Influence of subunit composition of rabbit liver ferritin on its clearance from plasma

1. Purified rabbit liver ferritin was separated into fractions composed of 87% light molecular weight, and 72% high molecular weight subunits. 2. Radiolabeled ferritin fractions, injected intravenously into rabbits, showed no differences in clearance kinetics. 3. Differences found in ferritin clearance from plasma do not depend on the subunit composition of different molecular species.     

Turnover of prolyl hydroxylase and an immunologically related protein in rabbit tissue

The turnover rates of prolyl hydroxylase and immunologically related (cross reacting) protein were examined using labeled leucine as precursor or by measuring the decay of elevated prolyl hydroxylase and immunologically cross-reacting protein back to basal levels. Prolyl hydroxylase and immunologically cross-reacting protein were purified from neonatal rabbit skin at various times following the administration of [³H]leucine. Prolyl hydroxylase was purified by affinity chromatography. Immunologically cross-reacting protein was purified by antibody precipitation from the dialyzed 70% (NH₄)SO₄ supernatants and subsequent electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide slab gels. The radioactivity of the species isolated, which corresponded to the two major subunits of prolyl hydroxylase, was used in the turnover studies of immunologically cross-reacting protein. The peak incorporation of label into prolyl hydroxylase was found to be 12 h while for immunologically cross-reacting protein this occured within 2 h. The loss of radioactivity from these protein pools denotes an apparent $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for prolyl hydroxylase of 73 h and a $\text{1}\text{2}$ for immunologically cross-reacting protein of 53 h. From the specific activity of free skin leucine pools, the effect of reutilization could be corrected and a true $\text{t}\text{1}\text{2}$ for prolyl hydroxylase of 45 h was determined. The $\text{t}\text{1}\text{2}$ values of these proteins were determined by a second method in which prolyl hydroxylase and immunologically cross-reacting protein in the aorta and liver of adult male rabbits were elevated by daily epinephrine-thyroxine treatment for 12 days. The decline of prolyl hydroxylase and immunologically cross-reacting protein with termination of treatment in the aorta denotes values of 42 h for enzyme and 53 h for immunologically cross-reacting protein. Calculated enzyme κ_d values, by both methods, indicate that breakdown of enzyme does not account for tissue immunologically cross-reacting protein.     

Effects of cholestyramine on receptor-mediated plasma clearance and tissue uptake of human low density lipoproteins in the rabbit

This study examines the effects of cholestyramine (2 g/day) on the plasma clearance and tissue uptake of human low density lipoprotein (LDL) in rabbits. 1,2-Cyclohexanedione modification of human LDL abolishes its recognition by high affinity cell membrane receptors in vitro and delays its plasma clearance in comparison to native LDL. Consequently, the difference between the fractional rates of catabolism of simultaneously injected native and cyclohexanedione-treated LDL is an index of in vivo receptor-mediated clearance of the lipoprotein. When human 125I-LDL and 131I-cyclohexanedione-treated LDL were injected into rabbits, 44% of the lipoprotein was cleared from the plasma by the receptor mechanism. Various tissues were removed from the animals at the end of the turnover study and their relative uptakes of 125I native and 131I-cyclohexanedione-treated LDL were measured. All exhibited receptor activity to some extent, incorporating more native than cyclohexanedione-modified LDL. The greatest receptor activity per g of tissue was found in lymph nodes, spleen, and liver and, in terms of whole organ uptake, the liver played a major role in LDL catabolism. Treatment of the rabbits with cholestyramine lowered the circulating LDL cholesterol level by promoting its clearance (120%, p < 0.001) via the receptor pathway. This was associated with a virtual doubling of receptor-mediated incorporation of the lipoprotein into the liver. These results suggest that the drain which cholestyramine induces in the hepatic cholesterol pool promotes LDL receptor activity in this organ and thereby lowers the level of circulating LDL.     

Turnover of the plasma membrane proteins of hepatoma tissue culture cells.

The turnover of the plasma membrane proteins of hepatoma tissue culture cells was examined by three different methods--loss of polypeptides labeled in situ by lactoperoxidase-catalyzed iodination, loss of membrane polypeptides labeled with amino acid precursors, and loss from the membrane of fucose-labeled polypeptides. In both logarithmically growing and density-inhibited cells the proteins of the membrane are degraded with a half-life of about 100 hours. This is longer than the half-life of total cell protein, 50 to 60 hours, and longer than the doubling time of the cells, about 30 hours. Similar values for the rate of degradation of the membrane proteins were obtained by each of the three techniques. The same fucose-labeled polypeptides are present in the microsomal and the plasma membrane fractions of hepatoma tissue culture cells as analyzed by electrophoresis in dodecyl sulfate-acrylamide gels. But the fucose-labeled polypeptides were lost from the microsomal fraction at a faster rate than from the plasma membrane. Autoradiographic and double labeling techniques using 125I and 131I, or [3H]leucine and [14C]leucine were used to measure the relative rates of degradation of the proteins in the plasma membrane. All of the leucine-labeled polypeptides and the iodinated polypeptides had similar rates of degradation. These results support a model for the biogenesis of the plasma membrane in which the proteins are incorporated and removed in large structural units.     

The formation of ferritin from apoferritin. Kinetics and mechanism of iron uptake

Ferritin has a high capacity as an iron store, incorporating some 4500 iron atoms as a microcrystalline ferric oxide hydrate. Starting from apoferritin, or ferritin of low iron content, Fe²⁺ and an oxidizing agent, the uptake of iron can be recorded spectrophotometrically. Progress curves were obtained and the reconstituted ferritin was shown by several physical methods to be similar to natural ferritin. The progress curves of iron uptake by apoferritin are sigmoidal; those for ferritins of low iron content are hyperbolic. The rate of iron uptake is dependent on the amount of iron already present in the molecule. The distribution of iron contents among reconstituted ferritin molecules is inhomogeneous. These findings are interpreted in terms of a crystal growth model. The surface area of the crystallites forming inside the protein increases until the molecule is half full, and then declines. This surface controls the rate at which new material is deposited. The experimental results can best be accounted for by a two-stage mechanism, an initial slow `nucleation' stage, which is apparently zero order with respect to [Fe<sup>2+</sup>], followed by a more rapid `growth' stage. The rate of Fe²⁺ oxidation is increased in the presence of apoferritin as compared with controls. Ferritin can therefore be regarded as an enzyme to which the product remains firmly attached. The protein appears to increase the rate of `nucleation'. The apparent zero order of this stage suggests the presence of binding sites on the protein, which are saturated with respect to Fe²⁺. These sites are presumed also to be oxidation sites. The oxidation and subsequent formation of the ferric oxide hydrate may proceed according to one of three alternative models.     

Calmodulin regulation of the ATP-dependent calcium uptake by inverted vesicles prepared from rabbit synaptosomal plasma membranes

Calmodulin has been shown to activate the ATP-dependent Ca2+ uptake in inside-out vesicles which have been prepared from rabbit synaptosomal plasma membranes by the methodology of Gill et al. (Gill, D.L., Grollman, E.F. and Kohn, L.D. (1981) J. Biol. Chem. 256, 184-192). Following extensive washings of these membranes with EGTA/EDTA solutions, the Ca2+ uptake activity demonstrated an affinity for calmodulin of 30 nM and an affinity for Ca2+ of 2 microM. The activity was completely inhibited by the anticalmodulin compound R24571 (Ki congruent to 8 microM). The molecular weight of the ATPase molecule, revealed by a combination of the [125I]calmodulin overlay technique and [32P]phosphoenzyme electrophoresis, was 145 000. The overlay technique also revealed that the mechanism of activation is via a direct binding of calmodulin to the pump molecule.     

Selective uptake of cholesteryl ester from high density lipoproteins by plasma membranes of adipose tissue

The interaction between high density lipoproteins (HDL) and adipose tissue is an important pathway for cholesterol and cholesteryl ester flux. In intact fat cells, a disproportionately greater net uptake of cholesteryl ester occurs subsequent to lipoprotein binding than would have been predicted from a consideration of holoparticle uptake alone. To characterize the early events in this process, cholesteryl hexadecyl ether, a nonmetabolizable, accumulative marker of cholesteryl ester, was incorporated into canine HDL2, and its uptake by omental adipocyte plasma membranes was measured in relation to the binding of HDL2, which in this animal species is enriched in apolipoprotein A-I and free of apolipoprotein E. The dose-response profile for HDL2 binding was consistent with a single lipoprotein binding site at all concentrations of HDL2, whereas uptake of cholesteryl ester from HDL2 was biphasic, suggesting a high affinity site at low HDL2 concentrations and a low affinity site at high lipoprotein concentrations. Pronase treatment stimulated binding twofold and this was accompanied by a parallel twofold stimulation of cholesteryl ester uptake. EDTA, on the other hand, reduced binding and uptake of cholesteryl ester by 20%, indicating partial dependence upon divalent cations. The proportion of HDL2 cholesteryl ester accumulated by plasma membranes relative to HDL2 protein bound was not altered by either pronase or EDTA, despite the fact that these agents had opposite effects upon binding. In dissociation studies, a portion of membrane-associated HDL2 did not equilibrate with exogenous HDL2 and a greater proportion of the cholesteryl ester failed to dissociate. A stepwise mechanism for cholesteryl ester uptake, involving (i) saturable, high affinity HDL2 binding to cell surface sites, (ii) vectoral, HDL2 concentration-dependent delivery of cholesteryl ester to the membrane, and (iii) cholesteryl ester sequestration into a nonexchangeable membrane compartment, appears to be independent of metabolic energy or cell processing.     

Purification of a specific repressor of ferritin mRNA translation from rabbit liver

The synthesis of ferritin is regulated at the translation level in coordination with iron availability. Under conditions of low iron, translation of ferritin mRNA is repressed and the majority of ferritin mRNA is non-polysomal. Upon an increase in iron, translation of ferritin mRNA is derepressed resulting in as much as a 50-100-fold increase in the rate of ferritin synthesis. This regulation is mediated at least in part by a specific translational repressor which binds to a conserved sequence, the iron responsive element, located in the 5'-untranslated region of ferritin mRNA. In this communication we report the purification of such a repressor from rabbit liver. This repressor, which we call the "ferritin repressor protein," has an apparent molecular mass of 90 kDa when analyzed by gel filtration chromatography. It inhibits translation of ferritin mRNA in a highly specific fashion when added to a wheat germ lysate programmed with liver poly(A+) mRNA. In addition, it binds specifically to sequences contained within the first 92 nucleotides of ferritin mRNA, most likely the iron responsive element. Analysis of highly purified repressor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it is composed primarily of a single polypeptide of approximately 90 kDa. Elution of this 90-kDa polypeptide from a sodium dodecyl sulfate gel followed by renaturation and analysis for repressor activity shows that it both binds to the 5'-untranslated region of ferritin mRNA and represses its translation in vitro.     

A long term survey of plasma and tissue ferritin in mice fed on iron deficient diets

1. Changes in plasma and tissue ferritin concentrations were surveyed in mice fed on iron deficient diets (Fe, 2.4 micrograms/g) from the age of 8 to 30 weeks by an enzyme-immunoassay for mouse ferritin. Values were compared with those in mice on control diets (Fe, 110 micrograms/g) and mice with blood loss. 2. In mice on iron deficient diets, while plasma ferritin remained at normal level, ferritin in the spleen and kidney decreased, but that in the liver increased as much as twice of that in the control. 3. Mice with blood loss revealed a drastic reduction of ferritin in every tissue and plasma examined.     

Serum iron, serum ferritin and tissue ferritin during development in ducks

Serum ferritin and tissue ferritin from kidney, heart, small intestine, spleen and liver from ducks during development from 16 to 112 days of age were measured by radioimmunoassay using rabbit anti-duck liver ferritin antibodies and goat second antibody. Serum iron concentration and tissue ferritin iron content are given. Serum ferritin concentration, tissue ferritin and ferritin iron content increase gradually during development. The decrease in all these parameters at 8 weeks of age might be due to molting.     

Haem binding to ferritin and possible mechanisms of physiological iron uptake and release by ferritin.

Haem binding to horse spleen ferritin and Pseudomonas aeruginosa bacterioferritin has been studied by spectroscopic methods. A maximum of 16 haems per ferritin molecule, and 24 haems per bacterioferritin molecule, has been shown to bind. The influence of the bound haem on the rate of reductive iron release has been investigated. With a range of reductants and in the absence of haem the rate of release varied with the reductant, but in the presence of haem the rate was both independent of the reductant and faster than with any of the reductants alone. This indicates the rate-limiting step for iron release in the absence of haem was electron-transfer across the protein shell. Based on the results obtained with the in vitro assay system and from a consideration of data currently in the literature, plausible schemes for ferritin and bacterioferritin iron uptake and release are described.     

Effects of dietary factors on iron uptake from ferritin by Caco-2 cells

Biofortification of staple foods with iron (Fe) in the form of ferritin (Ft) is now possible, both by conventional plant breeding methods and transgenic approaches. Ft-Fe from plants and animals is absorbed well (25-30%) by human subjects, but little is known about dietary factors affecting its absorption. We used human intestinal Caco-2 cells and compared Fe absorption from animal Ft and FeSO4 to determine the effects of inhibitors and enhancers, such as phytic acid, ascorbic acid, tannic acid, calcium and heme. When postconfluent cells were coincubated with 59Fe-labeled (1 microM) FeSO4 and dietary factors, at different molar ratios of dietary factor to Fe (phytic acid:Fe, 10:1; ascorbic acid:Fe, 50:1; tannic acid:Fe, 50:1; calcium:Fe, 10:1 and hemin:Fe, 10:1), all inhibited uptake from FeSO4, except ascorbate, confirming earlier studies. In contrast, these dietary factors had little or no effect on Fe uptake from undigested Ft or Ft digested in vitro at pH 4, except tannins. However, results after in vitro digestion of Ft at pH 2 were similar to those obtained for FeSO4. These results suggest that Fe uptake occurs from both undigested as well as digested Ft but, possibly, via different mechanisms. The Fe-Ft stability shown here could minimize Fe-induced oxidation of Fe-supplemented food products.     

Dynamic equilibria in iron uptake and release by ferritin

The function of ferritins is to store and release ferrous iron. During oxidative iron uptake, ferritin tends to lower Fe²⁺ concentration, thus competing with Fenton reactions and limiting hydroxy radical generation. When ferritin functions as a releasing iron agent, the oxidative damage is stimulated. The antioxidant versus pro-oxidant functions of ferritin are studied here in the presence of Fe²⁺, oxygen and reducing agents. The Fe²⁺-dependent radical damage is measured using supercoiled DNA as a target molecule. The relaxation of supercoiled DNA is quantitatively correlated to the concentration of exogenous Fe²⁺, providing an indirect assay for free Fe²⁺. After addition of ferrous iron to ferritin, Fe²⁺ is actively taken up and asymptotically reaches a stable concentration of 1–5 m. Comparable equilibrium concentrations are found with plant or horse spleen ferritins, or their apoferritins. After addition of ascorbate, iron release is observed using ferrozine as an iron scavenger. Rates of iron release are dependent on ascorbate concentration. They are about 10 times larger with pea ferritin than with horse ferritin. In the absence of ferrozine, the reaction of ascorbate with ferritins produces a wave of radical damage; its amplitude increases with increased ascorbate concentrations with plant ferritin; the damage is weaker with horse ferritin and less dependent on ascorbate concentrations.     

Influence of heat treatment on rabbit liver ferritin

In order to enhance the stability of beta-galactosidase, we conjugated the enzyme with dextran T-10 (Mr approx. 10 000). The conjugate contained 9-10 mol dextran/mol protein (beta-galactosidase, Mr 68 000), and the specific activity retained after conjugation was 90 +/- 4% (n = 3) of the initial activity. Uptake and degradation of native and conjugated beta-galactosidase in isolated hepatocytes and nonparenchymal liver cells was studied. There was a marked increase in stability against degradation in both cell types when beta-galactosidase was conjugated with Dextran. The degradation of dextran-conjugated enzyme was reduced by 35% in hepatocytes and by 43% in nonparenchymal cells, after 80 and 40 min, respectively, as compared with the free enzyme. However, there was insignificant difference between the uptake of native and conjugated enzyme into the liver cells. Upon intravenous infusion into rats, native and conjugated enzyme were cleared from plasma with only a slight difference in the clearance rate. The observed stability of dextran-conjugated beta-galactosidase towards cellular degradation was in accordance with the in vitro experiments. The conjugate showed marked thermal stability at 50 degrees C and enhanced resistance towards proteolysis by the broad specific protease subtilopeptidase A. This demonstrates that dextran conjugation may be used as a means of stabilizing lysosomal enzymes for therapeutic purposes.