Eitan Bogin

Effect of Parathyroid Hormone on Osmotic Fragility of Human Erythrocytes

The survival of erythrocytes (RBC) is shortened in uremia, and it has been shown that calcium influx into RBC evoked crenation and increased their rigidity. The high blood levels of parathyroid hormone (PTH) may augment entry of calcium into RBC and hence affect their integrity. We examined the effect of PTH on osmotic fragility of human RBC and investigated the mechanisms through which PTH interacts with RBC. Both the amino-terminal (1-34) PTH and the intact (1-84) PTH, but not the carboxy-terminal (53-84) PTH, produced significant increases in osmotic fragility. This effect was abolished by prior inactivation of the hormone. There was a dose-response relationship between both moieties of PTH and the increase in osmotic fragility. This action of PTH required calcium, was mimicked by calcium ionophore, and was partially blocked by verapamil. PTH caused significant influx of (45)Ca into RBC, which was not associated with potassium leak. The hormone did not affect water content of RBC. Scanning electron microscopy revealed that the incubation of RBC with PTH was associated with the appearance of membrane filamentous extensions, which anchor RBC together. Inhibition of glycolytic activity of RBC with NaF or inhibition of Na-K-activated ATPase with ouabain did not abolish the effect of PTH on osmotic fragility. PTH did not stimulate RBC Na-K-activated ATPase or Mg-dependent ATPase but caused marked and significant stimulation of Ca-activated ATPase. The basal activity of the RBC adenylate cyclase was low and PTH produced only a modest stimulation of this enzyme. Both cyclic AMP and dibutyryl cyclic AMP had no effect on osmotic fragility. THE DATA INDICATE THAT: (a) the RBC is a target organ for PTH, (b) the hormone increases osmotic fragility of RBC, and (c) this effect of PTH is due to enhanced calcium entry into RBC. We suggest that the increased calcium influx may affect the spectrin-actin of the cytoskeletal network of the RBC and may alter the stability and integrity of the cell membrane. This action of PTH on the RBC could be, at least in part, responsible for the shortened survival of RBC in uremia, and assign a new role for PTH in the pathogenesis of the anemia of uremia.

Oxidative phosphorylation in fractionated bacterial systems XLIII. Coupling factors associated with the NAD+-linked electron transport pathway

Oxidative phosphorylation with the cell-free system from Mycobacterium phlei is dependent on the presence of both the particulate and soluble fractions. While the particles contain the bulk of the respiratory components, the soluble fraction contains a number of dehydrogenases which are necessary to initiate electron transport and protein components which are necessary for the coupling process. The supernatant components necessary for restoration of phosphorylation were resolved into two different coupling factors which appear to operate at different phosphorylative sites. The purified coupling factors appear to be protein in nature. The phosphorylation restored on addition of the purified coupling factors to the electron transport particles was found to be sensitive to uncoupling agents. The purified coupling factors were found to contain some ATPase, adenylate kinase, 14C-ADP-ATP and 32Pi-ATP exchange activities. Antibodies prepared to either of the coupling factors was found to be specific for the coupling protein to which it was prepared. In addition, the antibodies were found to inhibit the phosphorylation elicited by that coupling factor. An additional coupling factor was found to be bound to the electron transport particles. The particulate bound factor was released from the particles by treatment with 1.0 m urea. The level of phosphorylation was increased by a combination of the different coupling factors. The coupling factors had no significant effect on the level of oxidation.

Exogenous NADH oxidation and particulate fumarate reductase in Mycobacterium phlei.

Abstract Bacteria contain a number of distinct pathways that are capable of oxidizing NADH. Only one of the NAD + -linked pathways, that of the electron transport particles, has been shown to be linked to the generation of energy-rich phosphate bonds. Regulation of the phosphorylative pathway associated with the oxidation of NADH by the system from Mycobacterium phlei appears to be controlled by the level of NAD + , AMP, and inorganic phosphate. The phosphorylative and nonphosphorylative pathways were inhibited by NAD + ; however, relief of inhibition of NADH oxidation by NAD + was found to occur only with the phosphorylative pathway on addition of inorganic phosphate and AMP. Kinetic studies indicated that the inhibition by NAD + on NADH oxidation by either pathway was allosteric in nature; however, AMP and inorganic phosphate alter only the kinetic pattern exhibited by the phosphorylative pathway. In addition, fumarate which is one of the chief carbon sources used to support the growth of M . phlei , was found to stimulate the cell-free phosphorylative pathway associated with NADH oxidation. Under these conditions fumarate was found to accept electrons from cytochrome b reduced by NADH and to form succinate. The oxidation of cytochrome b by fumarate and the formation of succinate from fumarate were inhibited by hydroxyquinoline- N -oxide. The fumarate reductase appeared to be localized in the electron transport particles and was cytochrome b -Iinked.

The effect of trypsin and heat treatment on oxidative phosphorylation in Mycobacterium phlei

The electron transport particles from Mycobacterium phlei contain particle-bound coupling factors and require the addition of soluble coupling factors for restoration of phosphorylation. However, exposure of the particles to heat (50°) or treatment with trypsin increased the P/O ratio with succinate or with NAD+-linked substrates. In addition, the heat treated particles no longer required the soluble coupling factors for phosphorylation, but the trypsin-treated particles required this addition for maximal levels of phosphorylation. A combination of treatment with trypsin and heat resulted in higher levels of phosphorylation than either treatment alone. The phosphorylation observed in both cases was sensitive to uncoupling agents.

Multiple cytochromes b in Mycobacterium phlei

Abstract Electron transport particles from M. phlei contain at least 3 different active forms of cytochrome b, one reduced by NADH, with a λmax at 563 nm (bN563), and the other two reduced by either succinate or NADH, with λmax at 559 and 563 nm (bS559) and (bS563). Low temperature λmax for cytochrome b reduction with NADH or succinate are described. During steady state only bS563 was observed with succinate. In the presence of ATP, succinate reduced an increased amount of a b563. A branching of the NAD+-linked pathway and a convergence at the level of cytochrome c is suggested, with only one branch accessible to succinate.

Biochemical changes associated with the osmotic fragility of young and mature erythrocytes caused by parathyroid hormone in relation to the uremic syndrome.

The effect of parathyroid hormone at concentrations found in uremic patients on erythrocytes (RBC) from newborn and adult rabbits was studied in relation to the fragility pattern in hypotonic salt solutions and the activities of Ca- and Mg-dependent ATPases. Median osmotic fragility of RBC from newborn rabbits was significantly lower than in mature rabbits. Parathyroid hormone (PTH) stimulated to a greater extent the mean osmotic fragility in RBC from newborn rabbits, than in those from adults. Similarly, the hormone stimulated to a much greater extent the Ca-ATPase but not the Mg-ATPase in RBC from the newborn rabbits, in comparison to those from adult rabbits. PTH, which is greatly elevated in the blood of patients with chronic renal failure, may be one cause of the anemia seen in these patients, and its effect, which is mediated by Ca-ATPase activity, is stronger on young RBC. There were significant morphological changes in the young RBC caused by PTH, as seen with scanning electron microscopy.

Interchangeability of coupling factors from bacterial and mammalian origin

Abstract Bacterial and mammalian preparations capable of oxidative phosphorylation can be separated into a particulate fraction containing the electron transport chains and a soluble fraction containing coupling proteins necessary for restoration of phosphorylation. The addition of coupling factors of bacterial origin to submitochondrial particles was found to replace the requirement for mammalian coupling factors for restoration of phosphorylation. Coupling factors obtained from light beef heart mitochondria were found to restore phosphorylation to the electron transport particles of Mycobacterium phlei . The level of phosphorylation restored by the heterologus coupling factors was found to be lower than that restored by the native coupling factor; however, the phosphorylation restored by either coupling factor was found to be sensitive to uncoupling agents.

Oxidative phosphorylation in fractionated bacterial systems. XLII. The effect of coupling factors on urea-treated particles from M. phlei.

Abstract The electron transport particles from Mycobacterium phlei are capable of coupling phosphorylation to the oxidation of succinate or generated NADH. Treatment of the particles with urea resulted in the loss of the phosphorylative activity without any effect on the oxidation of either substrate. Addition of a factor (BCF 1 ) released by urea treatment of particles to the urea-treated particles restored phosphorylation to the original level with succinate as the electron donor. This factor, however, was not effective when exogenous NADH (1) or ascorbate-TPD were used as substrates. The addition of a combination of the factor released by urea treatment and the soluble coupling factor (BCF 3 ) resulted in an increase in the level of phosphorylation. In contrast, with ascorbate and TPD as the electron donors only BCF 3 was capable of restoring phosphorylation. The restored phosphorylation was found to be sensitive to uncoupling agents to the same degree as that observed with these agents in the unresolved system. Antibodies prepared to the BCF 3 fraction were found to inhibit the phosphorylation elicited by this factor and had no effect on the phosphorylation restored by the other coupling factors (BCF 1 and 2 ). Evidence has been obtained which indicates that the factor released by urea treatment of the particles may participate in the reaction(s) common to both the succinate and NAD + -linked pathways and that it is different from the soluble coupling factors.

Extraparticulate Chain Interaction between Different Electron Transport Particles

Interaction or cross-linking between the respiratory chains of the electron transport particles of bacterial origin occurs with a mixture of active and inactive particles. Interaction between bacterial particles and liver sub-mitochondrial particles also occurs. Irradiation of the bacterial particles at 360 nanometers resulted in the destruction of quinone and consequent loss of ability of reduced nicotinamide adenine, dinucleotides to reduce cytochromes b, c1, c, and a plus a3. A mixture of both irradiated and untreated particles in the presence of the reduced dinucleotide resulted in the reduction of cytochromes c and a plus a3, in an amount equivalent to the total concentration of these cytochromes in both types of particles. In contrast, the amount of cytochrome b reduced was equivalent to half the particle concentration or to that observed with the active particles alone. The rate of reduction of cytochromes c and a plus a3 with the mixture of particles was similar to that with the active particles alone. The interaction or cross-linking between the particulate respiratory chains of bacteria or of bacterial and mammalian systems occurs after cytochrome b and before or at cytochrome c.