linear competitive inhibitor 2,4-diaminopteridine suggest that the carboxyl group is concerned with the binding of dihydrofolate and its analogues to the enzyme. This conclusion is confirmed by the result that a group with a pK value of 6.7 must be protonated for the binding of methotrexate. It is proposed that the binding involves the formation with N-5 of dihydrofolate or N-1 of methotrexate of a hydrogen bond which has considerable ionic character and which lies within a hydrophobic environment. Further, it is suggested that the same hydrogen acts as an auxiliary catalyst which facilitates hydride transfer from NADPH to dihydrofolate for its conversion to tetrahydrofolate. Evidence to support this suggestion comes from the finding that the V profile is similar to the V/K profile except that the pK of the group which must be protonated for maximum enzyme activity is shifted upward by about 2 pH units. Such an increase in a pK value is consistent with the formation of a hydrogen ionic bond in the ternary enzyme-NADPH-dihydrofolate complex. The results of inactivation experiments with trinitrobenzenesulfonate appear to indicate that a lysine residue is necessary to maintain the enzyme in its active conformation.

Hydrogen-Ion Concentration↗
PubMed1980

A kinetic method for determining dissociation constants for metal complexes of adenosine 5'-triphosphate and adenosine 5'-diphosphate.

A general kinetic method is described for determining the dissociation constants of metal-ATP complexes that act as inhibitory substrate analogues for any enzyme that utilizes MgATP(2-). The usefulness of the procedure is illustrated by the results obtained from studies of the inhibition of hexokinase by lanthanide-ATP (LnATP) complexes. At relatively low concentrations of Mg2+, these complexes act as linear competitive inhibitors with respect to MgATP(2-). In the presence of higher, fixed concentrations of Mg2+, however, double reciprocal plots of the inhibition by LnATP vs. MgATP are nonlinear, and the data can be used to determine the ratio of the dissociation constants for the LnATP and MgATP complexes. As values are available for the dissociation constant of MgATP under a variety of conditions, that for any LnATP complex can be calculated. The dissociation constant for EuATP at pH 8.0 is 0.16 microM, while that for GdATP is 0.91 microM at pH 6.0, 0.087 microM at pH 7.95, and 1 microM at pH 8.65. Between pH 6 and 8, the ratio of the dissociation constants for GdATP and MgATP(2-) remains constant, and thus, within this range of pH, the lanthanide species involved must be Gd3+ and GdATP-. The method can also be applied to the determination of dissociation constants for inhibitory metal-ADP complexes if MgADP- is used as the variable substrate.

Adenosine Diphosphate↗
PubMed1980

Interaction of metal(III)-adenosine 5'-triphosphate complexes with yeast hexokinase.

In the presence of glucose, yeast hexokinase is specifically and strongly inhibited by all MIIIATP (M = metal) complexes that do not hydrolyze at neutral pH, as long as the ionic radius of the metal is less than 0.89 A. Ki values vary from the micromolar range (0.16 microM for AlATP at pH 7, for example) to as low as 13 nM for LuATP. With glucose and fructose, the tightly bound complexes also show reversible, slow binding behavior, but with poor substrates, little or no change in inhibition constant with time is observed. The kinetics of citrate as an activator of the hexokinase reaction are consistent with its reaction with AlATP present as a contaminant in commercial ATP to form Al citrate. The complex of Al(III) with citrate is 5 orders of magnitude more stable than AlATP, whose Kd is 0.7 microM at pH 7. ATP that has been treated with excess EDTA and adsorbed on and eluted from charcoal is free of aluminum, and citrate no longer affects the kinetics of the hexokinase reaction. Glycerokinase is also specifically inhibited by trivalent metal ATP complexes (Ki = 4 microM at pH 7 for AlATP).

Adenosine Triphosphate↗
PubMed1980

Instability in tyrR strains of plasmids carrying the tyrosine operon: isolation and characterization of plasmid derivatives with insertions or deletions.

The transformation of tyrR strains of Escherichia coli with multicopy plasmids which carry the tyrosine operon gave rise to modified plasmids with either insertions or deletions. The effect of each of these insertions or deletions was to decrease the level of expression of this operon. It is proposed that plasmid instability arose as a direct consequence of the metabolic effects of an overproduction of the enzymes coded for by the tyrosine operon. The results have significant implications for the cloning of genes that are repressed by the product of a regulatory gene. Since the predominant plasmid modification observed was the insertion of an IS1 element near the regulatory region of the tyrosine operon, the results also suggest a role for IS1 elements in the regulation of gene expression.

DNA Transposable Elements↗
PubMed1979

Methotrexate, a high-affinity pseudosubstrate of dihydrofolate reductase.

Investigations have been made of the slow, tight-binding inhibition by methotrexate of the reaction catalyzed by dihydrofolate reductase from Streptococcus faecium A. Quantitative analysis has shown that progress curve data are in accord with a mechanism that involves the rapid formation of an enzyme-NADPH-methotrexate complex that subsequently undergoes a relatively slow, reversible isomerization reaction. From the Ki value for the dissociation of methotrexate from the E-NADPH-methotrexate complex (23 nM) and values of 5.1 and 0.013 min-1 for the forward and reverse rate constants of the isomerization reaction, the overall inhibition constant for methotrexate was calculated to be 58 pM. The formation of an enzyme-methotrexate complex was demonstrated by means of fluorescence quenching, and a value of 0.36 muM was determined for its dissociation constant. The same technique was used to determine dissociation constants for the reaction of methotrexate with the E-NADP and E-NADPH complexes. The results indicate that in the presence of either NADPH or NADP there is enhancement of the binding of methotrexate to the enzyme. It is proposed that methotrexate behaves as a pseudosubstrate for dihydrofolate reductase.

Kinetics↗
PubMed1979

The use of steady-state rate equations to analyse progress curve data.

Analysis of progress curves for enzyme-catalyzed reactions has been made by using a procedure that does not require the derivation of complex integrated rate equations. The method involves conversion of progress curve data to reaction velocities that are then fitted to the appropriate differential rate equation. Application of the procedure to data obtained for the reaction catalyzed by aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1), showed that the resulting values for the kinetic parameters agreed well with those obtained by conventional progress curve analysis (Duggleby, R.G. and Morrison, J.F. (1978) Biochim. Biophys. Acta 526, 398--409).

Aspartate Aminotransferases↗
PubMed1979

Enzyme-enzyme interaction and the biosynthesis of aromatic amino acids in Escherichia coli.

The technique of affinity chromatography has been used to demonstrate that enzymes involved in the biosynthesis of tyrosine and phenylalanine in Escherichia coli undergo reversible interactions. Thus it has been shown that the aromatic amino acid aminotransferase (aromatic-amino-acid: 2-oxoglutarate amino-transferase, EC 2.6.1.57) reacts specifically with chorismate mutaseprephenate dehydrogenase (chorismate pyruvate mutase, EC 5.4.99.5 and prephenate: NAD+ oxidoreductase (decarboxylating), EC 1.3.1.12) in the absence of reactants and with chorimate mutase-prephenatedehydratase (prephenate hydro-lyase (decarboxylating), EC 4.2.1.51) in the presence of phyenylpyruvate. Tyrosine causes dissociation of the aminotransferase: mutasedehydrogenase complex while dissociation of the aminotransferase-mutasedehydratase complex occurs on omission of phenylpyruvate. Only the active form of chorismate mutase-prephenate dehydrogenase participates in complex formation.

Amino Acids↗
PubMed1978

Progress curve analysis in enzyme kinetics: model discrimination and parameter estimation.

The method of progress curve analysis for enzyme-catalyzed reactions (Duggleby, R.G. and Morrison, J.F. (1977) Biochim. Biophys. acta 481, 297--312) has been extended to a two substrate, reversible reaction through the use of enzyme-catalyzed recycling of one of the products. The reaction investigated was that catalyzed by aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) and the product, alpha-ketoglutarate was recycled to glutamate using NADH and NH4Cl in the presence of glutamate dehydrogenase. The values determined for the kinetic parameters of the aminotransferase were found to agree well with those obtained from steady-state velocity measurements. The standard errors of the parameters, as calculated by the procedure originally described, were found to underestimate the observed variation between different experiments. Therefore, a procedure of data compression was devised which leads to more realistic values for standard errors. The compressed data obtained with aspartate aminotransferase have been fitted to the integrated rate equations that describe a variety of kinetic mechanisms. The best fit was obtained with the Ping-Pong model which is applicable to the aspartate aminotransferase reaction. Thus, progress curve analysis may be used to determine the kinetic mechanism of, and values of the kinetic parameters associated with, an enyzme-catalyzed reaction.

Aspartate Aminotransferases↗
PubMed1978

The purification and properties of the aspartate aminotransferase and aromatic-amino-acid aminotransferase from Escherichia coli.

A simple and convenient procedure is described for the isolation in good yield of two amino-transferases from various strains of Escherichia coli. On the basis of their substrate specificities one of the enzymes has been classified as an aromatic amino acid aminotransferase and the other as an aspartate aminotransferase, but both act on a wide range of substrates. Pyridoxal phosphate is bound more strongly to the aspartate aminotransferase than to the aromatic amino transferase which cannot be fully re-activated after removal of the prosthetic group. Both enzymes are composed of two subunits which appear to be identical.

Amino Acids↗
PubMed1978

Chorismate mutase-prephenate dehydratase from Escherichia coli: active sites of a bifunctional enzyme.

The relationship between the active sites of the bifunctional enzyme chorismate mutase-prephenate dehydratase has been examined. Steady-state kinetic investigations of the reactions with chorismate or prephenate as substrate and studies of the overall conversion of chorismate to phenylpyruvate indicate that there are two distinct active sites. One site is responsible for the mutase activity and the other for the dehydratase activity. Studies of the overall reaction using radioactive chorismate show that prephenate, which is formed from chorismate, dissociates from the mutase site and equilibrates with the bulk medium before combining at the dehydratase site. No evidence was obtained for direct channeling of prephenate from one site to the other, or for any strong interaction between the sites.

Binding Sites↗
PubMed1978

Kinetic studies on the reactions catalyzed by chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes.

Steady-state kinetic techniques have been used to investigate each of the reactions catalyzed by the bifunctional enzyme, chorismate mutase-prephenate dehydrogenase, from Aerobacter aerogenes. The results of steady-state velocity studies in the absence of products, as well as product and dead-end inhibition studies, suggest that the prephenate dehydrogenase reaction conforms to a rapid equilibrium random mechanism which involes the formation of two dead-end complexes, viz, enzyme-NADH-prephenate and enzyme-NAD+-hydroxyphenylpyruvate. Chorismate functions as an activator of the dehydrogenase while both prephenate and hydroxyphenylpyruvate acted as competitive inhibitors in the mutase reaction. By contrast. bpth NAD+ and NADH function as activators of the mutase. Values of the kinetic parameters associated with the mutase and dehydrogenase reactions have been determined and the results discussed in terms of possible relationships between the catalytic sites for the two reactions. The data appear to be consistent with the enzyme having either a single site at which both reactions occur or two separate sites which possess similar kinetic properties.

Allosteric Regulation↗
PubMed1978

Role of the Escherichia coli aromatic amino acid aminotransferase in leucine biosynthesis.

Strains of Escherichia coli that lack the branched-chain amino acid amino-transferase because of mutations in the ilvE gene had no growth requirement for leucine when the cells contained the aromatic amino acid aminotransferase that is the product of the tyrB gene. The presence of leucine increased the generation time of these cells and decreased the specific activity of the aromatic amino acid aminotransferase. It is concluded that this enzyme functions efficiently in leucine biosynthesis and can be repressed by leucine as well as by tyrosine.

Caproates↗