Tetrazolium reduction and nitrogenase activity in heterocystous blue-green algae

Fay, P.; Kulasooriya, S. A.

doi:10.1007/bf00409133

Cited by 35 publications

(15 citation statements)

References 21 publications

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“…There were a variety of factors which might have contributed to this low activity, including osmotic effects, cold inactivation during the centrifugation, and shearing during the initial preparation which results in shortened filaments. There is some evidence that shearing decreases rates of acetylene reduction (9).…”

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The Azolla, Anabaena azollae Relationship

Peters¹,

Mayne²

1974

Plant Physiol.

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This manuscript reports results of initial studies on acetylene reduction by the symbiotic association. It provides evidence that the symbiotic alga is the agent of acetylene reduction and, if acetylene reduction is a valid assay of the ability to fix N,. the site of N, fixation.MATERIALS AND METHODS Azolla plants, nutrient solutions, growth conditions, isolation of the symbiotic algae, and Chl determinations were as described in thr preceding paper (20).Chemicals DCMU was obtained as Diuron from the E. 1. duPont de Nemours Company. Polyvinylpyrrolidone was obtained from the Sigma Chemical Company. All other chemicals we reagent grade. The gas mixtures were obtained from Matheson Gas Products.Acetylene Nmduction Assays. Three to five fronds were incubated under dciher 1% CO,, 1% acetylene in argon (microaerophilic comlidions) (24) or with oxygen added to 21'% (v/v) (aerobic conditions) in calibrated 25-ml Erlenmeyer flasks fitted with serum caps and containing :5 ml of the desired nutrient solution. The flasks were evacuated and flushed prior to filling to a slightly positive pressure. The flasks were illuminated from the bottom with a bank of General Electric white fluorescent lighs. Screens were used to lower the inteasty for the light intensity curve. The intensity for all other assays was maintained at approximately 750 ft-c. Incubations wee carried out at 23 C.The gas phase was analyzed for ethylene by gas chmnatography using a Hewlett-Packard (Model 700) dual flame gas chromatograph with a hydrogen flame ionization detector and a 3.2 mm, 2-m long column containing 80 to 100 mesh Pora-820 www.plantphysiol.org on May 9, 2018 -Published by Downloaded from

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confidence: 99%

The Azolla, Anabaena azollae Relationship

Peters¹,

Mayne²

1974

Plant Physiol.

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“…The beterocysts are a major site of reductive nitrogen fixation in the algal filaments (7,18,19) and reduce triphenyltetrazolium chloride much more rapidly than do vegetative cells (6,13). Heterocysts isolated by means of a French press lack a photosystem II (4).…”

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confidence: 99%

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

Winkenbach¹,

Wölk²

1973

Plant Physiol.

138

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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)

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“…was performed by incubating culture aliquots with 2 mM INT for 10 min prior to microscopy (Fay & Kulasooriya, 1972). GFP was excited at 480 nm, and fluorescence was captured at 525 nm.…”

Section: Methodsmentioning

confidence: 99%