Preliminary studies on the primary structure of rat liver dihydropteridine reductase

Cyanogen bromide cleavage of reductively alkylated homogeneous rat liver dihydropteridine reductase afforded several peptide fragments identifiable by polyacrylamide electrophoresis of which 6 (CB-1 to CB-6) could be individually isolated by C8 reverse phase HPLC. Each was characterised by N-terminal amino acid analysis and sequence information was derived for CB-1, CB-4 and CB-6. The blocked N-terminal of the holoenzyme was identified as pyroglutamate and the C-terminal sequence was obtained by sequential degradation.     

Structure-function relationship of NAD(P)H:quinone reductase: characterization of NH2-terminal blocking group and essential tyrosine and lysine residues

The amino terminal blocked peptide of rat liver NAD(P)H:quinone reductase (DT-diaphorase) was determined by amino acid sequence analysis and by mass spectrometry. The mature protein is composed of 273 amino acids and contains an acetylated amino terminus, which was not identified by previous cDNA analysis. The enzyme was inactivated by p-nitrobenzenesulfonyl fluoride (NBSF) or 2,4,6-trinitrobenzenesulfonate (TNBS) with pseudo-first-order kinetics. These studies suggest that essential tyrosine and lysine may be present in the active site of this enzyme. The NBSF inhibition was protected by 1-naphthol and 1-naphthylamine, but not by NAD+. However, TNBS inhibition was not prevented by the naphthalene derivatives or NAD+. Specific peptides labeled with NBSF or TNBS were isolated by high-performance liquid chromatography and were sequenced. These analyses revealed that the NBSF-labeled tyrosine resides in a predominantly hydrophobic region and TNBS-labeled lysine in a predominantly hydrophilic region.     

Amino acid sequence of human plasma apolipoprotein C-II from normal and hyperlipoproteinemic subjects

The complete amino acid sequence of human plasma apolipoprotein C-II isolated from normal individuals and a subject with type V hyperlipoproteinemia has been determined. Apo-C-II contains 79 amino acids with the following amino acid composition: Asp4, Asn1, Thr9, Ser9, Glu7, Gln7, Pro4, Gly2, Ala6, Val4, Met2, Ile1, Leu8, Tyr5, Phe2, Lys6, Arg1, Trp1. Cleavage of apo-C-II by cyanogen bromide produced three peptides designated as CB-1 (9 residues), CB-2 (51 residues), and CB-3 (19 residues). Two peptides, CT-1 (50 residues) and CT-2 (29 residues), which overlapped the cyanogen bromide peptides, were obtained by tryptic digestion of citraconylated apo-C-II at the single arginine residue. The amino acid sequence of apo-C-II was obtained by the automated phenyl isothiocyanate degradation of intact apo-C-II and the peptides produced by cleavage of apo-C-II by cyanogen bromide and trypsin. Phenylthiohydantoins were identified by high performance liquid chromatography and chemical ionization-mass spectrometry. The amino acid sequence of apo-C-II from the normal subject was identical with the apo-C-II isolated from the hyperlipoproteinemic subject.     

Role of aspartate-37 in determining cofactor specificity and binding in rat liver dihydropteridine reductase

Full-length rat dihydropteridine reductase (DHPR) cDNAs have been combined with a prokaryotic expression vector and introduced into Escherichia coli. Transformed bacteria express dihydropteridine reductase immunoreactive proteins and demonstrate conversion of quinonoid dihydropteridines to their tetrahydro forms. Several recombinant enzymes have been purified to homogeneity and biochemical studies have been carried out comparing their properties with those exhibited by the rat liver enzyme. The optimal reaction conditions, kinetic constants, and stability are similar for the recombinant and naturally occurring enzyme. The results indicate that the nonmutant recombinant rat DHPR is an authentic replica of the natural protein and that the characteristics of DHPR activity are determined by a single gene product and do not require specific modification via the eukaryotic cell. In addition to the wild type, three specific mutagenic forms of the reductase, A-6-V, W-104-F, and D-37-I, and an additional abbreviated structure have also been formed. Each of the products exhibits reductase activity, although they show varied affinities for their cofactor, NADH, and less stability to chromatography, dialysis, and concentration than the wild-type enzyme. The N-terminal sequence contains a classic NADH binding region between amino acids 9 and 36, and Asp 37 is essential for binding the cofactor as is shown by the approximately 20-fold increase in dissociation constant for the D-37-I mutant and diminished kcat (approximately 43 s-1 compared to 156 s-1 for the wild-type enzyme). The results indicate that the DHPR cofactor binding site is similar to typical dinucleotide requiring dehydrogenases such as lactic acid and liver alcohol dehydrogenase.     

Identification and characterization of an NADPH-cytochrome P450 reductase derived peptide involved in binding to cytochrome P450

The amino acids of cytochrome P450 reductase involved in the interaction with cytochrome P450 were identified with a differential labeling technique. The water-soluble carbodiimide EDC (1-ethyl-3-[3- (dimethylamino)propyl]-carbodiimide) was used with the nucleophile methylamine to modify carboxyl residues. When the modification was performed in the presence of cytochrome P450, there was no inhibition in the ability of the modified reductase to bind to cytochrome P450. However, subsequent modification of the reductase in the absence of cytochrome P450 caused a fourfold increase in the Km and an 80% decrease in kcat/Km (relative to the reductase modified in the first step), for the interaction with cytochrome P450. These effects are attributed to the modification of approximately 3.2 mol of carboxyl residues per mole of reductase. Tryptic peptides generated from the modified reductase were purified by reverse phase high-performance liquid chromatography and characterized. Amino acid sequencing and analysis suggest that the peptide which contains approximately 40% of the labeled carboxyl residues corresponds to amino acid residues 109-130 of rat liver NADPH-cytochrome P450 reductase. One or more of the seven carboxyl containing amino acids within this peptide is presumably involved in the interaction with cytochrome P450.     

α-Crystallin. Fractionation of subunits and sequence studies on an isolated polypeptide

alpha-Crystallin was carboxymethylated with radioactive iodoacetic acid in the presence of 7.6m-urea and then separated into six major fractions by chromatography on DEAE-cellulose in 7m-urea. Based on the amino acid compositions, specific radioactivities and sodium dodecyl sulphate-gel electrophoresis of the fractions, it was concluded that alpha-crystallin contains at least four different subunits: DU1A and DU1B, containing no cysteine; a third component represented by DU2B and DU3 containing one cysteine one cysteine residue per subunit; and DU4, which probably contains two residues of cysteine per subunit. Subunit DU1A was shown to be of sufficient purity for sequence studies. Cyanogen bromide cleavage yielded two peptides, CB-1 and CB-2, in approximately equal amounts as expected. The sum of the molecular weights and amino acid compositions of the peptides were both in excellent agreement with the results obtained for subunit DU1A. The amino acid sequence of the first sixteen residues of peptide CB-1 is: Ser-Leu-Thr-Lys-Asp-Phe-Asp-Glu-Val-Asn-Ile-Asp-Val-Ser-His-Phe-. The sequence of the first seventeen residues of peptide CB-2 is: Asp-Ile-Ala-Ile-Ser-His-Pro-Trp-Ile-Arg-Pro-Ser-Phe-Phe-Glu-Phe-His-. The N-terminal sequence of subunit DU1A was shown to be N-acetylmethionine followed by peptide CB-2.     

An antibody and anti-idiotypic antibody against the extra signal peptide of pre-aspartate aminotransferase

A peptide (extra signal peptide) comprising amino acids 1-29 of pig liver pre-mitochondrial aspartate aminotransferase (p-mAAT) was synthesized chemically. The peptide was found to block the import of rat liver p-mAAT into rat liver mitochondria. An antibody raised against the peptide immunoprecipitated rat liver p-mAAT synthesized in a rabbit reticulocyte cell-free translation system. These results suggested that the extra signal peptide sequence of p-mAAT is essential for import of p-mAAT into the mitochondria and that there is structural homology between the extra signal peptides of pig and rat liver p-mAAT. An anti-idiotypic antibody against the peptide was also prepared and purified by affinity chromatography on an Affi-Gel 10 anti-peptide IgG column and was then characterized.     

Purification and characterization of rat skeletal muscle fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase

Fructose-6-phosphate,2-kinase:fructose-2,6-bis-phosphatase from rat skeletal muscle has been purified to homogeneity, and its structure and kinetic properties have been determined. The Mr of the native enzyme was 100,000 and the subunit Mr was 54,000. The apparent Km values of fructose-6-P,2-kinase for Fru-6-P and ATP were 56 and 48 microM, respectively. The apparent Km value for Fru-2,6-P2 of fructose-2,6-bis-phosphatase was 0.4 microM, and the Ki for Fru-6-P was 12.5 microM. The enzyme was bifunctional, and the phosphatase activity was 2.5 times higher than the kinase activity. The enzyme was not phosphorylated by cAMP-dependent protein kinase. The amino acid composition of the skeletal muscle enzyme was similar to that of the rat liver enzyme, and the carboxyl terminus sequence (His-Tyr) was the same as that of the liver enzyme. The tryptic peptides generated from the liver and skeletal muscle enzymes were identical except for two peptides. A peptide corresponding to nucleotides 14-28 of the rat liver enzyme was not detected in the skeletal muscle enzyme. A peptide whose amino acid sequence was Thr-Ala-Ser-Ile-Pro-Gln-Phe-Thr-Asn-Ser-Pro-Thr-Met-Val-Ile-Met-Val-Gly-Leu-Pro - Ala-Arg was also isolated. This peptide was the same as that of rat liver enzyme (nucleotides 31-52) containing the phosphorylation site except in the muscle enzyme two amino terminus amino acids, Gly-Ser(P), have been altered to Thr-Ala. Thus, the rat skeletal muscle enzyme is very similar in structure to the rat liver enzyme except for the lack of possibly one peptide and the lack of a phosphorylation site by the substitution of the target Ser with Ala.     

Chromogranin B (secretogranin I), a putative precursor of two novel pituitary peptides through processing at paired basic residues

During the course of reversed-phase high-pressure liquid chromatography (RP-HPLC) purification of the 7B2 peptide originally isolated in our laboratory from human pituitary gland extracts, two novel peptides were identified and purified to homogeneity. The complete amino acid sequence of the first one was established in 1985 and recently found to be entirely homologous to positions 420-493 of the just published chromogranin B sequence. This peptide, denoted GAWK, could originate from chromogranin B following specific cleavage at the basic amino acids flanking both termini of GAWK. Moreover, another peptide isolated in our laboratory from the same source and denoted CCB has been discovered and its sequence is also part of the same chromogranin B molecule. Here again, this peptide, occupying positions 597-653 and located at the COOH-terminal region of chromogranin B, could derive from specific processing at basic amino acids, Arg-Lys-Lys, present at positions 594-596. In a manner reminiscent of the relationship between pancreastatin and chromogranin A, it is proposed that both GAWK and CCB are produced from chromogranin B after specific processing at basic amino acids. These data are thus in favor of a putative role of chromogranins as precursors to potentially bioactive peptides.     

Multiple phosphorylation sites of rat liver glycogen synthase

Rat liver glycogen synthase was purified to homogeneity by an improved procedure that yielded enzyme almost exclusively as a polypeptide of Mr 85,000. The phosphorylation of this enzyme by eight protein kinases was analyzed by cleavage of the enzyme subunit followed by mapping of the phosphopeptides using polyacrylamide gel electrophoresis in the presence of SDS, reverse-phase high-performance liquid chromatography and thin-layer electrophoresis. Cyclic AMP-dependent protein kinase, phosphorylase kinase, protein kinase C and the calmodulin-dependent protein kinase all phosphorylated the same small peptide (approx. 20 amino acids) located in a 14 kDa CNBr-fragment (CB-1). Calmodulin-dependent protein kinase and protein kinase C also modified second sites in CB-1. A larger CNBr-fragment (CB-2) of approx. 28 kDa was the dominant site of action for casein kinases I and II, FA/GSK-3 and the heparin-activated protein kinase. The sites modified were all localized in a 14 kDa species generated by trypsin digestion. Further proteolysis with V8 proteinase indicated that FA/GSK-3 and the heparin-activated enzyme recognized the same smaller peptide within CB-2, which may also be phosphorylated by casein kinase 1. Casein kinase 1 also modified a distinct peptide, as did casein kinase II. The results lead us to suggest homology to the muscle enzyme with regard to CB-1 phosphorylation and the region recognized by FA/GSK-3, which in rabbit muscle is characterized by a high density of proline and serine residues. A striking difference with the muscle isozyme is the apparent lack of phosphorylations corresponding to the muscle sites 1a and 1b. These results provide further evidence for the presence of liver- and muscle-specific glycogen synthase isozymes in the rat. That the isozymes differ subtly as to phosphorylation sites may provide a clue to the functional differences between the isozymes.     

A bradykinin-potentiating peptide (peptide K12) isolated from the venom of Egyptian scorpion Buthus occitanus

A nontoxic peptide with bradykinin-potentiating activity was isolated from the dialyzed venom of the scorpion Buthus occitanus by reverse-phase high performance liquid chromatography (RP-HPLC). The pharmacological activity of the peptide was bioassayed by its ability to potentiate added bradykinin (BK) on the isolated guinea pig ileum as well as the isolated rat uterus for contraction. Moreover, the peptide potentiates in vivo the depressor effect of BK on arterial blood pressure in the normotensive anesthetized rat. Chemical characterization of the peptide was also performed. The amino acid composition of the peptide showed 21 amino acid residues per molecule including three proline residues. The amino acid sequence of the purified peptide was confirmed by mass spectrometry. Either N- or C-terminal ends were free. The sequence does not show a homology with bradykinin-potentiating peptides isolated from either scorpion or snake venoms. Furthermore, we did not find a significant sequence homology between the sequence of the isolated peptide and any of proteins or peptides in GenPro or NBRF data banks. The peptide also inhibited angiotensin-converting enzyme (ACE), and could not serve as substrate for the enzyme. It could be concluded that the mechanism of bradykinin-potentiating peptide (BPP) activity may be due to ACE inhibition.     

Amino acid sequence of the b subunit of human factor XIII, a protein composed of ten repetitive segments

Factor XIII is a plasma protein that participates in the final stages of blood coagulation. The complete amino acid sequence of the b subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gt11 cDNA library prepared from human liver mRNA was screened with an affinity-purified antibody against the b subunit of human factor XIII. Nine positive clones were isolated from 2 X 10(6) phage and plaque-purified. The largest cDNA insert was sequenced and shown to contain 2180 base pairs coding for a portion of the leader sequence (19 amino acids), the mature protein (641 amino acids), a stop codon (TGA), a 3' noncoding region (187 nucleotides), and a poly(A) tail. When the b subunit of human factor XIII was digested with cyanogen bromide, nine peptides were isolated by gel filtration and reverse-phase high-performance liquid chromatography. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 299 amino acid residues were identified. These amino acid sequences were in complete agreement with the amino acid sequence predicted from the cDNA. The b subunit of factor XIII contained 10 repetitive homologous segments, each composed of about 60 amino acids and 4 half-cystine residues. Each of these repeated segments is a member of a family of repeats present in human beta 2-glycoprotein I, complement factor B, and haptoglobin alpha 1 chain. Three potential Asn-linked carbohydrate attachment sites were also identified in the b subunit of factor XIII.     

Amino acid sequence of the nucleotide binding region of chloroplast coupling factor 1

The labeling of chloroplast coupling factor 1 by 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) was studied. When the enzyme was incubated with approximately 10 microM BzATP and 6 mM MgCl2 at pH 7.9 for approximately 20 min and passed through two Sephadex G-50 centrifuge columns, three BzATP molecules were bound per coupling factor molecule. Photolysis of radioactive enzyme-bound BzATP followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the BzATP bound primarily to the beta-polypeptide. If unbound BzATP was not removed by centrifuge columns prior to photolysis, significant labeling of the alpha-polypeptide also occurred. After photolysis, the BzATP-labeled enzyme was treated with trypsin, and two radioactive peptides were isolated by high-performance liquid chromatography on a C18 column. The two peptides were sequenced and found to correspond to amino acids 360-378 and 393-397 of the beta-polypeptide. For the sequence 360-378, two specific amino acids were found to be radioactive (Tyr-362 and Asp-369). This region of the polypeptide is highly conserved in several different species and probably corresponds to part of the nucleotide binding region of the catalytic site. In the case of amino acids 393-397, a very low level of radioactivity was found for all amino acids. The significance of this peptide for the binding of nucleotides to coupling factor 1 could not be established.     

Catalytic site of rat liver and bovine heart fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Identification of fructose 6-phosphate binding site

Fructose-6-P binding sites of rat liver and bovine heart Fru-6-P,2-kinase:Fru-2,6-bisphosphatase were investigated with an affinity labeling reagent, N-bromoacetylethanolamine phosphate. The rat liver enzyme was inactivated 97% by the reagent in 60 min, and the rate of inactivation followed pseudo-first order kinetics. The bovine heart enzyme was inactivated 90% within 60 min, but the inactivation rate followed pseudo-first order up to 80% inactivation and then became nonlinear. The presence of fructose-6-P retarded the extent of the inactivation to approximately 40% in 60 min. In order to determine the amino acid sequence of the fructose-6-P binding site, both enzymes were reacted with N-bromo[14C]acetylethanolamine-P and digested with trypsin; radiolabeled tryptic peptides were isolated and sequenced. A single 14C-labeled peptide was isolated from the rat liver enzyme, and the amino acid sequence of the peptide was determined as Lys-Gln-Cys-Ala-Leu-Ala-Leu-Lys. A major and two minor peptides were isolated from bovine heart enzyme whose amino acid sequences were Lys-Gln-Cys-Ala-Leu-Val-Ala-Leu-Lys, Arg-Ile-Glu-Cys-Tyr-Lys, and Ile-Glu-Cys-Tyr-Lys, respectively. In all cases, N-bromoacetylethanolamine-P had alkylated the cysteine residues. The amount of bromo[14C]acetylethanolamine-P incorporated into rat liver and beef heart was 1.3 mol/mol of subunit and 2.1 mol/mol of subunit, respectively, and the incorporations in the presence of Fru-6-P were reduced to 0.34 mol/mol of subunit and 0.9 mol/mol of subunit, respectively. Thus, the main fructose-6-P binding site of rat liver and bovine heart enzymes was identical except for a single amino acid substitution of valine for alanine in the latter enzyme. This peptide corresponded to residues 105 to 113 from the N terminus of the known amino acid sequence of rat liver enzyme, but since the complete sequence of bovine heart enzyme is not known, the location of the same peptide in the heart enzyme cannot be assigned.     

Trypanothione reductase of Trypanosoma congolense: gene isolation, primary sequence determination, and comparison to glutathione reductase

The gene encoding trypanothione reductase, the redox disulfide-containing flavoenzyme that is unique to the parasitic trypanosomatids (Shames et al., 1986), has been isolated from the cattle pathogen Trypanosoma congolense. Library screening was carried out with inosine-containing oligonucleotide probes encoding sequences determined from two active site peptides isolated from the purified Crithidia fasciculata enzyme. The nucleotide sequence of the gene was determined according to the dideoxy chain termination method of Sanger. The structural gene is 1476 nucleotides long and encodes 492 amino acids. We have identified the active site peptide containing the redox-active disulfide, a peptide corresponding to the histidine-467 region of human erythrocyte glutathione reductase, as well as the flavin binding domain that is highly conserved in all disulfide-containing flavoprotein reductase enzymes. Alignment of five tryptic peptides (80 residues) isolated from the C. fasciculata trypanothione reductase with the primary sequence of the T. congolense enzyme showed 88% homology with 76% identity. Additionally, a sequence comparison of the glutathione reductase from Escherichia coli or human erythrocytes to T. congolense trypanothione reductase reveals greater than 50% homology. A search for the amino acid residues in the primary sequence of trypanothione reductase functionally active in binding/catalysis in human erythrocyte glutathione reductase shows that only the two arginine residues (Arg-37 and Arg-347), shown by X-ray crystallographic data to hydrogen bond to the GS1 glutathione glycyl carboxylate, are absent.     

cDNA cloning, functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein

A 64 kD protein was enriched from rat liver mito-chondria during the purification of choline dehydro-genase (CHDH)[1]. Homologous comparison and func-tional experiments demonstrated that the protein was electron-transfer flavoprotein-ubiquinone oxidoreduc-tase protein (ETF-QO). The N-terminal sequence determination of rat liver ETF-QO protein purified by various methods did not provide unequivocal result. However, when the protein was digested with V8 protease, peptide fragments could b…     

Rat liver carboxylesterase: cDNA cloning, sequencing, and evidence for a multigene family

A cDNA clone was isolated from a rat liver lambda gt11 expression library by screening with polyclonal antibodies raised against a rat liver microsomal carboxylesterase. This clone of 1.8 kb contained an open reading frame encoding a mature protein of 531 amino acids with a predicted molecular weight of 58,084. The 5' portion of the clone coded for 9 amino acids of a putative signal peptide. The 3' end of the clone included an untranslated region and a poly (A) tail. Carboxylesterase active site regions, five potential N-linked glycosylation sites, and 2 postulated cystine disulfide bridges were found in the cDNA-deduced amino acid sequence. Sequences obtained from tryptic peptides and the NH2-terminus of the purified native carboxylesterase were aligned with the deduced amino acid sequence, and the overall identity was 84%. Southern blot analysis suggested the presence of multiple genes. Thus it is concluded that we have cloned a rat liver carboxylesterase, and that this enzyme is a member of a multigene family.     

Physarum polycephalum expresses a dihydropteridine reductase with selectivity for pterin substrates with a 6-(1', 2'-dihydroxypropyl) substitution

Physarum polycephalum is one of few non-animal organisms capable of synthesizing tetrahydrobiopterin from GTP. Here we demonstrate developmentally regulated expression of quinoid dihydropteridine reductase (EC 1.6.99.7), an enzyme required for recycling 6,7-[8H]-dihydrobiopterin. Physarum also expresses phenylalanine-4-hydroxylase activity, an enzyme that depends on dihydropteridine reductase. The 24.4 kDa Physarum dihydropteridine reductase shares 43% amino acid identity with the human protein. A number of residues important for function of the mammalian enzyme are also conserved in the Physarum sequence. In comparison to sheep liver dihydropteridine reductase, purified recombinant Physarum dihydropteridine reductase prefers pterin substrates with a 6-(1', 2'-dihydroxypropyl) group. Our results demonstrate that Physarum synthesizes, utilizes and metabolizes tetrahydrobiopterin in a way hitherto thought to be restricted to the animal kingdom.     

Isolation and characterization of nascent albuminyl peptides synthesized in vivo from rat liver.

A procedure is described for the isolation of nascent albuminyl peptides from rat liver polysomes which is based on the isolation of total peptidyl tRNA by ion-exchange chromatography on ECTEOLA cellulose followed by immuno-affinity chromatography employing monospecific anti-albumin antibodies immobilized on Sepharose 4B. Identity of the isolated nascent albuminyl peptides was assayed by tryptic peptide fingerprint analysis. Quantitation and determination of the specific activity of the nascent albuminyl peptides, labeled in vivo with l-[14c]leucine, were made by subjecting the peptides to acid hydrolysis, dansylation and resoultion of the amino acids by thin-layer chromatography, and determination of the specific activity of dansyl leucine.     

cDNA cloning,functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein

A membrane-bound protein was purified from rat liver mitochondria. After being digested with V8 protease, two peptides containing identical 14 amino acid residue sequences were obtained. Using the 14 amino acid peptide derived DNA sequence as gene specific primer, the cDNA of correspondent gene 5′-terminal and 3′-terminal were obtained by RACE technique. The full-length cDNAthat encoded a protein of 616 amino acids was thus cloned, which included the above mentioned peptide sequence. The full length cDNA was highly homologous to that of human ETF-QO, indicating that it may be the cDNA of rat ETF-QO. ETF-QO is an iron sulfur protein located in mitochondria inner membrane containing two kinds of redox center: FAD and [4Fe-4S] center. After comparing the sequence from the cDNA of the 616 amino acids protein with that of the mature protein of rat liver mitochondria, it was found that the N terminal 32 amino acid residues did not exist in the mature protein, indicating that the cDNA was that of ETF-QOp. When the cDNA was expressed in Saccharomyces cerevisiae with inducible vectors, the protein product was enriched in mitochondrial fraction and exhibited electron transfer activity (NBT reductase activity) of ETF-QO. Results demonstrated that the 32 amino acid peptide was a mitochondrial targeting peptide, and both FAD and iron-sulfur cluster were inserted properly into the expressed ETF-QO. ETF-QO had a high level expression in rat heart, liver and kidney. The fusion protein of GFP-ETF-QO co-localized with mitochondria in COS-7 cells.