Ferritin synthesis on polyribosomes attached to the endoplasmic reticulum

Ferritin Synthesis on Polyribosomes Attached to the Endoplasmic Reticulum Maria C. Linder, Navid Madani, Rachel Middleton, A. Miremadi, Gaetano Cairo, Lorenza Tacchini, Luisa Schiaffonati, and Emilia Rappocciolo MCL, NM, RM, AM. Department of Chemistry and Biochemistry, California State University-Fullerton, Fullerton, California U.S.A.-GC, LT, LS, ER. Istituto di Patologia Generale de1 l’llniversita degli Studi, Centro di Studio sulla Patologia Cellulare de1 CNR, Milan, Italy

ABSTRACT The evidence that ferritin is synthesized both on free polyribosomes and on polyribosomes attached to the endoplasmic reticulum is reviewed. Evidence that some ferritin is secreted from cells after synthesis on bound polyribosomes was found to be inconclusive.

INTRODUCTION Fertitin is best known for its function in the detoxification and storage of iron within cells. In response to increased iron, ferritin protein and ferritin iron accumulate within cells, such as the hepatocytes and Kupffer cells of the liver [see Ref. 11. The

mechanism of this iron regulation primarily involves control by iron of ferritin mRNA translation [see Kiihn and Her&e, this volume, for review]. The ferritin produced is in the form of a symmetrical shell, within which iron atoms are deposited as ferric oxyhydroxide [see Andrews et al., this volume, for review]. It is found mainly in the cytosol of cells like those of the liver, and it has been assumed that this ferritin is synthesized on free polyribosomes, as this is where mRNAs for soluble intracellular proteins are thought to be translated [see Ref. 21. However, ferritin is found not just within cells but also extracellularly in the blood plasma, and some of the extracellular “serum ferritin” differs from intracellular ferritin. Some of it binds to concanavalin A, indicating the presence of carbohydrate.

Address reprint requests to: Dr. Gaetano Cairo, Istituto di Patologia Generale dell’Universita degli Studi, Centro di Studio sulla Patologia Cellulare de1 CNR, Via Mangiagalli 31, 20133 Milano, Italy. Journal of Inorganic Biochemistry, 47.229-240 (1992) 0 1992 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010

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Moreover, Cragg et al. [see Ref. 31 showed that ferritin isolated from human serum (with hemochromatosis) contained glycosylated subunits of mr 23,000; and more recently, Goode et al. [see Refs. 4 and unpublished work] have found that ferritin isolated from serum of normal horses contains subunits of mr 28,ooO as well as larger subunits, while being smaller overall, with a molecular mass of 160,000 (compared with about 480,000 for “standard” intracell~ar ferritins). In addition, serum ferritin has a much lower iron content (Fe:protein about 2% [see Ref. 51) than intracellular, iron-storage ferritin (lo-30%). The presence of ferritin in serum raises the question of whether it is secreted or accidentally leaked from damaged cells especially since it increases in conditions such as inflammation and cancer [see Refs. 5-71. If ferritin were secreted, it would be expected to be synthesized on polyribosomes attached to the endoplasmic reticulum (ER), so that the ferritin could enter the channels of these organelles, be glycosylated, incorporated into the vesicles of Golgi, and exocytosed. Konijn et al. [see Ref. 81 showed that (ER)-bound polyribosomes from rat liver incorporated radioactive amino acid into a different form of ferritin in vitro than fenitin made by free polyribosomes. More recently, Linder et al. [see Ref. 91 found that heart tissue contained significant amounts of a low-iron 7 S ferritin which had striking similarities to ferritin isolated from serum of normal horses. This led to the development of a procedure for separating free and ER-bound polyribosomes from rat heart tissue and the demonstration that in this tissue about half of the ferritin mRNA was associated with the ER-bound polyribosomes [see Ref. lo]. Schiaffonati et al. [see Ref. 111 also demonstrated the presence of ferritin mRNA on ER-bound polyribosomes from livers of rats treated with turpentine to induce inilammation; and Campbell et al. [see Ref. 121 showed that inflammation changed the distribution of ferritin message between the mRNP and polyribosomes in both the liver and the spleen. The work described here further addresses the concept that ferritin is synthesized on ER-bound polyribosomes by several tissues, and considers how this may or may not be related to secretion of ferritin and changes in serum ferritin that occur in conditions of inflammation and cancer. MEXHODS For the studies by the California group, adult female Sprague-Dawley rats (Simonson Laboratories, Gilroy, CA) were either untreated, injected i.p. with 0.3 ml, 0.9% NaCl, or ferric ammonium citrate (350 pg Fe), or intramuscularly with 0.25 ml turpentine in both thighs, 2 and 12 hr before sacrifice. Rats were killed by exsanguination under pentobarbital anesthesia, as previously described [see Ref. 131. Tumor-bearing rats and their controls were of the Fischer CDF strain (National Cancer Institute, Bethesda, MD). Dunning mammary tumor DMRA-5A was implanted subcutaneously in the flank [see Ref. 131. Tissues from pairs of rats were harvested and processed in parallel. In the Italian studies, the rats were albino males of the Wistar strain (about 200 g). Some were injected i.p. with 400 rg of Fe/100 g body weight as ferric ammonium citrate, controls with ammonium citrate alone, and killed 5 hr later. For separation of free and ER-bound polyribosomes in studies by the California group, dilute post-nuclear supematants of tissue were fractionated on discontinuous sucrose gradients, containing layers of 2.0 M, 1.8 M, and 1.38 M sucrose, as previously described [see Ref. lo], with RNAsin (Promega, Madison, WI) present

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throughout the gradient. RNA was extracted from portions of the gradient containing the purified bound and free polyribosomes, as well as from the upper 5~ of the gradient (designated the mRNP fraction), for slot-blot hybridization with P-cDNA for rat L ferritin, all as previously described. Control probes were for rat tubulin and ceruloplasmin. In studies by the group in Milan, membrane-bound and free polyribosomes were prepared by the method of Reference 14, as modified by Schiaffonati et al. [see Ref. 1I]. Preparation of microsomes and washing with high salt were as described by Adesnik and Maschio [see Ref. 151 except that the concentration of NaCl was 0.3 M. Polyribosomal or microsomal RNA, extracted with phenol-chloroform [see Ref. 111 was applied to electrophoresis under denaturing conditions (20 pg aliquots in 1.2% agarose/formaldehyde gels) and blotted to Hybond C Extra nitrocellulose filters (Amersham). Baked filters were prehybridized and hybridized to 32P-labeled probes prepared from clones for rat L and H ferritin subunits (kindly provided by H. N. Munro; [see Refs. 16, 171) as described in Ref. 18. DNA probes were labeled by nick translation with an Amersham kit. Quantitation of autoradiograms was by laser densitometry (LKB) making sure that film exposure was in the linear range. Values were corrected for the amount of rRNA loaded into each lane based on densitometer quantification of ethidium bromide-stained 28 and 18 S RNA on the filters. Cell-free protein synthesis was performed by addition of rat liver ER-bound polyribosomes (0.5 A,,) to rabbit reticulocyte lysates @omega), and incubation (60 min, 30”) in the presence of “S-methionine (40 &i/25 ~1). In some cases, 2 ~1 portions of canine pancreatic microsomal membranes (Promega) were included in the mixture. In the protease protection studies, aliquots of the incubated mixture were digested with 0.3 mg/ml proteinase K (Boehringer) in 10 mM CaCl, +- 2% Triton X-100, for 1 hr at 0”, before immunoprecipitation (see below). Microinjection and culturing of oocytes were carried out as follows. Plasmid (pSP64T) containing human H subunit cDNA [see Ref. 191 was linearized with EcoRl and transcribed in vitro at 40” for 1 hr. The reaction mixture contained 5 pg DNA, 40 units SP6 polymerase, 40 mM Tris pH 7.9,6 mM MgCl,, 10 mM DTT, 2 mlvl spermidine, 0.5 mM ATP, GTP, CTP, UTP, 0.5 mM m’-GpppG, and 100 units RNAsin. The reaction was stopped by digestion with DNAse (2 units; 15 min, 37”), and total RNA was extracted with phenol-chloroform, precipitated with ethanol and dissolved in water at a concentration of about 1 mg/ml. Manually dissected Xenopus oocytes (Blades Biological, Edenbridge, Kent, U.K.) were each microinjetted with about 40 ng of RNA, then incubated for 2 hr with 5 pCi3H-leucine (60 Ci/mmole) in Barth’s saline solution. (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO,, 0.82 mM MgSO,, 0.33 mM Ca(NO,),, 0.41 mM CaCl,, 10 mM Hepes, pH 7.6). After a 24 hr chase with cold (10 n&I) leucine, medium was saved and oocytes were homogenized in 20 ml!4 Tris, pH 7.6, containing 0.1 M NaCl, 50 mM Mg acetate, 1% Triton X-100, and 1 mM PMSF (lO~l/oocyte). Homogenates were briefly centrifuged to remove yolk aggregates before analysis of the translation products. Duplicate samples were analyzed for total TCA precipitable radioactivity [see Ref. 201 and for incorporation of radioactivity into ferritin. For the latter, samples were incubated overnight with monoclonal antibody to the human H ferritin subunit or with polyclonal antibody to rat liver fenitin [see Ref. 111 in 50 mM Tris-HCl (pH 7.6), containing 150 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, and 0.25% gelatin. Antigen-antibody complexes were harvested by incubation with protein A-Sepharose beads (Pharmacia) for 2 hr at 4”, washed 4 x with incubation buffer,

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resuspended in SDS sample buffer and analyzed on 15% polyacrylamide-SDS gels [see Ref. 211. Gels were processed for fluorography [see Ref. 221 and exposed to Amersham Hyperfilm MP. Relative amounts of immunoprecipitated radioactivity were quantitated by densitometry. Ferritin in rat serum was assayed with a “Western” type of ELISA, by the California group. For this, 250 ~1 portions of serum, diluted 2-4-fold in 0.9% NaCl, were slot-blotted onto nitrocellulose membranes (BRL, Bethesda, MD), and these were incubated successively with rabbit gamma globulin (made against horse spleen ferritin that contained only 19 and 21 k subunits), followed by goat anti-rabbit IgG, etc., using a biotin-streptavidin amplified ELISA kit system (Bio-Rad, Richmond, CA).

RESULTS Fischer rats bearing subcutaneous implants of the fast-growing mammary tumor, Dunning DMBA-SA, were used by the California group as a model to study the effect of cancer on ferritin mRNA distribution to free and ER-bound polyribosomes, as well as to study tissue ferritin content. Generally, rats were sacrificed when their tumors were of a large size (> 20 g). Control rats were sacrificed and their tissues were processed in parallel. In the case of the tumors, only grossly viable tissue was assayed. Table 1 shows clearly that the livers of these female rats, with or without tumors, had a high total iron and ferritin iron content, while the tumors had much less. Based on Fe:protein ratios of the pure ferritins from such tissues, it was calculated that there was also much less ferritin protein in the tumors than in liver (or spleen). This is not surprising, since the liver is a major iron-storing organ. Figure 1 shows the relative concentration of ferritin mRNA in RNA extracted from three different fractions of tissues from tumor-bearing rats. In all three tissues, the concentration of ferritin mRNA was highest on the ER-bound polyribosomes, and least in the mRNP fraction. Of even greater interest, however, was the finding that the concentration of ferritin message in tumor RNA was very high, and was in fact considerably higher than that of liver (and spleen) RNA. This is particularly striking in view of the fact that the tumor had relatively little ferritin iron and ferritin protein. The difference in ferritin mRNA concentration could not be attributed to a large difference in tissue RNA concentration, as recoveries of RNA from all three tissues was within the same range, per g. The presence of so much message on the

TABLE 1. Iron and Ferritin Content of Tissues from Pairs of Rats With and Without Implanted Tumors Total Fe (rcgh.3)

Ferritin Fe Mll3)

Fe&in

Protein

(mg/g)

Liver Controlrats Tmnor-bearing rats Tumor(viableportion)

410,389 293,430 37,32

242,205 173, 178 15, 15

0.87,0.82 0.69,0.71 0.13.0.13

Calculations of ferritinproteincontent were based on Fe:protein ratios of 0.25 and 0.12 for liver and tumor ferritins detemdned in earlier experiments.

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FIGURE 1. Concentration of ferritin mRNA in RNA from free (P) and ER-bound (B) polyribuscmes and the mRNP (R) fraction of livers, spleens, and tumors of tumor-bearing rats. Autoradiographs of slot blots hybridized with 32P-cDNA for rat L-ferritin for tissues of pairs of rats processed in parallel.

bound polyribosomes of the tumor, and the relatively low concentration of ferritin in the tumor both suggest that the tumor is secreting ferritin into the blood. Freliminary evidence that levels of ferritin were increased in the serum of these same tumor-bearing rats is shown in Figure 2. Clearly, the slots containing serum from the tumor-bearing rats (T) developed a much greater reaction to ferritin antibody than those with serum from unimplanted control rats (C). The potentially contrasting effect of iron and inflammation on the distribution of ferritin rnRNA in liver was also examined. Iron induction results in the accumulation

FIGURE 2. Concentration of serum ferritin in rata with (T) and without (C) implanted tumors, determined by a “Western-type” ELBA.

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of ferritin iron and ferritin protein intracellularly [see Refs. 5, 231. Intlammation lowers the level of circulating iron in the blood plasma [see Ref. 241, and also stimulates liver ferritin synthesis [see Refs. 6, 71. However, although it was verified that liver ferritin synthesis was increased 12 hr after turpentine treatment (Madam and Linder, unpublished work), turpentine appeared to have little impact on ferritin and iron accumulation in the liver, at least within 24 hr after injection. [Ferritin iron concentrations were 233 f 28, 261 f 24, and 224 f 26 (Means + SD, N = 4), at 0, 12, and 24 hr after turpentine injection, consistent with earlier studies [see Ref. 61.3 Figure 3 summariz es the data of Madani and Linder (unpublished) on the relative concentrations of ferritin mRNA in RNA extracted from free and ER-bound polyribosomes and the RNP fraction of liver, 12 hr after injection of turpentine, 2 hr after injection of ferric ammonium citrate, or in untreated or saline-treated controls. In untreated animals there was RNA on both the free and bound polyribosomes, and a significant portion in the mRNP fraction. In acutely iron-treated rata, the highest concentration of message was in RNA from the free polyribosomes, supporting the concept that ferritin made for intracellular storage of iron is synthesized on free cytoplasmic polyribosomes. In contrast, inflammation induced by turpentine (at least at 12 hr), resulted in the highest concentration of ferritin message on the ER-bound polyribosomes. This indicates that iron and inflammation have different effects on the distribution of ferritin message intracellularly, and suggests that infIammation is promoting synthesis of ferritin for secretion from the liver. These findings were borne out by others in which post-mitochondrial supematants were applied to linear (550%) sucrose gradients, and the RNA extracted from fractions obtained after sedimentation were slot-blotted and hybridized with ‘*PcDNA for ferritin. No detergent was added, so polyribosomes bound to the ER would tend to sediment more slowly than comparably large free polyribosomes. The unpublished data of Madam and Linder are summarixed in Figure 4. Again, it is clear that each condition gave a different result. Ferritin mRNA from livers of control rats was most abundant in fractions 2 and 3, that from in&med rats (at 12 hr) was further down the gradient, and that from iron-treated rats (at 2 hr) was at the bottom of the gradient, again indicating a differential effect of iron and inflammation on the distribution of ferritin mRNA within liver cells. (It is of course not clear whether we are here dealing with differences ascribable only to hepatocytes and/or also to Kupffer cells.)

5

‘;

!! E 8

so l

s

so

2

40

0 i

E g r LL

20

f 3 d

0

Control

+ Iron

+

Turp

0

FIGURE 3. Relative concentrations of ferritin mRNA in liver mRNP (R), free polyribosomes (F’) and ERbound (B) polyribosome fractions of RNA from rats untreated, treated with iron for 2 hr. or treated with turpentine for 12 hr. Summary of densitometric data obtained from slot blots (as in Fig. 1) from unpublished work of Madani and Linder. (Means for g- 10 determinations; **OP< 0.01 for difference from values for untreated (9, and iron treated (0) rats, respectively .)

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8 5

1

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Control

I+

furp

B-

+

Iron

5r” t

4-

0' 3: = J

2-

B l-

12

3

4

5

6

7

FIGURE 4.

Distribution of fen-kin mRNA along linear sucrose gradients after sedimentation of liver post-mitochondrial supematants, without detergent. Average autoradiographic values for samples from control rats, or those treated with turpentine (12 hr), or iron (2 hr). Fractions are from the top (1) to the bottom (7) of the gradient.

The Milan group also studied the distribution of ferritin mRNA to free and ER-bound polyribosomes from livers of rats by Northern analysis [Tacchini, et al., unpublished work]. Figure 5 shows that ferritin mRNA was detected in RNA extracted from the ER-bound polyribosomes, and that this was the case whether 32P-cDNA probes for L and H ferritin were applied. As in studies by the California group, ferritin mRNA was on the bound polyribosomes even of untreated rats. However, it appeared to be increased by iron treatment, the increase being somewhat greater on the free polyribosomes (here 5 hr after iron injection). This suggests that the mRNA on the bound polyribosomes is also affected by iron. (This was not seen by the California group in the studies with liver 2 hr after iron injection and may reflect differences occurring over time.) To confirm that the ferritin mRNA was truly bound to the ER and not just a contaminant, attempts were also made to wash out the message by treatment with a high salt solution [see Ref. 151 known to release loosely

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MMF + -

-

m

t= B I

-

L +

5

FIGURE 5. Effect of high-salt treatment and iron injection of rats on distribution of ferritin mRNA associated with rat liver microsomes (M) or free (F) and membrane-bound (E3) polyribosomes. Autoradiographs of Northern blots (20 ug RNA/slot) hybridized with rat L and H ferritin cDNAs.

bound polyribosomes from membranes. Figure 5 shows that significant amounts of ferritin mRNA remained firmly bound to the ER membranes. If ferritin is synthesized on bound polyribosomes for secretion, the translated product should enter the channels of the ER, where it could be glycosylated and also protected from proteases on the outside of the membranes. Indeed, in classic studies, insertion of the translated polypeptide into ER channels was deduced from the results of experiments in which it was demonstrated that the polypeptides synthesized could be protected at least partially from proteolysis (by exogenous proteases) when microsomal vesicles were included with the polyribosomes in an in vitro translation system. This approach was taken by the Milan group. Figure 6 shows that there were

no changes in the apparent molecular weight of ferritin subunits produced after supplementation of the translation mixture with microsomal inembranes, suggesting the absence of signal sequence cleavage and/or glycosylation. Similarly (Fig. 6), the presence of added microsomal membranes did not protect the ferritin subunits synthesized from proteinase K digestion. Immunoprecipitation of albumin from an aliquot of the same translation reaction showed that it was correctly cleaved and protected (data not shown). This suggests there was no co-translational insertion of ferritin into ER channels. However, as only a small portion of the product is usually protected in such experiments, the conclusion is less certain. Another approach to the matter of ferritin secretion was begun by the group in Milan, using oocytes of Xenopus laevis into which mRNA from human H ferritin was injected. Such oocytes have been shown by others to faithfully and efficiently synthesize, process, and secrete heterologous proteins [see Ref. 251. Upon analysis of the radioactive intra- and extracellular proteins synthesized by the microinjected oocytes, using SDS-PAGE and fluorography, +immunoprecipitation with H subunit-specific antibody, ferritin was detected only intracellularly (Fig. 7) and repre-

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FIGURE 6. In vitro translocation assays for ferritin synthesized by membrane-bound polyribosomes added to reticulocyte lysates, in the absence and presence of canine pancreatic microsomal membranes (m). Fluorographs of translation products immunoprecipitated with ferritin antibody and separated in SDS-PAGE electrophoresis. Translation products were digested with proteinase K (pk) in the absence (lanes 3, 4) or presence (lane 5) of detergent (det).

sented 40-50X of the total protein synthesized. Extracellular ferritin was not detected even in immunoprecipitates of culture medium from 15 oocytes (incubated for more than 24 hr). DISCUSSION Most of the findings presented here support the concept that ferritin is secreted rather than leaked by cells into the blood plasma. Apart from the structural differences between cell and serum ferritins already noted in previous studies and the evidence that serum ferritin is at least partially glycosylated, we have presented extensive evidence that ferritin mRNA is indeed found on polyribosomes that translate proteins while bound to the endoplasmic reticulum, and that ferritin synthesis by such bound polyribosomes was actually occurring. It is well-known and well-accepted that proteins synthesized there are either incorporated into endocytic vesicles for secretion and/or incorporated into membranes that eventually join the cell surface (or are part of other organelles) [see Ref. 21. Indeed, there appear to be no examples of proteins synthesized on the ER where there is evidence that secretion (or membrane insertion) does not occur. In addition, we have presented evidence that in two conditions in which serum ferritin concentrations are known to rise (intlammation and cancer), there is an increased association of ferritin mRNA with the ER-bound polyribosomes of the liver (as well as a shift in ferritin message to polyribosomes in the spleen [see Ref. 121. Moreover, in the case of tumor-bearing rats, there is a high concentration of ferritin mRNA on the ER-bound polyribosomes also of the tumor, and little accumulation of ferritin in the tumor cells, especially relative to liver. This strongly suggests that the ferritin synthesized by the tumor is being released rather than retained by the tumor cells, and that both the liver and the tumor are secreting increased amounts of ferritin that could account for the increase in serum ferritin occurring in cancer.

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kd

1

2

3

4

5

FIGURE 7. Synthesis of human H ferritin subunits by Xenopu LT OOCJ &.s microinjected with mRNA. Immunoprecipitated translation products of oocytes injecte xi with human H mRIUA(lanes 2, 3, 5), or preproendothelin mRNA (lane 4). Lau1e 1, molecular weight markers; 2, immunoprecipitate of homogenatefrom l/2 oocyte; 3,4, whole: homogenate from l/2 oocyte; 5, immunoprtxipitateof medium from 15 oocytes.

The association of ferritin message with the ER is not fortuitous or due to contamination with free polyribosomes. It could not be washed out of the ER with high salt solutions, and it was verified that a good separation of free and bound polyribosomes had occurred, using probes for mRNA of other proteins that are known to be retained (tubulin, NADH cytochrome b, reductase) or secreted (albumin, ceruloplasmin, in liver; atrial natriuretic factor, in heart; see Reference 10, also Madani and Linder and Tacchini et al. unpublished work). [The method developed by the California group was also validated using radiolabeled free and ER-bound polyribosomes, and showed only 3-5 96 cross-contamination [see Ref. lo].]

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15. M. Adesnik and F. Maschio, Eur. J. Biochem. 114: 271 (1981). 16. E. A. Leibold, N. Aziz, A. J. P. Brown, and H. N, Munro, .I. Biol. Chem. 259: 4327 (1984). 17. M. T. Murray, K. White, and H. N. Munro, Proc. Nat/. Acad. Sci. USA 84: 7438 (1987). 18. G. Cairo, L. Tacchini, L. Schiaffonati, E. Rappocciolo, E. Ventura, and A. Pietrangelo, Biochem. J. 264: 925 (1989). 19. F. Costanzo, M. Columbo, S. Staempfli, C. Santoro, F. Marone, R. Frank, H. Delius and R. Cortese, EMBO J. 3: 23 (1984). 20. R. J. Mans, and G. D. Novelli, Arch. Biochem. Biophys. 94: 48 (1961). 21. U. K. Laemmly, Nature 227: 680 (1970). 22. R. A. Laskey, and A. D. Mills, Eur. .I. Biochem. 56: 335 (1975). 23. J. W. Drysdale, and H. N. Munro, J. Biol. Chem. 241: 3630 (1966). 24. M. C. Linder, Nutritional Biochemistry and Metabolkm, in M. C. Linder, Ed., Elsevier, New York, 1991, pp. 215-276. 25. J. J. Heikkila, ht. J. Biochem. 22: 1223 (1990). Received January 27, 1992; accepted February 4, 1992