Ferritin: The role of aluminum in ferritin function

Neurobiologyof Aging, Vol. 12, pp. 413-418. ©Pergamon Press plc, 1991. Printed in the U.S.A.

0197-4580/91 $3.00 + .00

Ferritin: The Role of Aluminum in Ferritin Function 1'2 J A M E S T. F L E M I N G 3 A N D J A Y A N T G . J O S H I 4

Department of Biochemistry, The University of Tennessee, Knoxville, TN 37996-0840 R e c e i v e d 1 A u g u s t 1990; A c c e p t e d 12 A p r i l 1991 FLEMING, J. T. AND J. G. JOSHI. Ferritin: The role of aluminum in ferritin function. NEUROBIOL AGING 12(5) 413-418, 1991 .--We previously showed that human brain ferritin (HBF) binds aluminum (A1) in vivo and in vitro and HBF isolated from Alzheimer's brain had more AI bound compared to aged matched controls (7). To further understand the role ferritin may play in AI neurotoxicity, we have studied in vitro the effect of AI on the function of human ferritin isolated from Alzheimer's (AD) and normal brain tissue, and compared the results with other mammalian ferritins. AI 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 > > human liver > rat brain > 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 AI. Several mammalian ferritins were compared for their total iron uptake: horse spleen = human liver > human brain (normal) = human brain (AD) ferritin. In 20 mM HEPES (pH 6.0) buffer holoferritin is more resistant to precipitation by AI than apoferritin suggesting that holoferritin is a better chelator for nonferrous metal ions. Aluminium

Iron

Ferritin

been suggested to be the only biologically relevant species (27), the highly hydrated species of aluminum have also been reported to alter the conformation of proteins (37). Recent studies have implicated A1 in several central nervous system disorders including amyotrophic lateral sclerosis (10), dialysis encephalopathy (2) and Alzheimer's disease (AD) (6). In brain tissue from AD patients, A1 has been found associated with the neurofibrillary tangles (31) and aluminosilicates have been found at the core of senile plaques (3). While it is not clear whether or not A1 is the primary cause of the neuropathology, a recent epidemiological study of 4,100 people in England found individuals who lived in areas with high A1 water levels had a 50% greater chance of developing Alzheimer's disease (28). Previously we reported that ferritin exists in significant concentration in brain tissue, that ferritin isolated from rats fed 100 IxM A1 in the drinking water have more A1 bound to ferritin compared to control animals, and the ferritin isolated from the brains of AD patients had more A1 bound compared to agematched controls (7). Gerber and Conner (11) reported that antiserum made to human ferritin preferentially labels oligodendrocytes in grey and white matter of human brain. Using a similar technique Grundke-Iqbal observed ferritin within astroglia,

THE ubiquitous iron (Fe) storage protein ferritin (Mr - 4 8 0 , 0 0 0 ) is composed of 24 subunits of at least two classes, heavy (H) and light (L), with molecular weights of approximately 21,000 and 19,000 in mammals. The subunits form a protein shell which can store up to 4500 iron atoms per ferritin molecule as nontoxic ferric hydroxyphosphate (1). In addition to storing iron, we have suggested that ferritin also participates in metal detoxification (20). Ferritin binds V ÷ 2 and Tb ÷ 3 in vitro and Cu + 2, Zn ÷ 2, Pb + 2, Cd ÷ 2, Be + 2 and A1 in vitro and in vivo (7, 23, 26, 29, 32, 33, 41). Rats, whose ferritin synthesis in liver was increased 5-fold by Fe +3 injection, survived otherwise toxic doses of Be ÷ 2 (23). Ferritin protected and reversed the in vitro inhibition of N a + K ÷ ATPase, alkaline phosphatase and phosphoglucomutase by Be ÷2 (33). There has been increasing concern over the rise in AI in water due to acid rain. Normally stored in the soil as insoluble aluminosilicates and aluminoxides, A1 leaching dramatically increases with a slight decrease in water pH (27). Organisms that evolved in an environment with low A1 levels are now exposed to and must deal with this potential toxin. A1 forms several different complexes in water; the concentrations of particular species are dependent upon the pH of the environment. Although A13 + has

tParts of this publication have been presented at the following meetings: (1) Joshi, J. G.; Fleming, J. T.; Sczekan, S. VIIIth Intemational Conference on Proteins of Iron Transport and Storage, Quebec, Canada, 1987; (2) Fleming, J. T.; Joshi, J. G. FASEB J. 2:A766; 1988, Las Vegas, USA; (3) Joshi, J. G.; Fleming, J. T. Ist International Meeting on the Molecular Mechanisms of Metal Toxicity and Carcinogenicity, Urbino, Italy, 1988. 2The biologically functional species of aluminum remains controversial. Therefore, aluminum is abbreviated as A1 to represent all biologically active species. 3j. T. Fleming submitted this paper toward partial fulfillment of the requirements for the doctorate of philosophy degree at the University of Tennessee. Present address: Center For Environmental Biotechnology, 10515 Research Dr., Bldg. A, Suite 200, Knoxville, TN 37932-2562. 4Requests for reprints should be addressed to Dr. Jayant G. Joshi, Department of Biochemistry, University of Tennessee, Knoxville, TN 37996-0840.

413

414

FLEMING AND JOSHI

particularly microglia, in areas containing neurofibrillary degeneration and amyloid accumulation (12). Furthermore, ferritinpositive processes were associated with all stages of plaque formation in both AD and normal hippocampal sections, but the intensity of staining and proliferation of processes was greater in the AD sections (12). This report describes the effect of A1 on the functions of ferritin in vitro. METHOD

The water used was passed through two ion exchangers and the resistance was greater than 1.5 M ohms. Ultra-pure HNO 3 was obtained from Baker. Metal standards used for atomic absorption spectroscopy were obtained from Sigma and were used within the period of the expiration date. Horse spleen ferritin, thioglycolic acid, Naphthol Blue Black, HEPES and Tris base were obtained from Sigma. Chelex-100 was obtained from BioRad. NAD(P)H-FMN-oxidoreductase was obtained from Boehringer Mannheim (West Germany). Nitrogen "Zero grade" was obtained from MG Industries (Knoxville, TN) and passed over a heated copper coil to remove any residual oxygen. Two histopathologically examined human brains were available. They were obtained through the Cole Neuroscience Foundation; the age, sex and cause were as follows: 1) 69 years, female, atrial fibrillation and mitral stenosis (normal); 2) 79 years, female organic brain syndrome with senile plaques and neurofibrillary tangles characteristic of AD. Upon autopsy, the brains were removed, and stored at - 8 0 ° C . Unstripped rat brains were obtained from Pel-Freeze (Rogers, AK) and kept frozen at - 2 0 ° C . All other chemicals were of the highest purity obtainable. All glassware and plasticware was first rinsed with 2% ultra-pure HNO 3 followed by a rinse with double deionized water. Statisticai analysis was performed using a Hewlett Packard HP-41 T-statistics program.

Isolation of Brain Ferritin Rat brain, human brain, and human liver ferritin were isolated as follows. Frozen brain tissue was sliced into 1 cm sections and immediately homogenized in 5 ml/g of 0.1 M TrisHC1 buffer pH 7.4 with 0.01% pentylmethylsulfonylfluoride to inhibit proteases. The homogenate was centrifuged at 20,000 x g for 20 min to pellet cellular debris, after which the pellet was again homogenized and centrifuged. The supernatant was heated in a water bath, with vigorous stirring, to 80°C, immediately cooled to 10°C in an ice bath, and centrifuged at 20,000 x g for 20 min. The pH of the supernatant was adjusted to 5.0 with 1.0 M acetic acid, stirred for 1 h at 4°C, and centrifuged at 20,000 x g for 20 min. Solid ammonium sulfate was slowly added to the supernatant, with stirring, to a final concentration of 40%, the solution was stored overnight at 4°C, and, subsequently, centrifuged at 20,000 x g for 45 min. Solid ammonium sulfate was added to the supernatant to a final concentration of 50%, stored overnight at 4°C and centrifuged at 20,000 x g for 45 min. The pellet from the 40-50% ammonium sulfate precipitate was redissolved in a minimal volume of 50 mM Na2HPO4 buffer pH 7.0, 0.02% NaN 3, applied to a 1.8 x 75 cm sephacryl S-300 (Pharmacia) gel filtration column, and eluted with the same buffer. Two ml fractions were collected and the iron-rich fractions, as determined by atomic absorption, were pooled and concentrated in a stirred cell using a 30 kDa cut-off filter (Amicon PM-30). A sucrose step gradient was prepared by pipetting the following sucrose solutions into a 13.5 ml ultracentrifuge tube: 1 ml 80%, 2 ml 50%, 2 ml 30%, 2 ml 20%, 2 ml 15%, 2 ml 5%. One ml of the iron-rich concentrate was layered on the sucrose gradient

and centrifuged at 35,000 rpm for 4 h in a Beckman 50 Ti ultracentrifuge rotor. After centrifugation, the 80% sucrose fraction was collected, the sucrose removed by washing with 10 ml of 50 mM Na2HPO 4 buffer pH 7.0, 0.02% NaN 3 in a stirred cell using a PM-30 filter, concentrated to - 1 ml and stored at 4°C. The purity of the isolated ferritin was determined by electrophoresis on 5% nondenaturing acrylamide gels as previously described (7). Routinely, over 90% of the protein in such preparations migrated as a single band (500 kDa) with some minor bands of higher molecular weight. Also, after SDS-PAGE, the purified protein resolved into H and L subunits in a ration of 70% to 30% respectively as previously observed (7). Ferritin concentrations were determined by multiplying by 0.7 the Lowry value obtained colorimetrically using bovine serum albumin as a standard (24). This correction factor was derived by comparing the ferritin protein value obtained colorimetrically with that determined by Kjeldahl nitrogen analysis (33). Human and rat brain ferritins were stored at a concentration of 1-2 mg/ml in 50 mM Na2HPO 4 buffer pH 7.0, 0.1% NaN 3. Human liver ferritin was stored at a concentration of - 1 0 mg/ml in the same buffer.

AI Loading Up to 5 mg of holoferritin from human brain was incubated for 1 h with 0.1 mM EDTA in 20 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) (pH 5.0) followed by dialysis against 2 g Chelex in 1 1 of 20 mM HEPES (pH 5.0) buffer to remove unbound nonferrous metals. Apoferritin was prepared by removing iron from holoferritin by reduction with 1% thioglycolic acid in 0.1 M CH3COONa (pH 4.8) buffer followed by dialysis in 20 mM HEPES (pH 5.0) buffer containing Chelex-100 (40). These ferritin preparations did not contain detectable Cd or AI as judged by atomic absorption spectrophotometry (7). Subsequently, 50 Ixg of these ferritins were incubated with 3, 6, 12, 18, 24 and 30 ~g of these ferritins 0.4 mM A1C13.6H20 in 20 mM HEPES (pH 5.0) to a final volume of 1 ml. The concentration of ferritin in these solutions was therefore 0.1 p.M (2.4 txM in ferritin subunits) and the concentrations of the A1 solutions were 1.2, 2.4, 4.8, 7.2, 9.6 and 12 txM to yield A1/ferritin subunit molar ratios of 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 respectively. Any unbound A1 was removed by dialysis first against 20 mM HEPES (pH 5.0) and then against 20 mM HEPES (pH 7.0) in inverted microcentrifuge tubes (30). The ferritin solutions were then centrifuged to remove any precipitated protein and the actual A1 bound to ferritin was verified by atomic absorption (7).

The Rate of Iron Loading Several preparations of ferritin were made with varying ratios of A1/ferritin subunit as described above. To 50 I-~g of these ferritins in 0.1 M HEPES (pH 7.0) buffer 1.2 ILl of 0.05 M Fe(NH4)2(SO4)2.6H20 was added to bring the total volume to 0.5 ml. The concentration of ferritin was, therefore, 0.2 txM and the concentration of Fe +2 was 1.2 p~M to give a 6 mol Fe+2/ mol ferritin ratio. This amount of iron was chosen to approximate initial iron loading conditions (40). The rate of iron loading was then determined by measuring the change in absorbance at 310 nm over 30 s (35) in a Beckman DU-7 spectrophotometer, and subtracting the blank rate obtained by the auto-oxidation of Fe + 2 alone.

Iron Uptake The iron loading capacities of ferritin preparations were determined by sequentially adding ferrous iron in 200 mol Fe+3/

ALUMINUM AND FERRITIN'S FUNCTION

mol ferritin aliquots to give a final Fe + 2/ferritin ratio of 5000. An aliquot of 1.6 i~1 of a 25 mM solution of a Fe(NH4) 2 (SO4)2.6H20 in water degassed with N 2 was added to 100 ixg of ferritin in 0.05 M HEPES, 20 mM KIO3, 80 mM NazS203 (pH 7.0) to a final volume of 1 ml. The concentration of ferritin was, therefore, 0.2 IxM and the concentration of Fe +2 was 40 IxM to give a 200 Fe ÷ 2/ferritin molar ratio (25). Fe + 2 aliquots were added at 10-min intervals until a Fe ÷ 2/ferritin ratio of 5000 was obtained. After the addition of iron the protein was dialyzed against the same HEPES buffer in microcentrifuge tubes as described above and centrifuged for 2 min at 5000 x g at room temperature in a Brinkmann centrifuge. The iron in the supernatant was quantified by atomic absorption and the protein in the supernatant determined (24).

Iron Release The rate of iron release from several mammalian ferritins was determined as described by Jones et al. (19). In addition, several human brain ferritin preparations with varying A1/subunit ratios were prepared and used to determine if A1 would affect the in vitro rate of iron release. In a typical experiment a 470 ~1 solution containing 2.5 mM FMN and 2.5 mM ct,a-bipyridyl in 0.22 M HEPES (pH 7.4) buffer was pipetted into a cuvette and degassed for 30 s with N 2. Ten txl of a 0.12 M NADH solution was added to give a final concentration of 2.5 mM. NAD(p)HFMN (0.06 units) oxidoreductase was added in 10 Ixl to give a total volume of 490 p,1. The cuvette was then sealed with parafilm and allowed to incubate for 1 h at room temperature. The solution was then again flushed with N 2, a 10 p~l aliquot containing 5-20 p~g of ferritin loaded with 0, 0.5, 1, 2, 3, 4 and 5 A1/ferritin subunit was added and the increase in absorbance at 522 nm was monitored over a period of 10 min.

Precipitation of Ferritin by AI A freshly prepared 1.0 mM A1C13.6H20 solution in water (2.4, 4.8, 9.6, 14.4, 19.2 and 24 pal) was added to 100 ixg of apo- and holoferritin in 20 mM HEPES (pH 6.0) in a final volume of 0.5 ml. The concentration of the ferritin was 0.4 txM (9.6 IxM in ferritin subunits) and the concentration of A1 solutions were 4.8, 9.6, 19.2, 28.4, 38.4 and 48 p,M to give A1/ ferritin subunit molar ratios of 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 respectively. After 2 h at room temperature the samples were centrifuged in a Brinkmann centrifuge at 5000 x g for 2 min and the supernatant assayed for protein (24). RESULTSAND DISCUSSION

Iron Loading and Uptake Studies on the kinetics of iron uptake by apoferritin have shown that hyperbolic curves were obtained when low Fe/ferritin ratios were used. The hyperbolic curve represents the initial phase of iron binding at specific sites within the apoferritin shell, after which the bound Fe + 2 is oxidized to Fe + 3. Several investigators have shown that cations such as Zn ÷2, Ni ÷2, Hg +2, Cd ÷2, Co ÷2 and Mg ÷2 inhibit iron uptake by competing for the iron binding site (29,41). To determine if A1 would affect iron loading into human brain ferritin, aliquots of human brain apoferritin were preloaded with various A1/subunit rations, unbound A1 was removed and the amount of A1 bound to the protein was quantified. These samples were then used to determine the in vitro rate of iron loading. This modification differs from earlier studies (29,41) where the metal ion was simply added to a ferritin solution, thus

415

permitting even unbound metal ions to compete with iron for the binding sites. A1 concentrates in the brain and bones of rats fed A1 citrate or AI(OH) 3 (38). A1C13 was used as the source of A1 ion in this study because we have previously demonstrated in vitro binding of A1 to ferritin using A1C13 (7,36). While AI citrate is more soluble than A1C13, in preliminary experiments, we were not able to demonstrate binding of A1 to ferritin using A1 citrate. In addition we have previously reported the in vivo binding of A1 to ferritin obtained from rats fed 100 IxM A1CI3 in their drinking water (7). The question of whether or not the AI concentrations used in these experiments are physiologically realistic is inappropriate; A1 is differentially concentrated in different areas of the brain (21). The local A1 concentration can be significantly higher than the concentration of A1 in the circulatory system (25,31). We have, however, previously demonstrated the physiological or in vivo relevance of the amounts of A1 loaded to ferritin in this study; brain ferritin isolated from rats fed 100 p~M A1C13 in their drinking water had an A1/ferritin subunit ratio of 5.0 (7). As seen in Fig. 1, A1 caused a concentration-dependent decrease in the initial rate of iron loading into ferritin. Replotting the data as 1/v vs. bound A1 resulted in too much scatter to say with assurance whether or not the effect is competitive (data not shown). The observed inhibition was not an experimental artifact produced by the formation of an Al(et,ct-dipyridyl) 3 complex because in aqueous solutions such a complex decomposes spontaneously (39). The effect could not have been due to A1induced apoferritin aggregation because the Al-loaded ferritins were dialyzed and centrifuged before the iron experiment was performed. In vitro the effect of A1 on the rate of iron loading was very similar for human apoferritins from normal or AD brain. In a separate series of experiments, human holoferritin from normal and AD brain was loaded with A1, excess A1 removed and the resulting samples assayed for iron and A1. The results showed that the iron content did not differ significantly after A1 loading in vitro (Table 1). We then compared in vitro the maximum capacity of various mammalian ferritins to store iron (Table 2). Consistent with earlier observations (35), in all instances, oxidative storage of iron into ferritin (Fe + 2 ~ Fe ÷ 3) precipitated significant amounts of protein (Table 2). The amount precipitated was not related to the subunit size because horse spleen as well as human liver ferritin contain predominantly L chains. As determined by SDS-PAGE, human brain ferritin has 70% H and 30% L chain (7). The predominance of H chain subunits may account for the significantly lower capacity of brain ferritin to store iron (36). There was no significant difference between the capacities for ferritin from AD and normal human brain tissue.

Iron Release Jones et al. found a 5-fold increase in the rate of dihydroFMN-mediated iron release from horse spleen ferritin compared to horse heart ferritin (19). They suggested that the decreased iron release rate of horse heart ferritin may be due to a greater restriction of dihydroflavin diffusion to the iron core caused by the presence of larger H subunits compared with the smaller L subunits of horse spleen ferritin. Our iron release data for human and rat ferritins is consistent with this hypothesis (Table 3). Human brain ferritin (70% H-chain) has a Vm (the maximal rate of iron release) less than one-half that of human liver ferritin (p<0.05). A comparison of the iron release rates for rat brain and rat liver ferritin reveals that rat brain, with 26% H, 42% M and 30% L (8) has a 65% greater V m compared to rat liver fer-

416

FLEMING AND JOSHI

0.20-

TABLE 2

(.9

IN VITRO IRON UPTAKE OF HUMAN BRAIN (AD), HUMAN BRAIN Z

(NORMAL), HUMAN LIVER AND HORSE SPLEEN FERRrrlNS

©

(D

<.... (5 Z

D

,<

!

o,

Initial Fe (mol Fe/mol ferritin)

0.15 O

Ferritin

O /,,

Ii

0.10 -

O

<

Horse Spleen Human Liver Human Brain (AD) Human Brain (N)

2366 2700 1661 1489

Final Fe* (mol Fe/mol ferritin) 3915 4147 2855 2678

± --± ±

Precipitated Protein %

182 117t 268:~ 186

48 25 23 24

@ 0.05 0.0

i 0.5

I 1.0

4 1.5

I

2.0

AI+g/FERRITIN SUBUNIT

FIG. 1. Effect of A1 on the in vitro rate of iron loading into human brain (AD), human brain (normal) and horse spleen ferritins. Fifty ixg of human brain (AD), human brain (N) and horse spleen ferritin was incubated with 3, 6, 12, 18, 24 and 30 Ixl of 0.4 mM AICI3.6H20 in 20 mM HEPES (pH 5.0) to a final volume of 1 ml. The concentration of ferritin in these solutions was, therefore, 0.1 ixM (2.4 ixM in ferritin subunits) and the concentrations of the AI solutions were 1.2, 2.4, 4.8, 7.2, 9.6 and 12.0 ixM to yield Al/ferritin subunit molar ratios of 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 respectively. The ferritin solutions were dialyzed to remove any unbound AI, centrifuged to remove any precipitated protein and the amount of A1 bound to ferritin verified by atomic absorption. To 50 ixg of these ferritins in 0.1 M HEPES (pH 7.0) buffer, 1.2 ill of 0.5 mM Fe(NH4)2(SO4)2.6HzO was added to bring the total volume to 0.5 ml. The concentration of ferritin was, therefore, 0.2 IXM and the concentration of Fe ÷2 was 1.2 ixM to give a 6 mol Fe+2/mol ferritin ratio. Rates are plotted as A31o/min/mg protein. Each point represents the mean of two determinations. (O) human brain ferritin (normal); (A) human brain ferritin (AD); (C)) horse spleen ferritin.

ritin ( p < 0 . 0 5 ) w h i c h is 38% H c h a i n (14). T h e differences in the o b s e r v e d rates o f iron release a m o n g the m a m m a l i a n ferritins further underscores the importance o f functional differences attributable to ferritin tissue heterogeneity and probably to isoferritins. Figure 2 s h o w s that A1 did not affect the rate o f iron release from either A D or n o r m a l h u m a n brain ferritin. T h e rates o f release are e x p r e s s e d per n m o l o f initial Fe ÷ 3 present to allow c o m p a r i s o n o f s a m p l e s with varying iron content. Apparently, A1 binds to the ferric h y d r o x y p h o s p h a t e core in s u c h a w a y as

TABLE 1

*Each value represents the average of three individual determinations expressed as means -_+ s.d. ~Significantly greater than human brain ferritin (p<0.001). :~Not significantly different from human brain ferritin (N). A 25 mM solution of Fe(NH4)2(SO4)2.6H20 in water degassed with N 2 (1.6 ix) was added to 100 ixg ferritin in 0.05 M HEPES, 20 mM KIO3, 80 mM Na2S203 (pH 7.0) to a final volume of 1 ml. The concentration of ferritin was, therefore, 0.2 ixM and the concentration of Fe +2 was 40 ixM to give a 200 Fe +2/ferritin molar ratio. Fe ÷ 2 was added to ferritin in 200 Fe +2/ferritin molar aliquots until a final 5000 Fe ÷ 2/ferritin molar ratio was obtained.

not to interfere with the Fe + 3 reduction reaction.

Precipitation of Ferritin by Al Aliquots of apo- and holoferritin f r o m h u m a n brain were incubated with increasing concentrations o f A1CI3.6H20 and the percent precipitated protein w a s determined at each AI c o n c e n tration (Fig. 3). Holoferritin was m o r e resistant to precipitation by A1 c o m p a r e d to apoferritin. Similar results were obtained with horse spleen ferritin (data not shown). T h i s s u g g e s t s that there are probably A1 binding sites even on the exterior o f the protein that permit aggregation. In the case o f holoferritin, the

TABLE 3 IN VITRO RATE OF IRON RELEASE FROM HUMAN BRAIN

(NORMAL AND AD), RAT BRAIN, RAT LIVER, HUMAN LIVER AND HORSE SPLEEN FERRITINS

Ferritin

Initial Fe (mol Fe/mol ferritin)

Horse Spleen

1328

Rat Brain

1391

Rat Liver

1065

Human Liver

1750

Human Brain (AD)

1661

Human Brain (N)

1489

IRON ANALYSIS OF AI LOADED HUMAN BRAIN FERRITINS

Alzheimer' s

Normal

0.5 1.0 2.0 3.0 4.0 5.0 mean -- s.d.

1121.9 1164.9 967.8 740.2 -1186.4 1036 ± 186.3

1100.0 1302.9 928.4 -1084.5 854.9 1054 ± 173.5

*Aluminum bound is expressed as tool Al/mol ferritin subunit. +Iron is expressed as mol Fe/mol ferritin molecule.

101.4 ± 15.9 (n = 4) 21.1 ± 4.0t (n = 4)

Fe/Ferritint A1/Subunit*

Vm*

13.0 ± 7.2 (n = 3) 33.4 ± 5.3~: (n = 5) 12.l ± 2.6§ ( n = 10) 14.0 ± 2.7 (n = 10)

*V m expressed as nmol of Fe(II) released per rain per nmol of initial Fe(III). Each value represents the average of the number of individual determinations in parentheses expressed as means - s.d. tSignificantly greater than rat liver ferritin (p<0.05). ~Significantly greater than human brain ferritin (p<0.05). §No significant difference was found between AD and normal human brain ferritin.

A L U M I N U M AND FERRITIN'S FUNCTION

417

20 It. 0

E O.

E

;t 0

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IO

#

5

E 0 I

00

I0

I

t

I

,.

2.0

3.0

4.0

50

0

I

2

3

4

5

AI~a/ Ferritin Subunit

A I'a / Ferritin Subunit FIG. 2. Effect of A1 on the rate of in vitro iron release from human brain ferritin (normal and AD). Four hundred and seventy V,1 of a 2.5 mM FMN, 2.5 mM a,ot-bipyridyl in 0.22 M HEPES (pH 7.4) buffer was pipetted into a cuvette and degassed for 30 s with N 2. Ten l~l of a 0.12 M NADH solution was added to give a final concentration of 2.5 mM. NAD(P)H-FMN oxidoreductase (0.6 units) was added in 10 I~1 to give a total volume of 490 V,1. The cuvette was then sealed with parafilm and allowed to incubate for 1 h at room temperature. The solution was then again flushed with N 2, a 10 ~1 aliquot containing 5-20 p,g ferritin loaded with 0, 0.5, 1, 2, 3, 4 and 5 A1/ferritin subunit was added and the increase in absorbance at 522 nm was monitored over a period of 10 min. Vr~, is expressed as nmol of Fe(II) released per min per nmol of initial Fe(III). Each value represents the average of five individual determinations expressed as means-s.d. (A) human brain ferritin (AD); (&) human brain ferritin (normal).

bound A1 is transferred to the iron core or preferentially binds to the core thus preventing the precipitation of the protein. That holoferritin is more resistant to precipitation by A1 compared to apoferritin confirms the suggestion that the iron core participates in A1 binding (36). No explanation is at hand to explain why A1 reduces only the rate of ion uptake but not its release. Several investigators have suggested that rearrangement of ferritin bound metals occurs over time on the basis of changes in difference spectra (40). If rearrangement of the core does not take place, a decrease in the initial rate of iron release may not be expected. In our studies, the ferritin-A1 complexes were stored at 4°C for several days before the iron release studies were performed and, therefore, had several days to undergo rearrangement. In addition, the ferritin preparations were dialyzed to remove free and loosely bound A1. It is conceivable that any A1 on the surface of the core may be removed by dialysis and that the remaining bound A1 would be within the core matrix. If such is the case, the release of iron near the bound A1 would be slower than that on the surface of the iron core. This possibility was not explored. Because ferritin iron loading and release are entirely independent phenomena, the rate-limiting steps may be dissimilar for the two processes. Therefore, an effect of A1 on iron release may not necessarily be expected. The data presented here clearly establish that A1 binds to apoas well as holoferritin. As reported previously, the presence of phosphate in the iron core of ferritin permits binding of larger quantities of metal ions (7,35). The species of aluminum bound is unclear and continues to be a puzzling aspect of aluminum toxicity. At physiological pH the concentration of AI(OH) 4 - 1 is 106 more than A1 +3 and even at pH 6.0, the hydroxylated species predominate (27). Yet, glucose-6-phosphate dehydrogenase

FIG. 3. Effect of AI on the in vitro precipitation of human brain ferritin. A freshly prepared 1.0 mM AICI3.6H20 solution in water (2.4, 4.8, 9.6, 14.4, 19.2 and 24 I~1) was added to 100 p,g of apo- and holoferritin in 20 mM HEPES (pH 6.0) in a final volume of 0.5 ml. The concentration of the ferritin was 0.4 IxM (9.6 IxM in ferritin subunits) and the concentrations of the A1 solutions were 4.8, 9.6, 19.2, 28.4, 38.4 and 48 txM to give A1/ferritin subunit molar ratios of 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 respectively. Values are expressed as the percent of the original protein precipitated. (O) holo-human brain ferritin; (A) apo-human brain ferritin. from yeast (4) or pig and human brain (5), inactivated by preincubation with AIC13-6H20 at pH 7.0 is not reactivated by EDTA which is a strong chelator for A1 ÷ 3. Curiously, no inactivation was observed when the enzyme was exposed to a premixed solution of A1C13.6H20 and EDTA. These observations suggest that the hydroxides of aluminum may be active even in vivo and that the observed reduction of iron uptake by ferritin in the presence of A1 may be due to the binding of the hydroxylated species of A1 to ferritin. Our finding of a significant decrease in iron loading with as little as 0.5 A1/ferritin subunit, suggests that even the levels of A1 found associated with brain ferritin in vivo may adversely affect ferritin function. If more A1 is bound to the ferritin of AD patients compared to controls (7), this elevated A1 may compromise brain iron metabolism in vivo by interfering with iron storage. Accordingly, slow deposition of A1 in the brain and its subsequent binding to ferritin may precipitate ferritin if bound to apoferritin or generate ferritin molecules with a reduced rate of iron uptake. Iron is a well established inducer of ferritin synthesis (1). More recent studies show that in vivo AI increases the translatability of ferritin mRNA or rat brain polysomes (34). This is consistent with the elevated levels of ferritin observed in the brains of AD patients and Al-fed rats (7) and may represent an indirect consequence of A1 toxicity where more functional ferritin molecules were synthesized to sequester iron. In summary, the data presented here suggest that A1 may interfere with iron metabolism by affecting the primary function of ferritin, the storage and transport of iron. By stimulating ferritin synthesis, A1 may overload the tissue with ferritin. The brain regions such as the basal ganglia which contain high levels of iron (15) may, therefore, be more vulnerable to A1 toxicity. For example, iron from ferritin and hemosiderin, in the presence of oxygen produces hazardous free radicals (9) and A1 accelerates the rate of lipid peroxidation initiated by iron (13). Brain is a highly aerobic tissue. In vivo colocaiization of A1 and iron in the brain would make such areas more susceptible to oxidative damage (21). The consequences of such damage in producing specific neurological disorders remains to be established.

418

F L E M I N G A N D JOSHI

ACKNOWLEDGEMENTS This work was supported by the Robert and Monica Cole Neuroscience Foundation and the Council for Tobacco Research.

REFERENCES 1. Aisen, P.; Listowsky, I. Iron transport and storage proteins. Annu. Rev. Biochem. 49:357-393; 1980. 2. Alfrey, A. C. Dialysis encephalopathy syndrome. Annu. Rev. Med. 29:93-121; 1978. 3. Candy, J. M.; Klinowski, J.; Perry, R. H.; Perry, E. K.; Fairbairn, A.; Oakley, A. E.; Carpenter, T. A.; Atack, J. R.; Blessed, G.; Edwardson, J. A. Aluminosilicates and senile plaque formation in Alzheimer's Disease. Lancet 1:354-356; 1986. 4. Cho, Sung-Woo; Joshi, J. G. Inactivation of Baker's yeast glucose6-phosphate dehydrogenase by aluminum. Biochemistry 28:36133618; 1989. 5. Cho, Sung-Woo; Joshi, J. G. Inactivation of glucose-6-phosphate dehydrogenase isozymes from human and pig brain by aluminum. J. Neurochem. 53:616--621; 1989. 6. Crapper, D. R.; Krishnan, S. S.; Dalton, A. J. Brain aluminum distribution in Alzheimer's disease and experimental neurofibrillary degeneration. Science 180:511-513; 1973. 7. Fleming, J.; Joshi, J. G. Ferritin: Isolation of aluminum-ferritin complex from brain. Proc. Natl. Acad. Sci. USA 84:7866-7850; 1987. 8. Fleming, J. T. Studies in brain ferritin. Ph.D. Dissertation, Dept. of Biochemistry: University of Tennessee, Knoxville; 1989. 9. Fridovich, I. Superoxide dismutases. J. Biol. Chem. 264:77617764; 1989. 10. Garruto, R. M.; Fukatsu, R.; Yanagihara, D. C.; Gajdusek, D. C.; Hook, G.; Fiori, C. E. Imaging of calcium and aluminum in neurofibrillary tangle bearing neurons in parkinsonism dementia of brain. Proc. Natl. Acad. Aci. USA 81:1875-1879; 1984. 11. Gerber, M. R.; Conner, J. R. Do oligodendrocytes mediate iron regulation in the brain? Ann. Neurol. 26:95-98; 1989. 12. Grundke-Iqbal, I.; Fleming, J. T.; Lassman, H.; Iqbal, K.; Joshi, J. G. Ferritin is a component of the neuritic plaques (senile) in Alzheimer's disease. Acta Neuropathol. (Berl.) 81:105-110; 1990. 13. Guttridge, J. M. C.; Quinlan, G. J.; Clark, I.; Halliwell, B. Aluminum salts accelerate peroxidation of membrane lipids stimulated by iron salts. Biochim. Biophys. Acta 835:441--447; 1985. 14. Hazard, J. T.; Yokota, M.; Arosio, P.; Drysdale, J. W. Immunological differences in human isoferritins: Implications of the immunologic quantitation of serum ferritin. Blood 49:139-145; 1977. 15. Hill, J. M. The distribution of iron in brain. In: Youdin, M. B. H., ed. Brain iron: Neurochemical and behavioral aspects. New York: Taylor and Francis; 1988:1-24. 16. Hoy, T. G.; Jacobs, A. Changes in the characteristics and distribution of ferritin in iron-loaded cell cultures. Biochem. J. 193:87-92; 1981. 17. Jellinger, K.; Paulus, W.; Grundke-Iqbal, I.; Riederer, P.; Youdin, M. B. H. J. Neural Transm. 2:327-340; 1990. 18. Jones, B. M.; Worwood, M.; Jacobs, A. Isoferritins in normal leukocytes. Br. J. Haematol. 55:73-82; 1983. 19. Jones, T.; Spencer, R.; Walsh, C. Mechanism and kinetics of iron release form ferritin by dihydroflavins and dihydroflavin analogues. Biochemistry 17:4011-4017; 1978. 20. Joshi, J. G.; Clauberg, M. Ferritin: An iron storage protein with diverse functions. BioFactors 1:207-212; 1988. 21. Joshi, J. G. Aluminum, a neurotoxin which affects diverse metabolic reactions. BioFactors 2:163-169; 1990.

22. Levi, S.; Luzzago, A.; Cesareni, G.; Cozzi, A.; Franseschinelli, F.; Albertini, A.; Arosio, P. Mechanisms of ferritin iron uptake: Activity of the H-chain and deletion mapping of the ferro-oxidase site. J. Biol. Chem. 263:18086-18092; 1988. 23. Lindenschmidt, R. C.; Sendelbach, L. E.; Witschi, H. P.; Price, D. J.; Fleming, J.; Joshi, J. G. Ferritin and in vivo beryllium toxicity. Toxicol. Appl. Pharmacol. 82:344-350; 1986. 24. Lowry, O. H.; Rosebrough, N. H.; Farr, A. L.; Randall, R. J. Protein measurements with folin phenol reagent. J. Biol. Chem. 193: 265-280; 1951. 25. McLachlan, D. R. C. Aluminum and Alzheimer's disease. Neurobiol. Aging 7:525-532; 1986. 26. Macara, I. B.; Hoy, T. G.; Harrison, P. M. The formation of ferritin from apoferritin. Biochem. J. 135:785-789; 1973. 27. Martin, R. B. Bioinorganic chemistry of aluminum. In: Sigel, H., ed. Metal ions in biological systems. New York: Marcel Dekker, Inc.; 1988:1-57. 28. Martyn, C. N.; Barker, D. J. P.; Osmond, C.; Harris, E. C.; Ewardson, J. A.; Lacey, R. F. Geographical relation between Alzheimer's disease and aluminum in drinking water. Lancet 1:59-62; 1989. 29. Niederer, W. Ferritin: Iron incorporation and iron release. Experientia 26:218-220; 1970. 30. Overall, C. M. A microtechnique for dialysis of small volume solutions. Anal. Biochem. 165:208-214; 1987. 31. Perl, D. P.; Brody, A. R. Alzheimer's Disease: X-ray spectrometric evidence of aluminum accumulation in neurofibrillary tangle bearing neurons. Science 208:297-299; 1980. 32. Price, D. J.; Joshi, J. G. Ferritin: A zinc detoxicant and a zinc ion donar. Proc. Natl. Acad. Sci. USA 79:3116-3119; 1982. 33. Price, D. J.; Joshi, J. G. Ferritin: Binding of beryllium and other divalent metal ions. J. Biol. Chem. 258:10873-10880; 1983. 34. San-Marina, S.; Nicholls, D. M.; Kruck, T. P. A.; McLachlan, D. R. C. Aluminum effects on synthesis of brain metal binding proteins. FASEB J. 4:A1014; 1990. 35. Sczekan, S. R.; Joshi, J. G. Isolation and characterization of ferritin from soybeans (Glycine max). J. Biol. Chem. 262:1378013788; 1987. 36. Sczekan, S. R.; Joshi, J. G. Metal binding properties of phytoferritin and synthetic iron cores. Biochim. Biophys. Acta 990:8-14; 1989. 37. Siegel, N.; Huag, A. Aluminum interactions with aluminum. Biochem. Biophys. Acta 774:36-45; 1983. 38. Slanina, P.; Falkeborn, Y.; Frech, W.; Cedergren, A. Aluminum concentrations in the brain and bones of rats fed citric acid, aluminum citrate or aluminum hydroxide. Food Chem. Toxicol. 22:391397; 1984. 39. Tikhnov, V. N. Aluminum. In: Schmorak, J., trans. Analytical chemistry of aluminum. New York: John Wiley and Sons; 1973:23177. 40. Treffry, A.; Harrison, P. M. Spectroscopic studies on the binding of iron, terbium and zinc by apoferritin. J. Inorg. Chem. 21:9-20; 1984. 41. Wardeska, J. G.; Viglione, B.; Chasteen, N. D. Metal ion complexes of apoferritin. J. Biol. Chem. 261:6677-668; 1986.