| 1. |
- Gough, Simon, et al.
(författare)
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A new method for isolating physiologically active Mg-protoporphyrin
- 2007
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Ingår i: Plant Physiology and Biochemistry. - : Elsevier BV. - 1873-2690 .- 0981-9428. ; 45:12, s. 932-936
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Tidskriftsartikel (refereegranskat)abstract
- Mg-protoporphyrin monomethyl ester (MPE) is a biosynthetic intermediate of chlorophyll and converted by MPE cyclase to protochlorophyllide. Limited availability of MPE has so far hampered cyclase research. In a new, simplified, method MPE was prepared from freeze dried bchE mutant Rhodobacter capsulatus DB575 cells by extraction with acetone/H2O/25% NH3. Isolated MPE was identified by absorption and fluorescence spectroscopy, and its purity was analyzed by HPLC. The extracted MPE was dried and redissolved in buffered DMSO and its substrate activity is shown by enzymatic cyclase assays. A linear time course was observed for MPE conversion to protochlorophyllide by enzymes from barley etioplasts. Our innovation of freeze drying the R. capsulatus cells before extraction provides a high yield method for MPE, which is significantly faster and more reproducible than previous extraction methods.
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| 2. |
- Gough, Simon P, et al.
(författare)
-
A new method for isolating physiologically active Mg-protoporphyrin monomethyl ester, the substrate of the cyclase enzyme of the chlorophyll biosynthetic pathway.
- 2007
-
Ingår i: Plant Physiology and Biochemistry. - : Elsevier BV. - 0981-9428. ; 45:12, s. 932-6
-
Tidskriftsartikel (refereegranskat)abstract
- Mg-protoporphyrin monomethyl ester (MPE) is a biosynthetic intermediate of chlorophyll and converted by MPE cyclase to protochlorophyllide. Limited availability of MPE has so far hampered cyclase research. In a new, simplified, method MPE was prepared from freeze dried bchE mutant Rhodobacter capsulatus DB575 cells by extraction with acetone/H2O/25% NH3. Isolated MPE was identified by absorption and fluorescence spectroscopy, and its purity was analyzed by HPLC. The extracted MPE was dried and redissolved in buffered DMSO and its substrate activity is shown by enzymatic cyclase assays. A linear time course was observed for MPE conversion to protochlorophyllide by enzymes from barley etioplasts. Our innovation of freeze drying the R. capsulatus cells before extraction provides a high yield method for MPE, which is significantly faster and more reproducible than previous extraction methods.
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| 3. |
- Hansson, Mattias, et al.
(författare)
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PCR-mediated deletion of plasmid DNA
- 2008
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 375:2, s. 373-375
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Tidskriftsartikel (refereegranskat)abstract
- The PCR-mediated plasmid DNA deletion method is a simple approach to delete DNA sequences from plasmids using only one round of PCR, with two primers, and without ligation or purification prior to in vivo recombination. By using only PCR, the method is sequence independent and, as shown in this study, is applicable to various sizes of plasmids and deletions using minimal primer design.
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| 4. |
- Rzeznicka, Kamila, et al.
(författare)
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Xantha-l encodes a membrane subunit of the aerobic Mg-protoporphyrin IX monomethyl ester cyclase involved in chlorophyll biosynthesis
- 2005
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 102:16, s. 5886-5891
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Tidskriftsartikel (refereegranskat)abstract
- Chlorophyll biosynthesis is a process involving approximate to 20 different enzymatic steps. Half of these steps are common to the biosynthesis of other tetrapyrroles, such as heme. one of the least understood enzymatic steps is formation of the isocyclic ring, which is a characteristic feature of all (bacterio)chlorophyll molecules. In chloroplasts, formation of the isocyclic ring is an aerobic reaction catalyzed by Mg-protoporphyrin IX monomethyl ester cyclase. An in vitro assay for the aerobic cyclase reaction required membrane-bound and soluble components from the chloroplasts. Extracts from barley (Hordeum vulgare Q mutants at the Xantha-1 and Viridis-k loci showed no cyclase activity. Fractionation of isolated plastids by Percoll gradient centrifugation showed that xantha-1 and viridis-k mutants are defective in components associated with chloroplast membranes. The Xantha-1 gene, corresponding to Arabidopsis thaliana CHL27, Rubrivivax gelatinosus acsF, Chlamydomonas reinhardtii CRD1, and CTH1 and situated at the short arm of barley chromosome 3 (3H), was cloned, and the mutations in xantha-l(35), xantha-l(81), and xantha-l(82) were characterized. This finding connected biochemical and genetic data because it demonstrated that Xantha-1 encodes a membrane-bound cyclase subunit. The evidence suggests that the aerobic cyclase requires at least one soluble and two membrane-bound components.
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