530

Biochimica et Biophysica Acta, 5 4 3 ( 1 9 7 8 ) 5 3 0 - - 5 3 5 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 28651

USE OF IRON FROM TRANSFERRIN AND MICROBIAL CHELATES AS SUBSTRATE FOR HEME SYNTHETASE IN TRANSFORMED AND PRIMARY ERYTHROID CELL CULTURES

A. B A R N E K O W

* a n d G. W I N K E L M A N N

Institut fiir Biologie II, Lehrstuhl Mikrobiologie I, Universit~'t Tiibingen, D-74 Tiibingen, A u f der Morgenstelle 1 (F.R.G.) (Received March 15th, 1978)

Summary The enzymatic heme production in cell-free extracts of virus-transformed Friend erythroleukemia cells and primary bone marrow cells from rabbits has been measured by determining the activity of heme synthetase after addition of iron sulfate, transferrin or microbial iron chelates. In transformed cells the amounts of heme formed did not show significant differences independent of which substrate was offered. In cell-free extracts of primary bone marrow cells no increase of heine production could be observed.

Introduction Ferrochelatase or heme synthetase is the final enzyme in heme synthesis. It catalyses the incorporation of ferrous iron into porphyrins to form heme. Ferrochelatase is found in erythropoietic tissues, but also in liver mitochondria [1], microorganisms [2] and chloroplasts [3]. Iron as substrate for ferrochelatase is normally delivered by transferrin. Besides this naturally occurring chelator, microbial iron trihydroxamate complexes [4], which are excreted under iron-deficient conditions by various bacteria and fungi have proved to be good iron transport molecules in erythroid cell cultures [5]. Also the utilization of iron for hemoglobin synthesis supplied by sideramines could be observed in Friend virus-infected murine erythroleukemia cells [6]. This study investigates if there exists a specificity for iron reduction and iron incorporation into porphyrin by heme synthethase, as has been pointed out for the iron transport, or if the cell membrane itself is a limitating factor. Therefore, experiments were performed with cell-free extracts * Present address: Institut fiir Biochemie I, Universit~/t Heidelberg, D-69 Heidelberg, Im Neuenheimer Feld 328, G.F.R.

531 of a murine cell line of virus-transformed erythroleukemia [7] which could be induced to synthesis of hemoglobin, and with primary rabbit bone marrow. The ferrochelatase activity was determined after supplementing various iron chelators. Materials and Methods

Chemicals. Ferricrocin and fusigen were kindly provided b y Professor H. Diekmann, Hannover. Ferrirubin was from Professor W. Keller-Schierlein, ETH Zi]rich, Switzerland. Ferrioxamin B was from the stock of the Institut fiir Biologie II, Lehrstuhl Mikrobiologie I, Tiibingen. Crystalline protoporphyrin IX was a gift from Dr. S. Werner, Munich. Medium for cell cultures was purchased from LS-service, Munich. All other chemicals were obtained from Merck, Darmstadt or Boehringer Mannheim. Cell cultures. Friend virus-infected murine erythroleukemic cells (B8/3A) were kindly supplied b y Professor Dr. W. Ostertag, Max-Planck-Institut fiJr experimentelle Medizin, Abt. Molekulare Biologie, GSttingen. These cells were grown up to a cell density of 5 • 106 cells/ml in sterile culture flasks containing 20 ml minimum essential medium with Earle's salts, 1% L-glutamine (200 mM), 1% non-essential amino acids, 100 units penicillin G/ml and 15% fetal bovine serum, at 37°C. Then 0.3 ml cell suspension was transferred to 20 ml fresh medium. The passaging was performed every 3--5 days. The cells were induced to hemoglobin synthesis b y addition of 1.5% dimethylsulfoxide to the medium. After 4 days optimal hemoglobin synthesis occurs [8]. Primary, non-transformed bone marrow cells were obtained from 6-monthold rabbits. The femora were cracked open, and then the bone marrow delivered into serum-free medium. The cells were washed several time b y sedimentation in fresh medium, at 3000 rev./min for 3 min. Preparation o f the cell-free extracts. On the fourth day after induction to hemoglobin synthesis, cells were collected and washed with serum-free medium b y repeated sedimentation at 3000 rev./min. The transformed erythroleukemia cells and non-transformed rabbit bone marrow cells were then lysed in 0.1 M phosphate buffer, pH 8, containing 1% Triton X-100. The membranes were sedimented at 15 000 X g for 10 min. The supernatant was used for the ferrochelatase assay. Ferrochelatase assay. Iron porphyrin formation was measured spectrophotometrically following the hemochromogen m e t h o d of Porra and Jones [9]. The reaction mixture contained in a total volume of 4.6 ml 50 nmol protoporphyrin (in 0.1 M phosphate buffer with 1% Triton X-100), 3 ml cell-free extract, 100 nmol iron complex and 500 nmol NADH. Porphyrin and cell-free extract were pipetted into Thunberg tubes and the iron chelate and NADH solutions into the side-arm. Then the tubes were evacuated and flushed with N: five times and sealed under a slight positive pressure of N2. After equilibration at 37°C for 10 min, the contents of the side-arm were tipped into the porphyrin/enzyme solution to complete the reaction mixture. After 90 min incubation reaction was stopped b y placing the tubes in an ice-water bath for several minutes. The Thunberg tubes were then opened under nitrogen atmosphere and 1 ml 0.2 M iodoacetamide solution was immediately added. The tubes were

532 TABLE I COMPARISON INTO HEME

OF IRON INCORPORATION

OUT OF SIDERAMINES, TRANSFERRIN

AND FeSO4

Cell-free e x t r a c t s f r o m t r a n s f o r m e d r o u t i n e e r y t h r o l e u k e m i c cells, w h i c h had b e e n i n d u c e d to h e m o g l o b i n s y n t h e s i s b y M e 2 S O , w e r e i n c u b a t e d w i t h 50 n m o l p r o t o p o r p h y r i n , 1 0 0 n m o l i r o n c o m p l e x a n d 5 0 0 n m o l N A D H u n d e r n i t r o g e n a t m o s p h e r e f o r 90 r a i n at 37°C. F e r r o c h e l a t a s e w a s m e a s u r e d b y t h e p y r i d i n e h e m o c h r o m o g e n m e t h o d . See also M a t e r i a l s a n d M e t h o d s . R e s u l t s are m e a n s o f five d i f f e r e n t e x p e r i m e n t s (standard deviation ~2%). Iron substrate (riM)

Protein (rag)

Heine (nM)

S p e c i f i c a c t i v i t y × 10 -5 (#tool heine]rain per mg protein)

100 100 100 100 100 100 100 100

5.415 2.8 3.53 3.215 3.38 5.00 5.00 --

10.185 5.78 4.2 6.2025 6.9 8,825 ---

2.072 2.293 1.32 2.074 2.279 1.96 ---

f e r r i c r o cin fusigen ferrirubin ferrioxa-min B transferrin FeSO 4 FeSO 4 FeSO 4

closed and allowed to stand for 10 min in the ice-water bath prior to opening. 1.0 ml of pyridine, 1 ml 1 M NaOH were added; 3-ml aliquots of this solution added in each of t w o cuvettes. To one solution 0.005 ml 0.01 M K3FE(CN)6 was added and to the other about 1 mg Na2S204. The amount of heme formed was then determined by measuring the difference spectrum of the reduced minus oxidized pyridine hemochromogen at 541 and 557 nm using values of 20.7 [10] for Ae (raM) of protohemochromogen. Determination of protein. Concentration of protein in extracts was measured by the method of Lowry et al. [11]. Results Cell-free extracts from transformed erythroleukemic cells, which have been induced to hemoglobin synthesis by dimethylsulfoxide, show good heme formation when iron is added to the reaction mixture as iron sulfate, transferrin or sideramine-iron (Table I). Ferrioxamine B and ferrirubin, proved to be poor

T A B L E II COMPARISON OF IRON INCORPORATION

OUT OF SIDERAMINES AND FeSO 4 INTO HEME

Cell-free e x t r a c t s f r o m t r a n s f o r m e d r a b b i t b o n e m a r r o w w e r e i n c u b a t e d w i t h 50 n m o l p r o t o p o r p h y r i n , 1 0 0 n m o l i r o n c o m p l e x a n d 5 0 0 n m o l N A D H u n d e r n i t r o g e n a t m o s p h e r e f o r 90 r a i n at 37°C. F e r r o c h e l a t a s e w a s m e a s u r e d b y t h e p y r i d i n e - h e m o c h r o m o g e n m e t h o d . See also M a t e r i a l s a n d M e t h o d s . R e s u l t s a r e m e a n s o f five d i f f e r e n t e x p e r i m e n t s ( s t a n d a r d d e v i a t i o n < 2 % ) . Iron substrate (nM)

Protein (rag)

Heine (nM)

S p e c i f i c a c t i v i t y × 10 -5 (~mol heine/rain per mg protein)

100 ferricrocin 100 fusigen 100 FeSO 4

3.93 3.93 3.93 3.93

112 129 110 134

31.6 36.5 31.1 37.8

533

/ 10-

/

/

©'

2b

/

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4b

6b

8'0

1~0

Tim e(min) Fig. 1. T i m e - d e p e n d e n t h e m e f o r m a t i o n w i t h f e r ~ c r o c i n as i r o n d o n o r . C o n d i t i o n s as d e s c r i b e d in T a b l e I.

iron transport molecules, gave rise to significant amounts of heme formation when added to the reaction mixture. Assays with the addition of non-induced erythroleukemia cell-free extracts showed no heme formation during the incubation time of 90 min. Addition of iron sulfate or sideramine-iron as iron substrate has no effect. Thus, without addition of iron to the incubation medium no remarkable heme production could be pointed out. In ferrochelatase assays with cell-free extracts of primary bone marrow cells, the heme formation was much higher and did not increase, when iron was added as iron sulfate or chelate iron (Table II). These cells seem to have big iron stores, so that t h e y do n o t need additional iron for heme synthesis for 90 min. Also the absolute a m o u n t of heme produced is about 15 times higher than in ferrochelatase assays with erythroleukemia cells. To follow the heme production, time-dependent heme formation with ferricrocin as iron donor was performed (Fig. 1). A linear increase in heme concentration could be observed within the incubation time of 90 min. Discussion The experiments with cell-free extracts from transformed, erythroleukemic cells, which had been induced to hemoglobin synthesis, show that besides transferrin and iron sulfate also the sideramines fusigen, ferricrocin and ferrioxamin B can be used as iron substrates for enzymatic heme production. The control w i t h o u t addition of enzyme does n o t show any heme formation. The nonenzymatic heme production described by Tokunaga and Sano [12] and Kassner and Walchak [13] could be prevented by addition of Triton X-100. The use of sideramine-iron as substrate for synthesis of heme, requires the existence of a reductase, which is able to reduce the Fe 3÷ to Fe 2÷. The Fe 2+ is then incorporated into protoporphyrin to form heme. This reductase does n o t show any specificity regarding the different types of sideramines. Sideramines of the ferrichrome type, but also ferrioxamine B are reduced. Even transferrin, ferri-

534 oxamin B and ferrirubin, which had been proved to be poor iron transport molecules in transformed bone marrow cells [5] could deliver the iron in cellfree extracts. This might give further evidence, that the receptors on the cell membrane for transferrin uptake are not completely developed in transformed erythroleukemia cells. The small iron uptake rates with ferrioxamin B and ferrirubin seem to be caused by the membrane characteristics and not by the failure of utilization of the iron. Fastrich [14] also found a similar unspecificity of the reductase for the different trihydroxamates during studies on iron incorporation into protoporphyrin IX in Neurospora crassa. Barnes and Jones [15], who tested different sideramines as iron substrates for heme synthesis in rat reticulocytes and avian erythrocytes only found a reduction for ferrioxamin G. The results from bone marrow cells show that there must exist an iron pool or a way of regulation of the heme synthesis by intracellular concentrations of heme. Ponka and Neuwirt [16] established that the hemoglobin-synthesizing erythroid cells have a specific regulatory mechanism for controlling iron uptake rate. Heme inside and outside the cells inhibited the incorporation of iron in reticulocytes by a feedback regulation [17,18]. Heme does not decrease transferrin iron uptake, but the delivery of iron from transferrin [19]. This might be an account for the fact that primary bone marrow cells show no increase in heme synthesis, when iron is added as iron sulfate. The high rates of heme synthesis in primary bone marrow cells compared to transformed erythroleukemia cells show that heme does not decrease the utilization of intracellular non-heme iron for synthesis of heme, as reported by Ponka and Neuwirt [19]. As added iron sulfate also cannot increase heme synthesis, it cannot be excluded that also sideramine-iron can serve as substrate for heme synthesis, which cannot be measured under these circumstances. The results show that especially the sideramines ferricrocin and fusigen, which show very good iron transport behaviour and good intracellular utilization as iron substrate molecules for enzymatic heme synthesis, may be qualified as iron donors in erythroid systems. Acknowledgements This investigation was supported by the Deutsche Forschungsgemeinschaft. The authors are grateful to Dr. A.K. Walli for reading the manuscript.

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535 13 14 15 16 17 18 19

Kassner, R.J. and Walchak, H. (1973) Biochim. Biophys. A c t a 304, 294--303 Fastrich, D. (1974) Dissertation Universlt~t Ti~bingen, West Germany Barnes, R. and Jones, O.T,G. (1973) Biochlm. Biophys. Acta 304, 304--308 Ponka, P. and Neuwirt, J. (1974) Br. J. Haematol. 28, A n n o t a t i o n Ponka, P. and Neuwirt, J. (1969) Blood 33, 690--707 Ponka, P., Neuwirt, J. and Borov/t, J. (1974) Enzyme 17, 91--99 Ponka, P. and Neu wirt J, (1975) in Proteins of iron storage and t ra ns port in bi oc he mi s t ry and medicine, (Crichton, R.R., ed.), pp. 147--153, North-Holland Publ. Co., A m s t e r d a m