Isolation, partial characterization and localization of a dihydrolipoamide dehydrogenase from the cyanobacterium Synechocystis PCC 6803

Engels, Anke; Kahmann, Uwe; Ruppel, Hans Georg; Pistorius, Elfriede K.

doi:10.1016/s0167-4838(97)00025-3

Cited by 30 publications

(31 citation statements)

References 42 publications

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“…1B). However, in cyanobacteria a periplasmic form (61) and in soya bean a nodular form (62) have been found. In this report we show that dihydrolipoamide dehydrogenase (E3) shows dual localization in the acrosomal matrix and in the principal piece of the flagella of hamster spermatozoa, as we also observed for, the host complex, PDHc (Fig.…”

Section: Discussionmentioning

confidence: 99%

Novelty of the Pyruvate Metabolic Enzyme Dihydrolipoamide Dehydrogenasein Spermatozoa

Mitra¹,

Rangaraj

Shivaji

2005

Journal of Biological Chemistry

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Spermatozoa are cells distinctly different from other somatic cells of the body, capacitation being one of the unique phenomena manifested by this gamete. We have shown earlier that dihydrolipoamide dehydrogenase, a post-pyruvate metabolic enzyme, undergoes capacitation-dependent tyrosine phosphorylation, and the functioning of the enzyme is required for hyperactivation (enhanced motility) and acrosome reaction of hamster spermatozoa (Mitra, K., and Shivaji, S. (2004) Biol. Reprod. 70, 887-899). In this report we have investigated the localization of this mitochondrial enzyme in spermatozoa revealing non-canonical extra-mitochondrial localization of the enzyme in mammalian spermatozoa. In hamster spermatozoa, dihydrolipoamide dehydrogenase along with its host complex, the pyruvate dehydrogenase complex, are localized in the acrosome and in the principal piece of the sperm flagella. The localization of dihydrolipoamide dehydrogenase, however, appears to be in the mitochondria in the spermatocytes, but in spermatids it appears to show a juxtanuclear localization (like Golgi). The capacitation-dependent time course of tyrosine phosphorylation of dihydrolipoamide dehydrogenase appears to be different in the principal piece of the flagella and the acrosome in hamster spermatozoa. Activity assays of this bi-directional enzyme suggest a strong correlation between the tyrosine phosphorylation and the bi-directional enzyme activity. This is the first report of a direct correlation of the localization, tyrosine phosphorylation, and activity of the important metabolic enzyme, dihydrolipoamide dehydrogenase, implicating dual involvement and regulation of the enzyme during sperm capacitation.Spermatozoa, the haploid cells, are unique compared with other cells with respect to their morphology and functionality; needless to say the underlying signaling mechanisms in a functional spermatozoon are also unique. The metabolic pathways in a mature spermatozoon are compartmentalized (1-3), which probably enables them to survive in two different kinds of milieu, the male and the female reproductive tract. The residence time in the female reproductive tract is an obligatory event in the life cycle of a spermatozoon and has been termed "capacitation" (4, 5). During capacitation spermatozoa undergo multifaceted changes in aspects like metabolism, intracellular ion concentrations, plasma membrane fluidity, and thus membrane reorganization, intracellular pH, intracellular cAMP concentration, and generation of reactive oxygen species (6 -8). These cellular alterations during capacitation bring about three physiological changes: (a) hyperactivation (enhanced motility), (b) tyrosine phosphorylation in an array of proteins, and (c) acrosome reaction (release of the acrosomal contents); the first two events show a temporal correlation with capacitation, whereas acrosome reaction is taken to be the end point of capacitation (9).Protein tyrosine phosphorylation is considered to be a hallmark of sperm capacitation (10 -15), but only a few of thes...

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Section: Discussionmentioning

confidence: 99%

Novelty of the Pyruvate Metabolic Enzyme Dihydrolipoamide Dehydrogenasein Spermatozoa

Mitra¹,

Rangaraj

Shivaji

2005

Journal of Biological Chemistry

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“…Pollen was rinsed three times with EM buffer (50 mM KH 2 PO 4 ͞Na 2 HPO 4 , pH 7.0) and then fixed with 2.5% (wt͞vol) glutaraldehyde in EM buffer for 1 h, washed three times with EM buffer, and subsequently treated with 2% (wt͞vol) OsO 4 in distilled water for 1 h. After dehydration, embedding, and cutting, samples were placed onto 400-mesh nickel grids. Finally, samples were counterstained and examined with a Hitachi H500 electron microscope at 75 kV as described (33).…”

Section: Rna In Situ Hybridization and Nin88mentioning

confidence: 99%

Induction of male sterility in plants by metabolic engineering of the carbohydrate supply

Goetz

Godt

Guivarc’h

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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299

250

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Extracellular invertase mediates phloem unloading via an apoplastic pathway. The gene encoding isoenzyme Nin88 from tobacco was cloned and shown to be characterized by a specific spatial and temporal expression pattern. Tissue-specific antisense repression of Nin88 under control of the corresponding promoter in tobacco results in a block during early stages of pollen development, thus, causing male sterility. This result demonstrates a critical role of extracellular invertase in pollen development and strongly supports the essential function of extracellular sucrose cleavage for supplying carbohydrates to sink tissues via the apoplast. The specific interference with phloem unloading, the sugar status, and metabolic signaling during pollen formation will be a potentially valuable approach to induce male sterility in various crop species for hybrid seed production.

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“…Subsequently, the proteins were transferred to BA-85S nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany) as described previously (Michel et al, 1996). The antisera used were: anti-Lpd antiserum (Engels et al, 1997), anti-PsbA, anti-PsbO, and anti-AtpA antiserum (Tölle et al, 2002) as well as anti-PsaA/B antiserum (kind gift of Dr. Kruip, Lehrstuhl für Biochemie der Pflanzen, Ruhr-Universität Bochum, Germany). The anti-Slr1759 antiserum was raised in rabbits against two intrinsic synthetic peptides of 20 and 19 amino acids length.…”

Section: Sds-page and Immuno Blottingmentioning

confidence: 99%

“…6). The Lpd represents the E3 subunit of the pyruvate dehydrogenase complex which is mainly associated with the cytoplasmic membrane in Synechocystis PCC 6803 (Engels et al, 1997). This suggests that the amount of the cytoplasmic membrane located pyruvate dehydrogenase complex is higher in the mutant leading to a lower pyruvate concentration in Hik14.…”

Section: Analysis Of Selected Metabolitesmentioning

confidence: 99%

Physiological and Molecular Characterization of a Synechocystis sp. PCC 6803 Mutant Lacking Histidine Kinase Slr1759 and Response Regulator Slr1760

Nodop

Suzuki

Barsch

et al. 2006

Zeitschrift Für Naturforschung C

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The hybrid sensory histidine kinase Slr1759 of the cyanobacterium Synechocystis sp. strain PCC 6803 contains multiple sensory domains and a multi-step phosphorelay system. Immuno blot analysis provided evidence that the histidine kinase Slr1759 is associated with the cytoplasmic membrane. The gene slr1759 is part of an operon together with slr1760, encoding a response regulator. A comparative investigation was performed on Synechocystis sp. strain PCC 6803 wild type (WT) and an insertionally inactivated slr1759-mutant (Hik14) which also lacks the transcript for the response regulator Slr1760. The mutant Hik14 grew significantly slower than WT in the early growth phase, when both were inoculated with a low cell density into BG11 medium without additional buffer and when aerated with air enriched with 2% CO 2 . Since the aeration with CO 2 -enriched air results in a decrease of the pH value in the medium, the growth experiments indicated that Hik14 is not able to adjust its metabolic activities as rapidly as WT to compensate for a larger decrease of the pH value in the medium. No significant differences in growth between Hik14 and WT were observed when cells were inoculated with a higher cell density in BG11 medium or when the BG11 medium contained 50 mm Epps-NaOH, pH 7.5, to prevent the pH drop. This Hik14 phenotype has so far only been seen under the above defined growth condition. Results of photosynthetic activity measurements as well as Northern blot-, immuno blot-, and metabolite analyses suggest that the two-component system Slr1759/Slr1760 has a function in the coordination of several metabolic activities which is in good agreement with the complex domain structure of Slr1759. The direct targets of this two-component system have so far not been identified.

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Isolation, partial characterization and localization of a dihydrolipoamide dehydrogenase from the cyanobacterium Synechocystis PCC 6803

Cited by 30 publications

References 42 publications

Novelty of the Pyruvate Metabolic Enzyme Dihydrolipoamide Dehydrogenasein Spermatozoa

Novelty of the Pyruvate Metabolic Enzyme Dihydrolipoamide Dehydrogenasein Spermatozoa

Induction of male sterility in plants by metabolic engineering of the carbohydrate supply

Physiological and Molecular Characterization of a Synechocystis sp. PCC 6803 Mutant Lacking Histidine Kinase Slr1759 and Response Regulator Slr1760

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