Marchesini Norma, Ruiz Felix A, Vieira Mauricio, Docampo Roberto
Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802, USA.
J Biol Chem. 2002 Mar 8;277(10):8146-53. doi: 10.1074/jbc.M111130200. Epub 2001 Dec 17.
The mass-dense granules of Dictyostelium discoideum were shown to contain large amounts of phosphorus, magnesium, and calcium, as determined by x-ray microanalysis, either in situ or when purified using iodixanol gradient centrifugation. The high phosphorus content was due to the presence of pyrophosphate and polyphosphate, which were also present in the contractile vacuoles. Both organelles also possessed a vacuolar H(+)-ATPase, an H(+)-pyrophosphatase, and a Ca(2+)-ATPase, as determined by biochemical methods or by immunofluorescence microscopy. The H(+)-pyrophosphatase activity of isolated mass-dense granules was stimulated by potassium ions and inhibited by the pyrophosphate analogs aminomethylenediphosphonate and imidodiphosphate and by KF and N-ethylmaleimide in a dose-dependent manner. The mass-dense granules and the contractile vacuole appeared to contact each other when the cells were submitted to hyposmotic stress. Acetazolamide inhibited the carbonic anhydrase activity of the contractile vacuoles and prolonged their contraction cycle in a dose-dependent manner. Similar effects were observed with the anion exchanger inhibitor 4,4' -diisothiocyanatodihydrostilbene-2, 2' -disulfonic acid and the vacuolar H(+)-ATPase inhibitor bafilomycin A(1). Together, these results suggest that the mass-dense granules of D. discoideum are homologous to the acidocalcisomes described in protozoan parasites and are linked to the function of the contractile vacuole.
通过X射线微分析原位测定,或在使用碘克沙醇梯度离心法纯化后测定,发现盘基网柄菌的质量密集颗粒含有大量的磷、镁和钙。高磷含量是由于焦磷酸和多聚磷酸的存在,它们也存在于收缩泡中。通过生化方法或免疫荧光显微镜测定,这两种细胞器还都具有液泡H(+) -ATP酶、H(+) -焦磷酸酶和Ca(2+) -ATP酶。分离出的质量密集颗粒的H(+) -焦磷酸酶活性受到钾离子的刺激,并受到焦磷酸类似物氨基亚甲基二膦酸盐和亚氨基二膦酸盐以及KF和N -乙基马来酰亚胺的剂量依赖性抑制。当细胞受到低渗胁迫时,质量密集颗粒和收缩泡似乎相互接触。乙酰唑胺抑制收缩泡的碳酸酐酶活性,并以剂量依赖性方式延长其收缩周期。用阴离子交换抑制剂4,4' -二异硫氰酸二氢芪-2,2' -二磺酸和液泡H(+) -ATP酶抑制剂巴弗洛霉素A(1)也观察到了类似的效果。这些结果共同表明,盘基网柄菌的质量密集颗粒与原生动物寄生虫中描述的酸性钙小体同源,并与收缩泡的功能相关。