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R Brandsch

Publications and source records attributed to R Brandsch.

At least 19 recordsLinked to original sources
PubMed1998

Surface-promoted replication and exponential amplification of DNA analogues.

Self-replicating chemical systems have been designed and studied to identify the minimal requirements for molecular replication, to translate the principle into synthetic supramolecular systems and to derive a better understanding of the scope and limitations of self-organization processes that are believed to be relevant to the origin of life on Earth. Current implementations make use of oligonucleotide analogues, peptides, and other molecules as templates and are based either on autocatalytic, cross-catalytic, or collectively catalytic pathways for template formation. A common problem of these systems is product inhibition, leading to parabolic instead of exponential amplification. The latter is the dynamic prerequisite for selection in the darwinian sense. We here describe an iterative, stepwise procedure for chemical replication which permits an exponential increase in the concentration of oligonucleotide analogues. The procedure employs the surface of a solid support and is called SPREAD (surface-promoted replication and exponential amplification of DNA analogues). Copies are synthesized from precursor fragments by chemical ligation on immobilized templates, and then liberated and immobilized to become new templates. The process is repeated iteratively. The role of the support is to separate complementary templates which would form stable duplexes in solution. SPREAD combines the advantages of solid-phase chemistry with chemical replication, and can be further developed for the non-enzymatic and enzymatic amplification of RNA, peptides and other templates as well as for studies of in vitro evolution and competition in artificial chemical systems. Similar processes may also have played a role in the origin of life on Earth, because the earliest replication systems may have proliferated by spreading on mineral surfaces.

Chromatography, High Pressure Liquid↗
PubMed1998

Molecular cloning and tissue distribution of rat sarcosine dehydrogenase.

Sarcosine dehydrogenase (SarDH) is a mitochondrial flavoenzyme involved in the oxidative degradation of choline to glycine. The absence of SarDH activity in humans is genetically transmitted and is the cause of an amino acid metabolism disorder called sarcosinemia. Tryptic fragments of the purified enzyme from rat liver were subjected to Edman degradation and the sequences obtained were used to clone the cDNA encoding the full length protein. The deduced amino acid sequence of SarDH shares an overall similarity of 47% with dimethylglycine dehydrogenase (Me2GlyDH), another flavoenzyme involved in the mitochondrial choline catabolism with a similar FAD-binding domain. Covalent binding of FAD to SarDH was demonstrated by the observation of strong fluorescence at 530 nm under excitation at 450 nm of the enzyme immunoprecipitated under denaturing conditions from liver extracts. The localization of SarDH immunoreactivity in the mitochondrial matrix was confirmed by Western-blot analysis of purified mitochondrial fractions. Finally, the tissue distribution of SarDH was investigated by Northern-blot analysis of total RNA and Western-blot analysis of total protein from several rat tissues. A strong expression in the liver, but also in the lung, pancreas, kidney, thymus, and oviduct was observed. We therefore suggest that the enzymes of the choline catabolism pathway are important also for metabolism in nonhepatic tissues.

Amino Acid Sequence↗
PubMed1998

Anti-mitochondrial antibodies in patients with dilated cardiomyopathy (anti-M7) are directed against flavoenzymes with covalently bound FAD.

Anti-mitochondrial antibodies (anti-M7) in sera from patients with dilated cardiomyopathy and myocarditis recognize, besides mitochondrial antigens, bacterial sarcosine dehydrogenase. The common target antigen was identified as the covalently bound FAD of mitochondrial and bacterial flavoenzymes. Thus, anti-M7-positive serum reacted on Western blots exclusively with covalently flavinylated enzymes. The antigenic specificity of anti-M7 sera was reproduced by an antiserum raised in rabbits with 6-hydroxy-D-nicotine oxidase. The heart mitochondrial membrane antigen recognized by anti-M7 serum was identified as the flavoprotein subunit of succinate dehydrogenase, the antigens in rat liver mitochondrial matrix as the flavoenzymes dimethylglycine dehydrogenase and sarcosine dehydrogenase. Anti-M7 serum contained a specific anti-flavoenzyme antibody fraction. Nanomolar concentrations of FAD and riboflavin inhibited the immune reaction on Western blots and in ELISA, and incubation with FAD-agarose depleted the anti-M7 activity of the serum. N-terminally deleted dimethylglycine dehydrogenase proteins were only immunoprecipitated by anti-M7 sera when the FAD was covalently incorporated. An affinity constant (KD) of 10(-8) M was established for the anti-flavoenzyme antibodies by competitive ELISA. Of patients with cardiomyopathy and myocarditis, 36% and 25%, respectively, were anti-flavoenzyme-positive by Western blot and ELISA, but only two of 15 patients with other heart diseases and none of 50 healthy controls.

Animals↗
PubMed1998

The conformational change induced by FAD in covalently flavinylated 6-hydroxy-D-nicotine oxidase does not require (8alpha)FAD-(N3)histidyl bond formation.

The contribution of (8alpha)-(N3)histidyl bond formation to the conformation of covalently flavinylated proteins was investigated by trypsin treatment of wild type and mutant versions of a model enzyme, 6-hydroxy-D-nicotine oxidase (6-HDNO) of Arthrobacter nicotinovorans. In the absence of FAD, apo-6-HDNO exhibited a conformation exposing a protease accessible site. Holoenzyme formation through FAD-attachment to His71 induced a conformational change in the protein that shielded the trypsin recognition site. This conformational change, however, did not require FAD-histidyl bond formation since trypsin resistance was also exhibited by a 6-HDNO.Cys71 mutant protein which was unable to bind FAD covalently. Replacement of Arg67, an amino acid residue supposed to be essential in flavinylation, by Ala rendered the protein protease sensitive as did replacement of Pro73 by Ala. These amino acids apparently play an essential role in stabilizing the native protein conformation. The inability to reach the native conformation also prevented FAD attachment, indicating that a specific conformation of the protein is a prerequisite for FAD-histidyl bond formation. Deletion of Phe448 and Arg449 from the 458 amino acid residues-containing enzyme resulted in complete protease sensitivity, demonstrating that flavinylation takes place posttranslationally.

Flavin-Adenine Dinucleotide↗
PubMed1997

Molybdate-uptake genes and molybdopterin-biosynthesis genes on a bacterial plasmid--characterization of MoeA as a filament-forming protein with adenosinetriphosphatase activity.

A gene cluster consisting of homologs to Escherichia coli moaA, moeA, moaC and moaE, which encode enzymes involved in the biosynthesis of molybdopterin cofactor (MoCo), and to modA, modB and modC, which encode a high-affinity molybdate transporter, were identified on pAO1 of Arthrobacter nicotinovorans near genes of molybdopterin-dependent enzymes involved in nicotine degradation. This gene arrangement suggests a coordinated expression of the MoCo-dependent and the MoCo-biosynthesis genes and shows that catabolic plasmids may carry the transport and biosynthetic machinery for the synthesis of the cofactors needed for the functioning of the enzymes they encode. pAO1 MoeA functionally complemented E. coli moeA mutants. The overexpressed and purified protein, of molecular mass 44,500 Da, associated into high-molecular-mass complexes and spontaneously formed gels at concentrations above 1 mg/ml. Transmission electron microscopy and atomic force microscopy revealed that MoeA forms fibrilar structures. In the presence of Mg2+ MoeA exhibited ATPase activity (0.020 pmol ATP x pmol protein(-1) x min(-1)). ATP, ADP or AMP induced the disassembly of the MoeA fibers into aggregates. pAO1 MoeA shows 39% identity to the C-terminal domain of the rat neuroprotein gephyrin. Like gephyrin it binds to neurotubulin, but binds with preference to tubulin dimers.

Adenosine Triphosphatases↗
PubMed1997

IS1473, a putative insertion sequence identified in the plasmid pAO1 from Arthrobacter nicotinovorans: isolation, characterization, and distribution among Arthrobacter species.

A putative insertion sequence (IS1473) has been cloned and sequenced. The 1087-bp element was found between the moaA and the ndhA genes in the upstream region of the nicotine dehydrogenase (ndh) operon in the 160-kb pAO1 plasmid of Arthrobacter nicotinovorans. It is flanked by an imperfect repeat of 33 bp and carries two overlapping open reading frames which, by programmed -1 translational frameshifting, may produce a transposase of 36.735 Da with a pI = 10. 18. The deduced protein is similar to the transposases IS481 and IS1002 from Bordetella and IS476 from Xanthomonas campestris, all members of the IS3 family. The putative insertion element was found as a single copy in the pAO1 plasmid and absent on the chromosome of the A. nicotinovorans genome. Similar sequences were detected by hybridization on total DNA from Arthrobacter globiformis, Arthrobacter ramosus, and Arthrobacter ureafaciens.

Amino Acid Sequence↗
PubMed1996

Covalent attachment of FAD derivatives to a fusion protein consisting of 6-hydroxy-D-nicotine oxidase and a mitochondrial presequence. Folding, enzyme activity, and import of the modified protein into yeast mitochondria.

Autoflavinylation of 6-hydroxy-D-nicotine oxidase (6-HDNO) was successfully employed to modify the protein covalently with FAD derivatives. The model compounds N6-(2-aminoethyl)-FAD and N6-(6-carboxyhexyl)-FAD were spontaneously bound to a fusion protein consisting of the mitochondrial targeting sequence of Neurospora crassa F0-ATPase subunit 9 (Su9) attached to 6-HDNO. When translated in the rabbit reticulocyte lysate, Su9-6-HDNO was in the trypsin-sensitive apoenzyme form; when translated in the presence of flavins it adopted a trypsin-resistant conformation characteristic of the 6-HDNO holoenzyme. With flavin derivatives, Su9-6-HDNO exhibited approximately 50% of the 6-HDNO activity observed with FAD. The covalently modified Su9-6-HDNO was imported into Saccharomyces cerevisiae mitochondria with an efficiency equal to that of the apoenzyme. Apparently the increase in size and charge of the FAD moiety did not hamper translocation across the mitochondrial membranes. Yeast mutant ssc1-2 mitochondria deficient in mtHsp70 unfoldase activity imported the flavinylated Su9-6-HDNO protein. In mutant ssc1-3 mitochondria deficient in both mtHsp70 unfoldase and translocase activity Su9-6-HDNO was trapped as translocation intermediate; the Su9 presequence was passed to the matrix where it was proteolytically cleaved by the mitochondrial processing peptidase; (MPP); the translocation-arrested 6-HDNO moiety adopted a trypsin-sensitive conformation. Our results indicate that unfolding of the FAD-stabilized flavin-binding domain of 6-HDNO in passage through the mitochondrial general insertion pore does not require the activity of mtHsp70.

Animals↗
PubMed1996

MoaA of Arthrobacter nicotinovorans pAO1 involved in Mo-pterin cofactor synthesis is an Fe-S protein.

MoaA, involved in an early step in the biosynthesis of the molybdopterin cofactor (MoCo), has not yet been characterized biochemically and the reaction it catalyzes is unknown. We overexpressed MoaA from pAO1 of Arthrobacter nicotinovorans in Escherichia coli as a N-terminal fusion with either glutathione-S-transferase or a 6-histidine tag. The pAO1 encoded MoaA as well as the fusion proteins functionally complement E. coli moaA mutants. Here we show that purified MoaA contains approximately 4 microM Fe and approximately 3 microM acid-labile S/microM protein. EPR spectroscopy revealed a predominant signal at g(av) = 2.01, indicative of a [3Fe-xS] cluster.

Amino Acid Sequence↗
PubMed1996

The design of an alternative, covalently flavinylated 6-hydroxy-D-nicotine oxidase by replacing the FAD-binding histidine by cysteine and reconstitution of the holoenzyme with 8-(methylsulfonyl)FAD.

The cofactor of several flavoenzymes is autocatalytically bound to the polypeptide via a histidyl(N3)-(8alpha)-FAD linkage which makes the generation of apoenzyme difficult. We introduced an alternative covalent protein-FAD bond at the active site of 6-hydroxy-D-nicotine oxidase (6HDNO) by replacing the FAD-binding histidine with cysteine. The resulting mutant enzyme was expressed with noncovalently attached cofactor. Incubation with 8-(methylsulfonyl)FAD, and less efficiently with 8-chloro-FAD, resulted in the spontaneous replacement of the noncovalently bound FAD by the flavin derivative and the formation of an 8-(N-acetylcysteinyl)FAD linkage. The flavinylated 6HDNO.cys exhibited close to wild-type activity levels. This strategy may be generally applicable to the attachment of artificially designed flavin derivatives to the active site of covalently flavinylated enzymes.

Coenzymes↗
PubMed1996

Biogenesis of the covalently flavinylated mitochondrial enzyme dimethylglycine dehydrogenase.

Rat dimethylglycine dehydrogenase (Me2GlyDH) was used as model protein to study the biogenesis of a covalently flavinylated mitochondrial enzyme. Here we show that: 1) enzymatically active holoenzyme correlated with trypsin resistance of the protein; 2) folding of the reticulocyte lysate-translated protein into the trypsin-resistant, holoenzyme form was a slow process that was stimulated by the presence of the flavin cofactor and was more efficient at 15 degrees C than at 30 degrees C; 3) the mitochondrial presequence reduced the extent but did not prevent holoenzyme formation; 4) covalent attachment of FAD to the Me2GlyDH apoenzyme proceeded spontaneously and did not require a mitochondrial protein factor; 5) in vitro only the precursor, but not the mature form, of the protein was imported into isolated rat liver mitochondria; in vivo, in stably transfected HepG2 cells, both the precursor and the mature form were imported into the organelle; 6) holoenzyme formation in the cytoplasm did not prevent the translocation of the proteins into the mitochondria in vivo; and 7) lack of vitamin B2 in the tissue culture medium resulted in a reduced recovery of the precursor and the mature form of Me2GlyDH from cell mitochondria, suggesting a decreased efficiency of mitochondrial protein import.

Amino Acid Sequence↗
PubMed1996

[Laparoscopy in (apparently) benign ovarian tumors between benefit and catastrophy and the deceptive safety of laparoscopic lap sacs].

Endoscopic ovarian surgery is currently spreading tremendously, but also rather uncritically. The technical possibilities both of organ preserving and ablative endoscopic surgery are controversial, so that structuring of the indication for an endoscopic procedure as well as optimization of the endoscopic removal of ovarian tumor or adnexae is of utmost relevance. Therefore, a study was performed at the Department of Obstetrics and Gynaecology of the Heidelberg University with the following aims: Risk evaluation of operating into an ovarian malignancy at endoscopy for "presumably" benign cystic ovarian tumors in n = 100 cases in Heidelberg and literature survey Analysis of problems and complications during clinical application of laparoscopic lap sacs for removal of cystic adnexal tumors or adnexae (n = 50) Experimental examination of the risk of an endoscopic lap sacs to rupture during a procedure The risk of endoscopically operating into an ovarian cancer lies between 0.4 and 3% according to literature data. Despite maximal preoperative selection, mainly by ultrasound examination, in our group of 100 patients, in three women without preoperative signs of malignancy but with discreet intracystic structures in the ultrasound, an endoscopic adnexectomy with complete removal in a lap sac was performed, and though immediate section for microscopic examination was negative, final histology revealed one ovarian cancer la and 2 borderline tumors, same stage. In the time period analyzed, three further patients were referred to our center for secondary, delayed (median 3 months) staging after endoscopic procedures for presumed benign lesions. Clinical application of lap sacs proved the necessity of an intensive training. In 3 patients an intraperitoneal rupture of the sac occurred. Typical problems were volume discrepancies between sac respectively abdominal incision and tissue to be removed (28% of cases). The risk of rupture of the various lap sacs examined differed significantly (p < 0.05). Due to the complex pathological nature of cystic adnexal tumors, a 100% selection for the endoscopic approach is not feasible. Therefore, an endoscopic procedure should only be performed after optimal preoperative diagnosis and, in case of the slightest doubt, only if intraoperative microscopic examination is available, and the possibility to perform an immediate staging laparotomy. Sufficient information of the patient is relevant. Laparoscopic removal of tumor or adnexae should be performed in a lap sac. However, there is no absolute certainty of preventing spillage even with the lap sac, since not all endoscopic sacs available are of a sufficient quality.

Adult↗
PubMed1995

Folding, flavinylation, and mitochondrial import of 6-hydroxy-D-nicotine oxidase fused to the presequence of rat dimethylglycine dehydrogenase.

We analyzed the folding, covalent flavinylation, and mitochondrial import of the rabbit reticulocyte lysate-translated bacterial 6-hydroxy-D-nicotine oxidase (6-HDNO) fused to the mitochondrial targeting sequence of rat liver dimethylglycine dehydrogenase. Translation of 6-HDNO in FAD-supplemented reticulocyte lysate resulted in a protein that contained covalently incorporated FAD, exhibited enzyme activity, and was trypsin-resistant, a characteristic of the tight conformation of the holoenzyme. The attached mitochondrial presequence did not prevent folding, binding of FAD, or enzyme activity of the 6-HDNO moiety of the fusion protein (pre-6-HDNO). Pre-6-HDNO was imported into rat liver mitochondria and processed by the mitochondrial processing peptidase. Incubation of the trypsin-resistant pre-holo-6-HDNO protein with deenergized rat liver mitochondria demonstrated that upon contact with mitochondria, the protein was unfolded and became trypsin sensitive. Mitochondrial import assays showed that the unfolded pre-holo-6-HDNO with covalently attached FAD was imported into rat liver mitochondria. Inside the mitochondrion the holo-6-HDNO was refolded into the trypsin-resistant conformation. However, when pre-apo-6-HDNO was imported only part of the protein became trypsin resistant (approximately 20%). Addition of FAD and the allosteric effector glycerol 3-phosphate to apo-6-HDNO containing mitochondrial matrix was required to transform the protein into the trypsin-resistant conformation characteristic of holo-6-HDNO.

Amino Acid Sequence↗
PubMed1995

A pAO1-encoded molybdopterin cofactor gene (moaA) of Arthrobacter nicotinovorans: characterization and site-directed mutagenesis of the encoded protein.

A gene homologous to moaA, the gene responsible for the expression of a protein involved in an early step in the synthesis of the molybdopterin cofactor of Escherichia coli, was found to be located 2.7-kb upstream of the nicotine dehydrogenase (ndh) operon on the catabolic plasmid pAO1 of Arthrobacter nicotinovorans. The MoaA protein, containing 354 amino acids, migrated on an SDS-polyacrylamide gel with an apparent molecular weight of 40,000, in good agreement with the predicted molecular weight of 38,880. The pAO1-encoded moaA gene from A. nicotinovorans was expressed in E. coli as an active protein that functionally complemented moaA mutants. Its deduced amino acid sequence shows 43% identity to the E. coli MoaA, 44% to the NarAB gene product from Bacillus subtilis, and 42% to the gene product of two contiguous ORFs from Methanobacterium formicicum. N-terminal sequences, including the motif CxxxCxYC, are conserved among the MoaA and NarAB proteins. This motif is also present in proteins involved in PQQ cofactor synthesis in almost all the NifB proteins reported so far and in the fixZ gene product from Rhizobium leguminosarum. Mutagenesis of any of these three conserved cysteine residues to serine abolished the biological activity of MoaA, while substitution of the tyrosine by either serine, phenylalanine, or alanine did not alter the capacity of the protein to complement the moaA mutation in E. coli. A second Cys-rich domain with the motif FCxxC(13x)C is found close to the C-terminus of MoaA and NarAB proteins. These two Cys-rich sequences may be involved in the coordination of a metal ions. The pAO1 copy of moaA may not be unique in the A. nicotinovorans genome since the molybdopterin cofactor oxidation products were detected in cell extracts from a plasmidless strain.

Amino Acid Sequence↗
PubMed1994

Tissue specificity of rat mitochondrial dimethylglycine dehydrogenase expression.

Expression of mitochondrial dimethylglycine dehydrogenase (Me2GlyDH) was analysed in various tissues, liver cell types and developmental stages of the rat. Total RNA extracted from liver, spleen, brain, kidney, lung and heart was reverse-transcribed into cDNA and amplified with Me2GlyDH cDNA-specific oligonucleotides by PCR. Expression of the enzyme was observed mainly in liver and kidney. In addition, Me2GlyDH mRNA could be demonstrated in total RNA samples of lung, heart and brain but was barely detectable in spleen total RNA. In RNA prepared from 14-day rat embryos, Me2GlyDH-specific mRNA was clearly present. Among various liver cell types, besides hepatocytes, endothelial cells showed a high level of Me2GlyDH mRNA expression. There was no amplification product detectable in liver macrophages (Kupffer cells) and only a very faint one in fat-storing cells (Ito cells). Western blots confirmed at the protein level the predominant expression of the enzyme in liver and kidney, but Me2GlyDH protein was also present in the protein extract of lung, heart, spleen and brain. Immunohistochemical staining of liver slices with Me2GlyDH-specific antiserum revealed that expression of this enzyme is evenly distributed throughout the liver tissue. In the kidney, expression of the enzyme was located in the proximal tubule cells. Our results demonstrate that, contrary to the previously assumed liver-restricted expression, this enzyme is specifically expressed predominantly in the liver and kidney, but, in addition, it is detectable in many other tissues of the rat.

Aging↗
PubMed1994

Structural analysis and molybdenum-dependent expression of the pAO1-encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans.

The genes of nicotine dehydrogenase (NDH) were identified, cloned and sequenced from the catabolic plasmid pAO1 of Arthrobacter nicotinovorans. In immediate proximity to this gene cluster is the beginning of the 6-hydroxy-L-niotine oxidase (6-HLNO) gene. NDH is composed of three subunits (A, B and C) of M(r) 30,011, 14,924 and 87,677. It belongs to a family of bacterial hydroxylases with a similar subunit structure; they have molybdopterin dinucleotide, FAD and Fe-S clusters as cofactors. Here the first complete primary structure of a bacterial hydroxylase is provided. Sequence alignments of each of the NDH subunits show similarities to the sequences of eukaryotic xanthine dehydrogenase (XDH) but not to other known molybdenum-containing bacterial enzymes. Based on alignment with XDH it is inferred that the smallest subunit (NDHB) carries an iron-sulphur cluster, that the middle-sized subunit (NDHA) binds FAD, and that the largest NDH subunit (NDHC) corresponds to the molybdopterin-binding domain of XDH. Expression of both the ndh and the 6-hino genes required the presence of nicotine and molybdenum in the culture medium. Tungsten inhibited enzyme activity but not the synthesis of the enzyme protein. The enzyme was found in A. nicotinovorans cells in a soluble form and in a membrane-associated form. In the presence of tungsten the fraction of membrane-associated NDH increased.

Amino Acid Sequence↗
PubMed1994

Regulation of gene expression by cofactors derived from B vitamins.

Vitamins represent precursor molecules for the majority of cofactors essential for various enzyme activities. Is the expression of proteins that require a specific cofactor for their function correlated to its availability? Examples are presented and discussed of the involvement of vitamin-derived cofactors in the regulatory mechanisms controlling gene expression in bacteria and eukaryotes at the transcriptional and translational level.

Animals↗
PubMed1993

Cysteine to serine replacements in 6-hydroxy-D-nicotine oxidase. Consequences for enzyme activity, cofactor incorporation, and formation of high molecular weight protein complexes with molecular chaperones (GroEL).

6-Hydroxy-D-nicotine oxidase, an enzyme with FAD covalently attached to the protein, contains 6 cysteine residues in positions 45 (Cys1), 59 (Cys2), 136 (Cys3), 173 (Cys4), 260 (Cys5) and 433 (Cys6). Cys2, 3, 5, and 6 were replaced with serine by site-directed mutagenesis. The effects of these exchanges on enzyme activity, the autocatalytic incorporation of the cofactor, and the interaction of the mutant proteins with molecular chaperones were analyzed. The flavinylation of 6-hydroxy-D-nicotine oxidase is dependent on the presence of allosteric effectors, e.g. glycerol 3-phosphate or other phosphorylated tricarbon compounds. Replacement of Cys2 or Cys5 abolished this dependence. Covalent incorporation of FAD was reduced to an undetectable level in the Cys3 and Cys5 mutants. Replacement of Cys6 by Ser had no significant effect on enzyme activity and cofactor attachment. Deletion of two amino acids, Phe and Arg, situated 12 and 11 amino acid residues, respectively, from the carboxyl terminus of the protein, resulted in an inactive enzyme with no covalently bound FAD. This result indicates that almost the entire protein chain has to be synthesized before the cofactor can be incorporated, making a cotranslational flavinylation step rather unlikely. The distribution of the 6-hydroxy-D-nicotine oxidase polypeptide between the high molecular weight complexes and the free soluble form was analyzed by gel filtration on Sephacryl S-200. The wild-type holoenzyme as well as the wild-type apoenzyme were recovered in the eluent fraction of the column while the mutant proteins were retained in high molecular weight complexes, predominantly in those associated with GroEL, as revealed by immunoprecipitation. The extent of complex formation with this molecular chaperone depended on the position of the mutated Cys residue within the protein. Complex formation was highest with protein from the mutants Cys2 and Cys3, less with the Cys5, and absent with the Cys6 mutant protein. Thus, alterations in the amino-terminal part of the 6-hydroxy-D-nicotine oxidase appear more important for the interaction with molecular chaperones than alterations situated in the carboxyl-terminal part of the protein.

Amino Acid Sequence↗