A purification method and N-glycosylation sites of a 36-cysteine-containing, putative cell/cell adhesion glycoprotein gp64 of the cellular slime mold, Polysphondylium pallidum

We report the identification and purification of an endogenous carbohydrate-containing receptor of pallidin, the cell surface lectin implicated in mediating cell-cell adhesion in the cellular slime mold Polysphondylium pallidum. The receptor is identified in an aqueous extract of crude P. pallidum membranes as a potent inhibitor of the hemagglutination activity of pallidin. The inhibitor is purified to apparent homogeneity by affinity precipitation with pallidin followed by fractionation of the solubilized precipitate on Sepharose 4B. The hemagglutination inhibitor (HAI) is metabolically radiolabeled, indicating that it is a biosynthetic product of the amoebae and not an ingested food substance. The HAI is released into the extracellular medium by living, differentiated amoebae. This release is markedly facilitated by the addition of D-galactose, a specific saccharide that binds to pallidin. Hence, the HAI appears to have an in situ association with pallidin at the cell surface. Exogenously added HAI promotes the agglutination of differentiated amoebae in a gyrated suspension at very low concentrations. The results are consistent with a model of cell-cell adhesion in which the HAI is a multivalent, extracellular aggregation factor that is recognized by pallidin molecules on adjacent cells. The HAI would then be analogues to the aggregation factors identified in marine sponges.

Download Full-text

Purification and Properties of Acid Phosphatase I From the Cellular Slime Mold Polysphondylium pallidum

Canadian Journal of Biochemistry ◽

10.1139/o74-020 ◽

1974 ◽

Vol 52 (2) ◽

pp. 126-136 ◽

Cited By ~ 8

Author(s):

Ilona A. Horgen ◽

Paul A. Horgen ◽

Danton H. O'Day

Keyword(s):

Acid Phosphatase ◽

Gradient Centrifugation ◽

Gel Filtration ◽

Neutral Hydrogen ◽

Slime Mold ◽

Cellular Slime Mold ◽

Ph Optimum ◽

Purification Method ◽

Polysphondylium Pallidum ◽

Cellular Slime

A procedure for the purification of a phosphomonoesterase, designated as acid phosphatase I, from the cellular slime mold Polysphondylium pallidum is described. Ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography are utilized in this purification method. The enzyme was judged to be homogeneous by gel filtration and by acylamide gel electrophoresis. The molecular weight of the enzyme was estimated by gel filtration and density gradient centrifugation to be 150 000 daltons. Acid phosphatase I was shown to be relatively heat stable, and it lost no activity when kept at 4 °C, pH 7.35, for over 30 days. The pH optimum was 3.5, but the enzyme was found to be more stable when kept near neutral hydrogen ion concentrations. P. pallidum acid phosphatase I was most effective using the natural substrates, fructose-1,6-pbosphate, β-glycerolphosphate, and 5′-mononucleotides. Various compounds including known phosphatase inhibitors were tested as to their effect on the activity of the enzyme. The slime-mold acid phosphatase appears in many ways to be a typical acid phosphomonoesterase.

Download Full-text

Ammonia and the induction of microcyst differentiation in wild-type and mutant strains of the cellular slime mold Polysphondylium pallidum

Developmental Biology ◽

10.1016/0012-1606(82)90181-6 ◽

1982 ◽

Vol 92 (2) ◽

pp. 356-364 ◽

Cited By ~ 11

Author(s):

A.H.C. Choi ◽

D.H. O'Day

Keyword(s):

Slime Mold ◽

Cellular Slime Mold ◽

Wild Type ◽

Polysphondylium Pallidum ◽

Mutant Strains ◽

Cellular Slime

Download Full-text

Antisense RNA inactivation of gene expression of a cell-cell adhesion protein (gp64) in the cellular slime mold Polysphondylium pallidum

Journal of Cell Science ◽

10.1242/jcs.109.5.1009 ◽

1996 ◽

Vol 109 (5) ◽

pp. 1009-1016

Author(s):

S. Funamoto ◽

H. Ochiai

Keyword(s):

Gene Expression ◽

Cell Adhesion ◽

Antisense Rna ◽

Resistant Cell ◽

Cell Contact ◽

Cellular Slime Mold ◽

Adhesion Protein ◽

Cell Adhesion Protein ◽

Polysphondylium Pallidum ◽

Cell Cell

The gp64 protein of Polysphondylium pallidum has been shown to mediate EDTA-stable cell-cell adhesion. To explore the functional role of gp64, we made an antisense RNA expression construct designed to prevent the gene expression of gp64; the construct was introduced into P. pallidum cells and the transformants were characterised. The antisense RNA-expressing clone L3mc2 which had just been harvested at the growth phase tended to re-form in aggregates smaller in size than did the parental cells in either the presence or absence of 10 mM EDTA. In contrast, 6.5-hour starved L3mc2 cells remained considerably dissociated from each other after 5 minutes gyrating, although aggregation gradually increased by 50% during a further 55 minutes gyrating in the presence of 10 mM EDTA. Correspondingly, L3mc2 lacked specifically the cell-cell adhesion protein, gp64. We therefore conclude that the gp64 protein is involved in forming the EDTA-resistant cell-cell contact. In spite of the absence of gp64, L3mc2 exhibited normal developmental processes, a fact which demonstrates that another cell-cell adhesion system exists in the development of Polysphondylium. This is the first report in which an antisense RNA technique was successfully applied to Polysphondylium.

Download Full-text

Induction of encystment of Polysphondylium pallidum amoeba

Canadian Journal of Microbiology ◽

10.1139/m77-076 ◽

1977 ◽

Vol 23 (5) ◽

pp. 518-521 ◽

Cited By ~ 3

Author(s):

Joseph Lonski ◽

Nicholas Pesut

Keyword(s):

Slime Mold ◽

Cellular Slime Mold ◽

Polysphondylium Pallidum ◽

Cellular Slime

The induction of microcyst formation could be triggered in washed amoebae of the cellular slime mold Polysphondylium pallidum (strain-2) by the addition of 2 mM ethionine. Methionine at a ratio of 2:1 with ethionine would inhibit microcyst induction by ethionine. The involvement of polyamines in morphogenesis was also shown. Putrescine (0.02 to 0.1 M) induced the formation of microcysts, whereas spermidine (2 to 4 mM) was capable of causing a fourfold reduction in 0.05 M putrescine-induced microcysts but incapable of inhibiting microcyst induction by 0.08 M putrescine. Glycerol (0.5 M or 0.4 M) was also found to be an effective inducer of microcysts.

Download Full-text