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H J Bosma

Publications and source records attributed to H J Bosma.

10 recordsLinked to original sources
PubMed1998

Kinetics and specificity of reductive acylation of wild-type and mutated lipoyl domains of 2-oxo-acid dehydrogenase complexes from Azotobacter vinelandii.

The kinetics and specificity of reductive acylation of lipoyl domains derived from Azotobacter vinelandii 2-oxo-acid dehydrogenase complexes, catalysed by A. vinelandii and Escherichia coli complexes, have been investigated. With the wild-type pyruvate dehydrogenase complex from A. vinelandii the rate of reductive acetylation and deacetylation was studied by rapid mixing methods. The rate of reductive acetylation, 126 s(-1), corresponds well with the turnover rate derived from steady-state measurements. Deacetylation was rapid and specific for coenzyme A. No deacetylation was observed with reduced or oxidised lipoamide or with dithiothreitol. The rate of reductive acetylation of complex-bound lipoyl domains by pyruvate dehydrogenase (E1p) is at least 60 times higher than of free lipoyl domains under comparable conditions. This gain in catalytic rate indicates a large diffusion limitation of lipoyl domains when attached via the flexible linker segments to the complex, and illustrates the efficiency of substrate channeling in the multienzyme complex. The 2-oxo-acid dehydrogenases exhibit specificity for lipoyl domains in the reductive acylation reaction. The A. vinelandii lipoyl domain derived from the pyruvate dehydrogenase complex is a good substrate for A. vinelandii E1p, but not for A. vinelandii 2-oxoglutarate dehydrogenase (E1o), and vice versa. The A. vinelandii lipoyl domain of the pyruvate dehydrogenase complex is also, although at a lower rate, reductively acetylated by E. coli E1p and reductively succinylated by E. coli E1o. Likewise, the A. vinelandii lipoyl domain derived from the 2-oxoglutarate dehydrogenase complex is recognised by E. coli E1o, but not by E. coli E1p. This suggests that common determinants of the lipoyl domains exist that are responsible for recognition by the E1 components. On the basis of the observed specificity and lipoyl domain sequences and structures, an exposed loop of the A. vinelandii 2-oxoglutarate dehydrogenase complex lipoyl domain was subjected to mutagenesis. Although the reductive acylation experiments of mutants of the lipoyl domain indicate the importance of this loop for recognition, it is probably not the single determinant for specificity.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1984

The composition of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Does a unifying model exist for the complexes from gram-negative bacteria?

An improved purification procedure of the pyruvate dehydrogenase complex of Azotobacter vinelandii is described. This procedure minimizes losses of components and results in the isolation of the pure complex with a specific activity of 15-19 U/mg and an overall yield of 40%. The chain ratio of the three components was determined by covalent modification of the lysine residues with trinitrobenzene sulfonic acid, followed by separation of the components on sodium dodecyl sulfate gels. These determinations yielded an average chain ratio of 1.3:1:0.5 for E1:E2:E3 respectively. Based on E2 this corresponds with a minimum molecular mass of approximately 216 kDa. Because the molecular mass of the complex has been determined previously to be 800 +/- 50 kDa, it is concluded that the complex as isolated from A. vinelandii is based on a tetramer of E2 chains. The complex can be resolved into its individual components, which can be recombined to yield a fully active complex. Titration of E2E3 subcomplexes with E1 resulted in maximum complex activity at an E1/E2 ratio of 1.5-1.6. Similar titrations of E1E2 subcomplexes with E3 resulted in maximum activity at an E3/E2 ratio of 0.45-0.55. From these experiments it is concluded that the complex has maximum activity with a composition of three E1 dimers, one E2 tetramer and one E3 dimer. With excess of either E1 or E3 a decrease in activity is observed which indicates competition between these components for binding sites on E2. As shown before [Bosma, H.J., de Kok, A., Markwijk, B.W., and Veeger, C. (1984) Eur. J. Biochem. 140, 273-280], the isolated E2 component is composed of 32 peptide chains of 66 kDa each. Upon addition of E1 or E3, E2 dissociated into tetramers. Dissociation is complete upon the addition of four E1 dimers of four E3 dimers per E2 tetramer. Addition of E1 to saturated E2E3 subcomplex or E3 to saturated E1E2 subcomplex did not result in extra binding but rather in displacement of bound E3 or E1 respectively. It is therefore concluded that the binding sites of E1 and E3 to the E2 chains are either identical or so closely spaced that steric hindrance prevents simultaneous binding of both components. A model is presented based on the cubic structure of the isolated E2 component. In this model the 32 E2 peptide chains are arranged in tetramers in the corners of the cube. This model is discussed in connection with the existing model for the Escherichia coli complex.

Amino Acids↗
PubMed1984

The size of the pyruvate dehydrogenase complex of Azotobacter vinelandii. Association phenomena.

Sedimentation analysis and light-scattering studies indicate that the aggregation state of the pyruvate dehydrogenase complex of Azotobacter vinelandii in 50 mM potassium phosphate (pH 7.0) can be described in terms of a monomer-dimer equilibrium with a dissociation constant of 6.8 microM. The apparent molecular mass of the monomeric particle is 750 000-850 000 Da. The equilibrium is shifted to the monomeric species when pressure is applied on the system. Pressure-jump experiments yielded a relaxation time of about 70 ms. In the presence of 3% poly(ethylene glycol) 6000 and 10 mM MgCl2, further association takes place to a system that can be described in terms of dimer-tetramer-octamer equilibria. Upon applying a pressure of 80 MPa to this system these equilibria are shifted to the dimeric state but some monomer formation cannot be excluded. Release of pressure shows that the relaxation time of the dimer-tetramer equilibrium is less than 5 s, that of tetramer-octamer equilibrium is of the order of minutes. The isolated E2 component has a molecular mass of 2 000 000 +/- 100 000 Da; and thus consists of about 30 E2 peptide chains. Electron micrographs are similar to those of the E2 component of the Escherichia coli complex, which were interpreted as cubic structures with an octagonal symmetry. Upon addition of E1 to the pure E2 component, changes in the assembly occur and mixtures of large (E. coli-like, 22-45 S) and small (A. vinelandii-like, 11-18 S) subcomplexes are obtained. The two forms of the subcomplexes are in slow equilibrium (relaxation time 10-30 min). It is proposed that the E2 tetramer of the intact pyruvate dehydrogenase complex of A. vinelandii is represented by the corner structures of the isolated E2 component.

Azotobacter↗
PubMed1983

Biochemical changes during graded brain ischemia in gerbils. Part 1. Global biochemical alterations.

In Mongolian gerbils (Meriones unguiculatus) cerebral ischemia was produced by occlusion of the right common and the left external carotid arteries. Gerbils were classified according to their neurological appearance as "symptom-negative" (8 animals), "mild symptoms" (unilateral hemiparesis, 10 animals) and "severe symptoms" (hemiparesis and rolling seizures, 8 animals). Two hours after vascular occlusion various substrates and enzymes related to the energy-producing metabolism, were assessed in tissue samples from both hemispheres. In symptom-negative animals, the only change was a slight decrease of glycolytic intermediates in the right hemisphere. In animals with mild symptoms, the right hemisphere additionally exhibited an impairment of the redox and energy state and an enhancement of the activity of most enzymes of the glycolytic pathway, except hexokinase. In animals with severe symptoms, these changes were even more pronounced and affected--to a lesser degree--also the left hemisphere. The results obtained demonstrate that the neurological appearance of the animals after vascular occlusion correlates with the biochemical alterations and, therefore, can be used for estimating the density of graded ischemia.

Alanine Transaminase↗
PubMed1983

Biochemical changes during graded brain ischemia in gerbils. Part 2. Regional evaluation of cerebral blood flow and brain metabolites.

Regional changes of cerebral blood flow and biochemical substrates were assessed in the gerbil brain following different grades of cerebral ischemia. Ischemia was produced by occlusion of the right common carotid and left external carotid arteries. Gerbils were classified according to the severity of neurological symptoms as animals without, with mild and with severe neurological deficits. Brains were frozen in situ, sliced in 20-microns sections and processed for pictorial presentation of glucose and ATP, using bioluminescence techniques. Cerebral blood flow was determined in adjacent brain sections, using [14C]iodoantipyrine autoradiography. NADH fluorescence was recorded by illuminating the surface of the tissue block with ultraviolet light. Most animals without visible neurological symptoms exhibited reduced blood flow in circumscribed regions of cortex and basal ganglia of the right hemisphere without concomitant changes of biochemical substrates. In animals with mild neurological symptoms, blood flow in the right hemisphere was reduced, glucose and ATP decreased, and NADH fluorescence unhomogeneously enhanced. In animals with severe neurological symptoms blood flow was almost arrested in the right hemisphere and was distinctly reduced in the medial parts of the left hemisphere. The ischemic tissue was depleted from glucose and ATP, and exhibited bright NADH fluorescence. The severity of neurological symptoms, in consequence, correlated closely with both the degree and the size of biochemical lesions observed in the ischemic territory.

Adenosine Triphosphate↗
PubMed1982

Investigations on vasogenic and cytotoxic brain edema, comparing results from X-ray microanalysis and flame photometry.

In these studies vasogenic brain edema has been induced by implantation of rat glioma cells RGI 2.2 into BD-IX rats while cytotoxic edema pas produced by permanent regional ischemia in the mongolian gerbil. In the gerbil sodium concentration was raised from 201 meq/kg d.w. (dry weight) [p/b (peak/background) = 0] to 269 meq/kg d.w. (p/b = 0.25; 2 hours) and 651 meq/kg d.w. [p/b = 0.71; 24 hours), whereas potassium concentration decreased from 373 meq/kg d.w. (p/b = 1.69) to 337 meq/kg d.w. (p/b = 1.65) and 152 meq/kg d.w. (p/b = 0.53). In the rat tumor sodium and potassium concentrations were 279 meq/kg d.w. (p/b = 0.44) and 510 meq/kg d.w. (p/b = 1.94). Non-tumorous tissue contained 237 meq/kg d.w. (p/b = 0) and 517 meq/kg d.w. (p/b = 1.98). In addition X-ray microanalysis could show that chlorine behaves like sodium, whereas the concentration of phosphorus and sulphur remains nearly constant. X-ray microanalysis in this case proved to be useful in the localization and quantification of different elements. The main disadvantage, however, is the reduced sensitivity for light elements, e.g. sodium, which cannot be determined in normal brain.

Animals↗
PubMedDate not supplied

Selective vulnerability of hippocampus and disturbances of memory storage after mild unilateral ischemia of gerbil brain.

The effect of selective injury of hippocampal neurons on the consolidation of memory traces was studied in gerbils (meriones unguiculatus) after production of mild cerebral ischemia. The right carotid artery was permanently ligated, and animals without gross neurological deficits ("symptom-negative" gerbils) were selected. Eight days and eight weeks after vascular ligation, cell counts of hippocampal neurons were carried out and correlated with regional blood flow and the acquisition of operant behaviour. Eight days after carotid artery occlusion, learning behaviour was significantly impaired although the number of hippocampal neurons had not changed and blood flow had even increased above normal. After eight weeks, learning behaviour and blood flow were normal but now a significant loss of pyramidal neurons was present in the CA1 and CA2 sectors of the hippocampus. Our observations demonstrate that it is possible to detect subtle functional disturbances by appropriate behavioural investigation before manifestation of selective injury of the hippocampus. Recovery of integrative function, despite persistent cellular damage, provides further evidence for central nervous plasticity.

Animals↗