| 1. |
- ADLERCREUTZ, Patrick
(författare)
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On the importance of the support material for enzymatic synthesis in organic media : Support effects at controlled water activity
- 1991
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 199:3, s. 609-614
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Tidskriftsartikel (refereegranskat)abstract
- Enzymes were deposited on different porous support materials and these preparations were used to catalyze reactions in organic media. Reactions were carried out at specific water activities, achieved by equilibrating both the enzyme preparation and the substrate solution at the desired water activity before mixing them and thereby starting the reactions. The reaction rates obtained at the same water activity with different supports differed greatly, indicating a direct influence of the support on the enzyme. For horse liver alcohol dehydrogenase, Celite was the best support, and the reaction rate increased with increasing water activity. In the α‐chymotrypsin‐catalyzed alcoholysis of N‐acetyl‐l‐phenylalanine ethyl ester with 1‐butanol, high rates were again obtained with Celite, but with this support only about one third of the ethyl ester was converted to butyl ester, the rest was hydrolyzed. With the polyamide support, Accurel PA6, alcoholysis was the dominating reaction, and by using a low water activity (0.33), hydrolysis was completely suppressed while still maintaining a high alcoholysis activity. Controlled pore glass (CPG), derivatized with either hexyl or glucosyl groups, had quite different properties as enzyme supports. For horse liver alcohol dehydrogenase, glucose‐CPG was a much better support than hexyl‐CPG, and in the α‐chymotrypsin‐catalyzed reactions, glucose‐CPG favored hydrolysis, and hexyl‐CPG alcoholysis, at water activities exceeding 0.8. The results are discussed considering the absorption of water on the enzymes, on the supports and the solubility of water in the reaction media; all these parameters were measured separately.
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| 2. |
- Antuch, W, et al.
(författare)
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The NMR solution structure of a Kunitz-type proteinase inhibitor from the sea anemone Stichodactyla helianthus.
- 1993
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 212, s. 675-684
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Tidskriftsartikel (refereegranskat)abstract
- The solution structure of a 55-amino-acid Kunitz-type proteinase inhibitor, ShPI, purified from the Caribbean sea anemone Stichodactyla helianthus, was determined by NMR spectroscopy. Nearly complete sequence-specific 1H-NMR assignments were obtained at pH 4.6 and 36 degrees C, and stereo-specific assignments were determined for 23 pairs of diastereotopic substituents. A data set of 666 upper distance limit constraints and 122 dihedral angle constraints collected on this basis was used as input for a structure calculation with the program DIANA. Following energy minimization with the program OPAL, the average root-mean-square diviation (RMSD) of the 20 DIANA conformers used to represent the solution structure relative to the mean structure is 61 pm for all backbone atoms N, C alpha and C', and 106 pm for all heavy atoms of residues 2-53. This high-quality solution structure of ShPI has a nearly identical molecular architecture as the bovine pancreatic trypsin inhibitor (BPTI), despite a mere 35% of sequence similarity between the two proteins. Exchange rates measured for 48 out of the 51 backbone amide protons showed that the positions of 20 slowly exchanging amide protons correlate well with hydrogen bonds involving these protons in the energy-minimized solution structure. The solution structure of ShPI is compared to the four homologous proteins for which the three-dimensional structure is also available.
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| 3. |
- Ben Nasr, Abdelhakim, et al.
(författare)
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Streptokinase activates plasminogen bound to human group C and group G streptococci through M-like proteins
- 1994
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Ingår i: European Journal of Biochemistry. - 0014-2956. ; 222:2, s. 76-267
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Tidskriftsartikel (refereegranskat)abstract
- An ability to interact with plasminogen or plasmin could provide micro-organisms with a mechanism for invasion. Thus, group A, C and G streptococci secrete streptokinase which binds and activates plasminogen. Some streptococci also express surface structures which bind plasminogen without causing its activation. Plasminogen-binding surface proteins were extracted from one group C and one group G streptococcal isolate. Both proteins were found to bind plasmin, fibrinogen and serum albumin in addition to plasminogen. Gene fragments encoding the streptococcal proteins were amplified by PCR and were subsequently cloned and expressed in Escherichia coli. DNA sequence determination revealed for both genes open reading frames encoding proteins which contained repetitive domains and a carboxyl-terminal unrepeated region that were typical of M and M-like proteins. Though the amino-terminal regions of the group C and G streptococcal proteins demonstrated a rather high overall similarity between themselves, they were not similar to the variable regions of other M-like proteins with one exception: there was a 46% identity between the first 22 amino acids of the group G streptococcal protein and the corresponding sequence of PAM, the plasminogen-binding M-like protein of type M53 group A streptococci. Like the proteins extracted from the streptococci, the recombinant proteins bound plasminogen, fibrinogen and albumin. The three plasma proteins bound to separate sites on the streptococcal M-like proteins. Plasminogen bound by the group C and G streptococcal proteins was readily activated by streptokinase, providing evidence for a functional link between the secreted plasminogen-activator and proteins exposed on the bacterial surface.
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| 4. |
- Christakopoulos, Paul, et al.
(författare)
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Purification and Characterisation of an Extracellular β-Glucosidase with Transglycosylation and Exo-glucosidase Activities from Fusarium oxysporum
- 1994
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 224:2, s. 379-385
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Tidskriftsartikel (refereegranskat)abstract
- An extracellular β-glucosidase from Fusarium oxysporum was purified to homogeneity by gel-filtration and ion-exchange chromatographies. The enzyme, a monomeric protein of 110 kDa, was maximally active at pH 5.0–6.0 and at 60°C. It hydrolysed 1→4-linked aryl-β-glucosides and 1→4-linked, 1→3-linked and 1→6–linked β-glucosides. The apparent Km and kcat values for p -nitrophenyl β-d-glucopyranoside (4-NpGlcp) and cellobiose were 0.093 (Km), 1.07 mM (kcat) and 1802 (Km), 461.5 min-1 (kcat), respectively. Glucose and gluconolactone inhibited the enzyme competitively with Ki values of 2.05 mM and 3.03 μM, respectively. Alcohols activated the enzyme; butanol showed maximum effect (2.2-fold at 0.5 M) while methanol increased the activity by 1.4-fold at 1 M. The enzyme catalysed the synthesis of methylglucosides, ethylglucoside and propylglucosides, as well as trisaccharides in the presence of different alcohols and disaccharides, respectively. In addition, the enzyme hydrolysed the unsubstituted and methylumbelliferyl cello-oligosaccharides [MeUmb(Glc)n] but the rate of hydrolysis decreased with increasing chain length. Analysis of products released from MeUmb(Glc)n as a function of time revealed that the enzyme attacked these substrates in a stepwise manner and from both ends. Thus, β-glucosidase from F. oxysporum, with the above interesting properties, could be of commercial interest.
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| 5. |
- CHRISTENSSON, Anders, et al.
(författare)
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Complex formation between protein C inhibitor and prostate‐specific antigen in vitro and in human semen
- 1994
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 220:1, s. 45-53
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Tidskriftsartikel (refereegranskat)abstract
- Protein C inhibitor (PCI), a serine‐proteinase inhibitor first purified from human blood plasma, occurs at high concentrations (3–4 μM) in seminal fluid in both a high‐molecular‐mass and low‐molecular‐mass form. Immunochemical data have previously suggested that PCI in seminal plasma forms complexes with the most abundant serine proteinase in semen, prostate‐specific antigen (PSA). To provide a structural characterization of the PCI target, immunodetected as PSA, a procedure was developed to isolate low‐molecular‐mass and high‐molecular‐mass‐forms of PCI from seminal fluid. The high‐molecular‐mass form of PCI, recognized by monoclonal antibodies against PSA, was dissociated by alkaline treatment into the low‐molecular‐mass form of PCI and a 33‐kDa protein identified as PSA by 25 conclusive steps of N‐terminal sequence analysis. We developed a sensitive immunofluorometric assay (IFMA) to measure PCI‐PSA complexes in body fluids and investigated the rate at which purified PSA may form complexes with purified PCI. Formation of complexes detected by this IFMA and the appearance of SDS‐stable approximately 90‐kDa complexes paralleled loss of PSA activity recorded with chromogenic substrates. The rate of complex formation was slow compared to that reported for PCI and activated protein C, but was enhanced up to sixfold in the presence of heparin. Less than 10% of the initial PSA activity remained after 3 h incubation with a sevenfold molar excess of PCI and in the presence of heparin. In freshly collected ejaculates, the rate of PCI‐PSA complex formation measured by IFMA was similar to that observed between the purified proteins, and paralleled the appearance of SDS‐stable complexes by immunoblotting. During gel dissolution in freshly collected ejaculates, approximately 40% of immunodetected PCI becomes complexed to PSA. Although PCI is a slow inhibitor of PSA, complexes between PCI and PSA are detected at levels that correspond to an inactivation of up to 5% of the PSA activity in the ejaculate.
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| 6. |
- Eriksson, A M, et al.
(författare)
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Studies on the intracellular distributions of soluble epoxide hydrolase and of catalase by digitonin-permeabilization of hepatocytes isolated from control and clofibrate-treated mice.
- 1991
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Ingår i: European Journal of Biochemistry. - 0014-2956 .- 1432-1033. ; 198:2
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Tidskriftsartikel (refereegranskat)abstract
- Digitonin permeabilization of hepatocytes from control and clofibrate-treated (0.5% by mass, 10 days) male C57bl/6 mice was used to study the intracellular distributions of soluble ('cytosolic') epoxide hydrolase and of catalase. The following conclusions were drawn. (1) About 60% of the total soluble epoxide hydrolase activity in control mouse hepatocytes is situated in the cytosol. (2) The rest is not mitochondrial, but probably peroxisomal. (3) Of the total catalase activity in control mouse hepatocytes, 5-10% is found in the cytosol. (4) Treatment of mice with clofibrate increases the total hepatocyte activity of soluble epoxide hydrolase 4-fold, but does not influence the relative distribution of this enzyme between cytosol and peroxisomes. (5) The total catalase activity is increased 3.5-fold by clofibrate treatment and 15-35% of this activity is shifted from the peroxisomes to the cytosol.
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| 7. |
- GOLOLOBOV, Mikhail Y., et al.
(författare)
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The second nucleophile molecule binds to the acyl‐enzyme–nucleophile complex in α‐chymotrypsin catalysis : Kinetic evidence for the interaction
- 1993
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 217:3, s. 955-963
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Tidskriftsartikel (refereegranskat)abstract
- α‐Chymotrypsin‐catalyzed acyl tranfer was studied using three acyl‐group donors (Mal‐l‐Ala‐l‐Ala‐l‐PheOMe, Bz‐l‐TyrOEt and Ac‐l‐TrpOEt; Mal, maleyl; Bz, benzoyl; OMe, methyl ester; OEt, ethyl ester) and a series of amino‐acid amides. Most of the reactions studied can be described by the simplest kinetic model without the nucleophile binding to the acyl‐enzyme. The α‐chymotrypsin‐catalyzed transfer of the Mal‐l‐Ala‐l‐Ala‐l‐Phe group to the amides of L‐Phe and L‐Tyr showed a linear dependence of the partition constant, p, on the nucleophile concentration which can be interpreted by the hydrolysis of the acyl‐enzyme–nucleophile complex. The α‐chymotrypsin‐catalyzed transfer of the Bz‐l‐Tyr and Ac‐l‐Trp groups to several amino‐acid amides showed unusual behavior which can be interpreted by the kinetic model involving formation of a complex of the acyl‐enzyme with two nucleophile molecules. These observations can explain the conflicting conclusions concerning the kinetics of α‐chymotrypsin‐catalyzed acyl transfer evident in previous studies.
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| 8. |
- Hansson, Mats, et al.
(författare)
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Purification and characterisation of a water-soluble ferrochelatase from Bacillus subtilis
- 1994
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 220:1, s. 201-208
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Tidskriftsartikel (refereegranskat)abstract
- Bacillus subtilis ferrochelatase is encoded by the hemH gene of the hemEHY gene cluster and catalyses the incorporation of Fe2+ into protoporphyrin IX. B. subtilis ferrochelatase produced in Escherichia coli was purified. It was found to be a monomeric, water-soluble enzyme of molecular mass 35 kDa which in addition to Fe2+ can incorporate Zn2+ and Cu2+ into protoporphyrin IX. Chemical modification experiments indicated that the single cysteine residue in the ferrochelatase is required for enzyme activity although it is not a conserved residue compared to other ferrochelatases. In growing B. subtilis, the ferrochelatase constitutes approximately 0.05% (by mass) of the total cell protein, which corresponds to some 600 ferrochelatase molecules/cell. The turnover number of isolated ferrochelatase, 18-29 min-1, was found to be consistent with the rate of haem synthesis in exponentially growing cells (0.2 mol haem formed/min/mol enzyme). It is concluded that the B. subtilis ferrochelatase has enzymic properties which are similar to those of other characterised ferrochelatases of known primary structure, i.e. ferrochelatases of the mitochondrial inner membrane of yeast and mammalian cells. However, in contrast to these enzymes the B. subtilis enzyme is a water-soluble protein and should be more amenable to structural analysis.
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| 9. |
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| 10. |
- Liu, Y X, et al.
(författare)
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Tissue-specific and time-coordinated hormone regulation of plasminogen-activator-inhibitor type I and tissue-type plasminogen activator in the rat ovary during gonadotropin-induced ovulation.
- 1991
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Ingår i: European Journal of Biochemistry. - 0014-2956 .- 1432-1033. ; 195:2, s. 549-55
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Tidskriftsartikel (refereegranskat)abstract
- The plasminogen-activator system provides proteolytic activity in many biological processes. The regulation of plasminogen activation may occur at many levels including the synthesis and secretion of plasminogen activators (PA) and the specific inhibition of PA activity by inhibitors. PA-inhibitor type-1 (PAI-1) is an efficient inhibitor of tissue-type PA (tPA) and urokinase-type PA (uPA) that may therefore be instrumental for the control of plasminogen activation. To investigate if coordinated regulation of PA and PA inhibitors take place in vivo in response to physiological signals, we have examined the regulation of PAI-1 and tPA in the ovary during gonadotropin-induced ovulation. We found that PAI-1, as well as tPA activity and mRNA levels, were coordinately regulated by gonadotropins in a time-dependent and cell-specific manner, such that a surge of PA-activity was obtained just prior to ovulation. Both theca-interstitial and granulosa cells synthesized PAI-1, but their maximal PAI-1 expression occurred at different times during the periovulatory period, ensuring inhibition of proteolytic activity in ovarian extra cellular compartments both before and after ovulation. The coordinated regulation of tPA and PAI-1 in the ovary may fine-tune the peak of PA activity which may be important for the regulation of the ovulatory process.
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