Activities of Enzymes of the Oxidative and the Reductive Pentose Phosphate Pathways in Heterocysts of a Blue-Green Alga

Abstract

Preparations of heterocysts of Anabaena cylindrica Brunswick, N.J.) continuously illuminated with six fluorescent tubes (daylight, 15 w) and gassed with air to a density of 3.0 ,ug Chl/ml. The doubling time based on Chl was approximately 17 hr. Cultures were checked regularly for bacterial contamination by microscopic examination, and occasionally by subculture of 1 ml into 10 ml of culture medium supplemented with 1% glucose and 0.5% peptone. Every 24 hr 7 to 8 liters of the 12 liters of algal suspension were removed and replaced with fresh medium. Filaments concentrated by centrifugation (5 min, 1100g) were resuspended in a small volume of used growth medium and stored on ice for 1 to 3 hr. Harvest, concentration, and resuspension of filaments were accomplished within 30 min.Preparation of Cell-Free Extracts. Immediately prior to use, the concentrated suspension was centrifuged (2 min, 5000g) and resuspended either approximately 100 ug Chl/ml in ice-cold tris buffer (20 mm tris HCl, pH 7.6, containing 0.1 mm EDTA and 10 mm 2-mercaptoethanol), or approximately 250 ,ug Chl/ ml in ice-cold phosphate buffer (40 mm KH}P04-K,HPO4 pH 7.0). The dense suspension was cavitated at setting no. 3 of a Model S-125 Sonifier (Heat Systems, Inc., Melville, N.Y.), which was cooled with a water jacket (12 C). Cavitation for 5 to 11 sec/ml broke most of the vegetative cells and left the major portion of the heterocysts apparently intact. After 1 min/ml of cavitation, virtually all of the vegetative cells and heterocysts were broken. In certain experiments, the phosphate buffer was supplemented with 1.5 mM glucose-6-P and 3 mM MgCl,. Cell-free extracts were obtained by centrifugation for 4 or 5 min at 50OOg or 12,000g.For studies on the time course of solubilization of glucose-6-P dehydrogenase an algal suspension, initial volume 22 ml, was cavitated for 5 sec/ml and then for four successive periods of 3 sec/ml each. After each period of cavitation, a 1-ml aliquot was withdrawn and transferred into a 4-ml centrifuge tube at 0 C. The volume of suspension was then reduced to 12 ml, and cavitation was continued for a total of 1 min/ml. All centrifugations were carried out at 1 to 5 C.Heterocyst Preparations. Twenty-two-milliliter batches of algal suspension containing about 250 ,ug Chl/ml were cavitated for 11 sec/ml, left 10 min at 24 C, diluted with 60 ml of distilled water (2-4 C), and centrifuged for 2 min at 1500g.The pellets were combined and washed three times with 20 ml of growth medium (2-4 C). The final pellet was resuspended in phosphate buffer with supplements (Table IA). In another set of experiments (Table IB)

Proteomic analyses of the photoauto- and diazotrophically grown cyanobacterium Nostoc sp. PCC 73102

“…In contrast, G6PD protein levels in Nostoc symbionts are up-shifted under the dark heterotrophic conditions offered in plant symbiosis (Ekman et al, 2006). This result is expected since the OPP pathway is a major route for providing reductants (NADPH) to nitrogenase in heterocysts (Winkenbach & Wolk, 1973;Bothe & Neuer 1988). This illustrates the metabolic flexibility of the genus and the findings are supported by our data.…”

CARBON NUTRITION AND THE REGULATION OF UPTAKE HYDROGENASE ACTIVITY IN FREE‐LIVING AND SYMBIOTIC ANABAENA CYCADEAE

“…Heterocysts are differentiated cells physiologically unable to photoassimilate CO2 and dependent upon vegetative cells for import of photosynthetically produced carbohydrate which provides the reductant and other necessary requirements for effective fixation of nitrogen (Wolk, 1968;Winkenbach & Wolk, 1973). Such compartmentation may imply that, under photoautotrophic Nj-fixing conditions, heterocysts are carbon-limited.…”

Immunocytochemical Localization of Thioredoxins in the Cyanobacteria Anabaena cylindrica and Anabaena variabilis

“…To date, information on the distribution of proteins between vegetative cells and heterocysts (Lex & Carr, 1974;Winkenbach & Wolk, 1973;Apte et al, 1978;Neuer et al, 1983) has depended on the use of techniques of heterocyst isolation which involve selective disruption of vegetative cells. The main drawbacks of such techniques are that the heterocysts are at various stages of maturity and it is difficult to assess their 'intactness' and the degree of vegetative cell contamination.…”