Hemolytic anemias due to erythrocyte enzyme deficiencies.

Red blood cells can only fulfil their functions over the normal period of approximately 120 days with 1.7 x 10(5) circulatory cycles efficiently if they withstand external and internal loads. This requires ATP and redox equivalents, which have to be permanently regenerated by the energy and redox metabolism. These pathways are necessary to maintain the biconcave shape of the cells, their specific intracellular cation concentrations, the reduced state of hemoglobin with a divalent iron and the sulfhydryl groups of enzymes, glutathione and membrane components. If an enzyme deficiency of one of these metabolic pathways limits the ATP and/or NADPH production, distinct membrane alterations result causing a removal of the damaged cells by the monocyte-macrophage system. Most metabolic needs of erythrocytes are covered by glycolysis, the oxidative pentose phosphate pathway (OPPP), the glutathione cycle, nucleotide metabolism and MetHb reductase. Hereditary enzyme deficiencies of all these pathways have been identified; those that cause non-spherocytic hemolytic anemia are listed in Table 4. Their frequencies differ markedly both with respect to the affected enzyme and geographic distribution. Glucose-6-phosphate dehydrogenase enzymopathies (G6PD) are with more than 400 million cases by far the most common deficiency. The highest gene frequency has been found with 0.7 among Kurdish Jews. G6PD deficiencies are furthermore prevalent with frequencies of about 0.1 among Africans, Black Americans, and populations of Mediterranean countries and South East Asia. In Middle and Northern Europe the frequency of G6PD is much lower, and with approximately 0.0005, comparable with the frequency of pyruvate kinase (PK) enzymopathies, the most frequent enzyme deficiency in glycolysis in this area (Luzzatto, 1987; Beutler and Kuhl, 1990). The relationship between the degree of enzyme deficiency and the extent of metabolic dysfunction in red blood cells and other tissues depend on several factors: on the importance of the affected enzyme; its expression rate; the stability of the mutant enzyme against proteolytic degradation and functional abnormalities; the possibility to compensate the deficiency by an overexpression of the corresponding isoenzyme or by the use of an alternative metabolic pathway. Difficulties in estimating the quantitative degree of disorder in severe cases are due to the fact that these populations contain many reticulocytes, which generally have higher enzyme activities and concentrations of intermediates than erythrocytes. An alternative approach to predict metabolic changes is the analysis by mathematical modeling. Mathematical modeling of the main metabolic pathways of human erythrocytes has reached an advanced level (Rapoport et al., 1976; Holzhütter et al., 1985; Schuster et al., 1988). Models have been successfully employed to describe stationary and time-dependent metabolic states of the cell under normal conditions as well as in the presence of enzyme deficiencies. Figure 5 shows computational results of erythrocyte enzyme deficiencies. This analysis is based on the comprehensive mathematical model of the energy and redox metabolism for human erythrocyte presented in Fig. 6. Stationary states of the cell metabolism have been calculated by varying the activity of each of the participating enzymes by several orders of magnitude. To predict consequences of enzyme deficiencies a performance function has been introduced (Schuster and Holzhütter, 1995). It takes into account the homeostasis of three essential metabolic variables: the energetic state (ATP), the reductive capacity (reduced glutathione) and the osmotic state. From the data given in Fig. 5 one can conclude that generally the metabolic impairment resulting in deficiencies occurs earlier for enzymes with high control coefficients than for those catalyzing equilibrium reactions. On the other hand the flux curves of latter enzymes decrease more steeply below a critica

Oxygenation of biomembranes by mammalian lipoxygenases: the role of ubiquinone.

15-Lipoxygenase is implicated in the selective breakdown of mitochondria during red cell maturation by virtue of its capability of directly oxygenating phospholipids. To address the reason of the selectivity for mitochondria, we studied the reaction of pure rabbit 15-lipoxygenase with beef heart submitochondrial particles in vitro. This reaction is characterised by a loss of polyenoic fatty acids, the formation of phospholipid-bound hydroperoxy- and keto-polyenoic fatty acids, and oxidative modification of membrane proteins. The total oxygen uptake exceeds the formation of oxygenated polyenoic fatty acids several times. The excessive oxygen uptake was not inhibited by 3,5-di-tert-butyl-4-hydroxytoluene or by respiratory inhibitors, but was partly suppressed by superoxide dismutase plus catalase, salicylate, or mannitol. Pentane-extraction of the submitochondrial particles abolished the excessive oxygen uptake, whereas reconstitution with ubiquinone- 50 restored it. A marked excessive oxygen uptake did not occur during the analogous reaction with erythrocyte ghosts. It is proposed that ubiquinone-50 triggers the formation of hydroxyl radicals from 15-lipoxygenase-derived hydroperoxy-lipids via a Fenton-type reaction driven by ubisemiquinone radicals. A new prooxidative function of ubiquinone in the biologically programmed degradation of mitochondria in certain types of cells is proposed.

3,5-Di-t-butyl-4-hydroxytoluene (BHT) and probucol stimulate selectively the reaction of mammalian 15-lipoxygenase with biomembranes.

The lipophilic antioxidant 3,5-di-t-butyl-4-hydroxytoluene (BHT) and the structurally-related antiatherogenic drug probucol stimulate the oxygenation of mitochondrial membranes and erythrocyte ghosts by the rabbit 15-lipoxygenase as indicated by an increase in oxygen consumption as well as by an enhanced loss of polyenoic fatty acids and by the formation of specific lipoxygenase products in the membrane phospholipids. The oxygenation of linoleic acid, phospholipids and human low-density lipoproteins was not stimulated. With mitochondrial membranes, BHT causes a quenching of the 1-anilino-8-naphthalene sulfonate fluorescence. Thus, it is suggested that the stimulation of membrane oxygenation may be due to structural changes in the membranes leading to a better susceptibility of the polyenoic fatty acid residues towards lipoxygenase attack. Owing to this unexpected effect of the antioxidants, which is not related to their radical-scavenger capacity, care should be taken in interpreting experimental data on effects of BHT and probucol.

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.

Quantification of ATP-producing and consuming processes in quiescent pig spleen lymphocytes.

ATP production in quiescent pig spleen lymphocytes was estimated on the basis of their coupled respiration. ATP-consuming processes were assessed from the effects of inhibitors of protein synthesis, proteolysis, RNA synthesis, Na+ K(+)-ATPase and Ca(2+)-ATPase on respiration. About 95% of the total ATP consumption could be assigned to specific processes. More than 50% of the ATP produced appear to be consumed by the cation transport ATPases.

Lipoxygenases from soybeans and rabbit reticulocytes: inactivation and iron release.

Various inactivation methods were applied to lipoxygenases from soybean (isoenzyme 1) and rabbit reticulocytes to compare the inactivation behaviour of both enzymes and to elucidate the state of the iron which is known to be involved in the catalytic reaction of lipoxygenases: 1. Titration of the enzyme with mercury compounds shows that there are one or two SH groups responsible for the loss of activity in the presence of mercury. The SH groups seem not to be involved in the tight iron binding. 2. Inactivation by chelating agents such as o-phenanthroline or batho-phenanthroline sulfonic acid occurs only in the presence of reducing agents (mercaptoethanol and ascorbic acid). Our data support a co-oxidation mechanism. The complexation of iron by chelators is not the rate-limiting step. Both lipoxygenases show a very similar behaviour in this respect despite the fact that the reticulocyte enzyme requires the addition of trace amounts of copper ions for efficient inactivation. 3. Release of iron from the enzyme is also achieved by denaturation with guanidinium hydrochloride (Gu-HCl). In all cases, inactivation and release of iron were irreversible processes. 4. A sequence comparison for both animal and plant lipoxygenases shows strongly conserved amino acids, especially histidines and hydrophobic residues, which possibly may be involved in iron complexation and substrate binding.

Stimulation of rat red blood cell glycolysis by phenylhydrazine hydrochloride.

The effects of phenylhydrazine hydrochloride (5, 10 and 20 mM) on the energy metabolism of red cells of rats were studied. There were found an 1.3-2-fold increase of lactate formation, an up to seven-fold accumulation of pyruvate and of the flux via the oxidative pentose pathway. The stimulation of the glycolysis was caused by 1) a large decline of the ATP concentration and a corresponding increase of ADP and AMP, and 2) by the stimulation of the actual activities of the Na+,K(+)-ATPase and Ca+(+)-ATPase owing to a greater permeability of the plasma membrane. A significant increase of the Ca+(+)-influx was demonstrated. A decline of the sum of adenine nucleotides after 60 and 90 min of incubation with phenylhydrazine was not accompanied by accumulation of hypoxanthine.

[Influence of local anesthetics and narcotics on the energy metabolism of Ehrlich ascites tumor cells]. / Einfluss von Lokalanästhetika und Narkotika auf den Energiestoffwechsel von Ehrlich-Ascites-Tumorzellen.

The suitability of Ehrlich ascites tumour cells as a test object for potential drugs affecting ATP-producing or consuming processes directly or indirectly is made likely by actions of local anesthetics and narcotics on the energy metabolism of these cells. The influence of local anesthetics on cation transport and Ca+(+)-dependent processes as well as the inhibition of NADH-ubiquinone reductase by narcotics is demonstrated for Ehrlich ascites tumour cells, too.

New evidence for endoplasmic reticulum and Golgi apparatus in rabbit reticulocytes.

The incorporation of 14C-galactose and 14C-fucose in rabbit reticulocytes obtained by bleeding anaemia and separated according to their stage of maturation was studied. Only the youngest reticulocytes were shown to incorporate the two sugars with galactosylation being three times higher. The glycosylation behaved different from the synthesis of membrane proteins with respect to the dependence on temperature and time. Among the newly synthesized membrane proteins the signal sequence receptor (SSR), a compound of the system addressing protein translocation, was detected immunologically.

Maturation-dependent changes of the rat reticulocyte energy metabolism and hormonal responsiveness.

Significant reduction of total and coupled respiration as well as an increase of uncoupled respiration characterize the maturation of rat reticulocytes. ATP declines by 45%, whereas ADP increases by 70% and the ATP/ADP ratio decreases from 7.6 to 3.3. At the same time the oxygen-delivery capacity of red blood cells increases by 37% due to an increase of 2.3-BPG level. In the youngest reticulocytes the basic level of cAMP is 2-fold higher, whereas the maximal (-)-isoproterenol-induced accumulation of cAMP is 8-fold higher than in the fraction of most mature reticulocytes. Evidence for the presence of functionally active glucagon-receptors in reticulocytes is presented, with maturational inactivation/disappearance of R2-glucagon receptors.

Structure of the mRNA and of the gene coding for the rabbit erythroid 15-lipoxygenase.

The complete structure of the rabbit erythroid cell-specific 15-lipoxygenase mRNA and its gene was established by sequencing cDNA and genomic recombinants. The transcription initiation site was obtained by primer-extension sequencing. A presumptive promoter structure was characterized by sequencing 0.5 kb 5' to the transcription initiation site and by transfection experiments using constructs with the chloramphenicol transferase gene. The mRNA codes for a polypeptide of 662 amino acids. Its 3' untranslated region contains an intriguing repeated sequence of 10 copies with the consensus C4PuC3TCTTC4AAG which may be involved in its regulation during reticulocyte maturation. The transcription unit consists of 8.0 kb and is split like the gene for the leukocyte 5' lipoxygenase by 13 introns. Another interesting aspect in the structure of the 15-LOX gene is a highly conserved repeat in intron seven consisting of a unit of 54 nucleotides which is repeated eight times. Comparing the predicted amino acid sequence with those from other lipoxygenases published recently shows that lipoxygenases are a related group of enzymes which may have arisen from a common ancestral gene.

Catabolism of adenine nucleotides in rabbit blood cells.

The present study describes the effects of the inhibition of adenosine deaminase and of adenosine kinase on reticulocytes and erythrocytes of rabbits. Both in erythrocytes and in reticulocytes the degradation of adenine nucleotides proceeds via AMP----IMP----inosine----hypoxanthine. Under approximately physiological conditions the rate of degradation amounts in erythrocytes to 23 mumoles/l cells.h and in reticulocytes to 331 mumoles/l cells.h, respectively. In erythrocytes the formation of hypoxanthine corresponds closely to the degradation of adenine nucleotides in reticulocytes; the formation of hypoxanthine seems to exceed the degradation presumably mainly due to RNA degradation. Parallel to the primary deamination of AMP there is a primary dephosphorylation to adenosine of about 60 mumoles/l cells.h in erythrocytes and about 300 mumoles/l cells.h in reticulocytes. This pathway does not provide, however, any measurable contribution to the formation of hypoxanthine, because the adenosine formed is rephosphorylated via adenosine kinase almost completely.

Balancing of mitochondrial and glycolytic ATP production and of the ATP-consuming processes of Ehrlich mouse ascites tumour cells in a high phosphate medium.

A balance of energy budgeting of Ehrlich mouse ascites tumour cells including mitochondrial and glycolytic ATP production and about 80% of ATP consumption in a high phosphate medium is presented. In the share of glycolysis was about one-third of the total ATP production, more than twice that found in a low phosphate medium. The extent of a single energy reaction was assessed from the decrease of coupled oxygen consumption and lactate formation following the specific inhibition of this process. The inhibitory effects on coupled respiration and glycolysis were identical for the energy consuming processes measured: protein turnover, Na+/K(+)-ATPase, Ca2(+)-transport and RNA synthesis.

ATP-dependent peptide release from mitochondria of reticulocytes.

ATP-dependent release of TCA-precipitable peptides from mitochondria-containing stroma (MCS) is described. The process is independent of ubiquitin, but is sensitive to hemin and to heat treatment. Neither chloramphenicol nor EGTA inhibit. 50% of the activity is dependent on charged tRNA. The peptides released from MCS possess a molecular mass of about 1-5 kDa and are degraded to TCA-soluble compounds by a cytosolic protease system (fraction II) without ubiquitin.

The complete sequence of the rabbit erythroid cell-specific 15-lipoxygenase mRNA: comparison of the predicted amino acid sequence of the erythrocyte lipoxygenase with other lipoxygenases.

We report the complete sequence of the rabbit reticulocyte (RBC) 15-lipoxygenase (LOX) mRNA as deduced from (i) sequencing cDNA recombinants isolated by screening cDNA libraries or polymerase-chain-reactions, and (ii) the sequence originating from the transcription start point obtained by primer extension-sequencing reactions. Like the human leukocyte 5-LOX mRNA, the RBC 15-LOX mRNA contains a very short 5'-untranslated region with a long 3'-untranslated region. But, unlike the human leukocyte 5-LOX mRNA, the RBC 15-LOX mRNA contains an intriguing repeated sequence (ten copies with the consensus sequence C4PuC3TCTTC4AAG) just after the translational stop codon, which may be involved in its regulation during reticulocyte maturation. Comparison of the RBC 15-LOX mRNA sequence with those of the previously published human 5-LOX mRNA and the soybean 3-LOX gene shows only a few short regions of sequence similarity. However, the predicted amino acid sequences of the encoded LOX enzymes show certain conserved regions that are presumably involved in their catalytic activity, in particular a cluster of five conserved histidines that we predict chelate the iron moiety involved in the active site.

Maturation-dependent changes of the rabbit reticulocyte energy metabolism.

Rabbit reticulocytes were separated into four fractions of different maturity in order to investigate the changes of cellular respiration and glycolysis, adenine nucleotides, 2,3-biphosphoglycerate (2,3-BPG) as well as cyclic AMP level during the transition from the youngest to the most mature reticulocytes. A significant reduction of total oxygen consumption, mainly due to depression of coupled respiration was found. The decline of respiration was accompanied by a 2-fold increase of the rate of aerobic glycolysis indicating a reduced Pasteur effect during maturation. A decline of ATP and an increase of ADP concentration was found. The oxygen-delivery capacity of the red cells increased by about 26% caused by an increase of the 2,3-BPG level of about 2 mmol/l cells. Cyclic AMP level in the fraction of youngest reticulocytes was about 60-fold higher than that in mature rabbit erythrocytes. The biggest decline of cyclic AMP was registered during the transition from youngest to the intermediate stage of maturity.

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.

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.

Evidence for the appearance of a reticulocyte population low in lipoxygenase mRNA during the recovery from a phenylhydrazine-induced anemia in rabbits.

It is shown that during recovery from a phenylhydrazine-induced anemia in rabbits a selective decrease in lipoxygenase mRNA takes place with a corresponding shut-off of the synthesis of the enzyme. It is suggested that a new population, 'recovery'-reticulocytes, makes its appearance in the peripheral blood. Their cells are more mature than the stress macroreticulocytes. A cell-free system prepared from the recovery-reticulocytes exhibits low endogenous synthesis of non-globin polypeptides, even without nuclease treatment, but retains full capacity to be stimulated by exogenous mRNA.