The relationship between the two forms of glycogen phosphorylase in Dictyostelium discoideum

Abstract

Summary. The cellular slime mold, Dictyostelium disoideum, provides an ideal model system to study eukaryotic cell differentiation. In D. discoideum, glycogen degradation provides precursors for the synthesis of developmentally regulated structural products. The enzyme responsible for glycogen degradation, glycogen phosphorylase, exists in active and inactive forms. The active, or‘a’ form, is independent of 5′adenosine monophosphate (5′AMP) while the inactive, or‘b’ form, is 5′AMP‐dependent. The activity of the‘b’ form predominates early in development, while the activity of the‘a’ form peaks in mid‐late development; their combined specific activities remain constant at any point. Polyclonal antibodies raised to the purified forms of this enzyme showed low cross‐reactivity. The anti‐‘a’ serum reacted with a 104‐kDa protein that was associated with phosphorylase‘a’ activity; the anti‐‘b’ serum reacted with a 92‐kDa protein that was associated with phosphorylase‘b’ activity and weakly cross‐reacted with the 104‐kDa protein. Immunoblots of peptide maps of the purified enzyme forms showed that each antibody was specific for the proteolytic fragments of its respective antigen. We also demonstrated in vitro phosphorylation of the‘b’ form by an endogenous protein kinase. Cyclic AMP perturbation of intact cells caused induction of both phosphorylase‐‘a’ activity and the 104‐kDa protein. Immunotitration data suggested that the‘a’ form accumulates due to de novo protein synthesis, although this result must be interpreted with caution.

Developmental and cAMP-mediated regulation of glycogen phosphorylase 1 in Dictyostelium discoideum

“…Vegetative cells of Dictyostelium discoideum strain AX3 were cultured in HL5 as described previously (Naranan et al, 1988;Rogers et al, 1992). Dictyostelium strain AX3K was used for the reporter gene experiments.…”

“…After transfer, the visualization of gp-1 protein was done as previously described (Naranan et al, 1988), using polyclonal anti-gp-1 antibodies, a protein-A-peroxidase conjugate, and colorimetric antigen detection with 4-chloro-I-naphthol and hydrogen peroxide.…”

“…l), and our laboratory is examining the expression of two developmentally regulated glycogen phosphorylases in Dictyostelium. Glycogen phosphorylase-2 (gp-2) is expressed late in development, and the gp-2 mRNA and protein accumulate concomitantly with increases in gp-2 activity (Naranan et al, 1988;Rutherford et al, 1992). Glycogen phosphorylase-1 (gp-1) exhibits a more complex pattern of regulation.…”

“…Glycogen phosphorylase-1 (gp-1) exhibits a more complex pattern of regulation. gp-1 enzyme activity is maximal in vegetative amoebae, then decreases throughout the subsequent stages of development (Rutherford & Cloutier, 1986;Naranan et al, 1988). Although the gp-1 activity decreases, the level of gp-1 protein is constant during development (Naranan et al, 1988;Rogers et al, 1992).…”

Disruption of glycogen phosphorylase gene expression in Dictyostelium: Evidence for altered glycogen metabolism and developmental coregulation of the gene products

“…The second form of glycogen phosphorylase in Dicryosrelium, gp2, is a 106-kDa protein that is completely independent of 5' AMP for activity and does not appear to be phosphorylated in vivo or in vitro [30,311. Both the gp2 protein and the 3.3-kb gp2 transcript appear at about 8-10 h of development.…”