Proc. Nati. Acad. Sci. USA Vol. 77, No. 6, pp. 3715-3719, June 1980 Medical Sciences

Inborn errors of molybdenum metabolism: Combined deficiencies of and xanthine dehydrogenase in a patient lacking the molybdenum JEAN L. JOHNSON*, WILLIAM R. WAUD*t, K. V. RAJAGOPALAN*, MARINUS DURANf, FRITS A. BEEMERt, AND SYBE K. WADMANt *De~partment of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710; and tUniversity Children's Hospital, Het Wilhelmina Kinderziekenhuis, Nieuwe Gracht 137, Utrecht, The Netherlands Communicated by Philip Handler, March 28,1980

ABSTRACT A patient suffering from a combined deficiency MATERIALS AND METHODS of sulfite oxidase (sulfite dehydrogenase; sulfite:ferricytochrome c , EC 1.8.2.1) and xanthine dehydrogenase Analysis of Urinary Metabolites. Sulfite concentrations (xanthine:NAD+ oxidoreductase, EC 1.2.1.37) is described. The were estimated by using Merckoquant Sulfit-test strips (E. patient displays severe neurological abnormalities, dislocated Merck, Darmstadt, West Germany; also available from Scien- ocular lenses, and mental retardation. Urinary excretion of sulfite, thiosulfate, S-sulfocysteine, taurine, hypoxanthine, and tific Products). Thiosulfate was quantitated by using the method xanthine is increased in this individual, while sulfate and urate of S6rbo (9) with minor modifications, and sulfate was measured levels are drastically reduced. The metabolic defect responsible with a radiochemical procedure according to Miller et al. for loss of both activities appears to be at the level of (10). the molybdenum cofactor common to the two . Im- Amino acids were analyzed with a Technicon TSMI amino munological examination of a biopsy sample of liver tissue re- acid analyzer, using a standard procedure. For a quantitative vealed the presence of the xanthine dehydrogenase protein in near normal amounts. Sulfite oxidase apoprotein was not de- recovery of acidic amino acids the sample cartridges were tected by a variety of immunological techniques. The plasma loaded with a strong anion-exchange resin (Technicon type S molybdenum concentration was normal; however, hepatic chromobeads). The identities of S-sulfocysteine and taurine content of molybdenum and the storage pool of active molyb- were confirmed by high-voltage electrophoresis (40 V/cm; denum cofactor present in normal livers were below the limits formic acid/acetic acid/water, 15:10:75 V/V; pH 1.18). S- of detection. Fibroblasts cultured from this patient failed to Sulfocysteine can be distinguished from cysteic acid by its express sulfite oxidase protein or activity, whereas those from differing color with the ninhydrin/isatin staining reagent. the arents and healthybrother of the patient expressed normal Urinary purines and pyrimidines were analyzed by two-di- leve s of this enzyme. mensional thin-layer chromatography (11) and by quantitative Molybdenum is utilized in trace amounts by animal systems for cation exchange column chromatography (12). The same the functions of sulfite oxidase (sulfite dehydrogenase; sul- thin-layer chromatographic technique was used for the analysis fite:ferricytochrome c oxidoreductase, EC 1.8.2.1), xanthine of urinary hydantoin 5-propionic acid after loading with L- dehydrogenase (xanthine:NAD+ oxidoreductase, EC 1.2.1.37), histidine. Uric acid was measured with uricase (Leo Pharma- and aldehyde oxidase (aldehyde:oxygen oxidoreductase, EC ceutical, Ballerup, Denmark). Serum molybdenum concen- 1.2.3.1). All of these enzymes contain the metal as a complex trations were determined by neutron activation analysis (per- with an apparently identical organic moiety, the molybdenum formed by I. Lombeck, University Children's Hospital, Dus- cofactor (1). The complete structure of the cofactor has not been seldorf, West Germany). elucidated, but the presence of a pterin as a structural compo- Enzyme, Molybdenum, and Molybdenum Cofactor As- nent of cofactor isolated from chicken liver sulfite oxidase, milk says. Hepatic sulfite oxidase and xanthine dehydrogenase ac- xanthine oxidase, and Chlorella nitrate reductase has been tivities and molybdenum content were determined as described demonstrated (2). Of the molybdoenzymes that have been (13), using a sample of tissue obtained by surgical biopsy. characterized, nitrogenase alone contains a unique entity, the Xanthine dehydrogenase activity was also assayed by a fluo- iron-molybdenum cofactor (3), that is not functionally inter- rometric procedure using 2-amino-4-hydroxypteridine as changeable with the molybdenum cofactor (4). (14). Superoxide dismutase was assayed by Hosni Mutants defective in the ability to synthesize activity mo- Hassan of Duke University by its ability to inhibit xanthine lybdenum cofactor have been described in Aspergillus (5) and oxidase-mediated reduction of cytochrome c (15). Both cya- Neurospora (6). Inactive molybdenum enzymes are synthesized nide-sensitive (manganese-containing) and -insensitive (copper, in rats treated with large amounts of tungsten (7, 8). Normal zinc-containing) enzyme activities were measured. Monoamine levels of activity are restored upon molybdenum supplemen- oxidase activity was assayed with benzylamine (16) and alcohol tation. In this paper we report biochemical characterization of dehydrogenase with ethanol (17) as respective substrates. a human lacking functional molybdenum cofactor. The se- Glutamate dehydrogenase was assayed as described (18). verely retarded patient displays all the clinical features known Active molybdenum cofactor was assayed by its ability to to occur in both xanthine dehydrogenase deficiency and sulfite reconstitute sulfite oxidase activity in a preparation of demol- oxidase deficiency. ybdosulfite oxidase isolated from tungsten-treated rats (19) and by its ability to reconstitute nitrate reductase activity in extracts The publication costs of this article were defrayed in part by page of the Neurospora mutant nit-i (20). charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate t Present address: Southern Research Institute, 2000 Ninth Ave. S., this fact. Birmingham, AL 35205. 3715 Downloaded by guest on September 26, 2021 3716 Medical Sciences: Johnson et al. Proc. Nati. Acad. Sci. USA 77 (1980)

Table 1. Characteristic urinary excretion of uric acid, oxypurines, and cysteine metabolites in a patient with a combined deficiency of xanthine dehydrogenase and sulfite oxidase Urinary excretion, mmol/g of creatinine Creatinine, Uric Xan- Hypo- Thio- S-Sulfo- Date Conditions mg/liter acid thine xanthine Sulfite sulfate cysteine Taurine Sulfate March 28, 29, Normal diet 305 0.07 5.48 0.95 + 3.57 4.43 6.16 2.92 1977 Sulfur-restricted 215 3.33 2.51 1.35 3.47 diet June 13, 14, Restricted diet + 260 0.16 4.19 0.85 + 2.23 2.43 1.15 3.30 1978 oral Mo + NaHCO3 May 28, 29, Restricted diet + 330 <0.03 3.70 0.71 + 1978 Mo + NaHCO3 + allopurinol L-Cysteine I 125 + 6.38 3.20 4.56 4.48 loading* II 110 + 4.79 3.09 4.91 2.50 III 275 + 4.42 2.80 3.60 2.87 IV 865 + 5.85 3.04 5.82 3.67 Controls 4-10 0.06-0.25 0.02-0.07 Not 0.08 <0.02 <0.7 >7.5 detected * Loading was 100 mg of L-cysteine per kg of body weight. Urine was collected in 6-hr portions, numbered I-IV. Immunological Procedures. Antisera of high titer directed Typical tonic-clonic seizures have always been present. Her against human liver sulfite oxidase and rat liver xanthine de- electroencephalogram showed diffuse irregularities and on the hydrogenase were raised in rabbits as described (7). Immuno- pneumoencephalogram widened ventricles and periventricular globulins were purified by ammonium sulfate fractionation atrophy were seen. At the age of 3 years the patient has not (0-33%) and chromatography on DEAE-cellulose DE-52 achieved any developmental milestones. She is bedridden, does (Whatman). Immunodiffusion plates were purchased from not react to light or sounds, and is extremely hypertonic. Her Vega Chemicals (Tucson, AZ). Radioimmunoassay of sulfite length and weight are now at the 50th and 3rd percentiles, re- oxidase crossreacting material was performed by a procedure spectively. to be published elsewhere. Human liver sulfite oxidase was The diagnosis of sulfite oxidase deficiency plus xanthine iodinated with '25I by using the chloramine-T procedure, and dehydrogenase deficiency was first suspected after the finding a double antibody method was used to separate bound radio- of abnormal metabolites during a multi-chromatographic activity from free. screening of the patient's urine. These metabolites included Fibroblast Studies. Fibroblasts were cultured from punch sulfite, S-sulfocysteine, taurine, xanthine, hypoxanthine, and skin biopsy materials as described (21). Cells were lysed for uric acid (see Table 1). The metabolic profile was completed analysis by sonication in 5 vol of 10 mM potassium phosphate by analyzing the urinary excretion of thiosulfate and sulfate (see buffer, pH 7.8. The lysates were centrifuged at 20,000 X g for Table 1). 10 min. Samples of the supernatant fractions were used directly Attempts at Treatment. In her first year of life the patient for radioimmunoassay or subjected to gel filtration for sulfite received normal feedings with respect to her age. After the oxidase activity assay (21). diagnosis of sulfite oxidase deficiency had been made, a low- cysteine diet was prescribed, equivalent to 12 mg of cysteine RESULTS per kg of body weight. In order to counteract a possible sulfate Clinical Summary. The patient has shown feeding dif- deficiency extra oral Na2SO4 (700 mg/day) was given. No side ficulties almost from birth. On admission a small child (length effects of this administration were observed. Because of the and weight 10th percentile) was seen with an asymmetric skull presence of xanthine gravel in the diapers, allopurinol medi- and frontal bossing. Skull circumference was at the 50th per- cation was started (10 mg/kg per day) in an attempt to shift the centile. Eye abnormalities included bilateral dislocated ocular xanthine-to-hypoxanthine ratio to the more soluble hypoxan- lenses, enophthalmus, nystagmus, and a ring of Brushfield spots. thine. Because xanthine is more soluble at higher pH, the pa-

Table 2. Levels of molybdenum, molybdoenzymes, and molybdenum cofactor in liver tissue Xanthine Sulfite Sulfite oxidase Molybdenum cofactor, dehydrogenase oxidase crossreacting units/g Molybdenum, activity, activity, material, Sulfite Nitrate Subject Itg/g units/g units/g units*/g oxidase reductase Controls Adult 0.92 0.76 13.5 - 72 Adult 0.89 0.33 27.5 75 Child, 4 mo 0.38 0.46 13.5 14.9 55 Child, 6 mo 0.40 0.44 13.4 33 312 Patient <0.05 <0.05 <0.1 <0.1 <2 <50 * Based on a specific activity of 918 units per mg of human sulfite oxidase. Downloaded by guest on September 26, 2021 Medical Sciences: Johnson et al. Proc. Nati. Acad. Sci. USA 77 (1980) 3717 tient was given 250 mg of NaHCO3 four times daily. Finally (Table 2) indicated the presence of not more than 0.7% of the a trial with oral supplements of molybdenum [up to 200 of level found in a liver sample from a control individual of ap- Mo per day, given as (NH4)3MoO4] was made. None of the proximately the same age. In the control liver sample, the dietary changes or supplements of inorganic chemicals had any amount of crossreacting material detected by radioimmuno- effect on her clinical condition. In spite of attempts at treatment assay was in excellent agreement with the amount of active the frequency of the tonic-clonic seizures increased over the sulfite oxidase present. However, because the low value ob- years and the patient's neurological condition deteriorated. tained by radioimmunoassay of sulfite oxidase in the patient Neither the incidence of the xanthine calculi nor the xan- could represent either an extremely depleted level of normal thine-to-hypoxanthine ratio changed after allopurinol medi- protein or relatively high levels of an altered with low cation. reactivity in this particular assay, two other immunologic The effect of dietary changes on the metabolism of sulfur procedures were applied. amino acids was evident. Restriction of methionine and cysteine The sulfite oxidase molecule exists as a dimer of identical intake led to a decreased urinary output of abnormal sulfur subunits. Each subunit contains one cytochrome b5-type heme metabolites, whereas the opposite was observed after loading and one molybdenum cofactor moiety, but the two prosthetic with L-cysteine. This loading test did not provoke adverse groups are present on separate domains of the polypeptide chain clinical reactions like those reported by Irreverre et al. (22) in (23). The heme-binding domain contains those antigenic sites their patient with isolated sulfite oxidase deficiency. that bind antibodies inhibitory to the catalytic activity (un- Analysis of Hepatic Molybdenum and Molybdoenzymes. published observations). Thus, an intact b5 domain, preincu- As shown in Table 2, hepatic levels of sulfite oxidase and xan- bated with antiserum directed against sulfite oxidase, can de- thine dehydrogenase activities in the patient were below the plete the serum of all inhibiting antibodies and protect native limits of detection of the assay procedures employed. In addi- enzyme from inhibition during a subsequent incubation. By tion, atomic absorption analysis failed to detect any molybde- using this technique, it is possible to assay for the presence of num in the liver sample. By neutron activation analysis the an intact heme-binding portion of the sulfite oxidase molecule serum molybdenum concentration was found to be normal. The (13). The procedure was applied, using experimental conditions nonmolybdenum enzymes superoxide dismutase, glutamate as described (13); no protection was observed, indicating the dehydrogenase, monoamine oxidase, and alcohol dehydroge- absence of the heme domain antigen in the tissue sample. nase were found to be within normal limits. Sulfite oxidase crossreacting material was also sought by Various procedures were employed to test for the presence Ouchterlony double immunodiffusion. As shown in Fig. 1A, of inactive sulfite oxidase and xanthine dehydrogenase proteins. this technique also failed to detect any sulfite oxidase protein The possible presence of nonfunctional sulfite oxidase protein in the liver sample. capable of being activated by molybdate or by molybdenum Xanthine dehydrogenase apoprotein was readily observable cofactor was assayed as follows: 0.1 g of liver was homogenized on immunodiffusion plates (Fig. 1B). Precipitin bands formed in 0.5 ml of 10 mM potassium phosphate buffer, pH 7.4. An as lines of identity with active xanthine dehydrogenase present aliquot of the homogenate was diluted 1:10 in the same buffer in liver tissue from control individuals and appeared to be equal and incubated with 2 mM sodium molybdate at 370C for 45 in intensity. Bands were easily observable without staining but min. A second aliquot was diluted 1:4 in the same buffer and were intensified for photographic purposes by using an activity mixed with an equal volume of an extract of Escherichia coli stain that requires functional flavin and Fe/S centers but is (20). The mixture was incubated at 370C for 45 min. Active independent of the molybdenum cofactor (24). The xanthine sulfite oxidase could not be detected in either incubation mix- dehydrogenase protein present in the liver sample could not ture. be activated by incubation with molybdate or a source of mo- Radioimmunoassay of sulfite oxidase crossreacting material lybdenum cofactor. Demolybdoxanthine dehydrogenase syn-

A 1? B 3

2 / 2

1 i 1 '4

\ FIG. 1. Ouchterlony double immunodiffusion of liver extracts versus antisera directed against sulfite oxidase (A) and xanthine dehydrogenase (B). Samples were prepared for immunodiffusion as follows: 0.1 g of liver was homogenized in 0.5 ml of 10 mM potassium phosphate, pH 7.8, and centrifuged at 35,000 X g for 15 min. The supernatant fraction was lyophilized and resuspended in 30 ,ul of H20. Each well contained 10 ,ul of extract. Wells numbered 4 contained extracts from control liver; wells numbered 5 contained extracts of the liver from the patient. Wells 1, 2, and 3 contained purified human sulfite oxidase (A) or partially purified human xanthine dehydrogenase (B). Antisera were in the center wells. The plates were allowed to develop for 2 days at room temperature, soaked in 0.9% NaCl for 2 hr, and stained for protein with amido black (A) or for activity by using 0.25 mg of iodonitrotetrazolium violet and 0.2 mg of NADH per ml of 50 mM potassium phosphate, pH 7.8 (B). Downloaded by guest on September 26, 2021 .....;;,\ 3718 Medical Sciences: Johnson et al. Proc. Nati. Acad. Sci. USA 77 (1980) Table 3. Levels of sulfite oxidase activity and crossreacting containing metabolites were also in agreement with patterns material in cultured fibroblasts observed previously and emphasize the usefulness of these Sulfite oxidase characteristics in diagnosis of sulfite oxidase deficiency. Because Sulfite oxidase crossreacting xanthinuria in humans is known to be a relatively benign con- activity, material, dition (29), the presence of urinary xanthine stones is probably Subject units/mg units*/mg the only clinical manifestation of xanthine dehydrogenase Controls (n = 8) 0.0057-0.0230 deficiency in this patient. (average 0.0160) The finding of a combined deficiency of at least two mo- Patient <0.0008 <0.0008 lybdoenzymes in this individual must be considered a very rare Father 0.0094 0.0100 observation.§ Although a similar biochemical situation has been Mother 0.0089 0.0091 produced experimentally in the rat by tungsten administration Brother 0.0088 0.0088 (30) and has been observed in mutant fungal systems (5, 6), an inborn error of molybdenum metabolism leading to the si- * Based on a specific activity of 918 units per mg of human sulfite multaneous deficiency of two enzymes in a human is most oxidase. unusual. However, other biochemical systems are known in which one defect leads to the expression of two apparently thesized in livers of tungsten-treated rats has also resisted all nonrelated inborn errors of metabolism. For example, defects reconstitution attempts (unpublished observations). in cobalamin synthesis have been shown to result in the simul- Assays of Molybdenum Cofactor. In addition to the mo- taneous occurrence of methylmalonicacidemia and homocys- lybdenum cofactor present in the various molybdoenzymes, tinuria (31, 32). animal.tissues normally contain a storage pool of the cofactor The biochemical data obtained thus far have indicated that on the mitochondrial outer membrane (19). This pool of co- this patient is unable to synthesize active molybdenum cofactor. factor can be assayed by its ability to reconstitute sulfite oxidase It would appear likely that this results from a primary defect activity in preparations of demolybdoenzyme isolated from in an essential step in cofactor biosynthesis, although the exact livers of tungsten-treated rats (19) or by its ability to restore nature of the defect remains to be established. Recent studies nitrate reductase activity to extracts of the Neurospora mutant from this laboratory (2) have shown that the molybdenum co- nit-I (unpublished observations). As shown in Table 2, both of factor contains a pterin nucleus with an as yet unidentified these assay procedures failed to detect active cofactor in the 6-alkyl side chain. In addition, it is known that the pteridine of liver sample from the patient. Because the liver sample was the active cofactor is in a reduced state (2). It is not yet known devoid of molybdenum, 10 mM sodium molybdate was in- whether animals can synthesize the cofactor de novo or whether cluded in the reconstitution mixtures (20). all or part of it is a dietary requirement. Nonetheless, these Sulfite Oxidase Activity and Crossreacting Material in findings suggest that a biochemically complex synthetic path- Cultured Fibroblasts. Results of activity measurements and way would be required to produce active cofactor and that radioimmunoassays of sulfite oxidase in fibroblasts cultured defects at any of several steps could lead to aberrant cofactor from the patient, parents, and a normal older brother are synthesis and the resulting combined deficiency disease. Indeed, summarized in Table 3. Whereas both sulfite oxidase activity if the biosynthesis of the pterin ring of the molybdenum co- and crossreacting material were below limits of detection in the factor occurs by the same pathway as that of tetrahydrobiopt- fibroblasts from the patient, the levels found in other members erin, the phenylalanine hydroxylase cofactor (33), one might of the family were indistinguishable from normal. envision a more complex combined deficiency disease that Aldehyde Oxidase. Aldehyde oxidase is a molybdoenzyme includes phenylketonuria. It would thus be of considerable that resembles xanthine dehydrogenase in physical properties importance to examine atypical phenylketonuric patients (34) and overlaps the latter enzyme in substrate specificities (25). for deficiencies of sulfite oxidase and xanthine dehydroge- In addition, available data strongly suggest that this enzyme nase. also utilizes the molybdenum cofactor common to sulfite oxi- The apparent absence of sulfite oxidase apoprotein in both dase and xanthine dehydrogenase (1). However, the levels of liver and cultured fibroblasts from this patient is an interesting aldehyde oxidase in human liver are extremely low (26), and finding that requires further analysis-. Several possible expla- the lability of the enzyme in frozen samples has precluded di- nations for this observation come to mind. The possibility that rect assay of this enzyme in the liver sample from this patient. the primary defect in fact lies in the protein and leads secon- Aldehyde oxidase activity was tested in vivo with a L-histidine darily to absence of cofactor is unlikely because at least two of loading test (100 mg/kg of body weight). Healthy controls the other sulfite oxidase-deficient patients appear to lack the metabolize a small fraction of the administered dose to hy- apoprotein for the enzyme yet synthesize normal levels of active dantoin 5-propionic acid (27). Oxidation of the intermediate molybdenum cofactor (13, 21). A more likely alternative is that imidazolonepropionic acid to hydantoin propionic acid is cat- the molybdenum cofactor is required to specifically induce the alyzed by an enzyme tentatively identified as aldehyde oxidase synthesis of sulfite oxidase. However, the presence of apparently (28). The patient did not produce a measurable amount of normal levels of xanthine dehydrogenase apoprotein in the liver hydantoin propionic acid after L-histidine loading, which may sample would imply that the cofactor does not function as a be considered indirect evidence of aldehyde oxidase defi- positive regulator for this enzyme. A third possible explanation ciency. for absence of sulfite oxidase protein is that a deletion of genetic material required to direct synthesis of the molybdenum co- DISCUSSION factor may have simultaneously eliminated elements necessary for sulfite oxidase biosynthesis. And finally, it is entirely possible The patient described above is the fourth individual identified with a in the to active sulfite oxi- deficiency ability synthesize § During the preparation of this manuscript, two additional patients dase. Like the other three (21, 22), this patient exhibits symp- with combined deficiencies of sulfite oxidase and xanthine dehy- toms of mental retardation and neurological abnormalities and drogenase have come to our attention. One has been identified in has dislocated ocular lenses. Results obtained from this patient Paris by J.-M. Saudubray and one in London by S. Krywawych and concerning urinary and plasma levels of the various sulfur- F. Murray. Downloaded by guest on September 26, 2021 Medical Sciences: Johnson et al. Proc. Natl. Acad. Sci. USA 77 (1980) 3719

that molybdenum cofactor on the mitochondrial outer mem- 3. Shah, V. K. & Brill, W. J. (1977) Proc. Natl. Acad. Sci. USA 74, brane is essential for internalization or "packaging" of the na- 3249-3253. V. & W. Proc. scent sulfite oxidase in the mitochondrial intermembrane space. 4. Pienkos, P. T., Shah, K. Brill, J. (1977) Natl. Acad. Sci. USA 74,5468-5471. In the absence of this internalizing force, cofactorless sulfite 5. Pateman, J. A., Cove, D. J., Rever, B. M. & Roberts, D. B. (1964) oxidase in the cytosol may be perceived as a foreign protein and Nature (London) 201,58-60. be rapidly degraded. Or, cytosolic enzyme lacking molybde- 6. Nason, A., Antoine, A. D., Ketchum, P. A., Frazier, W. A. III & num cofactor may in addition be devoid of heme and in this Lee, D. K. (1970) Proc. Nati. Acad. Sci. USA 65,137-144. bare state be totally unrecognized by antiserum directed against 7. Johnson, J. L., Cohen, H. J. & Rajagopalan, K. V. (1974) J. Biol. the holoenzyme. Chem. 249, 5046-5055. The total absence of hepatic molybdenum in this individual 8. Johnson, J. L., Waud, W. R., Cohen, H. J. & Rajagopalan, K. V. raises the possibility that molybdate as such may not be present (1974) J. Biol. Chem. 249, 5056-5061. in human liver. If all molybdenum is normally present in co- 9. Sorbo, B. (1957) Biochim. Blophys. Acta 23, 412-416. factor form, either bound to molybdoenzymes or as the mito- 10. Miller, E., Hlad, C. J., Jr., Levine, A., Holmes, J. H. & Elrick, H. Clin. Med. chondrial storage pool, then absence of cofactor could result in (1963) J. Lab. 62,710-714. 11. Van Gennip, A. H., Van Noordenburg-Huistra, D. Y., de Bree, severe depletion of hepatic stores of the metal as observed in P. K. & Wadman, S. K. (1978) Clin. Chim. Acta 86,7-20. this patient. Such a situation does in fact obtain in the rat, in 12. Stoop, J. W., Zegers, B. J. M., Hendrickx, G. F. M., Siegenbeek which the total hepatic content of molybdenum can be ac- van Heukelom, L. H., Staal, G. E. J., de Bree, P. K., Wadman, counted for as molybdenum cofactor incQrporated into sulfite S. K. & Ballieux, R. E. (1977) N. Engl. J. Med. 296,651-655. oxidase and xanthine dehydrogenase and bound to the mito- 13. Johnson, J. L.- & Rajagopalan, K. V. (1976) J. Clin. Invest. 58, chondrial outer membrane (25). A final point to be considered, 551-556. however, is the possibility that the absence of active molybde- 14. Lowry, 0. H., Bessey, 0. A. & Crawford, E. J. (1949) J. Biol. num cofactor and sulfite oxidase protein are both in fact ram- Chem. 180,389-398. ifications of molybdenum deficiency. Normal levels of serum 15. McCord, J. M. & Fridovich, I. (1969) J. Biol. Chem. 244, molybdenum would rule against a dietary deficiency or an 6049-6055. 16. H. Y. K., D. R. & Hellerman, L. (1974) J. Biol. system. we cannot elimi- Chuang, Patek, aberrant intestinal uptake Although Chem. 249,2381-2384. nate the possibility of a specific defect in uptake of the metal 17. Kersters, K. & DeLey, J. (1966) Methods Enzymol. 9, 346- at the level of the liver cell (and fibroblast), it is more likely that 354. in the absence of functional cofactor to bind the metal, any 18. Beaufay, H., Bendall, D. S., Baudhuin, P. & de Duve, C. (1959) molybdenum taken up by the cells is rapidly exported. Blochem. J. 73,623-628. The data available presently do not establish whether the 19. Johnson, J. L., Jones, H. P. & Rajagopalan, K. V. (1977) J. Biol. combined deficiency in this patient is in fact a heritable dis- Chem. 252, 4994-5003. order. Because the molybdenum cofactor is normally synthe- 20. Amy, N. K. & Rajagopalan, K. V. (1979) J. Bacteriol. 140, sized in excess of requirements for known molybdoenzymes 114-124. (13), synthesis of decreased amounts of active cofactor by het- 21. Shih, V. E., Abroms, I. F., Johnson, J. L., Carney, M., Mandell, erozygous carriers of the defective gene may not be reflected R., Robb, R. M., Cloherty, J. P. & Rajagopalan, K. V. (1977) N. Engl. J. Med. 297, 1022-1028. as decreases in oxidase activity or protein. Only a direct sulfite 22. Irreverre, F., Mudd, S. H., Heizer, W. D. & Laster, L. (1967) assay of the storage pool of cofactor would reveal a difference. Biochem. Med. 1, 187-217. Unfortunately, sufficiently sensitive assays for cofactor levels 23. Johnson, J. L. & Rajagopalan, K. V. (1977) J. Biol. Chem. 252, in fibroblasts are not currently available. 2017-2025. It is clear that further studies on the structure of the molyb- 24. Lewis, N. J. & Scazzochio, C. (1977) Eur. J. Biochem. 76, denum cofactor and the reactions involved in its synthesis will 441-446. be required for a fuller understanding of the defect in the pa- 25. Krenitsky, T. A., Neil, S. M., Elion, G. B. & Hitchings, G. H. tient described above. In addition, studies of other patients with (1972) Arch. Biochem. Biophys. 150,585-599. combined deficiencies of molybdoenzymes would be of ex- 26. Johns, D. G. (1967) J. Clin. Invest. 46, 1492-1505. treme value in attempts to unravel the complex interactions 27. Niederwieser, A., Matasovic, A., Steinmann, B., Baerlocher, K. between the molybdenum cofactor and the various molyb- & Kempken, B. (1976) Pediatr. Res. 10, 215-219. 28. Payes, B. & Greenberg, D. M. (1968) Arch. Biochem. Biophys. doenzymes in which it serves. 125,911-917. The authors thank Prof. Dr. M. F. Niermeyer, Department of 29. Wyngaarden, J. B. (1978) in The Metabolic Basis of Inherited Clinical Genetics, Erasmus University, Rotterdam, The Netherlands Disease, eds. Stanbury, J. B., Wyngaarden, J. B. & Fredrickson, and Dr. Vivian Shih, Massachusetts General Hospital, Boston, for their D. S. (McGraw-Hill, New York), 4th Ed., pp. 1037-1044. help in culturing the fibroblasts used in this study. We also thank Dr. 30. Johnson, J. L., Rajagopalan, K. V. & Cohen, H. J. (1974) J. Biol. Nancy Amy of this laboratory (Duke Medical Center) for assistance Chem. 249, 859-866. in reconstitution assays with the Neurospora mutant nit-1. This work 31. Mudd, S. H., Levy, H. L. & Abeles, R. H. (1969) Biochem. Bio- was supported in part by Grant GM00091 from the National Institutes phys. Res. Commun. 35, 121-126. of Health (to K.V.R.). 32. Willard, H. F., Mellman, I. S. & Rosenberg, L. E. (1978) Am. J. Hum. Genet. 30, 1-13. 1. Johnson, J. L. (1980) in Molybdenum and Molybdenum-Con- 33. Kaufman, S. (1963) Proc. Natl. Acad. Sci. USA 50, 1085- taining Enzymes, ed. Coughlan, M. P. (Pergamon, Oxford), pp. 1093. 345-38. 34. Kaufman, S. (1979) in Chemistry and Biology of Pteridines, eds. 2. Johnson, J. L., Hainline, B. E. & Rajagopalan, K. V. (1980) J. Biol. Kisliuk, R. L. & Brown, G. M. (Am. Elsevier, New York), pp. Chem. 255, 1783-1786. 117-124. Downloaded by guest on September 26, 2021