The synthesis of glutamate and the control of glutamate dehydrogenase in pea mitochondria

The synthesis of glutamate from α-oxoglutarate and NH₄⁺ by pea seedling mitochondria has been demonstrated under certain defined but non-physiological conditions. Malate acts as a hydrogen donor for the synthesis of glutamate but isocitrate is more effective, whilst succinate, in the presence or absence of ATP, is a poor donor of hydrogen. Glutamate dehydrogenase has been purified from pea mitochondria and from the cytosol. The similarities between the two preparations are interpreted to mean that the soluble glutamate dehydrogenase is released from the mitochondria during isolation. The kinetics of the mitochondrial enzyme and the effect of various metabolites on its activity have been examined. The results are discussed in relation to the proposed role of this enzyme and it is suggested that the ratio NADH-NAD⁺ may play a role in the control of glutamate metabolism.     

The control of plant glutamate dehydrogenase by pyridoxal-5′-phosphate

The proposition that the nitrogen status of a plant is reflected by the ratio pyridoxal phosphate to pyridoxamine phosphate and that this ratio exerts a controlling influence on plant metabolism has been examined. The ratio pyridoxal phosphate to pyridoxamine phosphate has been shown to increase during nitrogen starvation. The inhibition of glutamate dehydrogenase by pyridoxal phosphate has been examined and the kinetics of inhibition are discussed in relation to the proposed control of metabolism.     

Establishment of the steady state in glucose-limited chemostat cultures of Klebsiella pneumoniae

To investigate the relationship between growth rate and concentration of the nutrient that limits growth, 'Klebsiella aerogenes' NCTC 418 (K. pneumoniae) was grown in a glucose-limited chemostat. The actual time required to establish a steady-state glucose concentration exceeded that expected theoretically. Apparently, there is a long-term adaptation of the cells to nutrient limitation. As yet, it is not clear whether this has a phenotypic or genetic origin. In the final steady state, the dependence of the growth rate on glucose concentration could be mathematically described equally well by a hyperbolic and by a logarithmic function.     

Ionization constants and thermodynamic parameters of histidine and derivatives

The pK_a values for the proton dissociation of carboxyl, imidazolium, and ammonium groups for histidine and ten of its derivatives were determined electrometrically at seven temperatures in the range 10–40°C. The ΔH and ΔS values were estimated from the temperature dependence of the dissociation constants of histidine and its derivatives. These results and the pK_a values compared in terms of inductive effect suggest an ion-dipole interaction between the protonated amino group and the unprotonated imidazole ring. The charge and the solvation effects of the neighboring groups are the main factors that determine the imidazole group pK_a in histidine and its studied derivatives. The N^τ-H tautomer is favored over the N^π-H by 1.6 kcal/mol, indicating that the inductive substituent effect at position 4 of the imidazole ring is the major component in determining this tautomeric preference.     

Immunocytochemical identification of neuroendocrine markers in human cardiac paraganglion-like structures.

Paraganglion-like structures (PLS) containing chromaffin-positive cells have been reported to be present in the adult human heart. The present work was initiated in order to evaluate the density of these structures in the interatrial septum and to study the presence of immunoreactivity of their cells to NSE and PGP 9,5 antibodies, two neuroendocrine markers. Six hundred 6-microns paraffin serial sections were obtained from the upper third of the interatrial septum from six adult human hearts. From 2 to 12 paraganglia were found in each case, and their principal cells stained positively with NSE and PGP 9,5 antibodies. Depending on how these PLS related to other cardiac structures, four different types were identified: Type I - "True paraganglia" (located adjacent to ganglia or nerve fibers); Type II - "Free paraganglia" (immersed in the interatrial adipose tissue, without evident connection to other structures); Type III - "Intraganglionic paraganglia" (located within the nervous ganglia); Type IV - "Intramyocardic paraganglia" (small nests of immunoreactive cells closely related to myocardiocyte bundles). These cardiac paraganglia, which probably belong to the visceral-autonomic group, may have a role in the regulation of the cardiac function and in the adaptive mechanisms of the heart. Its is also possible that they originate functioning and non-functioning tumours.     

Purification, characterization and redox properties of hydrogenase from Methanosarcina barkeri (DSM 800)

A soluble hydrogenase from the methanogenic bacterium, Methanosarcina barkeri (DSM 800) has been purified to apparent electrophoretic homogeneity, with an overall 550-fold purification, a 45% yield and a final specific activity of 270 mumol H2 evolved min-1 (mg protein)-1. The hydrogenase has a high molecular mass of approximately equal to 800 kDa and subunits with molecular masses of approximately equal to 60 kDa. The enzyme is stable to heating at 65 degrees C and to exposure to air at 4 degrees C in the oxidized state for periods up to a week. The overall stability of this enzyme is compared with other hydrogenase isolated from strict anaerobic sulfate-reducing bacteria. Ms. barkeri hydrogenase shows an absorption spectrum typical of a non-heme iron protein with maxima at 275 nm, 380 nm and 405 nm. A flavin component, identified as FMN or riboflavin was extracted under acidic conditions and quantified to approximately one flavin molecule per subunit. In addition to this component, 8-10 iron atoms and 0.6-0.8 nickel atom were also detected per subunit. The electron paramagnetic resonance (EPR) spectrum of the native enzyme shows a rhombic signal with g values at 2.24, 2.20 and approximately equal to 2.0. probably due to nickel which is optimally measured at 40 K but still detectable at 77 K. In the reduced state, using dithionite or molecular hydrogen as reductants, at least two types of g = 1.94 EPR signals, due to iron-sulfur centers, could be detected and differentiated on the basis of power and temperature dependence. Center I has g values at 2.04, 1.90 and 1.86, while center II has g values at 2.08, 1.93 and 1.85. When the hydrogenase is reduced by hydrogen or dithionite the rhombic EPR species disappears and is replaced by other EPR-active species with g values at 2.33, 2.23, 2.12, 2.09, 2.04 and 2.00. These complex signals may represent different nickel species and are only observable at temperatures higher than 20 K. In the native preparation, at high temperatures (T greater than 35 K) or in partially reduced samples, a free radical due to the flavin moiety is observed. The EPR spectrum of reduced hydrogenase in 80% Me2SO presents an axial type of spectrum only detectable below 30 K.