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Biomedical subjects

M J Boland

Publications and source records attributed to M J Boland.

32 records · Page 2Linked to original sources
PubMed1981

Glutamate synthase (NADH) from lupin nodules. Specificity of the 2-oxoglutarate site.

The binding of substrate analogues including potential alternative substrates, to glutamate synthase (NADH) (L-glutamate: NAD+ oxidoreductase (transaminating) E.C. 1.4.1.14) has been investigated by studying competitive inhibition with respect ot 2-oxoglutarate. Binding requires two terminal carboxyl groups on a C5 straight chain molecule although some C4 molecules bind weakly. Bulky substituents at C2 decrease or prevent binding. Glutarate, the most potent inhibitor, binds much less tightly than the substrate. A 2-oxo group in a molecule other than the substrate does not appear to contribute significantly to binding. None of the analogues was able to act as an alternative substrate.

Glutamate Synthase↗
PubMed1981

NADH-dependent glutamate synthase from lupin nodules. Reactions with oxidised and reduced 3-acetylpyridine-adenine dinucleotide.

3-Acetylpyridine-adenine dinucleotide, reduced form (AcPdADH) is able to act as an alternative reductant in glutamate-synthase-catalysed glutamate synthesis. In the AcPdADH-dependent reaction, kcat and Km values for the other substrates are fourfold lower than those for the NADH-dependent reaction, and Km for AcPdADH is about 3 microM. AcPdADH acts as a competitive inhibitor with respect to NADH in NADH-dependent glutamate synthesis, with a Ki of 1 microM. Glutamate synthase catalyses NADH-dependent reduction of AcPdAD+. This appears to proceed by a substituted-enzyme (ping-pong) mechanism, with competitive substrate inhibition by NADH at high levels. The Km values for this reaction are 1.4 microM for NADH and 14 microM for AcPdAD+ and kcat is 51 s-1; Ki for NADH is about 10 microM. The latter findings suggest that NADH is capable of reducing the enzyme molecule in the absence of other substrates and that a reduced form of the enzyme can exist in the absence of bound NADH.

Coenzymes↗
PubMed1980

The specificity of actinidin and its relationship to the structure of the enzyme.

The kinetic parameters kcat, Km and kcat/Km, have been determined for the actinidin-catalysed hydrolyses of N-substituted amino acid esters and amides and compared to the corresponding values for papain (EC 3.4.22.2). Substrates with aromatic N-substituents have lower kcat/Km values for actinidin (EC 3.4.22.14); the difference is much smaller for substrates with aliphatic substituents. The lower kcat/Km values for actinidin generally correspond to higher Km values suggesting that the strength of substrate binding differs between the two enzymes. This difference is explained in terms of the differences in the substrate binding sites found in X-ray crystallographic studies.

Binding Sites↗
PubMed1979

Kinetic mechanism of NADH-dependent glutamate synthase from lupin nodules.

From initial-rate studies, a partially random kinetic mechanism has been deduced for NADH-dependent glutamate synthase from lupin nodules. The mechanism involves compulsory binding of NADH as first substrate, followed by random-order binding of glutamine and 2-oxoglutarate. Patterns of inhibition by glutamate substantiate the mechanism. Dithionite was incapable of acting as an alternative reducing substrate although it is known to reduce the flavine groups of the enzyme. The implications of these results are discussed. Published rate equations for this type of mechanism were found to be unsatisfactory for this enzyme and suitable new equations are produced. These equations should have general application where the obligatory first substrate binds very tightly.

Dithionite↗
PubMed1978

The 12-lead electrocardiogram in exercise testing. A misleading baseline?

Mason and Likar reported a modified ECG lead system that largely eliminates intraexercise artifact without reportedly altering the configuration of the recorded leads. This system allows continuous monitoring of all 12 leads during and after exercise and has become the standard by which other systems are compared. We have found, however, that important changes may be seen between the baseline exercise ECG using the Mason-Likar modification and a simultaneous standard ECG. While these differences do not apparently affect the interpretation of the test, they may produce or obscure evidence of antecedent infarction on the baseline exercise ECG and thus preclude it from being considered interchangeable with a standard ECG.

Electrocardiography↗
PubMed1977

Enzymes of nitrogen metabolism in legume nodules. Purification and properties of NADH-dependent glutamate synthase from lupin nodules.

An NADH-dependent glutamate synthase has been purified 500-fold from the plant cytoplasm fraction of Lupinus angustifolius nodules. It consists of a single polypeptide chain, Mr 235000. The optimum pH is 8.5, at which Km values for 2-oxoglutarate, glutamine and NADH are 39 micrometer, 400 micrometer and 1.3 micrometer respectively. The catalytic centre activity is of the order of 70 s-1 and is independent of pH between 6.5 and 9.5. Glutamate synthase is inhibited by glutamic acid, oxaloacetic acid, aspartic acid and asparagine, all competitive with 2-oxoglutarate; and by NAD+, which is competitive with NADH. There is evidence of two flavine prosthetic groups per enzyme molecule.

Glutamate Synthase↗
PubMed1976

Isolated galactorrhea with normal serum prolactin levels: clinical implications.

Detailed endocrine-metabolic studies were performed on five women who were otherwise well but who had had inappropriate breast secretions for variable periods of time (three months to 16 years). Our results suggest that the presence of a lactose-containing breast secretion, which strictly defines galactorrhea, does not necessarily indicate a recognizable abnormality if normal hypothalamic-pituitary function is present. In these regularly menstruating women with isolated galactorrhea, we suggest a minimum initial evaluation, but careful long-term follow-up studies to identify those cases which may progress to the other recognized, more serious amenorrhea-galactorrhea syndromes.

Adult↗
PubMed1975

Purification and kinetic properties of chalcone-flavanone isomerase from soya bean.

The chalcone-flavanone isomerase from soya bean seed has been purified 8300-fold. A molecular weight of 15,600 plus or minus 1000 has been determined for the enzyme. Effects of iodoacetamide and sodium tetrathionate on the enzyme, and pH dependence of the catalytic step, indicate that a sulphydryl group is not involved in the reaction mechanism and the catalytic group is probably an imidazole side chain in the basic form. The kinetics of the isomerisation of isoliquiritigenin to liquiritigenin have been examined and show that at pH 7.6 the reaction is reversible with an equilibrium constant of 37 in favour of flavanone. A number of flavonoid compounds competitively inhibit the reaction, suggesting a possible regulatory role for this enzyme in flavonoid biosynthesis.

Chromatography, DEAE-Cellulose↗
PubMed1975

Pig heart lactate dehydrogenase. Binding of pyruvate and the interconversion of pyruvate-containing ternary complexes.

1. Lactate oxidation catalysed by pig heart lactate dehydrogenase was studied in the presence of inhibitory concentrations of pyruvate. Experimental results show the presence of an intermediate which occurs immediately after the hydride transfer step, but before the dissociation of pyruvate and the H+ produced by the reaction. The rate constant for pyruvate dissociation and the dissociation constant for pyruvate from the ternary complex differ from those obtained in pyruvate reduction experiments. 2. In single-turnover pyruvate reduction by pig heart lactate dehydrogenase at pH8.0 pyruvate can bind to the enzyme before a H+ is taken up, and the subsequent uptake of a H+ is governed by a step that is also rate-limiting for single-turnover and steady-state NADH oxidation. 3. Observation of various intermediates in the single-turnover pyruvate reduction experiments has made it possible to determine separately the dissociation constant and Km value for pyruvate at pH8.0, and also the catalytic turnover rate and Km for pyruvate under first-order conditions at different pH values. 4. Further studies on single-turnover pyruvate reduction carried out in 2H2O, or in water at low temperature, show another step which, under these conditions, is slower than that controlling H+ uptake and rate-limiting for NADH oxidation. A scheme is presented which explains these results.

Binding Sites↗