Abstract
The glycogen phosphorylase-2 (GP2) activity that appears during the cell differentiation of Dictyostelium was purified to homogeneity. The molecular weight of the nondenatured enzyme was 200,000 as determined by Sephacryl S-300 gel filtration and was 107,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native enzyme consists of two similar subunits. The intact protein was digested with trypsin and protease V8, and the resulting peptides were purified by microbore high pressure liquid chromatography. The peptides were sequenced, and oligonucleotides were constructed for polymerase chain reaction amplification of the GP2 gene from Dictyostelium genomic DNA template. The resulting polymerase chain reaction products were sequenced directly and were confirmed to encode portions of the GP2 gene. These fragments were used to probe a partial EcoRI genomic library for the remainder of the GP2 gene. The nucleotide sequence of the GP2-selected clones revealed an open reading frame of 2975 base pairs that was interrupted by two introns of 109 and 105 base pairs, respectively. The open reading frame encoded a protein of 992 amino acids with a calculated molecular mass of 112,500 Da and an isoelectric point of 6.4. An unusual sequence within the second exon of GP2, in which the triplet CAA was repeated 11 times, resulted in 11 in-frame glutamine residues of a possible 15 amino acids coded for by this region. The CAA repeat was transcribed, as shown by the sequence of cDNA. Comparison of the amino acid sequence of Dictyostelium GP2 to the phosphorylases from other organisms revealed that the Dictyostelium protein was 50 and 44% identical to yeast and rabbit muscle phosphorylases, respectively. Northern blot analysis showed that GP2 mRNA was absent in amebas and the early stages of development, reached a maximum level of expression at the slug stage, and then decreased in the terminal stages of development. Comparison of the mRNA expression with the appearance of GP2 enzyme protein and enzyme activity revealed that gp2 mRNA and a 113-kDa GP2 enzyme peptide were expressed concurrently at 10 h of development. However, enzyme activity did not appear until 18 h, coincident with a decrease in the level of the 113-kDa peptide and a corresponding increase in the amount of a 106-kDa GP2 peptide. Addition of cAMP to aggregation-competent cells in liquid culture resulted in the induction of GP2 mRNA, GP2 protein, and GP2 enzyme activity.
Highlights
The glycogen phosphorylase-2(GP2)activitythat petent cells in liquid culture resulted in the induction appears during the cell differentiation of Dictyoste- of GP2 mRNA, GP2 protein, and GPen2zyme activity
107,000on sodium dodecyl sulfate-polyacrylamidegel Cell differentiationin Dictyostelium proceeds through a electrophoresis, suggestingthat the native enzycmone- series of morphological changes inwhich single-celled amebas sists of two similar subunits
The nucleotide sequenceof the GP2-selected components of both stalk andspore cells [5, 6]. Because this clones revealed an open reading frame of 2975 base conversion of glycogen to cellulose is so closely coupled to cell pairs that was interruptedby two intronsof 109 and differentiationinthisorganism,the glycogen degradative
Summary
Described previously [11]; 1 unit of activity is the amountof enzyme catalyzing the synthesis of 1pmol of NADPH/min a t 23 "C. Cloning and Nucleic Acid Sequenciw-From the results of peptide purification by QMA, hydroxylapatite, and Sephacryl S-300 sequencing of protease-digested GP2, we found several sequencesthat were similar to those of rabbit muscle [17] and yeast [18]glycogen phosphorylases. Additional fragments of theGP2 coding region and flanking sequences were obtained by inverted PCR [20] anbdy partial EcoRI digestion of genomic DNA. These regions were 5' and 3' to the original PCR-amplified region as well as overlapping with it. Glycogen Phosphorylase-2 Gene in Ilictyostelium phy, removed two minor contaminantsfrom the hydroxylapatite eluate and yielded a homogeneous peptide as shown in
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