Abstract
The phospholipid biosynthetic enzyme activities: CDP-diglyceride synthetase, phosphatidylglycerophosphate synthetase, PGP phosphatase, phosphatidylserine (PS) synthase, PS decarboxylase, and S-adenosyl-L-methionine:phosphatidylethanolamine (AdoMet:PE) N-methyltransferase were detected in crude cell-free extracts of Rhodopseudomonas sphaeroides. CDP-diglyceride synthetase and phosphatidylglycerophosphate synthetase co-enriched with penicillin-binding protein activity, a known cytoplasmic membrane marker, throughout fractionation of cell-free extracts of both chemoheterotrophically and photoheterotrophically grown cells. PS decarboxylase also co-enriched with the cytoplasmic membranes in fractions derived from chemoheterotrophically and photoheterotrophically grown cells, but substantially greater quantities of PS decarboxylase activity was found in the chromatophores derived from photoheterotrophically grown cells than could be accounted for by cytoplasmic membrane contamination of this sample. PS synthase (60% of the recovered activity) and S-adenosyl-L-methionine:phosphatidylethanolamine N-methyltransferase (90% of the recovered activity) were found in the supernatant fraction after high speed centrifugation of crude cell lysates, suggesting that these enzyme activities were not tightly membrane associated. The localization of phospholipid biosynthetic enzyme activity in R. sphaeroides is discussed in terms of the biosynthesis of the photosynthetic membranes.
Highlights
CDP-diglyceridseynthetasep,hosphatidylglyceroteria which contain ICM in additionto CM and OM has not phosphate synthetase, PGP phosphatase, phosphati- beenextensively pursued for a variety of reasons, some of dylserine (PS) synthase, PS decarboxylase, and which areduetotechnical difficultiesassociatedwith cell
Tions derived from chemoheterotrophically andpho- throughout the cell cycle [10, 11].This burst of phospholipid toheterotrophicallygrown cells, but substantially insertionintothe ICM concurrent with theonset of cell greater quantities of PS decarboxylase activity was division is the result of the net transferof phospholipids from found in the chromatophores derived from photohet- a discrete site of cellular phospholipid synthesis to the ICM, erotrophically grown cellsthan could be accounted for rather than the denouo synthesis of phospholipids on the by cytoplasmic membrane contamination of this sam- ICM [9].A further demonstration of the segregation of ICM
The localization of phospholipid biosynthetic enzyme activity in R. sphaeroides is discussed in terms of the biosynthesis of the photosynthetic membranes
Summary
Medium, and Growth Conditions-R. sphneroides strain M29-5 (Leu-, Met-)was grown in asuccinicacid-based minimal medium supplemented with L-leucine and L-methionine as described by Lueking et al [7].Batchculturesin 6 liters of medium were routinely grown in 8-liter carboys at 30 “C. Chemoheterotrophic cells were continuously mixed and vigorously sparged (2 liters/min) with a mixture of oxygen/nitrogen/carbon dioxide (25:74:1). Lamp(Sylvania) and a bank of lumiline lamps(GeneralElectric) arranged on opposite sides of the carboy Both chemoheterotrophic and photoheterotrophic cells were harvested by centrifugation (7500 x g, 10 min) duringexponential growth a t approximate cell densities of 1 X lo9and 1.5 X IO9cells/ml, respectively. Unbroken cells were removed by centrifugation (10,000 X g, 10 min).The membranes (particulate fraction) were pelleted (150,000 X g, 1.5 h) andthesupernatant (soluble fraction) was stored at -20 "C. CDP-diglyceride synthetase activity was assayed by measuring incorporation of radioactive dCTP into trichloroaceticacid-precipitablematerial [24].PS synthase, PGP synthetase,and AdoMet:PE N-methyltransferase activities were quantitated by determining incorporation of radioactive serine, glycerol 3-phosphate, and methyl groups, respectively, into chloroformsoluble material [25,26,27]. All other reagents and solvents were of reagent grade and used without further purification
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
