Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration

Multienzyme reaction systems with simultaneous coenzyme regeneration have been investigated in a continuously operated membrane reactor at bench scale. NAD(H) covalently bound to polyethylene glycol with a molecular weight of 10⁴ [PEG-10,000-NAD(H)] was used as coenzyme. It could be retained in the membrane reactor together with the enzymes. L -leucine dehydrogenase (LEUDH) was used as catalyze for the reductive amination of α-ketoisocaproate (2-oxo-4-methylpentanoic acid) to L -leucine. Format dehydrogenease (FDH) was used for the regeneration of NADH. Kinetic experiments were carried out to obtain data which could be used in a kinetic model in order to predict the performance of an enzyme membrane reactor for the continuous production of L -leucine. The kinetic constants V_max and K_m of enzymes are all in the same range regardless of whether native NAD(H) or PEG-10,000-NAD(H) is used as coenzyme. L -leucine was produced continuously out of α-ketoisocaproate for 48 days; a maximal conversion of 99.7% was reached. The space-time yield was 324 mmol/L day (or 42.5 g/L day).     

Control of activity states of heart mitochondrial ATPase. Role of the proton-motive force and Ca2+

The ATPase complex of submitochondrial particles exhibits activity transitions that are controlled by the natural ATPase inhibitor (Gómez-Puyou, A., Tuena de Gómez-Puyou, M. and Ernster, L. (1979) Biochim. Biophys. Acta 547, 252–257). The ATPase of intact heart mitochondria also shows reversible activity transitions; the activation reaction is induced by the establishment of electrochemical gradients, whilst the inactivation reaction is driven by collapse of the gradient. In addition it has been observed that the influx of Ca²⁺ into the mitochondria induces a rapid inactivation of the ATPase; this could be due to the transient collapse of the membrane potential in addition to a favorable effect of Ca²⁺-ATP on the association of the ATPase inhibitor peptide to F₁-ATPase. This action of Ca²⁺ may explain why mitochondria utilize respiratory energy for the transport of Ca²⁺ in preference to phosphorylation. It is concluded that the mitochondrial ATPase inhibitor protein may exert a fundamental regulatory function in the utilization of electrochemical gradients.     

Role of Heme in Synthesis and Membrane Binding of Succinic Dehydrogenase in Bacillus subtilis

A 5-aminolevulinic acid-requiring mutant of Bacillus subtilis was isolated. When the mutant is shifted from medium containing 5-aminolevulinic acid to medium lacking this growth factor, the bacteria continued to grow at undiminished rate for about three generations. The membranes from these bacteria contained severely reduced amounts of cytochrome. The mutant was used to study the role of heme synthesis on synthesis and membrane binding of succinic dehydrogenase (SDH). The amount of SDH in whole-cell lysates in the soluble cytoplasmic fraction and in membranes was determined by one-dimensional (rocket) immunoelectrophoresis with an SDH-specific antiserum. After heme synthesis was blocked, the relative amount of SDH in the membrane decreased, whereas increasing amounts of SDH antigen were found in the cytoplasm. When heme synthesis was resumed on readdition of 5-aminolevulinic acid, the amount of membrane-bound SDH antigen increased at a much faster rate than net synthesis. During a 3-h growth period without 5-aminolevulinic acid, there was little change in the pattern of membrane proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioactively labeled membranes, as compared to membranes from control cultures. However, both the 65,000-dalton and the 28,000-dalton polypeptides of the SDH complex (L. Hederstedt, E. Holmgren, and L. Rutberg, J. Bacteriol. 138:370-376, 1979) were present in decreasing amounts in membranes from 5-aminolevulinic acid-starved bacteria. From these results we suggest that SDH in B. subtilis is synthesized as a soluble protein and becomes membrane bound only when it attaches to a site in the membrane, (part of) which is a cytochrome of b type.     

A MATHEMATICAL MODEL OF THROMBOPOIESIS IN RATS

A mathematical model of thrombopoiesis in rats is presented. This has four compartments; stem cells, megakaryocytes, thrombocytes and thrombopoietin. A high thrombopoietin concentration influences bone marrow proliferation in three ways. Firstly the stem cells are stimulated and a slow increase in megakaryocyte number follows. Secondly there are additional endomitoses in the (early) megakaryocytes resulting in an increase in megakaryocyte volume. Thirdly the megakaryocyte maturation time is shortened. The parameters of the model are determined from experimental values for the normal, maximum and minimum proliferation rates, maturation times and destruction rates. The model is tested by comparing simulated results for acute and chronic thrombocytopenia and thrombocytosis with experimental curves from the literature. The model and data agree within the limits of experimental error. Not all of the thrombopoietic regulatory system is known yet, so some important alternative hypotheses are investigated and compared with the model. Several hypotheses have been excluded in this way.     

Fluorescence-microscopical demonstration of a population of gastro-intestinal nerve fibres with a selective affinity for Quinacrine

Summary A population of nerve fibres in the gastro-intestinal tract of mice showing a high affinity for quinacrine was revealed by fluorescence microscopy. Similar results were obtained in rats and guinea pigs. Whole-mounts of sheets of the smooth muscle layer following incubation in 10^-6-10^-7 M quinacrine for 15–60 min revealed fine fluorescent varicose nerve fibers in the myenteric plexus of Auerbach both around nerve cell bodies and in the interconnecting strands. Many fibers were also present between the strands of the plexus, especially running parallel to the circular muscle layer. Such fibers were not seen in similarly quinacrine-incubated irides. A proportion of the cell bodies in Auerbach's plexus also showed quinacrine accumulation. These cells were apparently smaller neurons, sometimes with fluorescent processes. Intraperitoneal injections of quinacrine failed to demonstrate nerve fibers, but some cell bodies in Auerbach's plexus were positive. Subsequent paraformaldehyde treatment for monoamine visualization showed persistent adrenergic nerve terminals in the intestine and iris. These nerves seemed to be fewer and had a more yellow fluorescence than normally. The identity of the quinacrine-positive fibers is discussed with respect to recent suggestions that purinergic, substance P, enkephalin, and somatosin-containing nerves, in addition to adrenergic and cholinergic nerves, are present in the gut wall.Supported by the Swedish Medical Research Council (04X-03185). Magnus Bergvalls Stiftelse and Karolinska Institutets Fonder. For generous gifts of Mepacrine we thank Winthrop, Skärholmen, Stockholm, Sweden. The skilful technical assistance of Miss Gerd Boetius and Miss Maud Eriksson is gratefully acknowledged     

Formation of a functional adrenergic input to intraocular cerebellar grafts: Ingrowth of inhibitory sympathetic fibers

The intraocular transplantation technique was used to study the ingrowth of peripheral sympathetic adrenergic nerves from the iris into transplants of fetal rat cerebellum, and the possible function of these nerves. The transplants, grown in oculo for one-half to eight months, were analyzed by fluorescence histochemistry and electrophysiological techniques. Peripheral sympathetic adrenergic fibers from the iris were able to grow into the cerebellar transplants and arborize in a pattern similar to that in situ, appearing in all three cortical layers and the noncortical areas of the transplants. The density of visible nerves without pretreatment and after preincubation in 10⁻⁶ or 10⁻⁵ M α-methylnorepinephrine was comparable to mature rat cerebellum. The spontaneous discharge of the Purkinje cells in oculo was inhibited by microiontophoresis of norepinephrine (NE) and amphetamine in sympathetically innervated, as well as sympathectomized transplants denervated by ganglionectomy. The NE response was blocked by the adrenergic β-receptor blocker MJ-1999. GABA also inhibited the Purkinje cell activity while glutamate accelerated the discharge. Parenteral amphetamine inhibited Purkinje cell activity in sympathetically innervated transplants, but was ineffective in denervated transplants. The Purkinje cell spontaneous activity was inhibited by electrical stimulation of the NE fiber input through the cervical sympathetic trunk. This inhibition could be antagonized by parenteral reserpine or the β-adrenergic antagonist propranolol. The responses of the Purkinje cells within the transplants to drugs and transmitters mimic those of the adult rat in situ. In view of the fluorescence histochemical evidence for an ingrowth of peripheral sympathetic adrenergic fibers into the cerebellar transplants, and the results of stimulating the sympathetic trunk, it is suggested that peripheral adrenergic fibers may be able to establish functional connections with the Purkinje cells similar to the cerebellar adrenergic synapses normally formed in situ by fibers from the locus coeruleus.     

The topology of genome-scale metabolic reconstructions unravels independent modules and high network flexibility

The topology of metabolic networks is recognisably modular with modules weakly connected apart from sharing a pool of currency metabolites. Here, we defined modules as sets of reversible reactions isolated from the rest of metabolism by irreversible reactions except for the exchange of currency metabolites. Our approach identifies topologically independent modules under specific conditions associated with different metabolic functions. As case studies, the E.coli iJO1366 and Human Recon 2.2 genome-scale metabolic models were split in 103 and 321 modules respectively, displaying significant correlation patterns in expression data. Finally, we addressed a fundamental question about the metabolic flexibility conferred by reversible reactions: “Of all Directed Topologies (DTs) defined by fixing directions to all reversible reactions, how many are capable of carrying flux through all reactions?”. Enumeration of the DTs for iJO1366 model was performed using an efficient depth-first search algorithm, rejecting infeasible DTs based on mass-imbalanced and loopy flux patterns. We found the direction of 79% of reversible reactions must be defined before all directions in the network can be fixed, granting a high degree of flexibility.