Purification of and production of an antibody against a 63,000 Mr stimulus-sensitive phosphoprotein in Paramecium.

Murtaugh, Timothy J.; Gilligan, Diana M.; Satir, Birgit H.

doi:10.1016/s0021-9258(18)47789-7

Cited by 20 publications

(12 citation statements)

References 16 publications

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“…We also observed inactivation by Tris/HCl or by Polybuffer (chromatofocusing), in accordance with previous literature on yeast [35] and mammalian PGM [96]. However, during the isolation of 'parafusin' by DEAE-cellulose chromatography, gel filtration and chromatofocusing, either Tris/HCl or Polybuffer was used in the work with Paramecium mentioned [3,7]. (iii) 'Parafusin' is an acceptor for Glc-1-P transferred from UDP-glucose by Glc-phosphotransferase [5][6][7].…”

Section: Functional Implicationssupporting

confidence: 91%

“…PP63 ('parafusin') exists in several isoforms, with pl values between 5. 75 and 6.05 [1,3]. All the forms of different pI are extensively de-phosphorylated during synchronous exocytosis, including the most intensively phosphorylated form, of pI 5.95, which is also most intensely rephosphorylated after exocytosis [1].…”

Section: Introductionmentioning

confidence: 99%

“…All the forms of different pI are extensively de-phosphorylated during synchronous exocytosis, including the most intensively phosphorylated form, of pI 5.95, which is also most intensely rephosphorylated after exocytosis [1]. They all are sensitive to alkaline, but insensitive to acid, treatment, thus indicating serine/threonine phosphorylation [1,3]. We have evi-dence that PP63 isolated after 32p labelling in vivo is a substrate for protein serine/threonine phosphatases and kinases (R. Kissmehl, T. Treptau, H. W. Hofer and H. Plattner, unpublished work).…”

Section: Introductionmentioning

confidence: 99%

“…We have evi-dence that PP63 isolated after 32p labelling in vivo is a substrate for protein serine/threonine phosphatases and kinases (R. Kissmehl, T. Treptau, H. W. Hofer and H. Plattner, unpublished work). Isolation of PP63 [3] and its recent molecular cloning revealed several serine-phosphorylation sites [4]. On the other hand, it was reported that PP63 could be phosphoglycosylated in vitro by addition of glucose 1-phosphate (Glc-l-P), catalysed by an aGlc-1-P phosphotransferase utilizing UDPglucose, to a short chain of mannose residues 0-linked to a serine residue [5][6][7].…”

Section: Introductionmentioning

confidence: 99%

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A 63 kDa phosphoprotein undergoing rapid dephosphorylation during exocytosis in Paramecium cells shares biochemical characteristics with phosphoglucomutase

Treptau

Kissmehl

Wissmann

et al. 1995

Biochemical Journal

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We have enriched phosphoglucomutase (PGM; EC 5.4.2.2) approximately 20-fold from Paramecium tetraurelia cells by combined fractional precipitation with (NH4)2SO4, gel filtration and anion-exchange chromatography yielding two PGM peaks. Several parameters affecting PGM enzymic activity, molecular mass and pI were determined. Phosphorylation studies were done with isolated endogenous protein kinases. Like the 63 kDa phosphoprotein PP63, which is dephosphorylated within 80 ms during synchronous trichocyst exocytosis [Höhne-Zell, Knoll, Riedel-Gras, Hofer and Plattner (1992) Biochem. J. 286, 843-849], PGM has a molecular mass of 63 kDa and forms of identical pI. Since mammalian PGM activity depends on the presence of glucose 1,6-bisphosphate (Glc-1,6-P2) (which is lost during anion-exchange chromatography), we analysed this aspect with Paramecium PGM. In this case PGM activity was shown not to be lost, due to p-nitrophenyl phosphate-detectable phosphatase(s) (which we have separated from PGM), but also due to loss of Glc-1,6-P2. Like PGM from various vertebrate species, PGM activity from Paramecium can be fully re-established by addition of Glc-1,6-P2 at 10 nM, and it is also stimulated by bivalent cations and insensitive to chelating or thiol reagents. The PGM which we have isolated can be phosphorylated by endogenous cyclic-GMP-dependent protein kinase or by endogenous casein kinase. This results in three phosphorylated bands of identical molecular mass and pI values, as we have shown to occur with PP63 after phosphorylation in vivo (forms with pI 6.05, 5.95, 5.85). In ELISA, antibodies raised against PGM from rabbit skeletal muscle were reactive not only with original PGM but also with PGM fractions from Paramecium. Therefore, PGM and PP63 seem to be identical with regard to widely different parameters, i.e. co-elution by chromatography, molecular mass, phosphorylation by the two protein kinases tested, pI values of isoforms, and immuno-binding. Recent claims that PP63 ('parafusin') would not be identical with PGM specifically in Paramecium are critically evaluated. Since some glycolytic enzymes are discussed as being associated with the Ca(2+)-release channel in muscle sarcoplasmic reticulum, and since sub-plasmalemmal Ca2+ stores in Paramecium closely resemble sarcoplasmic reticulum, a possible function of PP63/PGM in exocytosis regulation is discussed, particularly since dephosphorylation strictly parallels exocytosis.

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Section: Functional Implicationssupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

A 63 kDa phosphoprotein undergoing rapid dephosphorylation during exocytosis in Paramecium cells shares biochemical characteristics with phosphoglucomutase

Treptau

Kissmehl

Wissmann

et al. 1995

Biochemical Journal

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“…This ciliated protozoan is an attractive model system' for the study of regulated secretion since massive and synchronous exocytosis can be initiated with an appropriate stimulus. In paramecium, parafusin, a cytoplasmic phosphoprotein of 63 kD, is the major protein to undergo dephosphorylation during exocytosis (Gilligan and Satir, 1982;Murtaugh et al, 1987;Satir et al, 1988). It appears to be rephosphorylated within seconds thereafter (Zieseniss and Plattner, 1985).…”

mentioning

confidence: 99%

Parafusin, an exocytic-sensitive phosphoprotein, is the primary acceptor for the glucosylphosphotransferase in Paramecium tetraurelia and rat liver.

Satir

Srisomsap

Reichman

et al. 1990

The Journal of Cell Biology

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Abstract. Parafusin, the major protein in Paramecium tetraurelia to undergo dephosphorylation in response to secretory stimuli, appears to be the primary acceptor for the glucosylphosphotransferase in this species based on five independent criteria: identical molecular size of 63 kD; identical isoelectric points in the phosphorylated state of pH 5.8 and 6.2; identical behavior in reverse-phase chromatography; immunological cross-reactivity with an affinity-purified anti-parafusin antibody; the presence of a phosphorylated sugar after acid hydrolysis. It appears likely that the dephosphorylation observed with secretion reflects the removal of otGlc-l-P from parafusin's oligosaccharides and is consistent, therefore, with a regulatory role for this cytoplasmic glycosylation event. The glucosylphosphotransferase acceptor in rat liver is also immunoprecipitated by the anti-parafusin antibody and is very similar in physical characteristics to the paramecium protein. This conservation suggests a role for parafusin in mammalian exocytosis as well, at a step common to both the regulated and constitutive secretory pathways.

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Aspects of signal transduction in stimulus exocytosis‐coupling in Paramecium

Satir

Busch

Vuoso

et al. 1988

J of Cellular Biochemistry

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This paper deals with the detailed mechanisms of signal transduction that lead to exocytosis during regulative secretion induced by specific secretagogues in a eukaryotic cell, Paramecium tetraurelia. There are at least three cellular compartments involved in the process: I) the plasma membrane, which contains secretagogue receptors and other transmembrane proteins, II) the cytoplasms, particularly in the region between the cell and secretory vesicle membranes, where molecules may influence interactions of the membranes, and III) the secretory vesicle itself. The ciliated protozoan Paramecium tetraurelia is very well suited for the study of signal transduction events associated with exocytosis because this eukaryotic cell contains thousands of docked secretory vesicles (trichocysts) below the cell membrane which can be induced to release synchronously when triggered with secretagogue. This ensures a high signal-to-noise ratio for events associated with this process. Upon release the trichocyst membrane fuses with the cell membrane and the trichocyst content undergoes a Ca2+-dependent irreversible expansion. Secretory mutants are available which are blocked at different points in the signal transduction pathway. Aspects of the three components mentioned above that will be discussed here include a) the properties of the vesicle content, its pH, and its membrane; b) the role of phosphorylation/dephosphorylation of a cytosolic 63-kilodalton (kDa)Mr protein in membrane fusion; and c) how influx of extracellular Ca2+ required for exocytosis may take place via exocytic Ca2+ channels which may be associated with specific membrane microdomains (fusion rosettes).

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Purification of and production of an antibody against a 63,000 Mr stimulus-sensitive phosphoprotein in Paramecium.

Cited by 20 publications

References 16 publications

A 63 kDa phosphoprotein undergoing rapid dephosphorylation during exocytosis in Paramecium cells shares biochemical characteristics with phosphoglucomutase

A 63 kDa phosphoprotein undergoing rapid dephosphorylation during exocytosis in Paramecium cells shares biochemical characteristics with phosphoglucomutase

Parafusin, an exocytic-sensitive phosphoprotein, is the primary acceptor for the glucosylphosphotransferase in Paramecium tetraurelia and rat liver.

Aspects of signal transduction in stimulus exocytosis‐coupling in Paramecium

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