Transferrin receptors on the human placental microvillous membrane.

A preparation of microvillous membrane vesicles from human placental syncytiotrophoblast binds transferrin to specific transferrin receptors. Transferrin binding to placental receptors is rapid, saturable, reversible, and specific. Approximately 2.5 X 10(13) receptors are present per milligram of membrane protein; the apparent association constant of transferrin for the placental receptor is 2.2 X 10(7) X M-1. No evidence for removal of iron from transferrin bound to intact membrane receptors was observed in these studies. Nonionic detergent solubilization and partial purification of the microvillous membrane transferrin receptor were carried out with preservation of the functional properties of the receptor.

Effects of Na+/H+ antiport and intracellular pH in the regulation of HL-60 cell apoptosis.

The roles of Na+/H+ antiport and intracellular pH in apoptosis of HL-60 cells were investigated here. We found that dimethyl amiloride, a specific Na+/H+ antiport inhibitor, induced intracellular acidification but not apoptosis; while sodium ionophore, monensin caused intracellular alkalinization as well as apoptosis in HL-60 cells. Br-A23187 and thapsigargin could induce a various degree of intracellular alkalinization through the stimulation of Na+/H+ antiport. Dimethyl amiloride blocked the intracellular alkalinization and inhibited apoptosis induced by Br-A23187 and thapsigargin. PMA also stimulated Na+/H+ antiport and induced intracellular alkalinization which was completely blocked by dimethyl amiloride and partially attenuated by PKC inhibitors. PMA could inhibit apoptosis in HL-60 cells. PMA-induced suppression of apoptosis was, however, not interfered by dimethyl amiloride, but could be abolished by PKC inhibitors. These results indicate that pHi alkalinization and/or the stimulation of Na+/H+ antiport, instead of intracellular acidification, are contributory to the induction of apoptosis. PMA-induced inhibition of apoptosis is not necessarily associated with intracellular alkalinization, but primarily due to activation of PKC. We suggest that stimulation of Na+/H+ antiport and pHi alkalinization act as facilitating factors in the induction of apoptosis.

Specific interaction between tetrandrine and Quillaja saponins in promoting permeabilization of plasma membrane in human leukemic HL-60 cells.

Spontaneous Ni2+ entry (leak), measured as fluorescence quench in fura-2-loaded HL-60 cells at the excitation wavelength of 360 nm, was strongly inhibited by tetrandrine (TET, 100 microM), a Ca2+ antagonist of Chinese herbal origin. Exposure of the cells for 5 min to saponins from Quillaja saponaria (QS, 30 microg/ml), surfactants well known to permeabilize the plasma membrane by complexing with cholesterol, promoted Ni2+ entry without causing fura-2 leak-out. Unexpectedly, TET caused an immediate (within 2.5 min) augmentation of QS-promoted Ni2+ entry; and a 5-min treatment with both TET and QS resulted not only in an enhanced Ni2+ entry, but also a fura-2 leak-out. Ginseng saponins (100 microg/ml) alone or together with TET did not cause such a permeabilization. Permeabilization induced by 1-3 microM digitonin, another cholesterol-complexing glycoside, could not be enhanced by TET. TET did not affect permeabilization induced by Triton X-100 (0.01%), a detergent which non-specifically disrupts the hydrophobic interaction at the plasma membrane. TET also did not enhance Ni2+ entry triggered by ionomycin (0.35 microM) or SK&F 96365 (20 microM). Further, it did not augment Ni2+ entry when the plasma membrane fluidity was modulated by changes of temperature (27-47 degrees C) or treatment with 5% ethanol. This QS-promoted Ni2+ entry could not be amplified by other lipophilic Ca2+ antagonists, such as diltiazem (100 microM) and verapamil (100 microM). The results hence indicate that TET enhanced Ni2+ entry (or permeabilization) elicited by QS treatment, but not other perturbations of the plasma membrane. We suggest that pore formation at the plasma membrane, a consequence of QS-cholesterol interaction, can be specifically enhanced by TET. Also, a comparative study of the effects of TET and its very close analogues, hernandezine and berbamine, reveals that the methoxyl group at the R2 position of TET appears to be crucial in enhancing QS-promoted Ni2+ entry.

The antigen-specific immunoglobulin G receptor is more sensitive to stimulation than the IgM receptor in transfected B cells.

A murine lymphoma cell line (M12.4) was transfected with immunoglobulin (Ig) genes encoding a T15+ (idiotype) IgM antibody or an idiotypically identical IgG antibody. Three transfectant clones of each class which showed similar (albeit distinguishable) levels of membrane expression of the transfected genes were used in the study. The response of each cell population to stimulation with anti-T15 antibodies was followed by measurement of the change in the intracellular Ca++ concentration. The IgG transfectants were found to be significantly more responsive to such stimulation than the IgM cells. In contrast, there was no difference in their response to a nonspecific reagent, the calcium ionophore A23187.

Production of anti-phosphorylcholine antibodies of the T15 idiotype in CBA/N xid mice: investigation of the defect using a T15 immunoglobulin transgene.

A notable defect in CBA/N xid mice is their relative inability to make antibodies to phosphorylcholine (PC), particularly those of the T15 idiotype which predominate in the anti-PC responses of immunologically normal mice. To investigate the basis of this defect, we introduced functionally rearranged genes encoding a T15+ PC-binding immunoglobulin G antibody into the germline of these animals. Expression of these genes in the xid cells was observed, shown by the existence of a distinct population of T15+ cells (3 x 10(6)) in the spleen of the transgenic animals, and the presence of PC-binding T15+ IgG antibodies (1-15 micrograms/ml) in the serum. Mixed antibody molecules were also found, however, which were composed of both transgene-encoded and endogenously-derived chains. Existence of the T15+ cells in these animals seemed normal, since these were not depleted (to any great extent) and were immunocompetent as well. The latter was shown by the increased T15+ antibody production in the transgenic animals when stimulated with a PC-associated thymus-independent type 1 (TI-1) antigen and anti-idiotype antibodies, but not with the pneumococcal TI-2 antigen. This is similar to the PC-specific (T15-) responsiveness of normal CBA/N xid mice. Based on these results, we argue that a reason why T15+ antibodies are not normally made by CBA/N xid animals is because T15+ genes are not utilized or, as with any T15+ precursors present, selected for in these animals, in contrast to normal mice where the Lyb-5 or CD5 cells (which are absent in CBA/N xid animals) are known to be specially endowed to make such antibodies.

Effects of protein kinase modulators on transferrin receptor expression in human leukaemic HL-60 cells.

The mRNA of transferrin receptor (TfR) is constitutively expressed in proliferating human leukaemic HL-60 cells. Treatment of HL-60 cells with phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, or dibutyryl-cyclic AMP (dbcAMP), a protein kinase A (PKA) activator, resulted in a 90% decrease in the level of TfR mRNA. Inhibition of TfR mRNA expression induced by 10 nM PMA and 100 microM dbcAMP was abolished by prior incubation of cells with 0.1-1.0 microM GF109203X, a PKC-specific inhibitor, and 1-10 microM H-89, a PKA-specific inhibitor, respectively. The blocking effects of GF109203X and H-89 were dose-dependent and complete at the highest concentrations of the inhibitors used. Although treatment of cells with GF109203X or H-89 alone did not alter the constitutive expression of TfR mRNA, incubation of cells with 30-100 nM staurosporine, a wide-spectrum protein kinase inhibitor, resulted in suppression of the constitutive expression of TfR mRNA in a dose-dependent manner. These results suggest that (i) the down-regulation of TfR mRNA expression during the differentiation of HL-60 cells can be mediated by activation of either PKC or PKA; (ii) the constitutive expression of TfR mRNA in proliferating HL-60 cells is staurosporine-sensitive and is probably maintained by protein kinase(s) other than PKC and PKA.

Dual effects of tetrandrine on cytosolic calcium in human leukaemic HL-60 cells: intracellular calcium release and calcium entry blockade.

1. Tetrandrine (TET, a Ca2+ antagonist of Chinese herbal origin) and thapsigargin (TSG, an endoplasmic reticulum Ca2+ pump inhibitor) concentration-dependently mobilized Ca2+ from intracellular stores of HL-60 cells, with EC50 values of 20 microM and 0.8 nM, respectively. After intracellular Ca2+ release by 30 nM TSG, there was no more discharge of Ca2+ by TET (100 microM), and vice versa. 2. Pretreatments with 100 nM rauwolscine (alpha 2-adrenoceptor antagonist), 100 nM prazosin (alpha 1-adrenoceptor antagonist), 10 nM phorbol myristate acetate (PMA, a protein kinase C activator) or 100 nM staurosporine (a protein kinase C inhibitor) had no effect on 100 microM TET-induced intracellular Ca2+ release. 3. After intracellular Ca2+ release by 30 nM TSG in Ca(2+)-free medium, readmission of Ca2+ caused a substantial and sustained extracellular Ca2+ entry. The latter was almost completely inhibited by 100 microM TET (IC50 of 20 microM) added just before Ca2+ readmission. In Ca(2+)-containing medium, 30 nM TSG caused a sustained phase of cytosolic Ca2+ elevation, which could be abolished by 100 microM TET. TET was also demonstrated to retard basal entry of extracellular Mn2+ and completely inhibit TSG-stimulated extracellular Mn2+ entry. 4. TSG-induced extracellular Ca2+ entry was insensitive to the L-type Ca2+ channel blocker, nifedipine (1 microM), but was completely inhibited by the non-selective Ca2+ channel blocker La3+ (300 microM). Depolarization with 100 mM KCl did not raise the cytosolic Ca2+ level. 5. These data suggest that (a) TET and TSG mobilized the same Ca2+ pool and TET-induced intracellular Ca2+ release was independent of protein kinase C activity and ox-adrenoceptor activation,and (b) TET blocked the voltage-insensitive Ca2+ entry pathway activated by TSG. These dual effects on HL-60 cells were also observed with hernandezine (HER), a TET-like compound and in another cell type, murine B lymphoma M12.4 cells.

Differential effects of phorbol ester on apoptosis in HL-60 promyelocytic leukemia cells.

The role of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) in apoptosis of HL-60 cells was investigated. PMA inhibited DNA fragmentation induced by thapsigargin (TG) and 4-bromo-calcium ionophore (Br-A23187). The inhibitory effect of PMA was concentration-related and was abolished by a specific PKC inhibitor, bisindolylmaleimide (GF109203X. In addition TG-induced apoptosis was decreased in cells in which PKC activity was down-regulated by long-term pretreatment with PMA. These results indicate that PKC activation by PMA inhibits HL-60 cell apoptosis induced by TG and Br-A23187, and that this inhibition is not influenced by the down-regulation of PKC. However, PMA did not inhibit DNA fragmentation induced by 1-beta-D-arabinofuranosylcytosine (Ara-C) and cycloheximide. PMA suppressed TG- or Br-A23187. Our results indicate that PKC participates in the regulation of apoptosis only by some pathways. Down-regulation of PKC is not responsible for the diverse effects of PKC activators on apoptosis. The effect of a PKC modulator on apoptosis is dependent upon interaction with individual apoptotic stimulus.

Dual effects of SK&F 96365 in human leukemic HL-60 cells. Inhibition of calcium entry and activation of a novel cation influx pathway.

The effects of the receptor-mediated Ca2+ entry blocker, SK&F 96365 on thapsigargin (TSG)-induced Ca2+ entry in fura-2-loaded HL-60 cells were studied. After Ca2+ release induced by 30 nM TSG, readmission of Ca2+ resulted in a sustained Ca2+ entry, which could be partially inhibited by 1-3 microM SK&F 96365. Surprisingly, SK&F 96365 at 30-100 microM, instead of causing a stronger inhibition, actually promoted Ca2+ entry. Furthermore, at 16-100 microM, this drug released intracellular Ca2+ on its own and induced Ca2+ entry upon readmission of Ca2+. This SK&F 96365-activated Ca2+ entry pathway was insensitive to nifedipine and, interestingly, accessible to Ni2+ and La3+. However, SK&F 96365 (30 microM) almost completely blocked (basal) Mn2+ entry and only caused 4.4% of the cells to be stained with trypan blue, strongly suggesting that the SK&F 96365-activated cation entry was not due to damage nor to a very nonselective permeabilization of the plasma membrane. These data indicate that low concentrations of SK&F 96365 inhibited Ca2+ entry and higher concentrations activated a novel cation entry pathway. Because these 2 opposing effects overlapped at an intermediate concentration (16 microM), which is within the range commonly used to block Ca2+ entry, cautious use of this Ca2+ antagonist appears to be warranted.

Spontaneous Mn2+ entry is specifically inhibited by calyculin A in human leukemic HL-60 cells.

The effects of calyculin A and other agents which enhance protein Ser/Thr phosphorylation, on the cytosolic free Ca2+ concentration ([Ca2+]i) and spontaneous Mn2+ entry were investigated in fura-2-loaded human leukemic HL-60 cells. Calyculin A (30 nM), a specific inhibitor of protein Ser/Thr phosphatase (PP) 1 and 2A, significantly decreased [Ca2+]i. By contrast, another structurally unrelated inhibitor of PP1 and 2A, okadaic acid (1 microM), caused a slight elevation in [Ca2+]i. Forskolin (30 microM), which could enhance protein kinase A activity by raising cAMP concentration, also caused a rise in [Ca2+]i. Phorbol myristate acetate (PMA, 300 nM), an activator of protein kinase C, did not have a significant effect on [Ca2+]i. Spontaneous entry of Mn2+ (a surrogate ion for Ca2+) was strongly inhibited by calyculin A, but not okadaic acid, forskolin or phorbol myristate acetate. Such inhibition was not significantly affected by staurosporine (300 nM), a non-specific inhibitor of protein Ser/Thr kinases. Our results suggest that calyculin A inhibited a plasmalemmal leak pathway to Mn2+ (and Ca2+), probably leading to a decrease in [Ca2+]i. Inhibition of spontaneous Mn2+ entry by calyculin A may depend on a specific protein phosphorylation pattern induced by staurosporine-insensitive protein kinase(s).

Seminal plasma transferrin and seminiferous tubular dysfunction.

Seminal plasma transferrin levels were assayed in 158 random semen samples collected from normospermic, oligospermic, and azoospermic men, and their relationships with seminal characteristics, sperm fertilizing capacity as assessed by the zona-free hamster ova penetration assay, and serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were evaluated. The concentrations of seminal plasma transferrin in azoospermic, oligospermic and postvasectomy samples were significantly lower than those in normospermic samples. Seminal plasma transferrin concentrations were similar in azoospermia due to obstruction of the reproductive tract or damage to the germinal epithelium. No significant difference in seminal plasma transferrin concentrations was observed in the groups of subjects with normal and elevated FSH or normal and elevated LH. A positive correlation was observed between seminal plasma transferrin concentration and sperm density and between total semen transferrin content and the total number of sperm in the ejaculate. There were no significant correlations between seminal plasma transferrin concentration and sperm motility, percent normal sperm, sperm fertilizing capacity, and serum FSH or LH concentration. The results indicate that seminal plasma transferrin is not a useful marker for Sertoli cell or seminiferous tubular dysfunction. In addition, it is doubtful that measurement of seminal plasma transferrin will yield additional information regarding the fertility potential of semen samples.

Studies on the forms of iron-transferrin released from rabbit reticulocytes.

Measurement of the distribution of the four species of transferrin, viz, apotransferrin, diferric transferrin and the two monoferric transferrin, before and after incubation of iron-rich rabbit transferrin with rabbit reticulocytes showed that not all transferrin released from the cells were in the form of apotransferrin. Instead, a mixture of all four species of the protein was released with apotransferrin and C-terminal monoferric transferrin being the major fractions. The buffer solution containing 125I-labelled transferrin showed a continuous gain in percentages in apotransferrin and C-terminal monoferric transferrin after each incubation with reticulocytes. The N-terminal monoferric transferrin, however, remained unchanged suggesting that in the process of transferrin uptake by cells, the diferric transferrin releases its iron from the acid-labile site at N-domain first before the other iron from the acid-stable site.

Roles of calcium in the regulation of apoptosis in HL-60 promyelocytic leukemia cells.

Increase in intracellular calcium concentrations ([Ca2+]i) is critical for the initiation of apoptosis in cells such as thymocytes and in other cells, calcium chelators may promote apoptosis. However, calcium modulators, such as calcium ionophore 4-bromo-calcium ionophore (Br-A23187) and thapsigargin (TG), induce apoptosis in different cells, including HL-60 cells in which the induction of apoptosis seems a calcium-independent process. These observations imply that the disturbance of calcium homeostasis is probably the most important factor in the regulation of apoptosis. In this article, reagents with different potencies of modulating calcium homeostasis were used to study the possible role of [Ca2+]i and the status of intracellular calcium stores in the causation of HL-60 cell apoptosis. We found that an increase in [Ca2+]i alone did not result in apoptosis, while the depletion of TG-sensitive calcium stores in the endoplasmic reticulum was closely related with the induction of apoptosis. In HL-60 cells, extracellular and intracellular calcium chelators promoted apoptosis. Calmodulin antagonist did not attenuate apoptosis induced by other reagents. Our results suggest that the depletion of Ca2+ stores is an important mean to modulate calcium homeostasis and that the mobilization of calcium (Ca2+) from intracellular stores, rather than an increase in [Ca2+]i, provides the signal for the induction of apoptosis in HL-60 cells.

Lactation increases prolactin receptor expression in spleen and thymus of rats.

Prolactin receptors (PRL-R) are widely expressed on cells of the immune system. During lactation, the increase in serum PRL levels may modify the gene expression of these receptors. Specific PRL binding sites and the expressions of both PRL-R-L and PRL-R-S forms in thymus and spleen of nulliparous control, 10-day postpartum lactating, and 10-day postpartum nonlactating rats were studied. A semi-quantitative RT-PCR technique was used to detect the PRL-R gene transcript in the tissues. Our results showed that specific PRL binding sites and PRL-R-L mRNA and PRL-R-S mRNA were present in the lymphoid tissues with the PRL-R-L mRNA predominant. Lactation markedly increased specific binding sites and PRL-R-L mRNA in both spleen and thymus and only PRL-R-S in spleen. No differences between nulliparous control and postpartum nonlactating rats were observed in any of the parameters measured. The parallel increase in specific PRL binding sites and PRL-R-L mRNA suggests that lactation up-regulates PRL-R expression in lymphoid tissues and may be beneficial to the maternal immune system.

bcl-2 protects HL-60 cells from apoptosis by stabilizing their intracellular calcium pools.

bcl-2 has been shown to enhance cell survival by inhibiting apoptosis induced under different circumstances. In this study we investigated the effects of bcl-2 overexpression on the homeostasis of subcellular organelles such as ER and mitochondria. In our study, HL-60/bcl-2 and control HL-60/neo cells were obtained by transfection of bcl-2 cDNA or the neomycin-resistant gene, respectively. Apoptosis was evaluated by both DNA fragmentation and flow cytometry qualitatively and quantitatively, and the intracellular calcium by Fura-2/AM. Thapsigargin (TG), a highly specific inhibitor of the ER-associated Ca2+ pump, and Br-A23187, a calcium ionophore, were used in this study. Our results showed that overexpression of bcl-2 significantly blocked TG- and Br-A23187-induced apoptosis in calcium containing buffer. Measurement of intracellular calcium showed that bcl-2 overexpression could reduce sustained elevation of cytosolic Ca2+ induced by these agents. However, in calcium-free medium, bcl-2 overexpression maintained Ca2+ uptake in ER of both TG- and Br-A23187-treated cells. Moreover, the depletion of Ca2+ by EGTA enhanced TG- and Br-A23187-induced apoptosis, and reduced the anti-apoptotic action of bcl-2, suggesting that cytosolic Ca2+ elevation may be required for optimal ER pool refilling. These findings suggest that bcl-2 facilitates and maintains the replenishment of Ca2+ in intracellular stores and, as a result, influences the intracellular calcium, thus protecting the cells from death. In addition, there were no cytochrome c release from mitochondria into the cytosol in TG- and Br-A23187- induced apoptosis, suggesting that cytochrome c release is not a universal phenomenon in the apoptotic process.

Redistribution of cytochrome c is not an essential requirement in C2-ceramide induced apoptosis in HL-60 cells.

bcl-2 has been shown to enhance cell survival by inhibiting apoptosis. The present study investigates the potential role of bcl-2 on apoptosis in HL-60 cells induced by different agents. HL-60/bcl-2 and control HL-60/neo cells were obtained by transfection of bcl-2 cDNA or the neomycin-resistant gene, respectively. Staurosporine (STS) promoted DNA fragmentation dose-dependently in the 6 h exposure assay while C2-ceramide was relatively slow in the induction of apoptosis (approximately 40% after 24 h) and required higher concentrations (> 20 microM). Caspases inhibitors, Ac-YVAD-cmk (100 microM) and zVAD-fmk (20 microM) had no effect on DNA fragmentation themselves. However, they blocked C2-ceramide-induced caspase-3 cleavage and apoptosis, but not the release of cytochrome c from the mitochondria. In addition, we found that both Ac-YVAD-cmk and zVAD-fmk failed to protect STS-induced apoptosis in HL-60 cells. Overexpression of bcl-2 inhibited STS and C2-ceramide induced cytochrome c redistribution, caspase-3 activation and apoptosis. These results suggest a protective role of bcl-2 in the regulation of apoptosis and cytochrome c release is unlikely to be involved in the final common pathway in apoptosis.

Telomerase activity is not related to apoptosis in leukemic cell lines.

Any deregulation of apoptosis or an escape from cellular senescence will drive the cells to neoplasia. It remains unclear whether there is a direct linkage between apoptosis and telomerase activity particularly in transformed cell lines. In the present study, we investigated the telomerase activities in three leukemic cell lines (HL-60, U937 and K562) after treating these cells with various doses of antitumor drugs, puromycin or actinomycin D (ActD). Our results showed that HL-60 cells underwent apoptosis rapidly when treated with either 20 microg/ml of puromycin or 5 microg/ml of Act D with more than 60% of the cells becoming apoptotic at 6 hrs and almost 100% at 12 hrs. But telomerase activity analyzed by TRAP assay in these apoptotic cells remained unchanged as compared with the untreated control cells suggesting that whether the cells were apoptotic or not, it had no effect on telomerase activity. However, if lower dosages of the drugs were used, that is, 0.5-1.5 microg/ml of puromycin or 0.01-0.5 microg/ml of Act D, a decrease in telomerase activity was observed at 24-48 hrs, and was completely undetectable at 72 hrs. This decrease in telomerase activity was dose- and time-dependent. The suppression of telomerase activity by low doses of these two drugs is probably due to the inhibitory effect of the drugs on protein translation or RNA transcription rather than direct inhibition of the telomerase activity. Flow cytometry analysis of the cell cycle of the drug-treated cells showed that these drugs unselectively induced apoptosis at all phases of the cell cycle and was unrelated to the changes in telomerase activity. Similar results were observed in U937 and K562 cells except that K562 cells underwent apoptosis more slowly than the former two cell lines.

The effect of pH on the kinetics of iron release from diferric ovotransferrin induced by pyrophosphate.

Pyrophosphate-induced iron release from diferric ovotransferrin follows biphasic kinetics in the pH range from 6.6 to 8.6 except at pH 8.0 where the kinetics become monophasic. The rates of formation of the four molecular species, Fe2OT, FeOTN, FeOTC, and ApoOT, were studied by urea gel electrophoresis and the four microscopic rate constants were calculated at various pH values. Below pH 8.0, these intrinsic rate constants for iron release from Fe2OT follow the order k2N greater than k1N greater than k2C greater than k1C. Each constant diminishes almost proportionally with an increase in pH with the faster rate constants being affected more by the fall in hydrogen ions than the slower ones. Around pH 8.0 the four rates are approximately equal, resulting in monophasic kinetics. However, the rate constants from the C-site become faster than that from the N-site at pH above 8.2. At low pH, there is a marked preference for iron to be released from the N-site rather than from the C-site and such preference becomes less distinct as pH increases. A rather weak positive cooperativity between the two sites is demonstrated in pH between 6.8 and 7.8. The ligand responsible for the transition from biphasic to monophasic kinetics at pH 8.0 is not known. It is possible that there are different anions such as [CO3(2-)] and [HCO3-] at the two iron-binding sites, which might explain the preferential rates of iron release from these sites during protonation.

Kinetics of pyrophosphate induced iron release from diferric ovotransferrin.

The kinetics of pyrophosphate-induced iron release from diferric ovotransferrin were studied spectrophotometrically at 37 degrees C in 0.1 M HEPES, pH 7.0. At high pyrophosphate concentrations, the kinetics are biphasic, indicating that the rates of iron release from the two, presumably noninteracting iron-binding sites of ovotransferrin are different. The pseudo-first-order rate constants for iron release from both the fast and slow sites exhibit a hyperbolic dependence on pyrophosphate concentrations. The data suggest that pyrophosphate forms complexes with the two iron-binding sites of ovotransferrin prior to iron removal. The stability constants of the complex formed with the fast site (Keqf) and slow site (Keqs) are 8.3 M-1 and 40.4 M-1, respectively. The first-order rate constants for the dissociation of ferric-pyrophosphate from the fast site (k2f) and the slow site (k2s) are 0.062 and 0.0044 min-1, respectively. Results from urea gel electrophoresis studies suggest that iron is released at a much faster rate from the N-terminal binding site of ovotransferrin. At high pyrophosphate concentration, only C-monoferric-ovotransferrin is detected during the course of iron release. At low pyrophosphate concentration, however, a detectable amount of N-monoferric-ovotransferrin is accumulated. This result is consistent with the kinetic finding that the site with a higher k2 (0.062 min-1) has a lower affinity toward pyrophosphate (Keq = 8.3 M-1) whereas the site with a lower k2 (0.0044 min-1) has a higher affinity for pyrophosphate (Keq = 40.4 M-1).

Will latent iron deficiency affect the mitochondrial iron-containing enzymes?

Since information pertinent to the effect of prelatent or latent iron deficiency on tissue iron is scare, the present study was aimed at producing this stage of iron deficiency in rats by phlebotomy and to determine whether the mitochondrial iron-containing enzymes, succinate dehydrogenase (SDH) and glycerophosphate dehydrogenase (GPDH) were affected. These phlebotomized rats showed a subclinical aneamic picture in the blood together with reduced plasma iron and storage iron in the spleen and liver, but an elevated plasma total iron-binding capacity (TIBC). Under this latent iron deficient state, the SHD in the heart and the skeletal muscle with mixed-fibre types (gastrocnemius and plantaris) but not the red (soleus) and white fibres (vastus lateralis) showed reduced activities. No significant changes in GPDH activities were found in these organs. This finding is consistent with our early report (Quisumbing et al., 1985) that even in mild iron deficiency, some loss of mitochondrial functions could have occurred and this could affect the muscular endurance. SDH was more affected by latent iron deficiency than GPDH.