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At least 685 records · Page 38Linked to original sources
PubMed1964

METABOLISM OF PENTOSES AND PENTITOLS BY AEROBACTER AEROGENES. I. DEMONSTRATION OF PENTOSE ISOMERASE, PENTULOKINASE, AND PENTITOL DEHYDROGENASE ENZYME FAMILIES.

Mortlock, R. P. (Michigan State University, East Lansing) and W. A. Wood. Metabolism of pentoses and pentitols by Aerobacter aerogenes. I. Demonstration of pentose isomerase, pentulokinase, and pentitol dehydrogenase enzyme families. J. Bacteriol. 88:838-844. 1964.-Aerobacter aerogenes PRL-R3 is capable of utilizing as sole substrates for energy and growth seven of the eight aldopentoses and all of the four pentitols. Growth upon media containing either d-xylose, l-arabinose, d-ribose, d-arabitol, or ribitol occurred within 24 hr at 26 C. When d-arabinose or l-arabitol were used as growth substrates, growth was complete within 2 days; 4 days were required for growth on d-lyxose or xylitol, and 3 to 4 weeks for growth upon l-xylose. The versatility of this strain of A. aerogenes is due to an ability to synthesize in the presence of appropriate carbohydrates (inducers) families of enzymes which catalyze the metabolism of the carbohydrates (i.e., families of pentitol dehydrogenases, aldopentose isomerases, and pentulokinases). The specificity of induction for members of the enzyme families was found to vary, and cross induction of enzyme activity was common, especially among the pentitol dehydrogenases. Ribitol dehydrogenase activity was detected in extracts of cells grown on all of the above carbohydrates with the exception of d-xylose, l-arabinose, and d-ribose. The ribitol dehydrogenase activity of xylitol-grown cell extracts was fivefold higher than the activity in extracts of ribitol-grown cells.

Alcohols↗
PubMed1990

Human 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase from placenta: expression in nonsteroidogenic cells of a protein that catalyzes the dehydrogenation/isomerization of C21 and C19 steroids.

The isolation, cloning, and expression of a cDNA insert complementary to mRNA encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase is reported. The insert contains an open reading frame encoding a protein of 372 amino acids, the initial 29 amino acids corresponding to the N-terminal sequence identified from the purified human placental microsomal enzyme. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental microsomal 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase, as detected by immunoblot analysis, and catalyzed the conversion of 17 alpha-hydroxypregnenolone to 17 alpha-hydroxyprogesterone, pregnenolone to progesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, very efficiently oxidized 5 alpha-androstan-3 beta,17 beta-diol to 5 alpha-dihydrotestosterone and, upon addition of NADH, reduced 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol. Thus, the dehydrogenation/isomerization steps of steroid biosynthesis can be catalyzed by a single polypeptide chain, which can metabolize all of the major physiological substrates.

17-alpha-Hydroxypregnenolone↗
PubMed2002

Evaluation of similarities in the cis/trans isomerase function of trigger factor and DnaK.

Two functionally redundant enzymes, trigger factor and the hsp70 chaperone DnaK, have been found to assist de novo protein folding in E coli. Trigger factor is a peripheral peptidyl prolyl cis/trans isomerase (PPIase) of the large subunit of the ribosome. In contrast, DnaK displays two catalytic features: the secondary amide peptide bond cis/trans isomerase (APIase) function supplemented by the ATPase site. APIases accelerate the cis/trans isomerization of nonprolyl peptide bonds. Both enzymes have affinity for an unfolded polypeptide chain. The diminished low temperature cell viability in the presence of trigger factor variants with impaired PPlase activity indicates that the enhancement of folding rates plays a crucial role in protein folding in vivo. For the DnaK-mediated increase in the folding yield in vitro, the minimal model for APlase catalysis involves the catalyzed partitioning of a rapidly formed folding intermediate as could be inferred from the DnaK/DnaJ/GrpE/ATP-assisted refolding of GdmCl-denatured luciferase. Using three different peptide bond cis/trans isomerization assays in vitro, we could show that there is no overlapping substrate specificity of trigger factor and DnaK. We propose that only if trigger factor recruits supplementing molecules is it capable of exhibiting functional complementarity with DnaK in protein folding.

Catalysis↗
PubMed1992

Simultaneous formation of native ribonuclease and proinsulin from their mixed S-sulfonated derivatives by protein disulfide isomerase.

Although the formation of native disulfide bonds of a protein from randomly linked disulfides by protein disulfide isomerase has been extensively studied, the possibility of simultaneous formation of the native proteins from a mixture has not been examined. It is shown in this paper that native ribonuclease and proinsulin can be nearly quantitatively formed by protein disulfide isomerase from a mixture of their S-sulfonated derivatives independent of the presence of each other.

Dithiothreitol↗
PubMed1992

An isoform of protein disulfide isomerase isolated from chronic myelogenous leukemia cells alters complex formation between nuclear proteins and regulatory regions of interferon-inducible genes.

We have previously shown that the electrophoretic mobility of complexes formed in vitro between nuclear proteins and the regulatory domains of interferon-inducible genes is altered by an extranuclear protein present in elevated levels in the myeloid cells of chronic myelogenous leukemia patients. Interferon-alpha reduces the level of this activity only in the cells of patients who are clinically sensitive to the antiproliferative effects of interferon-alpha. We have purified this protein to homogeneity and found it to be a 57-kDa protein which corresponds to an isoform of protein disulfide isomerase. Protein disulfide isomerase is an oxidoreductase which catalyzes the interconversion between the reduced and oxidized states of proteins which contain multiple sulfhydryl groups and disulfide bonds. These studies suggest that this protein may play an important role in the transcriptional activation of interferon-inducible genes, perhaps through redox mechanisms.

Base Sequence↗
PubMed1990

The cytosolic-binding protein for the immunosuppressant FK-506 is both a ubiquitous and highly conserved peptidyl-prolyl cis-trans isomerase.

We have recently isolated an abundant cytosolic protein from human T-cells which specifically binds the immunosuppressive agent, FK-506. The FK-506-binding protein (FKBP) is a member of a novel class of proteins possessing peptidyl-prolyl cis-trans isomerase activity. These proteins are believed to play an important role in accelerating the rate at which proteins fold into their native conformations. In the present study, we demonstrate that FKBP is not a lymphoid-specific protein, but is widely distributed and phylogenically conserved. FKBP, purified from three sources (a human T-lymphocyte cell line JURKAT, bovine calf thymus, and Saccharomyces cerevisiae) exhibit identical molecular weights, immunological cross-reactivities, and a high degree of NH2-terminal amino acid sequence homology. In addition, FKBP from all sources possesses peptidyl-prolyl cis-trans isomerase activity which can be specifically inhibited by FK-506. We conclude that FKBP may serve an important biological function in all eukaryotic cells.

Amino Acid Isomerases↗
PubMed1991

Preliminary characterization of a cloned neutral isoelectric form of the human peptidyl prolyl isomerase cyclophilin.

We report the cloning of a neutral isoelectric form of the human peptidyl prolyl isomerase, cyclophilin, its expression in Escherichia coli, and its purification and comparison to bovine thymus cyclophilin. The cloned protein exhibited a pI of approximately 7.8 and formed a simple 1:1 complex with cyclosporin A. This cloned form had a pI similar to that observed for the neutral isoform (pI approximately 7.4) of human splenocyte cyclophilin. The bovine thymus proteins exhibited anomalous behavior on CM-cellulose chromatography but were resolved into alkaline (pI approximately 9.3) isoforms and a new neutral (pI approximately 7.8) isoform by isoelectric focusing gel electrophoresis and ultimately into at least four discrete isoforms by capillary electrophoresis. For cyclosporin A binding we observe a Kd of approximately 160 nM for an electrophoretically heterogeneous preparation of the natural bovine protein and approximately 360 nM for the more homogeneous preparation of the cloned human neutral isoform. Stopped-flow measurements of the activation energies for peptidyl-prolyl isomerase activity indicate the recombinant human protein has an activation enthalpy of 3.67 kcal/mol and an activation entropy of -47.3 cal/K-mol for cis----trans isomerization.

Amino Acid Isomerases↗
PubMed1986

Microsomal enzymes of cholesterol biosynthesis from lanosterol. Solubilization and purification of steroid 8-isomerase.

Steroid-8-ene isomerase that catalyzes isomerization of delta 8- to delta 7-sterols has been solubilized from rat liver microsomes with a mixture of two detergents, octylglucoside and sodium taurodeoxycholic acid. During a 40-fold enrichment of the solubilized enzyme, other enzymes of cholesterol biosynthesis, endogenous lipids, and electron carriers are removed. A comparison of properties of the solubilized and partially purified isomerase with the membrane-bound enzyme shows they are essentially identical with respect to pH profile, effect of inhibitors and cofactors, substrate specificity, and Km values. Addition of phospholipid to the partially purified enzyme stimulates activity as much as 1.8-fold over control rates. Although the relative rate of isomerization of cholesta-8,24-dien-3 beta-ol is six times that observed with cholest-8-en-3 beta-ol, the delta 8 to delta 7 ratio at equilibrium is approximately equal. The reversibility of the reaction has been demonstrated by the direct conversion of cholest-7-en-3 beta-ol to cholest-8-en-3 beta-ol; at equilibrium the delta 7-isomer is predominant (19/1). The purified enzyme does not catalyze isomerization of cholesta-8,14-dien-3 beta-ol and cholest-8(14)-en-3 beta-ol under conditions that result in equilibrium mixtures of isomers from cholest-8(9)-en-3 beta-ol. These results are consistent with the earlier suggestion that delta 8(14)-sterols are neither formed nor metabolized by the same microsomal enzymes that catalyze transformation of lanosterol to cholesterol.

Animals↗
PubMed1989

Role of protein disulphide-isomerase in the expression of native proteins.

Formation of native disulphide bonds is a post-translational modification associated with the folding and assembly of secretory proteins. The process is catalysed within the lumen of the endoplasmic reticulum by the enzyme protein disulphide-isomerase (PDI), which is abundant in secretory cells and catalyses thiol: protein-disulphide interchange in vitro with very broad protein substrate specificity. The presence of PDI within microsomal vesicles is essential for efficient and rapid cotranslational disulphide bond formation during protein synthesis in vitro. The sequence of PDI is now known from several species, and shows the presence of two domains closely homologous to thioredoxins. Chemical modification data confirm the role of the thioredoxin domains in thiol:disulphide interchange activity, and structural models of these domains can be built based on homology with thioredoxin. Thus PDI is both strongly implicated in the process of native protein folding in vivo and well characterized at the molecular level. In addition to its disulphide-isomerase activity, PDI participates as a component of the enzyme prolyl-4-hydroxylase, and further functions have also been proposed.

Amino Acid Sequence↗
PubMed1989

Solubilization and partial purification of retinyl ester synthetase and retinoid isomerase from bovine ocular pigment epithelium.

Studies reported previously from this laboratory have demonstrated that membranes from the pigment epithelium of the vertebrate eye can transform free all-trans-retinol to 11-cis-retinol as well as 11-cis- and all trans-retinyl esters (Bernstein, P. S., Law, W. C., and Rando, R. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1849-1853; Bernstein, P. S., Law, W. C., and Rando, R. R. (1987) J. Biol. Chem. 262, 16848-16857; Fulton, B. S., and Rando, R. R. (1987) Biochemistry 26, 7938-7945). The congeneric retinals are also formed under conditions where retinol redox activity is present. Here we report the successful solubilization of both the retinyl ester synthetase and isomerase activities from the pigment epithelium membranes of the bovine eye. The zwitterionic detergent Zwittergent 3-14(N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate; cmc 0.012%) gave optimal solubilization of both activities. Three initial criteria for successful solubilization were used. First, high speed centrifugation (greater than 150,000 x g) left the activities in the supernatant. Second, the solubilized enzymatic activities were found in the included volume upon gel filtration. Finally, the solubilized activities were quantitatively passed through a 0.22-microns filter. Employing anion exchange and gel filtration chromatography results in a partial purification of the retinyl ester synthetase (approximately 189-fold). The solubilized retinoid isomerase is also partially purified (approximately 10-14-fold) following anion exchange chromatography. It is also shown that the membrane-bound and solubilized ester synthetase catalyzes the esterification of retinol using added lecithins as exogenous acyl donors. In addition, evidence is provided indicating that there is a positional selectivity for the acyl group transfer from the lecithin to retinol. The transfer occurs largely, if not entirely, from the 1-position of the lecithin.

Acyltransferases↗
PubMed1988

Characterization of the human gene for a polypeptide that acts both as the beta subunit of prolyl 4-hydroxylase and as protein disulfide isomerase.

A single polypeptide acts as the beta subunit of prolyl 4-hydroxylase and the enzyme protein disulfide isomerase and may also function as a cellular thyroid hormone binding protein. We report here that the human gene for this polypeptide is about 18 kilobase pairs and consists of 11 exons. The two thioredoxin-like regions are coded by exons 1-2 and 8-9, respectively. The codons for the two presumed active sites of protein disulfide isomerase, each a Cys-Gly-His-Cys sequence, are located 12 base pairs from the beginning of exons 2 and 9. The last 3 amino acids coded by exons 1 and 8 and the first 9 amino acids coded by exons 2 and 9, including a broken codon for Tyr, are identical in the respective exon-intron junctions. These regions are also highly homologous to the active sites of bacterial thioredoxins. The data suggest that evolution of this gene has involved exon shuffling and duplication of a two-exon unit, in which the internal exon-intron junctions have been entirely conserved. The region between exons 1-2 and 8-9 appears to contain other duplications. The 5' flanking sequences contain a TATA box, six CCAAT boxes, and other elements which may be involved in regulation of the cellular amounts of this polypeptide.

Amino Acid Sequence↗
PubMed1987

The influence of peptidyl-prolyl cis-trans isomerase on the in vitro folding of type III collagen.

Peptidyl-prolyl cis-trans isomerase was extracted from pig kidney cortex and partially purified. Enzyme activity was monitored against the cis-trans isomerization of succinyl-Ala-Ala-Pro-Phe-methylcoumaryl amide by means of a two-step process using chymotrypsin as the trans cleaving activity. The in vitro refolding of denatured type III collagen, which is rate-limited by the cis-trans isomerization of peptide bonds, was studied in the presence of peptidyl-prolyl cis-trans isomerase by optical rotatory dispersion and by resistance to tryptic digestion. A 3-fold increase in the initial rate of folding was observed compared to the uncatalyzed refolding. This rate increase is comparable to the rate increase found for the CT-phase in the refolding of urea-denatured ribonuclease A, but it is smaller than the increase in the rate of isomerization of succinyl-Ala-Ala-Pro-Phe-methylcoumarylamide.

Amino Acid Isomerases↗
PubMed1984

Enzymatic function in crystals of delta 5-3-ketosteroid isomerase. Catalytic activity and binding of competitive inhibitors.

Crystals of the steroid-metabolizing enzyme, delta 5-3-ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni, exhibit many enzymatic properties. Each enzyme subunit in the lattice binds a competitive inhibitor, progesterone, with the same stoichiometry (1:1) and affinity (KD = 6 X 10(-6) M) as the enzyme in solution. Another competitive inhibitor, 19-nortestosterone, competes with progesterone for the same binding sites in the crystal. The enzyme crystals catalyze the conversion of delta 5- to delta 4-ketosteroids, but because the enzyme is so efficient, and substrate diffusion into the crystal is so slow, substrate cannot penetrate deeply into the crystal before being converted to product. A general theoretical formulation is presented to account for the effects of substrate diffusion into enzyme crystals of different shapes and sizes. The dependence of apparent mean enzyme activity in steroid isomerase crystals as a function of crystal size is shown to be consistent with this theoretical formulation. These inhibitor binding and catalytic properties suggest that the enzyme is in an active conformation within these crystals.

Binding Sites↗
PubMed1980

Presence of epimerase activity in hamster liver corticosteroid side chain isomerase.

11-Deoxy-[21-3H]corticosterone ([21-3H]DOC) exchanged tritium with water in a reaction mediated by an enzyme preparation obtained from hamster liver. Both (21S)-[21-3H]- and (21R)-[21-3H]DOC participated in the exchange. Incorporation of tritium into water followed a hyperbolic path with time for the 21S epimer and a sigmoidal path for the 21R epimer. Separate enzymes did not appear to be involved. The system was also studied by measuring incorporation of deuterium from deuterated water into the steroid side chain. During incubation, DOC containing 2, 1, and no deuteria were all found. Their proportions changed with incubation time. The deuterium atoms were incorporated into the side chain sequentially and reversibly. It is proposed that (21S)-[21-3H]DOC directly exchanges tritium with water in a reaction catalyzed by an isomerase, and (21R)-[21-3H]DOC loses tritium only after prior enzyme-mediated epimerization to the 21S form. We had previously presented evidence that the isomerase reversibly converted the ketol side chain to the aldol form. Epimerization of the aldol isomer, 20 beta-hydroxy-3-oxopregn-4-en-21-al (isoDOC) by hamster liver enzyme to the 20 alpha-hydroxy isomer was shown directly. A model is presented which accounts for the kinetics of exchange of (21R)- and (21S)-[21-3H]DOC with water, interconversion of DOC and isoDOC, and the epimerization of isoDOC at C-20.

Animals↗
PubMed1994

Protein disulfide isomerase associates with misfolded human lysozyme in vivo.

Wild-type human lysozyme (hLZM) is quantitatively secreted into the media when expressed in mouse fibroblast cells, but some misfolded hLZMs are retained and rapidly degraded in a pre-Golgi compartment (Omura, F., Otsu, M., Yoshimori, T., Tashiro, Y., and Kikuchi, M. (1992) Eur. J. Biochem. 210, 591-599). To detect the association with misfolded hLZMs of cellular proteins involved in their folding, retention, and pre-Golgi degradation, a co-precipitation experiment was carried out using anti-hLZM antibody and metabolically labeled cell lysates, which were treated with a membrane-permeable cross-linking reagent. Here we report that protein disulfide isomerase associated in vivo with misfolded hLZMs, but not with the wild-type protein, and discuss the possible role of protein disulfide isomerase in the quality control of newly synthesized proteins in the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1994

Involvement of two sulfur atoms of protein disulfide isomerase and one sulfur atom of the DsbA/PpfA protein in the oxidation of mutant human lysozyme.

Protein disulfide isomerase (PDI) and the DsbA/PpfA protein catalyze the oxidation of mutant human lysozyme, L79CC81A, which has two native disulfide bonds, Cys6-Cys128 and Cys30-Cys116, a non-native Cys79-Cys95, and 2 free cysteine residues at positions 65 and 77. Oxidation of L79CC81A (R-form) yielded two isomers, L79CC81A-a (A-form) with tandem-linked Cys65-Cys77 and Cys79-Cys95, and L79CC81A-b (B-form) with cross-linked Cys65-Cys79 and Cys77-Cys95 (Kanaya, E., Ishihara, K., Tsunasawa, S., Nokihara, K., and Kikuchi, M. (1993) Biochem. J. 292, 469-476). PDI mainly enhanced the formation of the A- form in the absence of oxidized glutathione (GSSG); however, as the concentration of GSSG increased, it markedly accelerated the formation of the B-form. In contrast, the DspA/PpfA protein mainly enhanced the formation of the A-form, regardless of the presence or absence of GSSG. These results and the presumed spatial locations of Cys65, Cys77, and Cys79-Cys95 in the R-form suggest that 1 of the half-cystine residues in the active site of PDI and the DsbA/PpfA protein can react with 1 of the 2 free Cys residues of the R-form. The dependence on GSSG of the B-form formation with PDI can be explained by the formation of two transient intermolecular disulfide bonds between PDI and the R-form and the attack of GSSG by the resultant thiolate anion of Cys79 or Cys95. The independence of the reaction with the DsbA/PpfA protein from GSSG can be explained by the formation of one transient intermolecular disulfide bond. The possible formation of the two transient intermolecular disulfide bonds involving two sulfur atoms of PDI and 2 cysteine or half-cystine residues of the substrate could explain the high isomerase activity of PDI.

Binding Sites↗
PubMed1995

A genetically engineered fusion protein M4/TNF with increased bifunctional activity refolded in the presence of protein disulfide isomerase.

Recombinant DNA techniques were used to clone, to construct and to express a fusion protein M4/TNF in Escherichia coli. The fusion protein includes the chimeric F(ab')2 fragment (M4) recognizing the human tumor-associated TAG72 antigen and the tumor necrosis factor alpha (TNF) moiety. The M4/TNF purified from inclusion bodies of the bacteria homogenates was further solubilized in a denaturing buffer containing 6 mol l-1 guanidine and refolded in a refolding buffer. Our results showed that the M4/TNF refolded in a buffer containing 6 mmol l-1 oxidized glutathione (GSSG), 0.2 mmol l-1 dithioerythione (DTE) and 0.5 mumol l-1 protein disulfide isomerase (PDI) displayed a 4-fold higher anti-TAG72 immunoreactivity and a 5-fold higher TNF activity than that refolded in the same refolding buffer but without PDI. Our data thus indicates that the protein disulfide isomerase not only facilitates the correct formation of disulfide-bonds of the antibody molecule, but also the correct refolding of the TNF moiety in vitro.

Amino Acid Sequence↗
PubMed1996

Protein disulfide isomerase-catalyzed renaturation of ribonuclease A modified by S-thiolation with glutathione and cysteine.

Renaturation of modified ribonuclease A by protein disulfide isomerase was studied. The renaturation rate of fully S-thiolated ribonuclease A with glutathione, namely, ribonuclease A-glutathione mixed disulfide (RNase-SG) containing 8 moles of glutathione per mole of ribonuclease A (RNase-SG8), by protein disulfied isomerase (PDI) was more than three times faster than those of fully S-thiolated RNase with L-cysteine and scrambled ribonuclease A. Renaturation of RNase-SG species containing 7 or less glutathione was slower than that of RNase-SG8. These data seems to favor the hypothesis that S-thiolation of nascent proteins with glutathione may occur in the folding process during protein synthesis. The applicability of the present method consisted of chemical S-thiolation and PDI-catalyzed renaturation to the in vitro folding of recombinant cysteine-containing proteins is discussed.

Animals↗