Jayant G. Joshi

Ferritin: An expanded role in metabolic regulation

Synthesis of ferritin, a constitutive protein, is increased by iron. This protein is well recognized as a protein which detoxifies, stores and transports iron. The 24 subunits of ferritin assemble to form a protomer of Mr 480,000. This protein shell can sequester up to 4500 g atoms of iron as ferrichydroxyphosphate. Ferritin in vitro and in vivo binds other metal ions such as Cu, Zn, Cd, Pb, Be and Al. Next to Fe it binds large quantities of Be. Therefore, in vitro ferritin protects against and reverses the inhibition by Be of enzymes susceptible to this metal ion. Also, rats pretreated with Fe survive otherwise toxic levels of either pulmonary or intravenous exposure of Be. Liver ferritin from rats injected with Zn contains some of the injected metal ion. Incubation of such ferritin-zinc complex with zinc-requiring apoenzymes restores their activity. Fe(III) of ferritin is released only after its reduction to Fe(II) by a reductant. Incubation of phosphoglucomutase, a phosphoserine containing enzyme with ferritin and a reductant causes irreversible inactivation of the enzyme and removes 70% of its phosphate. Some other phosphoproteins are similarly inactivated but without the loss of the bound phosphate. Thus, uncontrolled release of iron from ferritin, in the presence of a reductant and oxygen can modify several biomolecules and can affect metabolic processes. A subclass of ferritin, acidic isoferritins, have been implicated in leukemia-associated inhibitory activity and has been suggested to inhibit production of Ia+ macrophage progenitors.

Ferritin: The role of aluminum in ferritin function

We previously showed that human brain ferritin (HBF) binds aluminum (Al) in vivo and in vitro and HBF isolated from Alzheimers brain had more Al bound compared to aged matched controls (7). To further understand the role ferritin may play in Al neurotoxicity, we have studied in vitro the effect of Al on the function of human ferritin isolated from Alzheimers (AD) and normal brain tissue, and compared the results with other mammalian ferritins. Al causes a concentration-dependent decrease in the initial rate of iron loading into apo-horse spleen and human brain ferritin and the rates were similar for ferritin isolated from both AD and normal brains. The rates of iron release of mammalian ferritins from different tissues were determined: horse spleen much greater than human liver greater than rat brain greater than human brain = rat liver ferritin. The rates of iron release of AD and normal human brain ferritin were similar and were unaffected by preloading with Al. Several mammalian ferritins were compared for their total iron uptake: horse spleen = human liver greater than human brain (normal) = human brain (AD) ferritin. In 20 mM HEPES (pH 6.0) buffer holoferritin is more resistant to precipitation by Al than apoferritin suggesting that holoferritin is a better chelator for nonferrous metal ions.

Metal-binding properties of phytoferritin and synthetic iron cores

The non-ferrous metal-binding properties of phytoferritin from Glycine max were investigated, using microfiltration to quantitate the binding of aluminum, beryllium, cadmium and zinc to the holoprotein. All of the metals tested were bound by the protein with similar affinities ( K d ≈ 10 −6 M), although the smaller metals (Al and Be) were bound in much higher numbers than the heavier Cd or Zn. Since apoferritin binds these metals poorly or not at all, we investigated the role of the iron core in metal binding by preparing synthetic iron cores (as models of the holoferritin core) and studied the binding of the non-ferrous metals to these cores. Our results indicate that these synthetic cores can bind other metals in a manner entirely analogous to holoferritin, particularly when inorganic phosphate has been previously incorporated into the core. The ferrous-hydroxy-oxide core was shown to have a high affinity for the phosphate anion ( K d ≈ 2.5·10 −9 M), and beryllium binding was dramatically reduced when phosphate anion was omitted. Thus, our data would implicate the phosphate anion in the iron core of ferritin as being responsible for the high amount of non-ferrous metal-binding seen in the ferritin molecule. These data are consistent with our previous work on mammalian ferritin [18], and indicate that ferritin is capable of binding, and perhaps detoxifying, deleterious cations.

Ferritin—A general metal detoxicant

Binding of nonferrous metal ions to ferritin was compared to that of the phosphate-free or phosphate containing synthetic iron cores. The Scatchard plots for the synthetic cores reveal a high affinity site for Cd, Zn, Be, and Al, with KD in the range 10−5–10−7M. Preloading the cores with phosphate increased the number of metal ions bound without altering the KD. The metal ions with smaller ionic radii (Be, Al) were bound in larger numbers than those with larger ionic radii (Cd, Zn).Ferritin isolated from soybean (Glycina max), horse spleen, and rat liver bound the metal ions in amounts larger than predicted from their iron core. Whereas the iron cores and their nonferrous metal ion complexes were insoluble, those in the protein shell remained in solution. Thus apoferritin precipitated with lower concentrations of aluminum than did holoferritin. Also, Al bound to apoferritin reduced the rate of iron loading into the protein.

Effect of long-term feeding of aluminium chloride on hexokinase and glucose-6-phosphate dehydrogenase in the brain

Rats were fed 100 microM AlCl3 for 1 year in their drinking water, then killed and their brains homogenized in 0.1 M Tris (pH 7.4). The 800 g supernatants were assayed for Al3+ and the activities of acetylcholine esterase (ACE), hexokinase and glucose-6-phosphate dehydrogenase (G6PDH). The concentrations of Al in the homogenates, as computed on the original brain for the control and Al fed group were 40 ng and 80 ng/g wet wt, respectively. The activity of ACE was the same in both groups but that of hexokinase and G6PDH in the Al-fed group was about 73% and 80%, respectively, of the control. Dialysis restored the G6PDH but increased the hexokinase of the control group 2-fold and that of Al-fed group 2.7-fold. Thus at this elevated level it was same in both groups. The contribution of Al from the undialysed homogenates during assay was too low to account for the inhibition. It is therefore suggested that a dialyzable inhibitor for hexokinase is normally present in the brain and that Al feeding increases its concentration to further inhibit the utilization of glucose.

Ferritin: protection of enzymatic activity against the inhibition by divalent metal ions in vitro

Ferritin binds a large quantity of Be2+ (Price D.J. and Joshi, J.G. J. Biol. Chem. 258 (1983) 10873) as well as other divalent metal ions. Therefore the ability of this protein to protect enzymes against or reverse the inhibition by metal ions was studied. Evidence presented here shows that the inhibition by Be2+ of the enzymes Na+K+ATPase, alkaline phosphatase and phosphoglucomutase is reversed by ferritin. Be2+ can be transferred reversibly between phosphoglucomutase and ferritin depending upon the relative concentrations of the 2 proteins. Ferritin also reactivated phosphoglucomutase inhibited by Zn2+, Cu2+, or Cd2+. Incubation of ferritin containing Be2+ with 4-10 fold molar excess of phosphoglucomutase (with respect to Be2+) removed 90% of the Be2+ from ferritin. The rates of inactivation of phosphoglucomutase by Be2+ donated by apoferritin or ferritin were identical. Based upon these observations it is suggested that Be2+ bound to the protein shell and to the iron core are in equilibrium with each other with the equilibrium favoring ferritin-Be2+ complex.

The human and bovine 14-3-3η protein mRNAs are highly conserved in both their translated and untranslated regions

14-3-3 proteins form a highly conserved protein family whose members have been shown to activate tyrosine and tryptophan hydroxylases, inhibit protein kinase C and possess phospholipase A2 activity in vitro. We have isolated and analyzed a 14-3-3 protein cDNA clone (H14-3-3) from a human fetal brain cDNA library and found it to possess a high level of sequence identity with the bovine 14-3-3 eta protein cDNA in both the translated and untranslated regions, suggesting the presence of cis-regulatory elements in the untranslated regions of these mRNAs. The proteins encoded by these two cDNAs are 98.4% identical. Two different sized RNA species, approx. 1.9 and 3.5 kb in size that are expressed in a variety of tissues hybridize with this cDNA. However, only the 1.9 kb RNA is detected in the fetal brain. Northern blot analysis of poly(A)+ RNA isolated from eight different human tissues shows that 14-3-3 protein mRNAs are expressed in many tissues in the body. In agreement with previous reports, the highest abundance of RNA hybridizing with this cDNA is seen in the brain.

Effects of chronic dietary aluminum on local cerebral glucose utilization in rats

Beginning at 4 weeks of age normal, male, Sprague-Dawley rats were reared on Purina Laboratory Chow and drinking water containing 100 microM AlCl3. After 2 years, local rates of cerebral glucose utilization were determined with the autoradiographic [14C] deoxyglucose method in the brain as a whole and in 25 brain regions in 6 treated rats and 4 age-matched controls. The results indicate that any effects of chronic aluminum in the diet on rates of cerebral glucose utilization are small. In the brain as a whole, the mean rate of glucose utilization in the aluminum-treated rats was 6% lower than that of the controls (p = 0.09). In 21 of the 25 brain regions examined mean rates of glucose utilization were generally lower in the aluminum-treated rats but in none of the region were the effects statistically significant.

Role of Aluminum and Iron in Brain Disorders

Aluminum and iron, the two most abundant elements in the earth’s crust, have very similar coordination chemistry1. Both form insoluble hydroxides at physiological pH. However, very early in evolution, living systems recognized the unique redox properties of iron and incorporated it in diverse biological reactions. Indeed, iron is essential to all forms of life. No such use has been discovered for aluminum and, until recently, it was considered harmless. Because all metal ions, including aluminum, are more soluble at acid pH, environmental insults such as acid rain have increased their bioavailability2,3. Living systems are yet to learn to cope with this new class of pollutants.

Insulin or increased concentration of zinc in liver, kidney or muscle does not alter the phosphoglucomutase activity in vivo

Injection of insulin into mice and rabbit has been reported to convert the latent form of phosphoglucomutase (E X Zn) to an active form (E X Mg) without any net increase in the protein associated with this enzyme (Hashimoto et al., Biochem. Biophys. Res. Commun., 27 (1967) 369 and Peck et al., J. Biol. Chem. 242 (1971) 1160). Because the injection of Cd in rats causes a 2-fold increase in Zn concentration in the liver, it appeared that such treatment in mice would increase the concentration of the latent enzyme and thereby enhance the insulin induced activation of the enzyme. The results however show that insulin or Cd treatment, alone or in combination do not alter the concentrations of the 2 forms of enzyme in liver, kidney or muscle of normal or fasted mice. In all instances the conversion of the latent enzyme to its activated form required preincubation with a chelating agent. Fractionation of the tissue homogenates showed that upon Cd injection the concentration of Zn in the fractions of Mr less and 1000 increased 6-fold in liver and 3-fold in kidney, that in muscle remained unchanged.