| 1. |
- Akerstrom, B., et al.
(författare)
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Structural relationship between α1-microglobulin from man, guinea-pig, rat and rabbit
- 1988
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Ingår i: European Journal of Biochemistry. - 0014-2956. ; 170:1-2, s. 143-148
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Tidskriftsartikel (refereegranskat)abstract
- Rabbit α1-microglobulin was purified from the urine of sodium-chromate-treated animals by the use of gel chromatography on Sephadex G-100 affinity chromatography on concanavalin-A - Sepharose and ion-exchange chromatography on DEAE-Sephadex. Rabbit α1-microglobulin had a molecular mass of 25.6 kDa on SDS/polyacrylamide gel electrophoresis. α1-microglobulin has previously been purified from the urine of humans, guinea-pigs and rats by similar methods, and the molecular masses of the four homologues were compared by SDS/polyacrylamide gel electrophoresis and gel chromatography in a denaturing medium. By these two methods the human homologue was 6 kDa and 3 kDa larger, respectively, than the other three proteins. Endoglycosidase F digestion of α1-microglobulin, followed by SDS/polyacrylamide gel electrophoresis, revealed three protein bands in the human α1-microglobulin sample, and only two bands in guinea-pig, rat and rabbit α1-microglobulin, with a gap between each band of 2.6-2.9 kDa. The amino-terminal amino acid sequences of the four homologues were determined and between 72% and 81% homology was seen. The five amino-terminal amino acids present in the other species were missing in guinea-pig α1-microglobulin. Our results indicate that human α1-microglobulin is substituted with two N-linked oligosaccharides, while only one is attached to each of the other α1-microglobulins, and that the extra glycosylamine-linked oligosaccharide in the human protein is attached to asparagine in position 17. Finally it is shown that all four homologues inhibit antigen stimulation of human lymphocytes, a finding which is consistent with our previous suggestion that the N-linked oligosaccharides carry the immunosuppressive activity of α1-microglobulin.
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| 2. |
- Fridén, H, et al.
(författare)
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Genetic and biochemical characterization of Bacillus subtilis mutants defective in expression and function of cytochrome b-558
- 1987
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 168, s. 695-701
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Tidskriftsartikel (refereegranskat)abstract
- Bacillus subtilis succinate dehydrogenase is bound to the cytoplasmic membrane by cytochrome b-558, a 23-kDa transmembrane protein which also functions as electron acceptor to the dehydrogenase. The structural gene for the apocytochrome, sdhC, has previously been cloned and sequenced. In this work the structure and translation of cytochrome b-558 was studied in different sdhC mutants. Mutant cytochrome was analyzed both in B. subtilis and after amplification in Escherichia coli. It is concluded that amino acid substitutions in the C-terminal half of the cytochrome can prevent the binding of succinate dehydrogenase without affecting membrane binding of the cytochrome protein or heme ligation. Mutagenesis of His-113 excludes this residue as an axial heme ligand. A base-pair exchange of G to A in the ribosome-binding sequence of sdhC was found to reduce cytochrome b-558 translation about tenfold in B. subtilis, whereas the mutation had no effect on translation in E. coli. Translation of the two succinate dehydrogenase genes from the sdhCAB polycistronic transcript does not seem to be coupled to translation of sdhC. Less than 10% of the wild-type amount of membrane-bound succinate dehydrogenase in B. subtilis still allows growth on non-fermentable substrate, but makes the dehydrogenase a limiting enzyme in the tricarboxylic acid cycle and leads to succinate accumulation.
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| 3. |
- Gügler, K, et al.
(författare)
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Elicitor-induced Tyrosine Decarboxylase in Berberine Synthesizing Suspension Cultures of Thalictrum rugosum
- 1988
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 170:3, s. 661-666
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Tidskriftsartikel (refereegranskat)abstract
- Tyrosine decarboxylase (EC 4.1.1.25) was induced in suspension cultures of Thalictrum rugosum by treatment with a yeast glucan elicitor. Maximum induction was observed at a carbohydrate concentration of 0.4 mg/g fresh weight of cells and maximum enzyme activity was reached 20 h after addition of elicitor. The enzyme was inducible in late exponential and early stationary growth phases. A good correlation between induced tyrosine decarboxylase activity and berberine biosynthesis has been established. It is suggested that tyrosine decarboxylase may be a key enzyme between primary and secondary metabolisms in the biosynthesis of norlaudanosoline-derived alkaloids.
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| 4. |
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| 5. |
- Kvassman, Jan, et al.
(författare)
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Mechanism of glyceraldehyde‐3‐phosphate transfer from aldolase to glyceraldehyde‐3‐phosphate dehydrogenase
- 1988
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 172:2, s. 427-431
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Tidskriftsartikel (refereegranskat)abstract
- The catalytic interaction of glyceraldehyde‐3‐phosphate dehydrogenase with glyceraldehydes‐3‐phosphate has been examined by transient‐state kinetic methods. The results confirm previous reports that the apparent Km for oxidative phosphorylation of glyceraldehydes‐3‐phosphate decreases at least 50‐fold when the substrate is generated in a coupled reaction system through the action of aldolase on fructose 1,6‐bisphosphate, but lend no support to the proposal that glyceraldehydes 3‐phosphate is directly transferred between the two enzymes without prior release to the reaction medium. A theoretical analysis is presented which shows that the kinetic behaviour of the coupled two‐enzyme system is compatible in all respects tested with a free‐diffusion mechanism for the transfer of glyceraldehydes 3‐phosphate from the producing enzyme to the consuming one.
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| 6. |
- Larsson, Karin M., et al.
(författare)
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Activity and stability of horse‐liver alcohol dehydrogenase in sodium dioctylsulfosuccinate/cyclohexane reverse micelles
- 1987
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 166:1, s. 157-161
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Tidskriftsartikel (refereegranskat)abstract
- Horse liver alcohol dehydrogenase (EC 1.1.1.1) solubilized in sodium dioctylsulfosuccinate (AOT)/cyclohexane reverse micelles was used for the oxidation of ethanol and reduction of cyclohexanone in a coupled substrate/coenzyme recycling system. The activity of the enzyme was studied as a function of pH and water content. The enzyme was optimally active in microemulsions prepared with buffer of pH around 8. An increase in enzymatic activity was observed as a function of increasing water content. The Km values for the substrates were calculated based on the total reaction volume. The apparent Km for ethanol in reverse micelles was about eight times lower as compared to that in buffer solution, whereas the Km for cyclohexanone was almost unaltered. Storage and operational stability were investigated. It was found that the specific activity of the alcohol dehydrogenase operating in reverse micellar solution was good for at least two weeks. The steroid eticholan‐3ß‐ol‐17‐one was also used as a substrate. In this case the reaction rate was approximately five times higher in a reverse micellar solution than in buffer.
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| 7. |
- Lawrence, D, et al.
(författare)
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Purification of active human plasminogen activator inhibitor 1 from Escherichia coli. Comparison with natural and recombinant forms purified from eucaryotic cells.
- 1989
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Ingår i: European Journal of Biochemistry. - 0014-2956 .- 1432-1033. ; 186:3, s. 523-33
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Tidskriftsartikel (refereegranskat)abstract
- Plasminogen activator inhibitor 1 (PAI-1) inhibits both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) and, therefore, is an important regulator of plasminogen activation. We have developed eucaryotic and procaryotic expression systems for PAI-1 and characterized the recombinant glycosylated and non-glycosylated products, together with a non-recombinant natural control, produced in the histosarcoma cell line HT 1080. For eucaryotic expression, the PAI-1 cDNA was stably transfected into chinese hamster ovary cells (CHO cells), while procaryotic expression in Escherichia coli was examined after inserting the DNA sequence encoding the mature PAI-1 protein into an inducible expression vector. Recombinant PAI-1 from CHO cells was purified approximately 50-fold in two steps and was indistinguishable from natural PAI-1. Between 3% and 4% of total cellular protein in the procaryotic expression system consisted of PAI-1, from which it was purified approximately 30-fold, with yields of between 15% and 20%. This PAI-1 formed 1:1 complexes with uPA and also with the single- and two-chain forms of tPA. Kinetic analysis demonstrated that the procaryote-produced PAI-1 had an inhibitory activity towards all three forms of PA that resembled that of natural PAI-1 with association rate constants of approximately 10(7) M-1 s-1. In contrast to PAI-1 from eucaryotic cells, the PAI-1 from E. coli had an inherent activity equal to that of guanidine/HCl-activated natural PAI-1. The activity could not be increased by treatment with denaturants suggesting that the latent form of PAI-1 was absent. However, at 37 degrees C the procaryote-produced PAI-1 lost activity at the same rate as natural PAI-1, with approximately 50% of the activity remaining after 3 h. This activity could be partially restored by treatment with 4 M guanidine/HCl. E. coli-derived PAI-1, added to human plasma and fractionated by Sephacryl S-200 chromatography, eluted in two peaks that were similar to those obtained with guanidine-activated PAI-1 from eucaryotic cells, suggesting that it bound to the PAI-1-binding protein (vitronectin).
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| 8. |
- Lundgren, B, et al.
(författare)
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Examination of the structural requirements for proliferation of peroxisomes and mitochondria in mouse liver by hypolipidemic agents, with special emphasis on structural analogues of 2-ethylhexanoic acid.
- 1987
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Ingår i: European Journal of Biochemistry. - 0014-2956 .- 1432-1033. ; 163:2
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Tidskriftsartikel (refereegranskat)abstract
- We have found here that there are clear structural requirements for peroxisome proliferation (monitored as increases in carnitine acetyltransferase activity, cyanide-insensitive palmitoyl-CoA oxidation, catalase and increases in the protein designated PPA 80) in mouse liver. From the investigation of ten structural analogues of 2-ethylhexanoic acid, it could be concluded that the most effective proliferators all have an ethyl group as the substituent on carbon 2 of the main chain, which consists of six carbons. The further observation from this group of compounds that a charged group is required for effective proliferation leads us to speculate that such a group is involved in the molecular mechanism as well. Many, but not all, of the effective peroxisome proliferators in a second group of compounds contain a phenoxy group, often with a substituted alpha carbon. Interestingly, the 2,4-dichlorophenoxyacetic and 2,4,5-trichlorophenoxyacetic acids are both effective peroxisome proliferators, but the closely related p-chlorophenoxyacetic acid is inactive in this respect, indicating that the chlorine atom at position 2 must be essential to the process in these cases. The results presented here also indicate that the structural requirements for proliferation of mitochondria are similar to those for proliferation of peroxisomes. Certainly, the most effective peroxisome proliferators also cause large increases in 'mitochondrial' protein and cytochrome oxidase activity, i.e. there is an obvious qualitative correlation.
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| 9. |
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| 10. |
- Nilsson, Magnus H. L., et al.
(författare)
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Isolation and characterization of a cDNA clone corresponding to bovine cellular retinoic-acid-binding protein and chromosomal localization of the corresponding human gene
- 1988
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 173:1, s. 45-51
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Tidskriftsartikel (refereegranskat)abstract
- A bovine adrenal cDNA library was constructed and a clone corresponding to cellular retinoic-acid-binding protein (CRABP) mRNA was isolated and sequenced. The insert of the clone corresponds to 75 bp of the 5′ untranslated portion, the whole translated and the complete 3′ untranslated portion of the bovine CRABP mRNA. A genomic Southern blot, probed with CRABP cDNA, indicated that only one copy of the gene is present in the human genome. Hybridizing bands in restricted chicken and fish DNA were also observed. Using the CRABP cDNA as probe we have located the human CRABP gene to chromosome 3 in hybridizations to mouse-human, hamster-human and rat-human cell hybrids. In situ hybridizations on rat testis cells probed with CRABP and cellular retinol-binding protein antisense mRNA indicate that both proteins are expressed in tubuli cells.
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