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PubMed1999

A method for detecting dopaminechrome isomerase activity on gels.

Melanogenesis involves oxidation of 3,4-dihydroxyphenylalanine (dopa) to dopachrome which then is converted into 5,6-dihydroxyindole by dopachrome isomerase. 5,6-Dihydroxyindole is oxidized to its quinone which in turn is metabolized nonenzymatically to melanin. In addition to dopachrome isomerase, a new dopminechrome isomerase activity involved in the conversion of dopaminechrome into 5,6-dihydroxyindole has been observed in the larva of Rhinoceros oryctes. This dopaminechrome isomerase differs from dopachrome isomerase in its electrophoretic mobility and substrate specificity. The present study reports a specific, sensitive and rapid staining method for detecting dopaminechrome isomerase activity after electrophoresis. Using this new method, the presence of the dopaminechrome isomerase activity, which is involved in melanogenesis, could easily be detected by staining tyrosinase embedded native gels in dopamine solution. Tyrosinase entrapped in the gels converts dopamine in dopaminechrome. The dopaminechrome isomerase separated in the gels catalyzes dopaminechrome to 5,6-dihydroxyindole which is oxidized further by tyrosinase to colored melanochrome. The dopaminechrome isomerase appears as a bluish purple band against a pink background.

Animals↗
PubMed1999

Peptidyl-prolyl cis-trans isomerases, a superfamily of ubiquitous folding catalysts.

Cyclosporine A therapy for prophylaxis against graft rejection revolutionized human organ transplantation. The immunosuppressant drugs cyclosporin A (CsA), FK506 and rapamycin block T-cell activation by interfering with the signal transduction pathway. The target proteins for CsA and FK506 were found to be cyclophilins and FK506-binding proteins, (FKBPs), respectively. They are unrelated in primary sequence, although both are peptidyl-prolyl cis-trans isomerases catalyzing the interconversion of peptidylprolyl imide bonds in peptide and protein substrates. However, the prolyl isomerase activity of these proteins is not essential for their immunosuppressive effects. Instead, the specific surfaces of the cyclophilin-CsA and FKBP-FK506 complexes mediate the immunosuppressive action. Moreover, the natural cellular functions of all but a few remain elusive. In some cases it could be demonstrated that prolyl isomerization is the rate-limiting step in protein folding in vitro, but many knockout mutants of single and multiple prolyl isomerases were viable with no detectable phenotype. Even though a direct requirement for in vivo protein folding could not be demonstrated, some important natural substrates of the prolyl isomerases are now known, and they demonstrate the great variety of prolyl isomerization functions in the living cell: (i) A human cyclophilin binds to the Gag polyprotein of the human immunodeficiency virus-1 (HIV-1) virion and was found to be essential for infection with HIV to occur, probably by removal of the virion coat. (ii) Together with heat shock protein (HSP) 90, a member of the chaperone family, high molecular weight cyclophilins and FKBPs bind and activate steroid receptors. This example also demonstrates that prolyl isomerases act together with other folding enzymes, for example the chaperones, and protein disulfide isomerases. (iii) An FKBP was found to act as a modulator of an intracellular calcium release channel. (iv) Along with the cyclophilins and FKBPs, a third class of prolyl isomerases exist, the parvulins. The human parvulin homologue Pin1 is a mitotic regulator essential for the G2/M transition of the eukaryotic cell cycle. These findings place proline isomerases at the intersection of protein folding, signal transduction, trafficking, assembly and cell cycle regulation.

Bacterial Proteins↗
PubMed1980

Resolution of protein disulphide-isomerase and glutathione-insulin transhydrogenase activities by covalent chromatography.

1. Protein disulphide-isomerase (EC 5.3.4.1) and glutathione-insulin transhydrogenase (EC 1.8.4.2) were resolved by covalent chromatography. Both activities, in a partially purified preparation from bovine liver, bind covalently as mixed disulphides to activated thiopropyl-Sepharose 6B, in a new stepwise elution procedure protein disulphide-isomerase is displaced in mildly reducing conditions whereas glutathione-insulin transhydrogenase is only displaced by more extreme reducing conditions. 2. This together with evidence for partial resolution of the two activities by ion-exchange chromatography, conclusively establishes that the two activities are not alternative activities of a single bovine liver enzyme. 3. Protein disulphide-isomerase, partially purified by a published procedure, has now been further purified by covalent chromatography and ion-exchange chromatography. The final material is 560-fold purified relative to a bovine liver homogenate; it has barely detectable glutathione-insulin transhydrogenase activity. 4. The purified protein disulphide-isomerase shows a single major band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to a mol.wt. of 57000. 5. The purified protein disulphide-isomerase has Km values for 'scrambled' ribonuclease and dithiothreitol of 23 microgram/ml and 5.4 microM respectively and has a sharp pH optimum at 7.5. The enzyme has a broad thiol-specificity, and several monothiols, at 1mM, can replace dithiothreitol. 6. The purified protein disulphide-isomerase is completely inactivated after incubation with a 2-3 fold molar excess of iodoacetate. The enzyme is also significantly inhibited by low concentrations of Cd2+ ions. These findings strongly suggest the existence of a vicinal dithiol group essential for enzyme activity. 7. When a range of thiols were used as co-substrates for protein disulphide-isomerase activity, the activities were found to co-purify quantitatively, implying the presence of a single protein disulphide-isomerase of broad thiol-specificity. Glutathione-disulphide transhydrogenase activities, assayed with a range of disulphide compounds, did not co-purify quantitatively.

Animals↗
PubMed1982

Production of glucose isomerase by genus Streptomyces.

Screening for glucose isomerases was done using 46 species and 63 strains of Streptomyces type cultures. Glucose isomerase was found in a wide range of these species. The intracellular glucose isomerase of S. olivochromogenes showed the highest fructose conversion ratio. The apparent extracellular glucose isomerase activities were observed for several strains of Streptomyces, Streptomyces venezuelae had the highest apparent extracellular glucose isomerase activity. The glucose isomerase yield was improved from 1.95 units/ml to 7.5 units/ml by induced mutation and culturing condition studies. This increase in glucose isomerase yield has made it possible to produce glucose isomerase industrially.

Aldose-Ketose Isomerases↗
PubMed1997

Sequence similarity between xylose isomerase and replicase: another TIM-barrel in the replicase structure?

The BLAST search using the strand beta 2 (46_GAHGVTFHDDDLIP) of the (alpha/beta)8-barrel of xylose isomerase from Streptomyces olivochromogenes resulted in retrieving the sequentially similar segment of replicase from garlic latent virus (692_GGHGIGFHRDD). The detailed analysis of the entire amino acid sequences of both xylose isomerase and replicase suggested that the polypeptide segment 644-1046 of replicase (the entire length of this enzyme is 1924 residues) could share the structure of xylose isomerase (20.7% identity using the entire sequence of xylose isomerase). The relatedness of replicase and xylose isomerase is supported by the fact that the sequence similarity can be observed along the whole sequence of xylose isomerase (386 amino acid residues). The sequence of replicase exhibits moreover the similarity with that of lycopene cyclase, an enzyme implicated in the beta-carotene biosynthesis, that was previously found to share similarity with xylose isomerase. Thus the relevant segment of replicase is predicted to adopt an (alpha/beta)8-barrel topology similar to that of xylose isomerase.

Aldose-Ketose Isomerases↗
PubMed1998

Site-directed mutagenesis identifies amino acid residues associated with the dehydrogenase and isomerase activities of human type I (placental) 3beta-hydroxysteroid dehydrogenase/isomerase.

3beta-hydroxysteroid dehydrogenase/steroid delta5-->4-isomerase (3beta-HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding human wild-type I (placental) and the human type I mutants - H261R, Y253F and Y253,254F. Western blots of SDS-polyacrylamide gels showed that the baculovirus-infected Sf9 cells expressed the immunoreactive wild-type, H261R, Y253F or Y253,254F protein that co-migrated with purified placental 3beta-HSD/isomerase (monomeric Mr=42,000 Da). The wild-type, H261R and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells (5.01). In kinetic studies with purified enzyme, the H261R mutant enzyme had no 3beta-HSD activity, whereas the Km and Vmax values of the isomerase substrate were similar to the values obtained with the wild-type and native enzymes. The Vmax (88 nmol/min/mg) for the conversion of 5-androstene-3,17-dione to androstenedione by the Y253F isomerase activity was 7.0-fold less than the mean Vmax (620 nmol/min/mg) measured for the isomerase activity of the wild-type and native placental enzymes. In microsomal preparations, isomerase activity was completely abolished in the Y253,254F mutant enzyme, but Y253,254F had 45% of the 3beta-HSD activity of the wild-type enzyme. In contrast, the purified Y253F, wild-type and native enzymes had similar Vmax values for substrate oxidation by the 3beta-HSD activity. The 3beta-HSD activities of the Y253F, Y253,254F and wild-type enzymes reduced NAD+ with similar kinetic values. Although NADH activated the isomerase activities of the H261R and wild-type enzymes with similar kinetics, the activation of the isomerase activity of H261R by NAD+ was dramatically decreased. Based on these kinetic measurements, His261 appears to be a critical amino acid residue for the 3beta-HSD activity, and Tyr253 or Tyr254 participates in the isomerase activity of human type I (placental) enzyme.

Amino Acid Sequence↗
PubMed1994

Purification, cDNA cloning and heterologous expression of human phosphomannose isomerase.

Phosphomannose isomerase catalyses the interconversion of fructose-6-P and mannose-6-P and has a critical role in the supply of D-mannose derivatives required for many eukaryotic glycosylation reactions. Three classes of enzymes possessing phosphomannose-isomerase activity have been identified in bacteria and lower eukaryotes. We have purified human phosphomannose isomerase to homogeneity from placental tissue. Protein sequence information obtained from internal fragments of the protein was used to design degenerate oligonucleotides which were used to amplify a fragment of a human phosphomannose-isomerase cDNA. A full-length cDNA was isolated from a human testes lambda gt11 library using this fragment as a probe. The cDNA encoded a protein with significant sequence identity to fungal and some bacterial phosphomannose isomerases but was unrelated to those from other bacteria. Based on amino acid sequence identity we propose a classification system for enzymes with phosphomannose-isomerase activity. The cDNA, under the control of the GAL1 promoter, was expressed in a Saccharomyces cerevisiae strain from which the native gene encoding phosphomannose isomerase had been deleted. The human enzyme was found to be able to functionally substitute for the yeast enzyme. Phosphomannose-isomerase mRNA was found in all human tissues tested but was more highly expressed in heart, brain and skeletal muscle. The cDNA was expressed in Escherichia coli permitting the isolation of pure recombinant protein which will be used for kinetic and structural studies.

Amino Acid Sequence↗
PubMed1997

Cloning, expression, purification and characterization of triosephosphate isomerase from Trypanosoma cruzi.

The gene that encodes for triosephosphate isomerase from Trypanosoma cruzi was cloned and sequenced. In T. cruzi, there is only one gene for triosephosphate isomerase. The enzyme has an identity of 72% and 68% with triosephosphate isomerase from Trypanosoma brucei and Leishmania mexicana, respectively. The active site residues are conserved: out of the 32 residues that conform the interface of dimeric triosephosphate isomerase from T. brucei, 29 are conserved in the T. cruzi enzyme. The enzyme was expressed in Escherichia coli and purified to homogeneity. Data from electrophoretic analysis under denaturing techniques and filtration techniques showed that triosephosphate isomerase from T. cruzi is a homodimer. Some of its structural and kinetic features were determined and compared to those of the purified enzymes from T. brucei and L. mexicana. Its circular dichroism spectrum was almost identical to that of triosephosphate isomerase from T. brucei. Its kinetic properties and pH optima were similar to those of T. brucei and L. mexicana, although the latter exhibited a higher Vmax with glyceraldehyde 3-phosphate as substrate. The sensitivity of the three enzymes to the sulfhydryl reagent methylmethane thiosulfonate (MeSO2-SMe) was determined; the sensitivity of the T. cruzi enzyme was about 40 times and 200 times higher than that of the enzymes from T. brucei and L. mexicana, respectively. Triosephosphate isomerase from T. cruzi and L. mexicana have the three cysteine residues that exist in the T. brucei enzyme (positions 14, 39, 126, using the numbering of the T. brucei enzyme); however, they also have an additional residue (position 117). These data suggest that regardless of the high identity of the three trypanosomatid enzymes, there are structural differences in the disposition of their cysteine residues that account for their different sensitivity to the sulfhydryl reagent. The disposition of the cysteine in triosephosphate isomerase from T. cruzi appears to make it unique for inhibition by modification of its cysteine.

Amino Acid Sequence↗
PubMed1990

Cloning and expression of the Clostridium thermosulfurogenes glucose isomerase gene in Escherichia coli and Bacillus subtilis.

The gene that encodes thermostable glucose isomerase in Clostridium thermosulfurogenes was cloned by complementation of glucose isomerase activity in a xylA mutant of Escherichia coli. A new assay method for thermostable glucose isomerase activity on agar plates, using a top agar mixture containing fructose, glucose oxidase, peroxidase, and benzidine, was developed. One positive clone, carrying plasmid pCGI38, was isolated from a cosmid library of C. thermosulfurogenes DNA. The plasmid was further subcloned into a Bacillus cloning vector, pTB523, to generate shuttle plasmid pMLG1, which is able to replicate in both E. coli and Bacillus subtilis. Expression of the thermostable glucose isomerase gene in both species was constitutive, whereas synthesis of the enzyme in C. thermosulfurogenes was inducible by D-xylose. B. subtilis and E. coli produced higher levels of thermostable glucose isomerase (1.54 and 0.46 U/mg of protein, respectively) than did C. thermosulfurogenes (0.29 U/mg of protein). The glucose isomerases synthesized in E. coli and B. subtilis were purified to homogeneity and displayed properties (subunit Mr, 50,000; tetrameric molecular structure; thermostability; metal ion requirement; and apparent temperature and pH optima) identical to those of the native enzyme purified from C. thermosulfurogenes. Simple heat treatment of crude extracts from E. coli and B. subtilis cells carrying the recombinant plasmid at 85 degrees C for 15 min generated 80% pure glucose isomerase. The maximum conversion yield of glucose (35%, wt/wt) to fructose with the thermostable glucose isomerase (10.8 U/g of dry substrate) was 52% at pH 7.0 and 70 degrees C.

Aldose-Ketose Isomerases↗
PubMed1995

xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana.

The xylA gene coding for xylose isomerase from the hyperthermophile Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 444 residues with a calculated molecular weight of 50,892. The native enzyme was a homotetramer with a molecular weight of 200,000. This xylose isomerase was a member of the family II enzymes (these differ from family I isomerases by the presence of approximately 50 additional residues at the amino terminus). The enzyme was extremely thermostable, with optimal activity above 95 degrees C. The xylose isomerase showed maximum activity at pH 7.1, but it had high relative activity over a broad pH range. The catalytic efficiency (kcat/Km) of the enzyme was essentially constant between 60 and 90 degrees C, and the catalytic efficiency decreased between 90 and 98 degrees C primarily because of a large increase in Km. The T. neapolitana xylose isomerase had a higher turnover number and a lower Km for glucose than other family II xylose isomerases. Comparisons with other xylose isomerases showed that the catalytic and cation binding regions were well conserved. Comparison of different xylose isomerase sequences showed that numbers of asparagine and glutamine residues decreased with increasing enzyme thermostability, presumably as a thermophilic strategy for diminishing the potential for chemical denaturation through deamidation at elevated temperatures.

Aldose-Ketose Isomerases↗
PubMed1996

Purification and cloning of a thermostable xylose (glucose) isomerase with an acidic pH optimum from Thermoanaerobacterium strain JW/SL-YS 489.

An unusual xylose isomerase produced by Thermoanaerobacterium strain JW/SL-YS 489 was purified 28-fold to gel electrophoretic homogeneity, and the biochemical properties were determined. Its pH optimum distinguishes this enzyme from all other previously described xylose isomerases. The purified enzyme had maximal activity at pH 6.4 (60 degrees C) or pH 6.8 (80 degrees C) in a 30-min assay, an isoelectric point at 4.7, and an estimated native molecular mass of 200 kDa, with four identical subunits of 50 kDa. Like other xylose isomerases, this enzyme required Mn2+, Co2+, or Mg2+ for thermal stability (stable for 1 h at 82 degrees C in the absence of substrate) and isomerase activity, and it preferred xylose as a substrate. The gene encoding the xylose isomerase was cloned and expressed in Escherichia coli, and the complete nucleotide sequence was determined. Analysis of the sequence revealed an open reading frame of 1,317 bp that encoded a protein of 439 amino acid residues with a calculated molecular mass of 50 kDa. The biochemical properties of the cloned enzyme were the same as those of the native enzyme. Comparison of the deduced amino acid sequence with sequences of other xylose isomerases in the database showed that the enzyme had 98% homology with a xylose isomerase from a closely related bacterium, Thermoanaerobacterium saccharolyticum B6A-RI. In fact, only seven amino acid differences were detected between the two sequences, and the biochemical properties of the two enzymes, except for the pH optimum, are quite similar. Both enzymes had a temperature optimum at 80 degrees C, very similar isoelectric points (pH 4.7 for strain JW/SL-YS 489 and pH 4.8 for T. saccharolyticum B6A-RI), and slightly different thermostabilities (stable for 1 h at 80 and 85 degrees C, respectively). The obvious difference was the pH optimum (6.4 to 6.8 and 7.0 to 7.5, respectively). The fact that the pH optimum of the enzyme from strain JW/SL-YS 489 was the property that differed significantly from the T. saccharolyticum B6A-RI xylose isomerase suggested that one or more of the observed amino acid changes was responsible for this observed difference.

Aldose-Ketose Isomerases↗
PubMed1990

Genetics and evolution of chloroplast isozymes of triosephosphate isomerase.

Triosephosphate isomerase is an ubiquitous and highly conservative dimeric enzyme, consisting of subunits of Mr 26,000-27,000. Plants usually contain one cytosolic and one plastid isozyme. While 2x wheats also contain one plastid isozyme, 4x wheats contain 3, and 6x cultural wheats contain five plastid isozymes. The multiplicity of the isozyme pattern in 6x wheats is explainable by the presence of three different genomes (AABBDD), each contributing a distinct triosephosphate isomerase gene (alpha',beta,delta), and by the formation of homodimeric and heterodimeric isozyme forms. While the beta beta-form was, as expected, also found in Aegilops speltoides which is regarded as donor of the B genome, the descent of the other genes for plastid triosephosphate isomerase did not occur in accordance with common contentions on the evolution of 6x cultural wheat and its presumptive ancestors. In the reciprocal intergeneric hybrids between wheat and rye, Secalotricum and Triticale, the patterns of both the cytosolic and the plastid-specific triosephosphate isomerases were biparentally inherited, indicating also that the plastid isozyme was nuclear-encoded. Data which are available about amino acid sequences and gene organization and immunological observations show that the cytosolic triosephosphate isomerase of plants is strongly related to other eukaryotic animal triosephosphate isomerase genes. Multiple evidence has been presented that the plastid- specific isozyme represents a distinct polypeptide and is specified by a distinct gene, relative to the cytosolic isozyme. Immunological comparisons indicate that the plastid isozyme shares homologies with the cytosolic isozyme but is not related to the enzyme from prokaryotic cyanobacteria or bacteria. To enable a more precise comparison, plastid triosephosphate isomerase has been cloned from a cDNA library from rye, and cDNA clones are being sequenced. The plastid enzyme of triosephosphate isomerase appears to have evolved from a duplication on an ancestral nuclear gene of the primordial plant cell. For other plastid-specific isozymes evidence exists that their genes were incorporated into the nucleus by gene transfer from a prokaryotic endosymbiont.

Biological Evolution↗
PubMed1956

Retinene isomerase.

Rhodopsin is formed by the condensation of opsin with a cis isomer of retinene, called neo-b. The bleaching of rhodopsin releases all-trans retinene which must be isomerized back to neo-b in order for rhodopsin to regenerate. Both retinene isomers are in equilibrium with the corresponding isomers of vitamin A, through the alcohol dehydrogenase system. An enzyme is found in cattle retinas and frog pigment layers which catalyzes the interconversion of all-trans and neo-b retinene. We call it "retinene isomerase." It is soluble in neutral phosphate buffer, and precipitates between 20 and 35 per cent saturation with ammonium sulfate. In the dark, the isomerase converts all-trans and neo-b retinene to an equilibrium mixture of 5 parts neo-b and 95 parts all-trans. With opsin present to trap neo-b, the isomerase catalyzes the synthesis of rhodopsin from all-trans retinene. This reaction, however, is too slow to account for dark adaptation. Retinene is isomerized by light, but too slowly to supply the retina with neo-b. In aqueous solution the pseudoequilibrium mixture contains about 15 per cent neo-b. When all-trans retinene is irradiated in the presence of isomerase, the rate of formation of neo-b is increased about 5 times, and the pseudoequilibrium shifted so that the mixture now contains about 32 per cent neo-b. The isomerase is specific for all-trans and neo-b retinene. It does not act on two other cis isomers of retinene, nor on all-trans or neo-b vitamin A. The role of the isomerase in vision appears to be as follows: in the light, as rhodopsin is bleached to opsin and all-trans retinene, the latter is in part converted to the neo-b isomer and stored in the pigment epithelium as neo-b vitamin A. During dark adaptation, the dominant process is the trapping by opsin of neo-b retinene supplied from stores of neo-b vitamin A, and the slow isomerase-catalyzed "dark" conversion of all-trans to neo-b retinene.

Animals↗
PubMed1991

Mitochondrial 3-2trans-Enoyl-CoA isomerase. Purification, cloning, expression, and mitochondrial import of the key enzyme of unsaturated fatty acid beta-oxidation.

3-2trans-Enoyl-CoA isomerase (EC 5.3.3.8) is a key enzyme in mitochondrial beta-oxidation of unsaturated fatty acids in bacteria, plant and animal cells. The enzyme was isolated from rat liver mitochondria and purified to homogeneity by two chromatographic steps. Partial polypeptide sequences of the 29 kDa protein were derived from cyanogen bromide, tryptic, Lys-C, and protease V8 fragments by Edman degradation. Peptide-derived synthetic oligonucleotides were used for the isolation of a 990 bp long isomerase-specific cDNA from rat liver cDNA libraries. 867 bp encode the 289 amino-acid residues of the preisomerase with a molecular mass of 32,254 Da. The 1.3-kb mRNA is most strongly expressed in skeletal muscle followed by liver, heart, kidney, and weakly expressed in spleen and brain. In vitro transcription and translation yielded a 32 kDa polypeptide which was immunoprecipitated by anti rat isomerase antibodies. In the presence of mitochondria the 32 kDa precursor isomerase was processed during mitochondrial import to the 29 kDa mature form of the 3-2trans-enoyl-CoA isomerase with 264 amino-acid residues (Mr 29,706). A N-terminal signal sequence of 25 amino-acid residues directs the import into the mitochondrial matrix and is cleaved in two successive steps passing through an intermediate form of Mr 30,475. The two cysteine residues in positions 142 and 148 of the preisomerase are present as free thiol groups as shown by derivatization of the mature, native protein with the fluorescent label N-(iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid. The mitochondrial 3-2trans enoyl-CoA isomerase shows significant homology and conserved amino-acid exchanges with the mitochondrial enoyl-CoA hydratase, the N-terminal domain of the bifunctional peroxisomal enoyl-CoA-hydratase:3-hydroxyacyl-CoA dehydrogenase and to extended domains of the alpha-subunit of the procaryotic beta-oxidation complex sharing enoyl-CoA isomerase, D(-)3-hydroxyacyl-CoA epimerase, enoyl-CoA hydratase and L(+)3-hydroxyacyl-CoA dehydrogenase activity, encoded by the fad B operon of E. coli.

Amino Acid Sequence↗
PubMed2000

A new mechanism for the control of phenoloxidase activity: inhibition and complex formation with quinone isomerase.

Insect phenoloxidases participate in three physiologically important processes, viz., cuticular hardening (sclerotization), defense reactions (immune reaction), and wound healing. Arrest or even delay of any of these processes compromises the survival of insects. Since the products of phenoloxidase action, viz., quinones, are cytotoxic, uncontrolled phenoloxidase action is deleterious to the insects. Therefore, the activity of this important enzyme has to be finely controlled. A novel inhibition of insect phenoloxidases, which serves as a new regulatory mechanism for control of its activity, is described. The activity of phenoloxidases isolated from both Sarcophaga bullata and Manduca sexta is drastically inhibited by quinone isomerase (isolated from Calliphora), an enzyme that utilizes the phenoloxidase-generated 4-alkylquinones. In turn, phenoloxidase reciprocated the inhibition of isomerase. By forming a complex and controlling each other's activity, these two enzymes seem to regulate the levels of endogenously quinones. In support of this contention, an endogenous complex consisting of phenoloxidase, quinone isomerase, and quinone methide isomerase was characterized from the insect, Calliphora. This sclerotinogenic complex was isolated and purified by borate extraction of the larval cuticle, ammonium sulfate precipitation, and Sepharose 6B column chromatography. The complex exhibited a molecular mass of about 620-680 kDa, as judged by size-exclusion chromatography on Sepharose 6B and HPLC and did not even enter 3% polyacrylamide gel during electrophoresis. The phenoloxidase activity of the complex exhibited a wide substrate specificity. Incubation of the complex with N-acetyldopamine rapidly generated N-acetylnorepinephrine, dehydro-N-acetyldopamine, and its dimers. In addition, transient accumulation of N-acetyldopamine quinone was also observed. These results confirm the presence of phenoloxidase, quinone isomerase, and quinone methide isomerase in the complex. Attempts to dissociate the complex with even trace amounts of SDS ended in the total loss of quinone isomerase activity. The complex does not seems to be made up of stoichiometric amounts of individual enzymes as the ratio of phenoloxidase to quinone isomerase varied from preparation to preparation. It is proposed that the complex formation between sequential enzymes of sclerotinogenic pathway is advantageous for the organism to effectively channel various reactive intermediates during cuticular hardening.

Animals↗
PubMed1996

S-glutathiolated hepatocyte proteins and insulin disulfides as substrates for reduction by glutaredoxin, thioredoxin, protein disulfide isomerase, and glutathione.

The disulfide-reducing activities of glutaredoxin, thioredoxin, protein disulfide isomerase, glutathione, and cysteine were directly compared with a mixture of hepatocyte 35S-glutathiolated proteins as the substrate. Dethiolation of individual 35S-labeled protein bands from the mixture of hepatocyte proteins was analyzed by SDS-PAGE. All of the 35S-labeled protein bands could be completely dethiolated by glutaredoxin, thioredoxin, protein disulfide isomerase, glutathione, or cysteine. On a molar basis glutaredoxin was over 10 times more effective than either thioredoxin or protein disulfide isomerase. Dethiolation rates of individual proteins varied in minor ways. For example, glutaredoxin dethiolated the 15-, 30-, and 48-kDa protein bands 3 to 4 times faster than the 27-, 28-, and 77-kDa bands. Glutaredoxins from pig liver or from bovine heart had the same specificity and similar activity. The rate of dethiolation by glutathione alone was low compared to the glutaredoxin-catalyzed process, but all 35S-labeled protein bands could be reduced by glutathione, cysteine, or dithiothreitol. Glutathione was clearly more effective than cysteine when these two thiols were compared on the basis of the concentration of thiolate anion available at neutral pH. Therefore, glutathione is a more specific reductant of S-glutathiolated proteins than is cysteine but it is much less effective than glutaredoxin. Since glutaredoxin activity in cells is 10 times higher than the concentration used in these experiments, ample activity is available to account for substantial rates of dethiolation in vivo. Thioredoxin is quite inefficient as a reductant of S-glutathiolated proteins, but it was reasoned that it might first reduce glutaredoxin, which then could reduce the S-glutathiolated protein. A combination of thioredoxin and glutaredoxin was effective. It is proposed that glutaredoxin is the principal agent responsible for protein dethiolation in vivo. The effectiveness of glutaredoxin, thioredoxin, and protein disulfide isomerase as reductants for protein disulfide bonds was examined with insulin as the substrate. Protein disulfide isomerase was very effective and thioredoxin was nearly as effective. Human thioredoxin was similar to Escherichia coli thioredoxin in reactivity and specificity. Glutaredoxin did not facilitate insulin reduction at equal concentrations. Thus, protein disulfide isomerase and thioredoxin are more effective than glutaredoxin as reductants of insulin protein disulfides. The apparent reduction potential of pig liver glutaredoxin (-0.159 +/- 0.004 V) was determined by measuring the amount of reduced glutaredoxin in equilibrium with mixtures of glutathione and glutathione disulfide. Glutaredoxin was a weaker reductant than E. coli thioredoxin (-0.260 V) and was similar to protein disulfide isomerase (-0.11 to -0.19 V). The role of these proteins as disulfide reductants is not determined solely by thermodynamic considerations. A glutathione binding site at the dithiol region of glutaredoxin may be of primary importance for its function in protein dethiolation, while a different specific peptide binding site in thioredoxin may be more suited to certain protein disulfide structures.

Animals↗
PubMed1978

Solubilization and separation of delta5,3beta-hydroxysteroid dehydrogenase and 3-oxosteroid-delta4-delta5-isomerase from bovine adrenal cortex microsomes.

A physical separation of delta5,3beta-hydroxysteroid dehydrogenase and 3-oxosteroid delta4-delta5-isomerase solubilized from bovine adrenocortical microsomes is described for the first time. The solubilization as well as the separation was carried out with a mixture of a detergent: a substituted betaine (Empigen BB/P) and sodium cholate. This latter detergent protects isomerase from complete inactivation by Empigen and is necessary for the recovery of a significant amount of soluble isomerase. Separation of dehydrogenase and isomerase was successfully accomplished by the use of a DEAE-Biogel A anion-exchanger. Dehydrogenase activity was eluted, while the isomerase was retained. Measurements of dehydrogenase activity with androst-5-en-3beta-ol-17-one, pregnen-3beta-ol-20-one and pregn-5-en-(3beta,17alpha)-diol-20-one and of isomerase activity with androst-5-en-(3,17)-dione and pregn-5-en-(3,20)-dione suggested that more than one isomerase and more than one dehydrogenase form were present.

17-Hydroxysteroid Dehydrogenases↗
PubMed1989

Distribution of retinol isomerase in vertebrate eyes and its emergence during retinal development.

Ocular tissue homogenates were incubated in darkness with [11,12-3H] all-trans retinol. Formation of radiolabeled 11-cis retinol was used as an index of isomerase activity and was determined by high-performance liquid chromatography. Isomerase was found in the eyes of cattle, human, rat, chicken, turtle, goldfish and frog, representing the mammals, birds, reptiles, bony fishes and amphibians. The enzyme was concentrated in the pigment epithelium (RPE). Variable activity was found in the retina, where the amount of radiolabeled 11-cis retinol formed under standard incubation conditions at protein concentrations of 0.03-1.08 mg/ml was 6.4 +/- 6.0% of that in the RPE-choroid. Using the same methodology, we could not detect isomerase in the retinas of three cephalopods (Octopus, Sepia and Loligo). In rats, isomerase was present at postnatal day 10 but not at postnatal days 0 and 4. Therefore, the expression in the RPE of retinol isomerase, which is essential for the formation of rhodopsin in the developing photoreceptors, is coordinated with the emergence of the rod outer segment in the retina. However, the continued expression of this enzyme in RCS rats does not depend on the presence of photoreceptors, because loss of photoreceptors was not associated with an absence of isomerase activity in RCS rats. Our findings suggest that a reciprocal flow of retinoids between the retina and the site of isomerase action in the RPE is a feature common to the visual cycle in all vertebrates.

Animals↗