| 1. |
- Renlund, Martin, et al.
(författare)
-
Free N-acetylneuraminic acid in tissues in Salla disease and the enzymes involved in its metabolism
- 1983
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 130:1, s. 39-45
-
Tidskriftsartikel (refereegranskat)abstract
- Salla disease is a lysosomal storage disorder of unknown etiology, characterized biochemically by increased urinary excretion of N-acetylneuraminic acid. This compound has now been shown to occur in abnormally large amounts in liver and cultured skin fibroblasts from these patients. Quantification of N-acetylneuraminic acid was performed using a new gas-chromatography/mass spectrometric single-ion method which is sensitive and specific. No abnormalities in the activity of several enzymes involved in sialic acid metabolism (N-acetylneuraminate:pyruvate lyase, neuraminidase, CMP-N-acetylneuraminate N-acylneuraminohydrolase and CTP:N-acyl-neuraminate cytidylyltransferase) were demonstrable. A possible explanation for the defect is a malfunctioning active transport of N-acetylneuraminic acid across the lysosomal membrane.
|
|
| 2. |
- Elbing, Karin, 1974, et al.
(författare)
-
Transcriptional responses to glucose at different glycolytic rates in Saccharomyces cerevisiae
- 2004
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:23-24, s. 4855-4864
-
Tidskriftsartikel (refereegranskat)abstract
- The addition of glucose to Saccharomyces cerevisiae cells causes reprogramming of gene expression. Glucose is sensed by membrane receptors as well as (so far elusive) intracellular sensing mechanisms. The availability of four yeast strains that display different hexose uptake capacities allowed us to study glucose-induced effects at different glycolytic rates. Rapid glucose responses were observed in all strains able to take up glucose, consistent with intracellular sensing. The degree of long-term responses, however, clearly correlated with the glycolytic rate: glucose-stimulated expression of genes encoding enzymes of the lower part of glycolysis showed an almost linear correlation with the glycolytic rate, while expression levels of genes encoding gluconeogenic enzymes and invertase (SUC2) showed an inverse correlation. Glucose control of SUC2 expression is mediated by the Snf1-Mig1 pathway. Mig1 dephosphorylation upon glucose addition is known to lead to repression of target genes. Mig1 was initially dephosphorylated upon glucose addition in all strains able to take up glucose, but remained dephosphorylated only at high glycolytic rates. Remarkably, transient Mig1-dephosphorylation was accompanied by the repression of SUC2 expression at high glycolytic rates, but stimulated SUC2 expression at low glycolytic rates. This suggests that Mig1-mediated repression can be overruled by factors mediating induction via a low glucose signal. At low and moderate glycolytic rates, Mig1 was partly dephosphorylated both in the presence of phosphorylated, active Snf1, and unphosphorylated, inactive Snf1, indicating that Mig1 was actively phosphorylated and dephosphorylated simultaneously, suggesting independent control of both processes. Taken together, it appears that glucose addition affects the expression of SUC2 as well as Mig1 activity by both Snf1-dependent and -independent mechanisms that can now be dissected and resolved as early and late/sustained responses.
|
|
| 3. |
- Eriksson, Torny, et al.
(författare)
-
Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma reesei) Cel7B catalytic module
- 2004
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:7, s. 1266-1276
-
Tidskriftsartikel (refereegranskat)abstract
- The catalytic module of Hypocrea jecorina (previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc(2)Man(5) was identified as the predominant N-glycan, although lesser amounts of GlcNAc(2)Man(7) and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However, partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations.
|
|
| 4. |
- Florén, Claes-Henrik, et al.
(författare)
-
Effects of fatty acid unsaturation on chylomicron metabolism in normal and hepatectomized rats
- 1977
-
Ingår i: Eur J Biochem. - : Wiley. - 0014-2956. ; 77:1, s. 23-30
-
Tidskriftsartikel (refereegranskat)abstract
- 1. Hepatectomized rats were injected intravenously with doubly labelled ([14C]linoleic acid and [3H]palmitic acid) thoracic duct lymphs from rats fed cream, triolein or corn oil. The disappearance of the radioactive fatty acids of different molecular triacylglycerol species and of phospholipids from plasma was studied.2. 73–93% of the injected triacylglycerols had been cleared from plasma within 15 min. At all stages of lipolysis the 3H/14C ratio of the plasma triacylglycerol was the same as in the injected material. If the cream chyle had been cooled to 4 °C before use there was, however, an enrichment of [3H]palmitic acid and of fully saturated triacylglycerols in the remnant particles formed.3. Only 38–50% of the radioactive chyle phosphatidylcholine was eliminated from plasma in 30 min. At this time most of the remaining phosphatidylcholine was, however, in other lipo‐protein classes than the chylomicron remnants.4. Also in intact rats data were obtained, indicating that the major portion of chylomicron phospholipids is transferred to other serum lipoproteins by exchange or net movement rather than being hydrolysed in the 1‐position by lipoprotein lipase or taken up intact by the liver.5. More of both the labelled fatty acids appeared in liver triacylglycerols in experiments with cream chyle than in experiments with corn oil chyle. Data were obtained suggesting that this may be due to a higher uptake of intact triacylglycerol as remnant particles.6. When linoleic acid is fed as a tracer dose in cream, a high proportion (16–36%) is incorporated into chyle phospholipids.
|
|
| 5. |
- GOLOLOBOV, Mikhail Y., et al.
(författare)
-
The second nucleophile molecule binds to the acyl‐enzyme–nucleophile complex in α‐chymotrypsin catalysis : Kinetic evidence for the interaction
- 1993
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 217:3, s. 955-963
-
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.
|
|
| 6. |
- Kang, J, et al.
(författare)
-
Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1 -> 3)-alpha-D-GalNAc
- 2004
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:23-24, s. 4939-4949
-
Tidskriftsartikel (refereegranskat)abstract
- The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional H-1 homonuclear and C-13-H-1 heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.
|
|
| 7. |
- Kvassman, Jan, et al.
(författare)
-
Mechanism of glyceraldehyde‐3‐phosphate transfer from aldolase to glyceraldehyde‐3‐phosphate dehydrogenase
- 1988
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 172:2, s. 427-431
-
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.
|
|
| 8. |
- Larsson, Karin M., et al.
(författare)
-
Activity and stability of horse‐liver alcohol dehydrogenase in sodium dioctylsulfosuccinate/cyclohexane reverse micelles
- 1987
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 166:1, s. 157-161
-
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.
|
|
| 9. |
- Malm, Johan, et al.
(författare)
-
Isolation and characterization of the major gel proteins in human semen, semenogelin I and semenogelin II
- 1996
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 238:1, s. 48-53
-
Tidskriftsartikel (refereegranskat)abstract
- Semenogelin I and semenogelin II constitute the major gel-forming proteins in human semen. The gel proteins were rapidly solubilized and separated from spermatozoa in ejaculates collected at pH 9.7 in buffer containing 4 mol/l urea and dithiothreitol. This protected the semenogelins from proteolytic degradation by prostate-specific antigen, and allowed their isolation by affinity chromatograph:g on heparin-sepharose. Semenogelins I and II were almost selectively retained and eluted partially separated in 0.25 mol/l NaCl. Further purification was achieved by chromatography on Superose. Approximately 10-20 mg semenogelin I and 2-5 mg semenogelin II were recovered from each sample with a purity exceeding 95% as judged by SDS/PAGE. The molecular mass of semenogelin I (49,958 Da) and the major form of semenogelin II (63,539 Da) measured by mass spectrometry was consistent with the reported cDNA data. The occurrence of a second, larger form of semenogelin II was due to asparagine-linked glycosylation. The amino-termini of the purified proteins were blocked, but digestion with pyroglutamate aminopeptidase enabled the identification of amino-terminal sequences consistent with the reported cDNA data. The amino acid compositions of the purified proteins were also consistent with those derived from cDNA data. The absorption coefficients (280 nm, 1%, 1 cm) for semenoaelins I and II. were 5.5 and 5.4, respectively, and the isoelectric point was above pH 9.5 for both proteins.
|
|
| 10. |
- Martinez Arias, Wilma, et al.
(författare)
-
Mechanism of NADH Transfer between Alcohol Dehydrogenase and Glyceraldehyde-3-Phosphate Dehydrogenase
- 1997
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 250:1, s. 158-162
-
Tidskriftsartikel (refereegranskat)abstract
- Steady-state and transient-state kinetic experiments have been performed to test the proposal that there is a direct (channelled) transfer of NADH from glyceraldehyde-3-phosphate dehydrogenase to alcohol dehydrogenase. The results lend no support to this proposal, but can be best explained in terms of a free-diffusion mechanism for NADH transfer between the two enzymes.
|
|
