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A lipoxygenase in rabbit reticulocytes which attacks phospholipids and intact mitochondria

Soluble lipoxygenases are known to occur in a variety of plants [1]. For a long time attempts to detect genuine soluble lipoxygenases in animal tissues were unsuccessful [1,2]. Only recently Nugteren has described a lipoxygenase in blood platelets but not in other tissues which preferentially attacks arachidonic acid [3]. In this report we describe a soluble lipoxygenase in rabbit reticulocytes. A remarkable feature of the lipoxygenase from reticulocytes is the ability to cause lysis of intact mitochondria by peroxidation of their lipids. Pure phospholipids are also attacked. It is proposed that the action of the lipoxygenase on mitochondria is involved in the degradation of mitochondria during the maturation process of the reticulocytes.

A reappraisal of the binding of cytosolic enzymes to erythrocyte membranes

Several cytosolic proteins have been shown to be associated with hypotonic erythrocyte ghosts via electrostatic interactions with the anion transport band 3 protein. This article considers the problems of demonstrating binding under physiological conditions and reviews the evidence for the relevance of enzyme binding to the membrane for the regulation of glycolysis. The hypotheses for the existence of topological and sequential multienzyme complexes of the glycolytic enzymes in erythrocytes are also discussed.

ATP-producing and consuming processes of ehrlich mouse ascites tumor cells in proliferating and resting phases

The extents of ATP-yielding and consuming processes in Ehrlich mouse ascites tumor cells during the proliferating and resting growth phase were compared. In the resting phase the total ATP production was decreased by one-third. The ATP supply by oxidative phosphorylation was drastically reduced, whereas the rate of glycolysis stayed nearly constant. All ATP-consuming processes investigated, i.e., protein turnover, Na+/K(+)-ATPase, Ca2(+)-ATPase, and RNA synthesis, were decreased proportionally with the total ATP consumption.

Cloning of a rabbit erythroid-cell-specific lipoxygenase mRNA

We report the isolation of cDNA recombinants representing part of the rabbit reticulocyte (immature red blood cell, RBC) lipoxygenase (LOX) mRNA. One cDNA predicts an amino acid (aa) sequence matching exactly the unique N-terminal 30-aa sequence of the purified enzyme. Further, the reticulocyte mRNA, hybrid-selected by this recombinant, can be translated in vitro to give a polypeptide that comigrates with the purified reticulocyte LOX and is recognized by affinity-purified anti-RBC LOX polyclonal antibodies. Southern blotting experiments hybridising the RBC LOX cDNAs available to total rabbit genomic DNA digested with various restriction enzymes gives a fairly simple hybridisation pattern under moderate stringency conditions: moreover, the same pattern is obtained with a cloned fragment of genomic DNA containing the RBC LOX gene. This indicates that the RBC LOX gene is unique in the genome and seems not to be very closely related to the genes encoding the other tissue LOXs. We also show by Northern transfer/hybridisation experiments that the RBC LOX mRNA is expressed only in the red cell lineage but not in white blood cells (bone marrow or spleen) or in other non-erythroid cells tested (e.g., brain and lung).

The stoichiometry of oxygen uptake and conjugated diene formation during the dioxygenation of linoleic acid by the pure reticulocyte lipoxygenase. Evidence for aerobic hydroperoxidase activity

Simultaneous measurements of oxygen uptake and conjugated diene formation (increase in the absorbance at 234 nm) during the dioxygenation of linoleic acid by the pure reticulocyte lipoxygenase gave a nearly theoretical stoichiometry of 1.1 in a temperature range from 5 to 30 degrees C and a wide range of concentrations of both oxygen and linoleic acid. At low concentrations of either oxygen or linoleic acid or both, secondary processes occurred such as linoleic acid-supported lipohydroperoxidase reactions leading to the disappearance of conjugated dienes and to the formation of oxodienes, linoleic acid dimers and epoxyhydroxy derivatives. Under these conditions marked deviations of the stoichiometry between oxygen uptake and conjugated diene formation appeared. The formation of conjugated oxodienoic fatty acids absorbing at 285 nm occurred only under conditions of high concentrations of linoleic acid and limiting oxygen supply. The results indicate that lipohydroperoxidase reactions catalyzed by the pure reticulocyte lipoxygenase do not only take place under strictly anaerobic conditions but also under conditions of limiting concentrations of either linoleic acid or oxygen or both.

Occurrence of the erythroid cell specific arachidonate 15-lipoxygenase in human reticulocytes

Human reticulocytes obtained from patients suffering from various haemolytic disorders convert exogenous [1-14C]-arachidonic acid to 15-hydroxy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid (15-HETE). Immunological studies (dot blot, Western blot) indicated that human reticulocytes contain a lipoxygenase which cross-reacts with a polyclonal antiserum against the rabbit reticulocyte lipoxygenase. Northern blotting with a cloned lipoxygenase cDNA probe shows that the specific mRNA is also present. Reaction of the lipoxygenase with submitochondrial particles caused inactivation of respiratory enzymes. The occurrence of an erythroid cell specific lipoxygenase of similar type in reticulocytes of various mammals and man suggests the general role of this enzyme in the maturational degradation of mitochondria.

The membrane association and dissociation of human glyceraldehyde-3-phosphate dehydrogenase under various conditions of hemolysis Immunochemical evidence for the lack of binding under cellular conditions

Several authors reported an association of the GAPD to the cell membrane of red cells [l--5]. The varying extent of this association suggested to us a dependence on conditions of hemolysis and recorded the question of its artefactional nature. Therefore we investigated the membrane association and dissociation of the GAPD by means of immunochemical and other techniques under various conditions of hemolysis.

On a slow inhibitory effect of free fatty acids on the respiratory chain of non-phosphorylating submitochondrial particles from beef heart.

Free fatty acids (FFA) exert a variety of inhibitory effects on the energy metabolism of mitochondria. They uncouple the oxidative phosphorylation [l-4], inhibit the ATP-Pi exchange [2,3], stimulate the Mg-dependent ATPase [ 1,2] and act as endogenous ionophores for potassium ions [ 51. Moreover, the CoA-esters of fatty acids are strong inhibitors of the adenine nucleotide translocase [6] and the pyridine nucleotide transhydrogenase system [7]. FFA exhibit an instantaneous multi-site inhibition of the electron transport system of non-phosphorylating submitochondrial particles (ETP) which is relieved or prevented by serum albumin [8,9]. In this report a new inhibitory effect of FFA is described which appears slowly.

Activation and desensitization of glycolysis by stimulation of adenylate cyclase in rat reticulocytes

Isoproterenol or forskolin induce a 10–15‐fold increase in concentration of cyclic AMP in rat reticulocytes as compared with the basal level of 2.3 ± 0.3 μM. Glycolysis is stimulated by both compounds transiently more than 2‐fold with a peak after 7.5 min followed by an exponential decline. The glycolytic rate in the presence of 1O μM isoproterenol or 10 μM forskolin did not return to basal levels within 60 min of incubation, but was depressed by as much as 50% under the influence of 100 μM forskolin. This phenomenon is designated as metabolic desensitization. The stimulation of glycolysis is probably due to activation of phosphofructokinase as well as to stimulation of Na+,K+‐ATPase. The diminished glycolytic flux during the period of metabolic desensitization is accompanied by a decline of glucose 6‐phosphate and in the presence of high concentrations of forskolin also by a decrease in glucose 1,6‐bisphosphate. A lower rate of influx of glucose is postulated.

Calcium exerts an indirect effect on ATP‐dependent proteolysis of rat liver mitochondria

The ATP‐dependent proteolysis of rat liver mitochondria prepared in electrolyte‐poor sucrose media requires the presence of Ca2+. Lanthanum, an inhibitor of Ca2+ uptake, inhibits the proteolysis. In contrast, proteolysis of mitochondria prepared in a salt medium does not require Ca2+, nor is it inhibited by lanthanum. It is concluded that Caa+ exerts its effect in an indirect manner, by causing swelling and thereby increasing the accessibility of the membrane proteins of the inner mitochondrial membrane.