Heme Biosynthesis in Friend Erythroleukemia Cells
Total Page：16
File Type：pdf, Size：1020Kb
Proc. Natl. Acad. Sci. USA Vol. 76, No. 2, pp. 833-836, February 1979 Cell Biology Heme biosynthesis in Friend erythroleukemia cells: Control by ferrochelatase (5-aminolevulinate synthase/induction by dimethyl sulfoxide/noninducible variant) TIM RUTHERFORD*, GEORGE G. THOMPSONt, AND MICHAEL R. MOOREt *Nuffield Department of Clinical Medicine, Radcliffe Infirmary, Oxford, OX2 6HE, England; and tUniversity of Glasgow, Department of Materia Medica, Stobhill General Hospital, Glasgow G21 3UW, Scotland Communicated by Sir Hans A. Krebs, December 5, 1978 ABSTRACT The activities of the enzymes of heme biosyn- We have studied the activity of all the enzymes of heme thesis (except protoporphyrin oxidase) have been followed synthesis (except protoporphyrin oxidase for which we do not during the induction of Friend cells in culture. All the enzyme activities increased after induction with dimethyl sulfoxide. The have a reliable assay in Friend cells). We show that the activity activities of the intermediate enzymes were much higher than of ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1), the final those of 5-aminolevulinate synthase [succinylCoA:glycine C- enzyme in the pathway, controls the rate of heme synthesis succinyltransferase (decarboxylating), EC 2.3.1.37], the initial during Friend cell induction, because (i) ferrochelatase is not enzyme, or ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), detectable in uninduced Friend cells, (ii) the increase in heme the final enzyme of the pathway. Ferrochelatase activity was synthesis after induction with Me2SO follows the increase in not detectable in the uninduced cell. 5-Aminolevulinate synthase ferrochelatase activity, not that of ALA and the activity increased during the first 24 hr of induction; porpho- synthase, (iii) bilinogen deaminase activity began to increase after 48 hr and cell growth medium contains traces of excreted protoporphyrin ferrochelatase activity, after 72 hr. However, the induction of but not of other porphyrins. heme synthesis followed the same time course as that of ferro- chelatase activity, not that of 6-aminolevulinate synthase ac- MATERIALS AND METHODS tivity. The cellular growth medium was found to contain traces Cell Lines and Cell Culture. Friend cell lines 707 clone of protoporphyrin but not of other porphyrins. Thus, ferroche- B10/1 and FwT 1/1 were a gift of P. R. Harrison. Cells were latase is shown to be rate limiting for heme synthesis during early stages of Friend cell induction. A Friend cell variant (Fw), grown in F12 medium supplemented with extra MEM amino which is not inducible except in the presence of exogenous acids and 10% fetal calf serum (GIBCO-Biocult). Cell numbers hemin, was also studied. Al the enzymes of heme synthesis were determined with an electronic cell counter (Coulter except ferrochelatase were inducible by butyric acid. Ferro- Electronics, model D). Me2SO (Merck) and n-butyric acid chelatase was not inducible by butyric acid or hemin plus bu- (Sigma) were added to cultures in logarithmic growth to con- tyric acid. These cells also excrete protoporphyrin. The failure centrations of 1.5% (vol/vol) and 2.0 mM, respectively, to in- to induce ferrochelatase activity is believed to be the cause of, duce differentiation. Control cultures were refed during each not a consequence of, the noninducibility of this cell line. experiment to maintain all cells in logarithmic growth. Friend cells are cultured erythroleukemia cells from Friend Enzyme Assays. Cell cultures were chilled on ice and cen- virus infected mice. When grown in the presence of dimethyl trifuged immediately (1500 rpm, 10 min). The cell pellets were sulfoxide (Me2SO) or other agents, the cells undergo consider- washed twice in ice-cold saline (150 mM NaCI) before being able erythroid maturation in vitro (1). This involves not only resuspended in 2 ml of ice-cold saline and lysed by ultrasonic the synthesis of hemoglobin, but also morphological, mem- homogenization (Polytron). Portions of the cell lysates were brane, and enzymic changes (1-6) typical of normal erythroid taken for enzyme assays and for protein determinations (12). differentiation. Thus, Friend cells provide a useful in vitro ALA synthase was assayed by a modification of the method system for studying the later stages of erythropoiesis (see refs. of Freshney and Paul (13). One hundred microliters of ho- 7 and 8 for recent reviews). mogenate was incubated with 100 Al of an incubation mixture The increase in heme synthesis stimulated by Me2SO (1) is containing 0.12 M phosphate buffer (pH 7.0), 0.5 M sucrose, reflected in increases in the activities of the enzymes of heme 40 mM MgCl2, 20 mM EDTA, 8 mM mercaptoethanol, and 1 synthesis: 6-aminolevulinate (ALA) synthase [succinyl-CoA: mM glycine, and with 200,uA of a reaction mixture containing glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37] 250,uCi of [2-14C] glycine in 0.5 mM glycine, 0.5 mM sodium (9), ALA dehydratase, and porphobilinogen deaminase [uro- malate, 3 mM MgCI2, 0.4 mM pyridoxal phosphate, and 0.05 porphyrinogen I synthase, porphobilinogen ammonia-lyase M phosphate buffer (pH 6.8) for 1 hr at 370C in a shaking water (polymerizing), EC 4.3.1.8] (10). ALA synthase, the first enzyme bath. The reaction was stopped by adding 200,ul of this mixture of the pathway, is usually taken to be the controlling rate-lim- to 40 Al of 50,M ALA in 0.5 ml of 1.5 M trichloroacetic acid. iting enzyme for heme synthesis. There is considerable evidence The protein precipitate was then removed by centrifugation, for this role of ALA synthase in human liver and for the control and 10 Al of the clear supernatant was spotted on Whatman of ALA synthase by hemin in rabbit and chicken reticulocytes 3MM paper for electrophoresis. ALA and glycine were sepa- (11). Thus heme synthesis in Friend cells would be expected to rated by high-voltage electrophoresis in 0.05 M potassium parallel the increase in ALA synthase activity. Instead, maxi- phthalate buffer, pH 4, at 2 kV for 1 hr at 4°C (Locarte high mum heme synthesis is delayed up to 48 hr from the induction voltage electrophoresis). The paper was then dried at the spots of ALA synthase activity (9, 10), suggesting control by other visualized with ninhydrin, cut out, and burnt in a sample oxi- factors. dizer (Intertechnique) prior to determination of f radioactivity on a Packard Tricarb f counter. Results were expressed as pmol The publication costs of this article were defrayed in part by page of ALA synthesized per mg of protein hr-1. charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: ALA, S-aminolevulinic acid; Me2SO, dimethyl sulfox- this fact. ide. 833 Downloaded by guest on September 28, 2021 834 Cell Biology: Rutherford et al. Proc. Natl. Acad. Sci. USA 76 (1979) ALA dehydratase (EC 4.2.1.24) was measured by the method of appropriate porphyrin standards. In all cases, an internal of Berlin and Schaller (14). standard of the diethyl ester of protoporphyrin was added. Porphobilinogen deaminase was assayed by the method of Frydman and Feinstein (15). Sonicated material (0.2 ml) was RESULTS AND DISCUSSION incubated with 0.1 ml of porphobilinogen (0.82 mM) and 1 ml Activities of Heme Biosynthetic Enzymes in Friend Cells of a solution of reduced glutathione (3.25 mM) in phosphate with or without Me2SO. Friend 707 cells were grown for 4 days buffer (83 mM, pH 7.65). This was incubated in stoppered tubes with or without Me2SO, and the enzymes of heme synthesis in the dark for 1 hr at 370C, and the reaction was stopped by were assayed. Results are shown in Table 1. All the enzymes the addition of 9 ml of 2:1 (vol/vol) ethyl acetate/acetic acid. were inducible by Me2SO. However, the activities of the in- This was centrifuged to remove the precipitate, and the pre- termediate enzymes were much higher than those of the first cipitate was re-extracted with 5-ml portions of 2:1 ethyl ace- and last enzymes in the pathway, ALA synthase and ferro- tate/acetic acid until no more porphyrin fluorescence was chelatase, respectively. These two enzymes are located in the observed. The total volume of extract was noted, and 0.4 ml was mitochondria. Coproporphyrinogen oxidase, which is also a mixed with 5 ml of 0.5 M HCl. The fluorescence in this sample mitochondrial enzyme, exhibited much higher activity. Thus was determined on a spectrofluorimeter to measure the for- the differences in observed activities are not simply between mation of uroporphyrin. mitochondrial and cytosol enzymes. The assay method for uroporphyrinogen decarboxylase (EC Ferrochelatase activity was not detectable in uninduced cells. 4.1.1.37), based on that of Frydman and Feinstein (15), utilizes We estimate the limits of detection to be 1/50th to 1/100th of two stages. The first, to produce uroporphyrinogen, the sub- the levels observed in induced cells. On this basis, ferrochelatase strate for the reaction, is based on the previous assay of por- activity might appear to be limiting. The results, however, must phobilinogen deaminase. For the first stage, 0.2 ml of lysate was be treated with caution, because in vitro results are subject to incubated as above for 1 hr at 370C. Then 0.19 ml of a solution artifact. In particular, ferrochelatase is an unstable enzyme, of potassium dihydrogen orthophosphate (0.4 mol/liter) was with requirements for anaerobic conditions and membrane added to the reaction mixture, and the incubation was contin- glycolipid (11). Nevertheless ferrochelatase activity could be ued for another hr at 370C. The reaction was stopped with 9 consistently detected in Me2SO-stimulated cells.