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PubMed1995

The evolution of sugar isomerases.

L-Arabinose isomerase (EC 5.3.1.4) catalyzes the isomerization of L-arabinose to L-ribulose. Here we report on the purification, kinetic mechanism and chemical mechanism of L-arabinose isomerase from Escherichia coli. The enzyme catalyzes the isomerization of L-arabinose to L-ribulose by a proton transfer mechanism, in contrast to xylose isomerase which uses a hydride transfer mechanism to perform a similar isomerization. Arabinose isomerase activity is metal dependent, although the enzyme can catalyze the exchange of the proton attached to carbon 2 of arabinose with the solvent in the absence of metal ion. Manganese(II) is the only metal ion which renders the enzyme active for the isomerization reaction. Arabinose isomerase has high substrate specificity for L-arabinose. The difference in chemical mechanism between xylose isomerase and arabinose isomerase suggests that these enzymes are not related by convergent evolution. This work also suggests that unless convergent evolution has been demonstrated, the mechanism of one enzyme may not give any insight into the mechanism of a second enzyme catalyzing the same reaction.

Aldose-Ketose Isomerases↗
PubMed2006

Bacitracin reveals a role for multiple thiol isomerases in platelet function.

The platelet-specific integrin alphaIIb beta3 has endogenous thiol isomerase activity associated with the CXXC motifs within the beta subunit. Using a highly purified form of bacitracin, a thiol isomerase inhibitor, we now provide further evidence of the functional significance of this enzymatic activity in integrin activation. In addition, we demonstrate a role for multiple thiol isomerases in platelet function. This bacitracin prevented platelet aggregation to thrombin and collagen, and directly inhibited alphaIIb beta3 activation, as detected by PAC-1 binding. In parallel, bacitracin inhibited the endogenous thiol isomerase activity of purified alphaIIb beta3 with a 50% inhibitory concentration of 15.5 micromol/l. In order to determine whether the effects of bacitracin are solely mediated by inhibition of integrin enzymatic activity, we examined integrin-independent indices of platelet activation. We found bacitracin inhibited both platelet secretion (CD62P and CD63) and thromboxane (TxA2) production, with complete inhibition at different concentrations. Thus, we demonstrated a role for multiple thiol isomerases in platelet function. Taken together, these studies support a role for the endogenous integrin thiol isomerase activity in activation of alphaIIb beta3 and highlight the novel regulation of platelet function by other, as yet undefined thiol isomerases.

Bacitracin↗
PubMed1995

Complex formation between mushroom tyrosinase and Manduca dopachrome isomerase.

Melanin biosynthesis in animals is initiated by the ubiquitously present tyrosinase and is aided by dopachrome isomerase. We have characterized a novel dopachrome isomerase (decarboxylating) from the hemolymph of Manduca sexta that generates a new quinone methide intermediate during melanogenesis (Sugumaran, M. and Semensi, V. (1991) J. Biol. Chem. 266, 6073-6078). This enzyme has the ability to form a complex with mushroom tyrosinase as judged by a number of physicochemical studies. The isomerase exhibited a marked inhibitory effect on tyrosinase and tyrosinase reciprocated by inhibiting the isomerase. While the isomerase showed no activity toward preformed dopaminechrome, it readily influenced the stability of dopaminechrome generated in situ by tyrosinase. Moreover, mushroom tyrosinase, which lacked specific binding to Concanavalin A Sepharose column, after complexing with the isomerase exhibited binding to this column. The complex formation also affected the pI value as well as mobility on a size exclusion column of these enzymes. Enzymes executing sequential metabolic transformation are known to form complexes called metabolons. Based on these above studies, it is concluded that both the enzymes involved in insect melanogenic pathway--phenoloxidase and dopachrome isomerase--are able to form a metabolon complex.

Animals↗
PubMed1987

Sequence of the Ampullariella sp. strain 3876 gene coding for xylose isomerase.

The nucleotide sequence of the gene coding for xylose isomerase from Ampullariella sp. strain 3876, a gram-positive bacterium, has been determined. A clone of a fragment of strain 3876 DNA coding for a xylose isomerase activity was identified by its ability to complement a xylose isomerase-defective Escherichia coli strain. One such complementation positive fragment, 2,922 nucleotides in length, was sequenced in its entirety. There are two open reading frames 1,182 and 1,242 nucleotides in length, on opposite strands of this fragment, each of which could code for a protein the expected size of xylose isomerase. The 1,182-nucleotide open reading frame was identified as the coding sequence for the protein from the sequence analysis of the amino-terminal region and selected internal peptides. The gene initiates with GTG and has a high guanine and cytosine content (70%) and an exceptionally strong preference (97%) for guanine or cytosine in the third position of the codons. The gene codes for a 43,210-dalton polypeptide composed of 393 amino acids. The xylose isomerase from Ampullariella sp. strain 3876 is similar in size to other bacterial xylose isomerases and has limited amino acid sequence homology to the available sequences from E. coli, Bacillus subtilis, and Streptomyces violaceus-ruber. In all cases yet studied, the bacterial gene for xylulose kinase is downstream from the gene for xylose isomerase. We present evidence suggesting that in Ampullariella sp. strain 3876 these genes are similarly arranged.

Actinomycetales↗
PubMed2004

Identification of an Archaeal type II isopentenyl diphosphate isomerase in methanothermobacter thermautotrophicus.

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in Streptomyces sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon Methanothermobacter thermautotrophicus encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in Salmonella enterica serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (idi) and 1-deoxy-D-xylulose 5-phosphate (dxs). The dxs gene was interrupted with a synthetic operon containing the Saccharomyces cerevisiae genes erg8, erg12, and erg19 allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His6-tagged M. thermautotrophicus type II IPP isomerase was produced in Escherichia coli and purified by Ni2+ chromatography. The purified protein was characterized by matrix-assisted laser desorption ionization mass spectrometry. The enzyme has optimal activity at 70 degrees C and pH 6.5. NADPH, flavin mononucleotide, and Mg2+ are required cofactors. The steady-state kinetic constants for the archaeal type II IPP isomerase from M. thermautotrophicus are as follows: K(m), 64 microM; specific activity, 0.476 micromol mg(-1) min(-1); and k(cat), 1.6 s(-1).

Amino Acid Sequence↗
PubMed1984

Anaerobic expression of maize glucose phosphate isomerase I.

Anaerobic treatment of maize seedlings results in the selective expression of 10 major and 10 minor polypeptides designated as anaerobic polypeptides (ANPs) (Sachs, M. M., Freeling, M., and Okimoto, R. (1980) Cell 20, 761-767). Enzymatic functions have been ascribed to two of these polypeptides, alcohol dehydrogenases I and II. This report identifies a third anaerobic polypeptide, a maize glucose phosphate isomerase. Glucose phosphate isomerase specific activity increased 60% in 24 h of anaerobiosis and to more than twice its original level in 72 h. Antiserum to spinach cytoplasmic glucose phosphate isomerase 1) was monospecific against maize protein, 2) reduced glucose phosphate isomerase activity in maize extracts by 60%, and 3) recognized a single polypeptide selectively labeled with [35S]methionine during anaerobiosis. This polypeptide co-purified with the major glucose phosphate isomerase of maize. Maize glucose phosphate isomerase co-migrated on two-dimensional polyacrylamide gels with ANP55, the anaerobic polypeptide with a molecular weight of 55,000 on sodium dodecyl sulfate-polyacrylamide gels. Electrophoretically distinguishable alleles of Phi 1 (phosphohexoisomerase-1 gene located at maize chromosome 1L-140) predictably altered the electrophoretic behavior of ANP55, thus proving that Phi 1 encodes ANP55 and that ANP55 is a glucose phosphate isomerase.

Alleles↗
PubMed1998

Purification and characterization of 9-hexadecenoic acid cis-trans isomerase from pseudomonas sp. strain E-3

A 9-hexadecenoic acid cis-trans isomerase (9-isomerase) that catalyzed the cis-to-trans isomerization of the double bond of free 9-cis-hexadecenoic acid [16:1(9c)] was purified to homogeneity from an extract of Pseudomonas sp. strain E-3 and characterized. Electrophoresis of the purified enzyme on both incompletely denaturing and denaturing polyacrylamide gels yielded a single band of a protein with a molecular mass of 80 kDa, suggesting that the isomerase is a monomeric protein of 80 kDa. The 9-isomerase, assayed with 16:1(9c) as a substrate, had a specific activity of 22.8 &mgr;mol h-1 (mg protein)-1 and a Km of 117.6 mM. The optimal pH and temperature for catalysis were approximately pH 7-8 and 30 degrees C, respectively. The 9-isomerase catalyzed the cis-to-trans conversion of a double bond at positions 9, 10, or 11, but not that of a double bond at position 6 or 7 of cis-mono-unsaturated fatty acids with carbon chain lengths of 14, 15, 16, and 17. Octadecenoic acids with a double bond at position 9 or 11 were not susceptible to isomerization. These results suggest that 9-isomerase has a strict specificity for both the position of the double bond and the chain length of the fatty acid. The enzyme catalyzed the cis-to-trans isomerization of fatty acids in a free form, and in the presence of a membrane fraction it was also able to isomerize 16:1(9c) esterified to phosphatidylethanolamine. The 9-isomerase was strongly inhibited by catecholic antioxidants such as alpha-tocopherol and nordihydroguaiaretic acid, but was not inhibited by 1, 10-phenanthroline or EDTA or under anoxic conditions. Based on these results, the possible mechanism of catalysis by this enzyme is discussed.

Journal Article↗
PubMed2005

Intrinsic isomerase activity of medium-chain acyl-CoA dehydrogenase.

Mitochondrial medium-chain acyl-CoA dehydrogenase is a key enzyme for the beta oxidation of fatty acids, and the deficiency of this enzyme in patients has been previously reported. We found that the enzyme has intrinsic isomerase activity, which was confirmed using incubation followed with HPLC analysis. The isomerase activity of the enzyme was thoroughly characterized through studies of kinetics, substrate specificity, pH dependence, and enzyme inhibition. E376 mutants were constructed, and mutant enzymes were purified and characterized. It was shown that E376 is the catalytic residue for both dehydrogenase and isomerase activities of the enzyme. The isomerase activity of medium-chain acyl-CoA dehydrogenase is probably a spontaneous process driven by thermodynamic equilibrium with the formation of a conjugated structure after deprotonation of substrate alpha proton. The energy level of the transition state may be lowered by a stable dienolate intermediate, which gains further stabilization via charge transfer with the electron-deficient FAD cofactor of the enzyme. This raises the question as to whether the dehydrogenase might function as an isomerase in vivo in conditions in which the activity of the isomerase is decreased.

Acyl Coenzyme A↗
PubMed1997

Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli.

Escherichia coli expressing the Erwinia carotenoid biosynthesis genes, crtE, crtB, crtI and crtY, form yellow-coloured colonies due to the presence of beta-carotene. This host was used as a visible marker for evaluating regulatory systems operating in isoprenoid biosynthesis of E. coli. cDNAs enhancing carotenoid levels were isolated from the yeast Phaffia rhodozyma and the green alga Haematococcus pluvialis. Nucleotide sequence analysis indicated that they coded for proteins similar to isopentenyl diphosphate (IPP) isomerase of the yeast Saccharomyces cerevisiae. Determination of enzymic activity confirmed the identity of the gene products as IPP isomerases. The corresponding gene was isolated from the genomic library of S. cerevisiae based on its nucleotide sequence, and was confirmed to have the same effect as the above two IPP isomerase genes when introduced into the E. coli transformant accumulating beta-carotene. In the three E. coli strains carrying the individual exogenous IPP isomerase genes, the increases in carotenoid levels are comparable to the increases in IPP isomerase enzyme activity with reference to control strains possessing the endogenous gene alone. These results imply that IPP isomerase forms an influential step in isoprenoid biosynthesis of the prokaryote E. coli, with potential for the efficient production of industrially useful isoprenoids by metabolic engineering.

Amino Acid Sequence↗
PubMed1987

Isolation and sequencing of the gene encoding delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni: overexpression of the protein.

We describe the cloning, sequencing, and overexpression of the steroid isomerase (3-oxosteroid delta 5-delta 4-isomerase, EC 5.3.3.1) gene of Pseudomonas testosteroni. A genomic library of P. testosteroni total DNA constructed from partial EcoRI digests ligated to a lambda gtWES vector was probed with a 23-base oligonucleotide mixture [ATGAAC(T)ACC(A,T)CCG(C,A)GAG(A)CAC(T)ATGAC] corresponding to the NH2-terminal sequence of steroid isomerase. Subclones derived from a recombinant phage containing a 5400-base-pair insert were sequenced and found to contain the expected 375-nucleotide open reading frame flanked at both ends by in-frame TGA termination codons. The DNA sequence agreed with the above 125-amino acid sequence except for codons 22, 24, 33, and 38, all of which encoded Asp rather than Asn, and codon 77, which encoded Glu rather than Gln. A 1370-base-pair fragment was inserted into pUC 19 plasmid vector and used to construct a strain of Escherichia coli JM 101 that overexpressed the isomerase gene in the presence of isopropyl beta-D-thiogalactopyranoside. Cytosolic extracts of this strain contained a major soluble protein that migrated with the steroid delta-isomerase subunit on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These cytosolic extracts had 10-50% of the specific activity of crystalline isomerase, depending on the method of preparation. The recombinant enzyme was crystallized in both monoclinic (flat plates) and hexagonal (bipyramids) crystal forms, described previously for the enzyme isolated from P. testosteroni. The kinetic properties of the crystalline recombinant enzyme, including specific activity, Km for 5-androstene-3,17-dione (340 microM), and Ki for the competitive inhibitor 19-nortestosterone (11.9 microM), agreed closely with the values reported for the isolated enzyme.

Amino Acid Sequence↗
PubMed1964

METABOLISM OF PENTOSES AND PENTITOLS BY AEROBACTER AEROGENES. II. MECHANISM OF ACQUISITION OF KINASE, ISOMERASE, AND DEHYDROGENASE ACTIVITY.

Mortlock, R. P. (Michigan State University, East Lansing), and W. A. Wood. Metabolism of pentoses by Aerobacter aerogenes. II. Mechanism of acquisition of kinase, isomerase, and dehydrogenase activity. J. Bacteriol. 88:845-849. 1964.-Aerobacter aerogenes PRL-R3 possesses the genetic information to synthesize, in the presence of the appropriate inducer, at least three members of the family of ketopentokinases, two members of the family of pentitol dehydrogenases, and two members of the family of aldopentose isomerases. That is, d-xylulokinase, d-ribulokinase, l-ribulokinase, ribitol dehydrogenase, d-arabitol dehydrogenase, d-xylose (--> d-xylulose) isomerase, and l-arabinose (--> l-ribulose) isomerase activities were detectable within 4 hr after addition of inducer. The possibility that mutation and selection are involved in the formation of l-xylulokinase, l-arabitol dehydrogenase, d-arabinose (--> d-ribulose) isomerase and d-lyxose (--> d-xylulose) isomerase could not be eliminated, because 11 hr or more of incubation after the addition of inducer were required before the appearance of these enzyme activities. d-Xylulokinase activity was induced in less than 2 hr when d-xylose or d-arabitol were inducers, but 45 hr were required for the appearance of activity when xylitol was the inducer, and 83 hr were required when d-lyxose was the inducer. Likewise, the time required for induction of ribitol dehydrogenase was 2 hr for ribitol, 12 hr for d-arabinose, and 45 hr for xylitol. The time required for the appearance of enzyme activity correlated with the time required for the beginning of cell growth and substrate utilization.

Alcohols↗
PubMed1989

Isopentenyl diphosphate:dimethylallyl diphosphate isomerase. An improved purification of the enzyme and isolation of the gene from Saccharomyces cerevisiae.

Isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IPP isomerase) is an enzyme in the isoprenoid biosynthetic pathway which catalyzes the interconversion of the primary five-carbon homoallylic and allylic diphosphate building blocks. We report a substantially improved procedure for purification of this enzyme from Saccharomyces cerevisiae. An amino-terminal sequence (35 amino acids) was obtained from a highly purified preparation of IPP isomerase. Oligonucleotide probes based on the protein sequence were used to isolate the structural gene encoding IPP isomerase from a yeast lambda library. The cloned gene encodes a 33,350-dalton polypeptide of 288 amino acids. A 3.5-kilobase EcoRI fragment containing the gene was subcloned into the yeast shuttle vector YRp17. Upon transformation with plasmids containing the insert, a 5-6-fold increase in IPP isomerase activity was seen in transformed cells relative to YRp17 controls, confirming the identity of the cloned gene. This is the first reported isolation of the gene for IPP isomerase.

Amino Acid Sequence↗
PubMed1986

The amino acid sequence of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B.

We have determined the primary structure of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B. The enzyme is a dimeric protein of two identical subunits, each consisting of a polypeptide chain of 131 residues and a Mr = 14,536. The intact S-carboxymethyl protein was sequenced from the NH2 terminus using standard automated Edman degradation and automated Edman degradation using fluorescamine treatment at known prolines to suppress background. The isomerase was fragmented using CNBr, trypsin, iodosobenzoic acid, and acid cleavage at aspartyl-prolyl peptide bonds. The peptides resulting from each fragmentation were separated by reversed-phase high performance liquid chromatography and sequenced by automated Edman degradation. The full sequence was deduced by the overlapping of the various peptides. A search for homologous proteins was performed. Only the oxosteroid isomerase from Pseudomonas testosteroni, an expected homology, was found to be similar. Comparison of the two proteins shows that the region of strongest homology is the region containing the aspartic acid at which steroidal affinity and photoaffinity reagents have been shown to react in the P. testosteroni isomerase. The P. putida isomerase contains 3 cysteines and 2 tryptophans, whereas the P. testosteroni isomerase lacks these amino acids. The two proteins are not highly conserved.

Amino Acid Sequence↗
PubMed1978

Characterization of the microsomal steroid-8-ene isomerase of cholesterol biosynthesis.

Rat liver microsomes contain an enzyme that catalyzes the isomerization of the nuclear double bond of steroids from the 8(9) position to the 7(8) position. The enzyme is most active with zymosterol, 5alpha-cholesta-8,24-dien-3beta-ol, which is a precursor of cholesterol. Properties of the microsomal isomerase have now been studied, and preliminary data are reported on both regulation of enzymic activity and first steps in the solubilization of the enzyme from membranes. After a brief lag period, the velocity of isomerase is relatively constant for about 5 min of incubation, and then isomerization subsides. The apparent Michaelis constant (52-70 micro M) is difficult to determine accurately, due to these complex kinetic changes. V(max) is 4.0-4.7 nmol/min per mg of microsomal protein. The apparent specific activity is more than ten times that of liver microsomal methyl sterol oxidase. The maximal specific activity of microsomal isomerase is approximately doubled when rats are fed an intestinal bile acid sequestrant, cholestyramine. Changes in specific activity of isomerase parallel changes in activities of other microsomal enzymes of cholesterol biosynthesis, such as 3-hydroxy-3-methylglutaryl-CoA reductase and 4-methyl sterol oxidase. Isomerase activity is destroyed by phospholipase A digestion, high concentration of bile salts, and solvent extraction, all of which are known either to remove phospholipid or to alter microsomal membrane integrity. On the other hand, isomerase remains active in the presence of a mild, nonionic detergent, Triton WR-1339; thus, solubilization with nonionic detergents is under study.

Animals↗
PubMed1982

Immobilization of glucose isomerase.

The immobilization of glucose isomerase (D-xylose ketol isomerase, EC 5.3.1.5) by covalently bonding to various carriers and by adsorption to ion exchange resins was attempted in order to obtain a stable immobilized enzyme which can be used for continuous isomerization of glucose in a column. Of the covalent bonding methods, the colloidal silica-glutaraldehyde method showed the highest binding capacity and gave the most stable immobilized glucose isomerase. The Ludox HS-30 bound glucose isomerase column showed a half-life of 24 days and an enzyme usage of 0.07 units per gram of isomerized sugar (d.s, fructose 45%). Of the resins used, the macromolecular type or porous type strongly basic anion exchange resins showed the highest binding capacity and gave the most stable immobilized glucose isomerase. The Amberlite IRA-904 resine-bound glucose isomerase showed a half-life of 23 days and an enzyme usage of 0.06 units per gram of isomerized sugar (d.s., fructose 45%). Based on the ease of the immobilization process, the possibility of carrier reuse and the extensive use already achieved by ion exchange resins in the sugar industry, IRA-904 resin was selected as the candidate for commercialization.

Adsorption↗
PubMed1991

A metal-mediated hydride shift mechanism for xylose isomerase based on the 1.6 A Streptomyces rubiginosus structures with xylitol and D-xylose.

The crystal structure of recombinant Streptomyces rubiginosus D-xylose isomerase (D-xylose keto-isomerase, EC 5.3.1.5) solved by the multiple isomorphous replacement technique has been refined to R = 0.16 at 1.64 A resolution. As observed in an earlier study at 4.0 A (Carrell et al., J. Biol. Chem. 259: 3230-3236, 1984), xylose isomerase is a tetramer composed of four identical subunits. The monomer consists of an eight-stranded parallel beta-barrel surrounded by eight helices with an extended C-terminal tail that provides extensive contacts with a neighboring monomer. The active site pocket is defined by an opening in the barrel whose entrance is lined with hydrophobic residues while the bottom of the pocket consists mainly of glutamate, aspartate, and histidine residues coordinated to two manganese ions. The structures of the enzyme in the presence of MnCl2, the inhibitor xylitol, and the substrate D-xylose in the presence and absence of MnCl2 have also been refined to R = 0.14 at 1.60 A, R = 0.15 at 1.71 A, R = 0.15 at 1.60 A, and R = 0.14 at 1.60 A, respectively. Both the ring oxygen of the cyclic alpha-D-xylose and its C1 hydroxyl are within hydrogen bonding distance of NE2 of His-54 in the structure crystallized in the presence of D-xylose. Both the inhibitor, xylitol, and the extended form of the substrate, D-xylose, bind such that the C2 and C4 OH groups interact with one of the two divalent cations found in the active site and the C1 OH with the other cation. The remainder of the OH groups hydrogen bond with neighboring amino acid side chains. A detailed mechanism for D-xylose isomerase is proposed. Upon binding of cyclic alpha-D-xylose to xylose isomerase, His-54 acts as the catalytic base in a ring opening reaction. The ring opening step is followed by binding of D-xylose, involving two divalent cations, in an extended conformation. The isomerization of D-xylose to D-xylulose involves a metal-mediated 1,2-hydride shift. The final step in the mechanism is a ring closure to produce alpha-D-xylulose. The ring closing is the reverse of the ring opening step. This mechanism accounts for the majority of xylose isomerase's biochemical properties, including (1) the lack of solvent exchange between the 2-position of D-xylose and the 1-pro-R position of D-xylulose, (2) the chemical modification of histidine and lysine, (3) the pH vs. activity profile, and (4) the requirement for two divalent cations in the mechanism.

Aldose-Ketose Isomerases↗
PubMed1995

Detection of dopachrome isomerase on gels and membranes.

Dopachrome isomerase, which mediates the conversion of dopachrome to dihydroxyindoles, is an enzyme affiliated with the melanogenic pathway found in most vertebrates and invertebrates. We have recently reported an activity-staining procedure for detecting this enzyme after electrophoresis on tyrosinease-embedded polyacrylamide gels using dopa [K. Nellaiappan, G. Nicklas, and M. Sugumaran, (1994) Anal. Biochem. 220, 122-128]. The usefulness of this method is limited to the availability of good quality of tyrosinase. To overcome the limitations of nonavailability as well as batch to batch variation in the specific activity of commercially available tyrosinase which will affect the staining, we have developed a new stain technique using chemically made dopachrome for the detection of dopachrome isomerase. The new method employs the use of dopachrome generated in situ by the oxidation of dopa with periodate. Dopachrome isomerase initially appears as a bluish-purple (melanochrome) band against an orange-red background. Eventual transformation of melanochrome to dark insoluble melanin polymer produces a black band corresponding to dopachrome isomerase activity. Continuous washing of the gel clears the background. Usefulness of this technique to stain dopachrome isomerase (a) in different organisms and (b) on polyacrylamide gels, agarose gels, and nitrocellulose membranes is demonstrated. In addition, conditions to stain the enzyme on nitrocellulose membranes using mushroom tyrosinase are described. Comparison of the effectiveness of tyrosinase versus periodate staining procedure reveals that the former method is superior to the latter in detecting dopachrome isomerase. The periodate method overcomes the problems associated with the quality of commercially available tyrosinase and its nonavailability in certain regions.

Collodion↗
PubMed1997

Expression, purification, and crystallization of two isozymes of 6-phosphoglucose isomerase of Bacillus stearothermophilus.

Two isozymes of 6-phosphoglucose isomerase (phosphoglucose isomerase A & phosphoglucose isomerase B), isolated from Bacillus stearothermophilus, have been overexpressed in Escherichia coli strain DF2145 and purified to homogeneity. Crystals of both isozymes have been obtained by the vapor diffusion method. The crystals of phosphoglucose isomerase A have unit cell dimensions a = b = 132.0 A, c = 183.6 A, and diffract to about 2.8 A resolution. An analysis of the reflection data indicates that the crystal system is hexagonal, space group P6122 or P6522. The crystals of phosphoglucose isomerase B complexed with 6-phosphogluconate belong to the orthorhombic space group I222 (or I212121), with cell dimensions a = 75.1 A, b = 95.7 A, c = 171.5 A, and diffract to a resolution of 2.3 A. These crystals promise to yield more detail for the substrate recognition and higher resolution structures of 6-phosphoglucose isomerase.

Crystallization↗