Chlorophyllase in Phaeodactylum tricornutum Photosynthetic Membranes. Extractability, Small-Scale Purification and Molecular Weight Determination by SDS-Gel-Electrophoresis

The extractability of chlorophyllase is used as an indicator for the way in which the enzyme is incorporated in Phaeodactylum tricornutum photosynthetic membranes. Whereas with various aqueous solutions no appreciable amount of chlorophyllase is washed from the membranes even after chloroform-pretreatment. the enzyme can be extracted with aqueous solutions after practically all the lipids have been removed by means of 80% acetone. The proteins in an aqueous extract of the membranes thus treated were separated by electrophoresis on a 4% polyacrylamide gel; extraction of the active protein from several gels yielded a purified chlorophyllase solution. As with the intramembraneous enzyme, activity of purified, solubilized chlorophyllase depends upon the combined presence of MgCl₂ and dithiothreitol. The enzyme is inactivated upon heating at 56°C for 5 min. After SDS-gel-electrophoresis of an enriched extract, enzyme activity could be localized in the gels. The molecular weight of SDS-treated chlorophyllase, or of its principal subunit, was estimated to be about 38 kilodaltons. The results are discussed in terms of the identity of prochlorophyllase.     

Comparison of dot blot with in-situ hybridization for the detection of Neisseria gonorrhoeae in urethral exudate

Dot blot hybridization and in-situ hybridization with DNA probes prepared from total genomic gonococcal DNA were compared with Gram staining and culturing for the detection of gonococci in urethral exudates. Fifteen of 60 patients were positive by at least one of the four methods. Gram staining and in-situ hybridization scored best with 13 positives but culture and dot blot hybridization yielded only 10 and 9 positives respectively. The failure to detect gonococci in culture can be explained by overgrowth in one case and possibly self medication with ampicillin before culture in another. The in-situ hybridization test is a fast and sensitive hybridization method for the detection of gonococci in urethral exudate from men.     

Chlorophyllase and Photsystem

• 1 Preliminary experiments showed that Triton X-100 at low concentrations (appr. 0.01%) induces reversible changes in some photosynthetic reactions in Phaeodactylum cells without influencing their absorption spectrum; presumably membrane permeability is effects. Although DC PIP can pass through the cell membrane in the presence of 0.01% Triton X-100, only a very low PS I activity, measured as MV photoreduction by DCPIP-ascorbate, is observed with whole cells.
• 2 MV photoreduction sensitized by membrane fragments isolated from Phaetdactylum, Euglena, Porphyridium and Synechococcus after French Press treatment is much higher with Phaeodactylum than with the other organisms. Spinach shroma membrane fragments show higher photosensitizing activity than grana fragments.
• 3 MV photoreduction in the above mentioned experiments is not influenced by DCMU (2 × 10^?6M) or sodium azide (0.01–0.05 M); KCN (4 × 10^?3M) has an inhibiting effect only with the blue-green alga Synechococcus. The reaction mechanism of chlorophyll-sensitized MV photoreduction is discussed.
• 4 Chlorophyll in an aqueous medium containing Triton X-100 (JsO.01%) sensitizes the photoreduction of MV by DCPIP ascorbate. A similar reaction is observed with chlorophyll combined with solubilized Phaeocdactylum chlorophyllase: in the latter case the presence of both MgCl₂ and DTT is required.
• 5 MV-photoreduction is concluded to be a very unreliable procedure for determining PS I activity in membranes if these membranes have been prepared in (the presence of detergents.
• 6 The results support the hypothesis that (prochlorophyllase is a PS I protein.
• 7 The capacity of PS I to reduce MV in vivo is discussed.