| 1. |
- Berggård, Tord, et al.
(författare)
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Calbindin D28k exhibits properties characteristic of a Ca2+ sensor.
- 2002
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 277:19, s. 16662-16672
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Tidskriftsartikel (refereegranskat)abstract
- Calbindin D28k is a member of the calmodulin super-family of Ca2+ -binding proteins and contains six EF-hands. The protein is generally believed to function as a Ca2+ buffer, but the studies presented in this work indicate that it may also act as a Ca2+ sensor. The results show that Mg2+ binds to the same sites as Ca2+ with an association constant of approximately 1.4 x 10(3) M-1 in 0.15 M KCl. The four high-affinity sites in calbindin D28k bind Ca2+ in a non-sequential, parallel manner. In the presence of physiological concentrations of Mg2+, the Ca2+ -affinity is reduced by a factor of two and the cooperativity, which otherwise is modest, increases. Based on the binding constants determined in the presence of physiological salt concentrations, we estimate that at the Ca2+ concentration in a resting cell calbindin D28k is saturated to 40-75% with Mg2+, but to less than 9 % with Ca2+. In contrast, the protein is expected to be nearly fully saturated with Ca2+ at the Ca2+ level of an activated cell. A substantial conformational change is observed upon Ca2+ binding, but only minor structural changes take place upon Mg2+-binding. This suggests that calbindin D28k undergoes Ca2+ -induced structural changes upon Ca2+ activation of a cell. Thus, calbindin D28k displays several properties that would be expected for a protein involved in Ca2+ -induced signal transmission and hence may function not only as a Ca2+ buffer, but also as a Ca2+ sensor. Digestion patterns resulting from limited proteolysis of the protein suggest that the loop of EF-hand 2, a variant site that does not bind Ca2+, becomes exposed upon Ca2+ binding.
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| 2. |
- Bjarnason, Bjarni, et al.
(författare)
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Enzyme flow immunoassay using a Protein G column for the screening of triazine herbicides in surface and waste water
- 2001
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Ingår i: Analytica Chimica Acta. - 0003-2670. ; 426:2, s. 197-207
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Tidskriftsartikel (refereegranskat)abstract
- A method for screening of triazine herbicides in surface and waste water is presented. The method is based on an enzyme flow immunoassay (EFIA) for the detection of the free fraction of a horse radish peroxidase (HRP)-labelled antigen (tracer). This was accomplished by trapping the bound tracer fraction in a Protein G column, allowing the residual free tracer fraction to pass and be detected spectrophotometrically after incubation with an enzyme substrate. As compared with detecting the bound tracer fraction this reduces the regeneration requirements of the Protein G column used for capturing the bound fraction and, therefore, reduces assay time. A polyclonal antibody directed against simazine showed no reactivity towards tracers that were thiopropionic acid derivatives of atrazine, simazine and terbutylazine. It had good sensitivity towards tracers using derivatives of 2-chloro-4,6-(alkylamino)-s-triazines such as atrazine and simazine. The highest sensitivity was obtained with an Et/Cl/N-C5-HRP tracer because this tracer could be used in combination with the lowest concentration of antibody. The detection limit was 0.1 μgl-1 with a linear range between 0.1 and 10 μgl-1 and an assay throughput of 12 h-1. Natural water samples from various locations in Russia were analysed for triazines and the results were compared with a previously developed fluorescein flow immunoassay for triazines. The results were further verified by supported liquid membrane (SLM) extraction combined with HPLC. The results show that the two immunoassays behave differently and that the sample matrix influences their performance, however, no false negative results were obtained. The possible reasons for the different results between the two immunoassays are discussed. (C) 2001 Elsevier Science B.V.
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| 3. |
- Carrino, David A, et al.
(författare)
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Age-related changes in the proteoglycans of human skin - Specific cleavage of decorin to yield a major catabolic fragment in adult skin
- 2003
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 278:19, s. 17566-17572
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Tidskriftsartikel (refereegranskat)abstract
- Dramatic changes occur in skin as a function of age, including changes in morphology, physiology, and mechanical properties. Changes in extracellular matrix molecules also occur, and these changes likely contribute to the overall age-related changes in the physical properties of skin. The major proteoglycans detected in extracts of human skin are decorin and versican. In addition, adult human skin contains a truncated form of decorin, whereas fetal skin contains virtually undetectable levels of this truncated decorin. Analysis of this molecule, herein referred to as decorunt, indicates that it is a catabolic fragment of decorin rather than a splice variant. With antibody probes to the core protein, decorunt is found to lack the carboxyl-terminal portion of decorin. Further analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry shows that the carboxyl terminus of decorunt is at Phe(170) of decorin. This result indicates that decorunt represents the amino-terminal 43% of the mature decorin molecule. Such a structure is inconsistent with alternative splicing of decorin and suggests that decorunt is a catabolic fragment of decorin. A neoepitope antiserum, anti-VRKVTF, was generated against the carboxyl terminus of decorunt. This antiserum does not recognize intact decorin in any skin proteoglycan sample tested on immunoblots but recognizes every sample of decorunt tested. The results with anti-VRKVTF confirm the identification of the carboxyl terminus of decorunt. Analysis of collagen binding by surface plasmon resonance indicates that the affinity of decorunt for type I collagen is 100-fold less than that of decorin. This observation correlates with the structural analysis of decorunt, in that it lacks regions of decorin previously shown to be important for interaction with type I collagen. The detection of a catabolic fragment of decorin suggests the existence of a specific catabolic pathway for this proteoglycan. Because of the capacity of decorin to influence collagen fibrillogenesis, catabolism of decorin may have important functional implications with respect to the dermal collagen network.
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| 4. |
- Ekström, Simon, et al.
(författare)
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Integrated microanalytical technology enabling rapid and automated protein identification
- 2000
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 72:2, s. 286-293
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Tidskriftsartikel (refereegranskat)abstract
- Protein identification through peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a standard technique, used in many laboratories around the world. The traditional methodology often includes long incubations (6-24 h) and extensive manual steps. In an effort to address this, an integrated microanalytical platform has been developed for automated identification of proteins. The silicon micromachined analytical tools, i.e., the microchip immobilized enzyme reactor (μ-chip IMER), the piezoelectric microdispenser, and the high-density nanovial target plates, are the cornerstones in the system. The μ-chip IMER provides on-line enzymatic digestion of protein samples (1 μL) within 1-3 min, and the microdispenser enables subsequent on- line picoliter sample preparation in a high-density format. Interfaced to automated MALDI-TOF MS, these tools compose a highly efficient platform that can analyze 100 protein samples in 3.5 h. Kinetic studies on the microreactors are reported as well as the operation of this microanalytical platform for protein identification, wherein lysozyme, myoglobin, ribonuclease A, and cytochrome c have been identified with a high sequence coverage (50-100%).
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| 5. |
- Heathfield, Terrence, et al.
(författare)
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Cleavage of fibromodulin in cartilage explants involves removal of the N-terminal tyrosine sulphate rich region by proteolysis at a site that is sensitive to MMP-13.
- 2004
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 279:8, s. 6286-6295
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Tidskriftsartikel (refereegranskat)abstract
- Integrity of cartilage fails in joint disease. The current work aimed to identify candidate active proteinases in joint diseases using an in vitro model for cartilage degradation induced by interleukin-1. A critical event in the process of cartilage destruction in joint disease is the failure of the collagen fiber network to maintain integrity. Proteins binding to the surface of the fibers are likely early points of failure. Fibromodulin, a member of the leucine-rich repeat protein family, is one predominant protein in cartilage and is known for its roles in the formation of collagen fibrils and sustained interaction with these formed fibers. Cleavage removes the tyrosine sulfate-rich region in the N terminus of fibromodulin. Whereas fibromodulin bound to collagen in tissue was digested, purified fibromodulin was not cleaved. In contrast an N-terminal 10-kDa fragment, Gln19-Lys98, of the protein generated by Lys-C digestion contains the cleavage site and was a substrate cleaved by the enzyme in medium from stimulated cultures. In solution, digestion of this substrate with matrix metalloproteinase (MMP)-2, -9, -8, and -13 demonstrated that only MMP-13 was capable to efficiently cleave it. The cleavage product obtained after MMP-13 digestion was identical to that observed in cleaved fibromodulin from cartilage explant cultures stimulated with interleukin-1. MMP-13 treatment of fresh articular cartilage also produced the fragment under study. The elucidation of the enzyme responsible for such cleavage may lead to treatment modalities involving its selective inhibition for patients suffering from arthritis. The known structure of the fragments permits the generation of neo-epitope antibodies to the cleavage site, which can be used to detect ongoing cartilage degradation in patients with arthritic disease, an important adjunct in monitoring disease progression, active disease, and efficacy of treatment.
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| 6. |
- Lorenzo, Pilar, et al.
(författare)
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Identification and characterization of asporin. a novel member of the leucine-rich repeat protein family closely related to decorin and biglycan
- 2001
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 276:15, s. 12201-12211
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Tidskriftsartikel (refereegranskat)abstract
- Asporin, a novel member of the leucine-rich repeat family of proteins, was partially purified from human articular cartilage and meniscus. Cloning of human and mouse asporin cDNAs revealed that the protein is closely related to decorin and biglycan. It contains a putative propeptide, 4 amino-terminal cysteines, 10 leucine-rich repeats, and 2 C-terminal cysteines. In contrast to decorin and biglycan, asporin is not a proteoglycan. Instead, asporin contains a unique stretch of aspartic acid residues in its amino-terminal region. A polymorphism was identified in that the number of consecutive aspartate residues varied from 11 to 15. The 8 exons of the human asporin gene span 26 kilobases on chromosome 9q31.1-32, and the putative promoter region lacks TATA consensus sequences. The asporin mRNA is expressed in a variety of human tissues with higher levels in osteoarthritic articular cartilage, aorta, uterus, heart, and liver. The deduced amino acid sequence of asporin was confirmed by mass spectrometry of the isolated protein resulting in 84% sequence coverage. The protein contains an N-glycosylation site at Asn(281) with a heterogeneous oligosaccharide structure and a potential O-glycosylation site at Ser(54). The name asporin reflects the aspartate-rich amino terminus and the overall similarity to decorin.
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| 7. |
- Miliotis, Tasso, et al.
(författare)
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Protein identification platform utilizing micro dispensing technology interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry
- 2000
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 886:1-2, s. 99-110
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Tidskriftsartikel (refereegranskat)abstract
- An integrated protein microcharacterization/identification platform has been developed. The system has been designed to allow a high flexibility in order to tackle challenging analytical problems. The platform comprises a cooled microautosampler, an integrated system for microcolumn HPLC, and a capillary reversed-phase column that is interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) system via a low internal volume flow-through microdispenser. The chromatographic separation is continuously transferred onto a MALDI target plate as discrete spots as the dispenser ejects bursts of droplets of the column effluent in a precise array pattern. A refrigerated microfraction collector was coupled to the outlet of the flow-through microdispenser enabling enrichment and re-analysis of interesting fractions. The use of target plates pre-coated with matrix simplified and increased the robustness of the system. By including a separation step prior to the MALDI-TOF-MS analysis and hereby minimizing suppression effects allowed us to obtain higher sequence coverage of proteins compared to conventional MALDI sample preparation methodology. Additionally, synthetic peptides corresponding to autophosphorylated forms of the tryptic fragment 485-496 (ALGADDSYYTAR) of tyrosine kinase ZAP-70 were identified at sensitivities reaching 150 amol. Copyright (C) 2000 Elsevier Science B.V.
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| 8. |
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| 9. |
- Nilsson, M., et al.
(författare)
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Prevention of domain swapping inhibits dimerization and amyloid fibril formation of cystatin C. Use of engineered disulfide bridges, antibodies, and carboxymethylpapain to stabilize the monomeric form of cystatin C
- 2004
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 279:23, s. 24236-24245
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Tidskriftsartikel (refereegranskat)abstract
- Amyloidogenic proteins like cystatin C and prion proteins have been shown to form dimers by exchange of subdomains of the monomeric proteins. This process, called "three-dimensional domain swapping," has also been suggested to play a part in the generation of amyloid fibrils. One variant of cystatin C, L68Q cystatin C, is highly amyloidogenic, and persons carrying the corresponding gene suffer from massive cerebral amyloidosis leading to brain hemorrhage and death in early adult life. The present work describes the production of two variants of wild type and L68Q cystatin C with disulfide bridges at positions selected to inhibit domain swapping without affecting the biological function of the four cystatin C variants as cysteine protease inhibitors. The capacity of the four variant proteins to form dimers was tested and compared with that of wild type and L68Q cystatin C. In contrast to the latter two proteins, all four protein variants stabilized by disulfide bridges were resistant toward the formation of dimers. The capacity of the two stabilized variants of wild type cystatin C to form amyloid fibrils was investigated and found to be reduced by 80% compared with that of wild type cystatin C. In an effort to investigate whether exogenous agents could also suppress the formation of dimers of wild type and L68Q cystatin C, a monoclonal antibody or carboxymethylpapain, an inactivated form of a cysteine protease, was added to systems inducing dimerization of wild type and L68Q cystatin C. It was observed that catalytic amounts of both the monoclonal antibody and carboxymethylpapain could suppress dimerization.
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| 10. |
- Önnerfjord, Patrik, et al.
(författare)
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Identification of tyrosine sulfation in extracellular leucine-rich-repeat proteins using mass spectrometry.
- 2004
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 279:1, s. 26-33
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Tidskriftsartikel (refereegranskat)abstract
- Multiple and variable tyrosine sulfation in extracellular class II leucine-rich repeat proteins/proteoglycans were characterized by mass spectrometry. The sulfogroup on tyrosine is labile and is released from peptides under normal mass spectrometric conditions. Thus, special approaches must be considered in order to identify this modification. By using a combination of mass spectrometry studies operating in negative and positive ion mode, tyrosine sulfation could be identified. In positive mode, the peptides normally appeared non-sulfated, whereas in negative mode a mixture of sulfated and non-sulfated species was observed. A combination of peptides released by different proteinases was used to obtain details on the locations of sulfate groups. Multiple tyrosine sulfates were observed in the N-terminal region of fibromodulin ( up to 9 sites), osteoadherin ( up to 6 sites), and lumican ( 2 sites). Osteoadherin contains two additional sulfated tyrosine residues close to its C terminus. We also identified an error in the published sequence of bovine fibromodulin, resulting in the replacement of Thr(37) by Tyr(37)-Gly(38), thus increasing its homology with its human counterpart.
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