Rutherford C L, Naranan V, Brickey D A, Sucic J F, Rogers P V, Selmin O
Biology Dept., Virginia Tech University, Blacksburg 24061.
Dev Genet. 1988;9(4-5):469-81. doi: 10.1002/dvg.1020090424.
A key step in the cellular differentiation of Dictyostelium is the degradation of glycogen to provide the precursors for synthesis of the structural end products of development. We have found that the enzyme that initiates this degradative pathway, glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase; EC 2.4.1.1), is developmentally regulated and exists as two forms. During the time course of development, a previously undescribed activity, the "b" form, decreases, while that of the "a" form increases. The "b" form is inactive unless 5'AMP is included in the reaction mixture. The two forms differ in their elution from DE52 cellulose, affinity constants, thermal stability, affinity for 5'AMP Sepharose, subunit molecular weight, and peptide maps. In crude extracts, anti-a antiserum stains a 104-kD protein that is associated with phosphorylase "a" activity and appears late in development, while anti-b antiserum stains a 92-kD protein that is associated with phosphorylase "b" activity and is present throughout development. We have also demonstrated in vitro phosphorylation of the "b" form by an endogenous protein kinase and a corresponding loss of 5'AMP dependence. If intact cells were exposed to exogenous cAMP, "b" activity decreased and was replaced by "a" activity, as well as the 104-kD protein band on SDS-PAGE. In order to determine if the two forms of the enzyme are different gene products, we screened lambda gt11 expression libraries with antibodies against the purified "a" and "b" forms. Three clones were found to be overlapping by Southern analysis. A yeast glycogen phosphorylase cDNA clone (gpy) and a human muscle glycogen phosphorylase clone (HM-11) cross-hybridized with the Dictyostelium inserts, and gpy shared a few common restriction fragments with the Dictyostelium clones on genomic blots. Northern analysis of Dictyostelium total RNA showed that the Dictyostelium inserts and gpy recognize an mRNA of 3.2 kb, while on poly A-enriched RNA, the yeast clone detects preferentially a 3.6-kb message.
盘基网柄菌细胞分化的关键步骤是糖原降解，以提供发育结构终产物合成的前体。我们发现启动这条降解途径的酶，糖原磷酸化酶（1,4-α-D-葡聚糖：正磷酸α-葡糖基转移酶；EC 2.4.1.1），受发育调控且以两种形式存在。在发育过程中，一种先前未描述的活性，即“b”形式，降低，而“a”形式的活性增加。“b”形式无活性，除非反应混合物中包含5'-AMP。这两种形式在从DE52纤维素上的洗脱、亲和常数、热稳定性、对5'-AMP琼脂糖的亲和力、亚基分子量和肽图谱方面存在差异。在粗提物中，抗-a抗血清可使一种与磷酸化酶“a”活性相关且在发育后期出现的104-kD蛋白显色，而抗-b抗血清可使一种与磷酸化酶“b”活性相关且在整个发育过程中都存在的92-kD蛋白显色。我们还证明了内源性蛋白激酶对“b”形式的体外磷酸化以及相应的5'-AMP依赖性丧失。如果完整细胞暴露于外源性cAMP，“b”活性降低并被“a”活性取代，同时SDS-PAGE上的104-kD蛋白条带也出现。为了确定该酶的两种形式是否为不同的基因产物，我们用针对纯化的“a”和“b”形式的抗体筛选了λgt11表达文库。通过Southern分析发现三个克隆相互重叠。一个酵母糖原磷酸化酶cDNA克隆（gpy）和一个人肌肉糖原磷酸化酶克隆（HM-11）与盘基网柄菌插入片段杂交，并且gpy在基因组印迹上与盘基网柄菌克隆共享一些共同的限制性片段。对盘基网柄菌总RNA的Northern分析表明，盘基网柄菌插入片段和gpy识别一个3.2 kb的mRNA，而在富含多聚A的RNA上，酵母克隆优先检测到一个3.6 kb的信息。
