Proc. Natl. Acad. Sci. USA Vol. 82, pp. 4517-4520, July 1985 Medical Sciences Deficiency of electron transfer flavoprotein or electron transfer flavoprotein:ubiquinone oxidoreductase in glutaric acidemia type II fibroblasts (inherited disease/acyl-CoA dehydrogenases/electroblotting/crossreacting material) FRANK E. FRERMAN* AND STEPHEN I. GOODMANt *Department of Microbiology, Medical College of Wisconsin, Milwaukee, WI 53226; and tDepartment of Pediatrics, University of Colorado Health Sciences Center, Denver, CO 80262 Communicated by Helmut Beinert, March 5, 1985 ABSTRACT Glutaric acidemia type II (GA II) is a human reported previously a >75% decrease of the characteristic genetic disorder. It has been suggested that the primary defect electron paramagnetic resonance signal (gz = 2.08) of the in this disorder is a deficiency of a protein involved in electron reduced iron-sulfur cluster of ETF:QO in liver submitochon- transport between the acyl-CoA dehydrogenases and the bc, drial particles from one GA II patient. These particles also complex of the mitochondrial respiratory chain. Antisera were lacked material that crossreacted with antibody to the por- raised to purified porcine electron transfer flavoprotein (ETF) cine ETF:QO (16). and electron transfer flavoprotein:ubiquinone oxidoreductase The work reported here extends our investigations to (ETF:QO). The antisera were used to detect the two electron cultured skin fibroblasts from the patient described previ- transferases in control and GA II fibroblasts by immunoblot- ously (16), five additional GA II patients, and parents of two ting. Fibroblasts from three unrelated GA II patients were GA II patients. These studies demonstrate that GA II can be deficient in immunologically detectable ETF:QO and extracts caused by a deficiency of ETF or ETF:QO. The data indicate from these three fibroblast lines contained no detectable that the genes for defective ETF:QO and a subunit of ETF ETF:QO catalytic activity. Fibroblasts from parents of two of are transmitted in an autosomal recessive fashion and provide these patients had ETF:QO activity intermediate between strong evidence for the specificity of the iron-sulfur flavo- activities in control fibroblasts and fibroblasts from the pa- protein. tients. These data indicate that the primary defect in these patients is a deficiency of ETF:QO and that the mode of METHODS transmission of the gene is autosomal recessive. Fibroblasts from two other patients with severe GA II had normal levels of Cell Culture. GA II fibroblast lines 1196 (1), 1730 (6), 1441 ETF-QO activity and antigen but were deficient in in- (7), 1716 (10), KH (2), and 1691 (16) were from unrelated munoreactive ETF. These findings show that GA II results patients who have been described previously. Fibroblasts from a deficiency of ETF in some patients and ETF:QO in were grown in Eagle's minimal essential medium containing others. In addition, these investigations provide strong evi- Earle's salts, 10% fetal bovine serum, nonessential amino dence for the specificity and physiological function of the acids, and antibiotics. iron-sulfur flavoprotein ETF:QO. Enzyme Preparations and Assays. Human fibroblast acyl- CoA dehydrogenases in the soluble fractions of sonically Glutaric acidemia type II (GA II) is an inherited metabolic disrupted cells were assayed fluorometrically as described by disease characterized by nonketotic hypoglycemia and the Frerman et al. (17). The sedimented membrane fractions accumulation and excretion of large amounts of several from these preparations were suspended in 10 mM Tris HCl organic acids (1-10). Most of these acids are clearly derived (pH 7.4) containing 0.5% Triton X-100 and used as the source from the acyl-CoA substrates of six mitochondrial of human fibroblast ETF:QO in enzymatic assays and in flavoprotein dehydrogenases (ref. 11; unpublished data)t that immunoblotting experiments (see below). Human liver transfer electrons to the mitochondrial respiratory chain via mitochondria were prepared by the procedure of Lemaster electron transfer flavoprotein (ETF) (12, 13) and ETF: and Hackenbrock (18) from material obtained by autopsy. ubiquinone oxidoreductase (ETF:QO), an iron-sulfur The liver mitochondria were suspended in 10 mM Hepes flavoprotein (14). Some patients also excrete sarcosine (2, 4), buffer (pH 7.8) containing 0.1 mM EDTA, 5% ethylene the substrate of another ETF-linked flavoprotein dehydrog- glycol, 2 mM phenylmethylsulfonyl fluoride (PhMeSO2F), 1 enase (15). The oxidation of substrate precursors of ETF- mM tosylamide-2-phenylethyl chloromethyl ketone (TPCK), linked acyl-CoA dehydrogenases by intact GA II fibroblasts and 1 mM tosyl-L-lysine chloromethyl ketone (TLCK) and is greatly impaired (1, 3, 9); however, in vitro assays of those disrupted by sonication for 60 sec at 4°C. The preparations primary flavoprotein dehydrogenases show normal catalytic were centrifuged at 100,000 X g for 1 hr at 4°C to obtain activities (2-4, 7). Whole cell oxidation by GA II fibroblasts soluble and particulate fractions, which were used as sources of succinate and pyruvate is also normal, indicating that of human ETF and ETF:QO, respectively. Mitochondrial electron flux from NADH and succinic dehydrogenases membrane fractions (7 mg ofprotein per ml), suspended in 10 through the bc, region of the respiratory chain to oxygen is mM Tris buffer (pH 7.4) containing 250 mM sucrose and 1 not defective (1, 3, 9). These observations have been inter- mM dithiothreitol, were extracted with 2.5% Triton X-100 for preted as suggesting that electron transfer from the primary flavoprotein dehydrogenases to the bc, complex of the Abbreviations: GA II, glutaric acidemia type II; ETF, electron respiratory chain is defective in GA II patients (3, 4). We have transferflavoprotein; ETF:QO, ETF:ubiquinone oxidoreductase; Q. ubiquinone; PhMeSO2F, phenylmethylsulfonyl fluoride; TPCK, tosylamide-2-phenylethyl chloromethyl ketone; TLCK, tosyl-L- The publication costs of this article were defrayed in part by page charge lysine chloromethyl ketone; mu, milliunits. payment. This article must therefore be hereby marked "advertisement" tHuman and pig liver glutaryl-CoA dehydrogenases reduce ETF to in accordance with 18 U.S.C. §1734 solely to indicate this fact. the anionic semiquinone. 4517 Downloaded by guest on September 27, 2021 4518 Medical Sciences: Frerman and Goodman Proc. Natl. Acad. Sci. USA 82 (1985) 30 min at 40C. The supernatant fraction obtained after mined spectrophotometrically, E436 = 13.4 mM-1 (13). centrifugation at 100,000 x g for 1 hr was used as the source ETF:QO was purified from pig liver submitochondrial par- of crude human ETF:QO for catalytic assays. ticles as described by Ruzicka and Beinert (14) and modified ETF:QO activity was assayed fluorometrically under by Beckmann and Frerman (31). The preparation gave a anaerobic conditions by following the decrease in fluores- single band on NaDodSO4/PAGE and isoelectric focusing in cence of oxidized ETF flavin in the ETF:QO-catalyzed 6% polyacrylamide gels in the presence of 0.05% Triton comproportionation of ETF hydroquinone (ETFhq) and oxi- X-100. General acyl-CoA dehydrogenase was prepared as dized ETF (ETFOX) that yields ETF semiquinone (ETFsq) described (32). Radioiodinated protein A (4.88 mCi/mg; 1 Ci (19): ETFox + ETFhq ± 2 ETFsq. This reaction will be = 37 GBq) was the generous gift ofKent Wilcox (Department discussed in detail elsewhere (unpublished data). Membrane of Microbiology, Medical College of Wisconsin) and was fractions of fibroblasts were extracted with 0.5% Triton prepared with the Bolton-Hunter reagent supplied by New X-100 in 10 mM Tris HCl (pH 7.4) and centrifuged at 100,000 England Nuclear (2000 Ci/mmol). x g. The pellet was reextracted and centrifuged as before and the two supernatant fractions were combined. The two RESULTS extractions solubilize 92-96% of ETF:QO activity. ETF hydroquinone was prepared by careful titration ofthe protein Table 1 shows that the specific activities of acyl-CoA with Na2S204 under anaerobic conditions. Assay mixtures dehydrogenases in five GA II fibroblast lines are not signifi- (about 0.75 ml) containing 2-amino-1,3-propanediol hydro- cantly different from those in control fibroblasts. Glutaryl- chloride (pH 8.6) and glucose were prepared in a fluorescence CoA dehydrogenase activity is also normal in the fibroblast cuvette sealed with a 1-cm-thick rubber stopper and made line 1441 (7) and liver mitochondria ofthe patient from whom anaerobic by evacuation and purging with argon; 3 units of the line 1691 was derived (unpublished data). Glutaryl-CoA glucose oxidase and 20 units of catalase were added. After dehydrogenase activity has been shown to be normal in line equilibration at 250C for 10 min, ETF hydroquinone and KH (2). These data are consistent with previous reports that anaerobic oxidized ETF were added with gas-tight syringes. individual acyl-CoA dehydrogenase activities are not defi- The final reaction mixtures, 0.8 ml, contained 20 mM 2- cient in GA 11(2-4, 7), which has been described as a multiple amino-1,3-propanediol hydrochloride, 20 mM glucose, glu- acyl-CoA dehydrogenase deficiency (7, 9, 10, 16). cose oxidase and catalase, 1.5 ,uM ETF hydroquinone, and Fig. 1 shows proteins in the particulate fractions from 0.3 uM oxidized ETF. The reactions were initiated by control and'GA II whole fibroblast sonicates that crossreact addition of the crude enzyme preparations (1-20 ,ug of with anti-porcine ETF:QO. Control fibroblast membranes fibroblast protein). Initial velocities were determined by (Fig. 1 A and B, lanes 3-5) contain crossreacting material with analyses of progress curves of fluorescence decay (20, 21). the same electrophoretic mobility as the oxidoreductase Reactions were carried out at 25°C with excitation at 342 nm; purified from porcine liver (Mr =69,000) (Fig. 1 A and B, lane emission was measured at 496 nm (21). One milliunit of 1) and a protein present in human liver submitochondrial activity is equivalent to 1 nmol of ETFOX reduced per min at particles (Fig.