| 11. |
- Eriksson, Svante, 1957, et al.
(författare)
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ROLE OF TYROSINE RESIDUE-264 OF RECA FOR THE BINDING OF COFACTOR AND DNA
- 1993
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 268:3, s. 1811-1816
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Tidskriftsartikel (refereegranskat)abstract
- The tyrosine fluorescence of the RecA protein is quenched by about 15% upon binding of the cofactor analog adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS). This quenching is not observed with a modified RecA in which the tyrosine residue at position 264 (Tyr-264) is replaced for alanine by site-directed mutagenesis, a modification which also results in a decrease of binding affinity of cofactor. This indicates that Tyr-264 is responsible for the fluorescence change and that the residue is close to or within the cofactor binding site. Upon DNA binding, a change of tyrosine fluorescence is observed both with the modified protein and with wild type RecA, indicating that DNA binding affects the environment of other tyrosine residues than Tyr-264. However, the change is significantly smaller in the modified protein, suggesting that both Tyr-264 as well as other residue(s) may be affected by the DNA binding. Changed fluorescence properties of the remaining tyrosine residues as a result of a slightly different DNA binding mode of the modified protein are also possible. Tyr-264 may be an important residue for the allosteric effect induced by the cofactor for the binding of DNA to RecA. In the recent crystal structure of RecA-ADP published by Story and Steitz (Story, R. M., and Steitz, T. A. (1992) Nature 355, 374-376), ADP is stacked with Tyr-103 and does not interact with Tyr-264. The fact that we observe no interaction of ATPgammaS with Tyr-103 (as evidenced from absence of fluorescence change) but instead with Tyr-264 may suggest an important conformational difference between the RecA complexes with, respectively, ADP and ATP.
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| 12. |
- Falkenberg, C, et al.
(författare)
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Isolation of rat serum alpha 1-microglobulin. Identification of a complex with alpha 1-inhibitor-3, a rat alpha 2-macroglobulin homologue
- 1990
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 265:27, s. 16150-16157
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Tidskriftsartikel (refereegranskat)abstract
- Alpha 1-Microglobulin (alpha 1-m), or protein HC, a low molecular weight plasma protein with immunoregulatory properties, was isolated from rat serum by affinity chromatography using Sepharose-coupled monoclonal anti-alpha 1-m antibodies. High molecular weight forms of alpha 1-m were then separated from the low molecular weight alpha 1-m by gel chromatography of the eluted proteins. The apparent Mr (28,000), the charge heterogeneity, the N-linked carbohydrate, and yellow-brown chromophore suggest that the low molecular weight alpha 1-m is the serum counterpart to urinary alpha 1-m, which was purified previously. A high molecular weight complex of alpha 1-m was also isolated by the gel chromatography. It was homogeneous as judged by nondenaturing polyacrylamide gel electrophoresis. The molecule was bound by antibodies against human alpha 2-macroglobulin, and experiments with antisera against the three alpha-macroglobulin variants in rat serum, alpha 1-macroglobulin, alpha 2-macroglobulin, and alpha 1-inhibitor-3 (alpha 1I3) suggested that alpha 1I3 was the complex-partner of alpha 1-m. An antiserum raised against high molecular weight alpha 1-m was then used to isolate the complex-partner of alpha 1-m from rat serum with affinity chromatography, and this molecule was positively identified as alpha 1I3 by its physicochemical properties. Gel chromatography of the alpha 1I3.alpha 1-m complex suggested a molecule with an Mr of 266,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, however, it migrated as three major molecular species with apparent molecular weights of 224,000, 205,000, and 194,000 and several minor species of both higher and lower molecular weights, suggesting a complex subunit structure. alpha 1-m and alpha 1I3 could be detected in all three major species by Western blotting, and NH2-terminal amino acid sequencing suggested a molar ratio of 1:1 of alpha 1-m and alpha 1I3 in all three species. alpha 1I3.alpha 1-m was colorless, did not show light absorbance beyond 300 nm which is typical of low molecular weight alpha 1-m and was electrophoretically homogeneous, suggesting that it lacks the chromophore. Finally, the serum concentrations of the alpha 1I3.alpha 1-m complex and free alpha 1-m were determined as 0.16 and 0.010 g/liter, respectively. Thus, alpha 1I3.alpha 1-m constitutes 1-3% of the total alpha 1I3 in rat serum (w/w) and approximately 60% of the total alpha 1-m.
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| 13. |
- Feng, P, et al.
(författare)
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The structure of the TATA-less rat tissue-type plasminogen activator gene. Species-specific sequence divergences in the promoter predict differences in regulation of gene expression.
- 1990
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 265:4, s. 2022-7
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Tidskriftsartikel (refereegranskat)abstract
- The genomic region carrying the rat tissue-type plasminogen activator (tPA) gene including its 5'-flanking sequence has been isolated and characterized by restriction enzyme analysis, Southern blotting, and DNA sequencing of all coding parts and the promoter region. The gene is approximately 25 kilobase pairs in size and comprises 14 exons separated by 13 introns. All the exon/intron boundaries agree with the GT-AG rule. The organization of the rat tPA gene is very similar to its human counterpart, and the location of the introns in the protein structure is identical to the human tPA gene. To characterize the promoter region, the transcription initiation site was identified by S1 nuclease protection experiments. A DNA fragment carrying 621 nucleotides of the 5'-flanking sequence was found to confer basal promoter activity and hormone responsiveness to a reporter gene construct in primary cultures of rat granulosa cells. Analysis of the rat tPA promoter sequence and a comparison with the human and mouse counterparts reveal several species-specific differences: the rat and mouse tPA promoters lack typical TATA and CAAT sequences found in the human tPA gene. Furthermore, the rat tPA promoter contains a consensus cAMP-responsive element shown to be required for cAMP responsiveness in eucaryotic genes. At the same position as the cAMP-responsive element in the rat gene, the mouse and human tPA genes have a 12-O-tetradecanoylphorbol-13-acetate-responsive element known to mediate activation by phorbol esters. The differences in the promoter sequences of the rat, mouse, and human tPA genes may have implications for the regulation of the tPA gene in different species.
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| 14. |
- Forsberg, Per-Olof, et al.
(författare)
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In vitro phosphorylation of human complement factor C3 by protein kinase A and protein kinase C : Effects on the classical and alternative pathways
- 1990
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 265:5, s. 2941-2946
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Tidskriftsartikel (refereegranskat)abstract
- Complement factor C3, recently found to contain covalently bound phosphate, was phosphorylated in vitro by cyclic AMP-dependent protein kinase (protein kinase A) and Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C). Both protein kinases phosphorylated the same serine residue(s) located in the C3a portion of the alpha-chain. In addition, protein kinase C phosphorylated the beta-chain to a lesser extent. Protein kinase A gave a maximal incorporation of 1 mol of phosphate/mol of C3 while that value with protein kinase C was 1.5 mol of phosphate/mol of C3. The velocity in pmol of [32P]phosphate/(min x unit kinase) was 20 times higher for protein kinase C than for protein kinase A although a 10 times lower ratio of protein kinase to C3 was used in the former case. The apparent Kmfor C3 was 2.6 µM when protein kinase C was used. The phosphorylated C3 was found to be more resistant to partial degradation by trypsin than unphosphorylated C3. It was also found that phosphorylation of C3 in the C3a portion of the alpha-chain inhibited both the classical and alternative complement activation pathways on an approximately stoichiometric basis.
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| 15. |
- Gullberg, M, et al.
(författare)
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Purification and characterization of a 19-kilodalton intracellular protein. An activation-regulated putative protein kinase C substrate of T lymphocytes.
- 1990
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 265:29
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Tidskriftsartikel (refereegranskat)abstract
- Activation of protein kinase C in T cells results in rapid phosphorylation of a 19-kDa intracellular protein termed 19K. We report the purification of 19K from human peripheral T cells and an internal 20-amino acid sequence determined from this protein. It is shown that 19K is a novel cytoplasmatic protein which is phosphorylated in vitro by partially purified protein kinase C. 19K-specific antibodies, raised by immunizing rabbits with purified protein, were used to show that the 19K is expressed, and phosphorylated in response to protein kinase C activation, in several cellular systems. These antibodies were also used to precipitate 19K from both [35S]methionine and 32Pi-labeled T cells. The data showed that 15 min of phorbol ester treatment has no effect on the rate of 19K synthesis but results in induction of 19K phosphorylation. However, we demonstrate, by Western blot analysis, that expression of 19K in primary peripheral T cells increased at least 10-fold over a period of 4 days after activation. The increase in 19K expression correlates with initiation of DNA synthesis, and in proliferating T cells 19K comprises approximately 0.2% of total cytoplasmatic protein. Thus, 19K is a novel putative protein kinase C substrate which is subject to activation associated up-regulation in human T cells.
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| 16. |
- Hansson, L, et al.
(författare)
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Recombinant human milk bile salt-stimulated lipase. Catalytic activity is retained in the absence of glycosylation and the unique proline-rich repeats.
- 1993
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 268:35, s. 26692-8
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Tidskriftsartikel (refereegranskat)abstract
- Human milk bile salt-stimulated lipase ensures efficient utilization of triacylglycerol by breast-fed infants. Cloning and sequencing of cDNA have revealed that the peptide chain consists of 722 amino acid residues showing only little homology to typical lipases. The sequence is identical to that of pancreatic carboxylic-ester hydrolase. The COOH-terminal part contains 16 proline-rich repeats of 11 residues with O-linked carbohydrate. The only N-linked sugar chain is situated close to the active-site serine. Using C127 cells and a bovine papilloma virus vector, high and stable expression of full-length lipase and of several variants, obtained by site-directed mutagenesis, was achieved. The produced proteins were purified and further characterized. Variants lacking all, or all but two, repeats were active with similar specific activity and the same bile salt dependence as the native milk enzyme. Changing the asparagine necessary for N-glycosylation gave the same principal results. Active recombinant full-length lipase was also produced in a bacterial system. We conclude that neither glycosylation (N- or O-linked) nor the proline-rich repeats are essential for catalytic activity or bile salt activation of human milk bile salt-stimulated lipase.
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| 17. |
- Holgersson, J, et al.
(författare)
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Blood group A glycolipid antigen expression in kidney, ureter, kidney artery, and kidney vein from a blood group A1Le(a-b+) human individual. Evidence for a novel blood group A heptaglycosylceramide based on a type 3 carbohydrate chain.
- 1990
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 265:34, s. 20790-8
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Tidskriftsartikel (refereegranskat)abstract
- Kidney, ureter, kidney artery, and kidney vein tissue were obtained from a single human transplant specimen. The donors erythrocyte blood group phenotype was A1Le(a-b+). Total non-acid glycolipid fractions were isolated and individual glycolipid components were identified by immunostaining thin layer plates with a panel of monoclonal antibodies and by mass spectrometry of the permethylated and permethylated-reduced total glycolipid fractions. The dominating glycolipids in all tissues were mono- to tetraglycosylceramides. In the kidney, ureter, and artery tissue less than 1% of the glycolipids were of blood group type, having more than 4 sugar residues. In contrast, 14% of the vein glycolipids were of blood group type, and the dominating components were type 1 chain blood group H pentaglycosylceramides and A hexaglycosylceramides. Trace amounts of structurally different blood group A glycolipids (type 1 to 4 core saccharide chains) with up to 10 sugar residues were found in the kidney, ureter, and vein tissues, including evidence for a novel blood group A heptaglycosylceramide based on the type 3 chain in the vein. The only detected A glycolipid antigen in the artery tissue was the blood group A difucosyl type 1 chain heptaglycosylceramide (ALeb) structure. Blood group Lewis and related antigens (Lea, Leb, and ALeb) were expressed in the kidney, ureter, and artery, but were completely lacking in the vein, indicating that the Le gene-coded alpha 1-4-fucosyltransferase was not expressed in this tissue. The X and Y antigens (type 2 chain isomers of the Lea and Leb antigens) were detected only in the kidney tissue.
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| 18. |
- Kim, Seog K., et al.
(författare)
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ROLE OF DNA INTERCALATORS IN THE BINDING OF RECA TO DOUBLE-STRANDED DNA
- 1993
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 268:20, s. 14799-14804
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Tidskriftsartikel (refereegranskat)abstract
- RecA protein can bind to double-stranded DNA even without the cofactor ATP if a DNA intercalator such as ethidium bromide is present (Thresher R. J., and Griffith, J. D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5056-5060). We have studied the structure and association kinetics of the ethidium-promoted DNA-RecA complex in order to understand the role of this intercalator in the DNA-RecA association process, information that could provide insight about the binding mechanism of RecA to DNA. Both linear dichroism and fluorescence measurements show that ethidium remains intercalated between the DNA bases in the RecA-DNA complex in the absence of ATP. Even in the presence of the ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), ethidium bromide shows some stimulating effect on the binding of RecA to DNA. The results indicate that the destacking of DNA bases is an important limiting step in the association of RecA to DNA (DNA is stretched in the ATPgammaS-RecA-DNA complex). In the presence of ATPgammaS, however, ethidium was extruded from DNA upon the binding of RecA. This result suggests that the binding mechanism of RecA to DNA may involve intercalation of one or more amino acid residues of RecA between the DNA bases. Such an intercalation would also be consistent with the stretching of DNA and the observation that the DNA bases remain in a (virtually stacked) perpendicular geometry (Takahashi, M., Kubista, M., and Norden, B. (1991) Biochemie (Paris) 73, 219-226; Norden, B., Elvingson, C., Kubista, M., Sjoberg, B., Ryberg, H., Ryberg, M., Mortensen, K., and Takahashi, M. (1992b) J. Mol. Biol. 226, 1175-1191).
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