Transport of lactate in Plasmodium falciparum-infected human erythrocytes.

The intraerythrocytic human malarial parasite Plasmodium falciparum produces lactate at a rate that exceeds the maximal capacity of the normal red cell membrane to transport lactate. In order to establish how the infected cell removes this excess lactate, the transport of lactate across the host cell and the parasite membranes has been investigated. Transport of radiolabeled L-lactate across the host cell membrane was shown to increase ca. 600-fold compared to uninfected erythrocytes. It showed no saturation with [L-lactate] and was inhibited by inhibitors of the monocarboxylate carrier, cinnamic acid derivatives (CADs), but not by the SH-reagent p-chloromercuriphenyl sulfonic acid (PCMBS). These results suggest that L-lactate is translocated through CAD-inhibitable new pathways induced in the host cell membrane by parasite activity, probably by diffusion of the acid form and through a modified native monocarboxylate:H+ symporter. Continuous monitoring of extracellular pH changes occurring upon suspension of infected cells in isoosmotic Na-lactate solutions indicates that part of the lactate egress is mediated by anionic exchange through the constitutive, but modified, anion exchanger. The transport of L-lactate across the parasite membrane is rapid, nonsaturating, and insensitive to either CADs or PCMBS, or to the presence of pyruvate. L-lactate uptake increased transiently when external pH was lowered and decreased when delta pH was dissipated by the protonophore carbonylcyanide m-chlorophenyl hydrazone (CCCP). These results are compatible with L-lactate crossing the parasite membrane either as the undissociated acid or by means of a novel type of lactate-/H+ symport.     

Stereoselective, nonenzymatic, intramolecular transfer of amino acids

Biological systems synthesize proteins with an almost exclusive use of L-amino acids and virtually none of the D isomer. There has been no satisfactory explanation for the origin of this use of the L isomer. Research presented here shows that at pH 5, transfer of phenylalanine from the adenylate anhydride to ester occurs and is 95-97% efficient for the L isomer and only about 50% efficient for the D isomer. The origin of the use of the L isomer, given D-ribose nucleotides, may be based in part on this stereoselectivity.     

The mechanism of lactate transport in human erythrocytes

Summary Lactate accumulates in human erythrocytes stored at 4°C in the presence of glucose. Efflux of lactate exhibits an activation energy of 22 kcal/mole and is markedly stimulated with increasing medium pH. Lactate influx into erythrocytes that were depleted of intracellular lactate by incubation at 37° at pH 8.0 was stimulated by decreasing medium pH. Under appropriate conditions the pH-dependent lactate flux was insensitive to 4-acetamido-4-isothiocyano-2,2-disulfonic stilbene or 4,4-diisothiocyano-2,2-disulfonic stilbene, inhibitors of the inorganic anion channel, while, e.g., inorganic phosphate transport was fully sensitive. These experiments as well as measurements of H⁺ movements associated with lactate fluxes demonstrate that lactate transport takes place via a specific monocarboxylate transporter (distinct from the inorganic ion channel) by a H⁺-lactate symport mechanism.     

Galactose metabolism of mammalian erythrocytes

Galactose metabolism in the red cells shows marked interspecies and even intraspecies variations. Red cells of guinea pig, dog and a group of rabbits metabolize galactose to a higher extent than those of other species, including human. In the rabbit, the difference in red cell galactokinase activity could not be correlated to the overall utilization of this sugar in the body.     

Phosphoribosylpyrophosphate in erythrocytes of ten mammalian species: concentration, synthesis and degradation.

1. The concentration of PRPP and the activity of PRPP synthetase have been measured in hemolysates from man and nine other mammalian species. PRPP synthetase activity was very low in dog hemolysate. 2. High concentrations of PRPP appeared to be associated with low levels of HGPRT activity, suggesting that HGPRT is the major pathway for utilization of PRPP in mammalian erythrocytes. 3. An alternative catabolic route for PRPP was observed in mammalian hemolysates, which seemed to be associated with acid phosphatase activity. The activity of acid phosphatase in mammalian hemolysates was measured.     

Haemolysis of various mammalian erythrocytes in sodium chloride, glucose and phosphate-buffer solutions.

Mouse, rat, rabbit, hamster, cow, pig, sheep, guinea-pig, dog and human erythrocytes were studied. A 0.9% or stronger solution of sodium chloride completely prevented haemolysis; sheep and pig erythrocytes appeared the more fragile, while human and dog erythrocytes were not haemolized in concentrations of 0.4% or more. Haemolysis of human, rabbit, cow, hamster, guineapig, pig and sheep erythrocytes was not observed in solutions of 0.4% or more of glucose. Except for sheep, human and dog erythrocytes, haemolysis was depressed in rate but not completely prevented by phosphate-buffer solution of pH 7.0.     

Studies of lactate dehydrogenase in the purified state and in intact erythrocytes.

Lactate dehydrogenase in intact erythrocytes was studied by observing isotope exchange between lactate and pyruvate by p.m.r. The inhibition of the enzyme in intact cells by both oxalate and pyruvate was found to be similar to that of the purified enzyme. The activity of the enzyme in intact cells indicates that the free solution NAD+ + NADH concentration in erythrocytes is about 10 microM whereas the total extractable NAD+ + NADH is about 80 nmol/ml of cell water.     

Injections of recombinant human erythropoietin increases lactate influx into erythrocytes.

Previous studies showed that erythropoietin not only increases erythrocyte production but is also essential in both the synthesis and the good functioning of several erythrocyte membrane proteins, including band 3. It is still unknown whether anion and/or H(+) fluxes are modified by erythropoietin. This study aimed to evaluate the effect of recombinant human erythropoietin (rHuEPO) injections on lactate transport into erythrocytes via band 3 and H(+)-monocarboxylate transporter MCT-1, two proteins involved in lactate exchange. Nine athletes received subcutaneous rHuEPO (50 U/kg body mass 3 times a week for 4 wk), and seven athletes received a saline solution (placebo group). All subjects were also supplemented with oral iron and vitamins B(9) and B(12). Lactate transport into erythrocytes was studied before and after the rHuEPO treatment at different lactate concentrations (1.6, 8.1, 41, and 81.1 mM). After treatment, MCT-1 lactate uptake was increased at 1.6, 41 (P < 0.01), and 81.1 mM lactate concentration (P < 0.001) although lactate uptake via band 3 and nonionic diffusion were unchanged. MCT-1 maximal velocity increased in the rHuEPO group (P < 0.05), reaching higher values than in the placebo group (P < 0.05) after treatment. Our results show that rHuEPO injections increased MCT-1 lactate influx at low and high lactate concentrations. The increase in MCT-1 maximal velocity suggests that rHuEPO may stimulate MCT-1 synthesis during erythrocyte formation in bone marrow.     

Oscillation of ion fluxes in mammalian erythrocytes. Mechanism of oscillation

The dependence of ionophore-induced oscillations in rat erythrocytes on various concentrations of A23187, FCCP and Ca2+ was analysed using ion-selective electrodes. The oscillations were shown to be independent of the extracellular concentration of carbonylcyanide p-trifluoromethoxyphenylhydrazone and Ca2+. The dependence of oscillations on the concentration A23187 was shown to be a threshold characteristic and represented by a bell-shaped curve. In the course of oscillations the redistribution of A23187 between cells and the incubation medium was demonstrated using high-speed centrifugation. A hypothesis for oscillatory-state generation in erythrocytes was suggested on the basis of pH-dependent changes of the Ca2+ ionophore A23187 content in cells. According to this hypothesis the H+ concentration within the external membrane-adjacent layer serves as a causative factor for induction of cyclic desorption of A23187 molecules from the cell membrane.     

Aminoacylation of undermethylated mammalian transfer RNA.

To study the role of 5-methylcytidine in the aminoacylation of mammalian tRNA, bulk tRNA specifically deficient in 5-methylcytidine was isolated from the livers of mice treated with 5-azacytidine (18 mg/kg) for 4 days. For comparison, more extensively altered tRNA was isolated from the livers of mice treated with DL-ethionine (100 mg/kg) plus adenine (48 mg/kg) for 3 days. The amino acid acceptor capacity of these tRNAs was determined by measuring the incorporation of one of eight different 14C-labeled amino acids or a mixture of 14C-labeled amino acids in homologous assays using a crude synthetase preparation isolated from untreated mice. The 5-methylcytidine-deficient tRNA incorporated each amino acid to the same extent as fully methylated tRNA. The tRNA from DL-ethionine-treated livers showed an overall decreased amino-acylation capacity for all amino acids tested. The 5-methylcytidine-deficient tRNA from DL-ethionine-treated mice were further characterized as substrates in homologous rate assays designed to determine the Km and V of the aminoacylation reaction using four individual 14C-labeled amino acids and a mixture of 14C-labeled amino acids. The Km and V of the reactions for all amino acids tested using 5-methylcytidine-deficient tRNA as substrate were essentially the same as for fully methylated tRNA. However, the Km and V were increased when liver tRNA from mice treated with DL-ethionine plus adenine was used as substrate in the rate reaction with [14C]lysine as label. Our results suggest that although extensively altered tRNA is a poorer substrate than control tRNA in both extent and rate of aminoacylation, 5-methylcytidine in mammalian tRNA is not involved in the recognition of the tRNA by the synthetase as measured by aminoacylation activity.     

Na K ATPase activity in mammalian erythrocytes

The effects of membranotropic substances--nonionic detergent Tween-20 and EDTA--on the activity and some properties of Na,K-ATPase from mammalian erythrocytes were studied. It was shown that pretreatment of whole erythrocytes with Tween-20 (5 mg/ml) allows a detection of the enzyme activity, which cannot be detected in intact cells. It was also found that erythrocyte ghosts with a high and stable activity of Na,K-ATPase can be obtained by injections of EDTA (1-2 mM) into the hemolysis medium. Although the enzyme activity in whole erythrocytes and their ghosts was detected by the use of various membranotropic agents, the type of the dependence of the Na,K-ATPase activity on MgCl2 and EDTA concentration in the incubation medium was essentially the same for both cell preparations, the optimal concentrations of MgCl2 and EDTA being 3 and 1 mM, respectively. A rise in MgCl2 concentration above 3 mM caused a decrease of the enzymatic activity. Simple techniques have been developed for the detection of the Na,K-ATPase activity in mammalian erythrocytes which allow the determination of a higher enzymatic activity than those described in literature.