Sethuraman A, Rao N N, Kornberg A
Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307, USA.
Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8542-7. doi: 10.1073/pnas.151269398. Epub 2001 Jul 10.
Endopolyphosphatases (Ppn1) from yeast and animal cells hydrolyze inorganic polyphosphate (poly P) chains of many hundreds of phosphate residues into shorter lengths. The limit digest consists predominantly of chains of 60 (P(60)) and 3 (P(3)) P(i) residues. Ppn1 of Saccharomyces cerevisiae, a homodimer of 35-kDa subunits (about 352-aa) is of vacuolar origin and requires the protease activation of a 75-kDa (674-aa) precursor polypeptide. The Ppn1 gene (PPN1) now has been cloned, sequenced, overexpressed, and deleted. That PPN1 encodes Ppn1 was verified by a 25-fold increase in Ppn1 when overexpressed under a GAL promoter and also by several peptide sequences that match exactly with sequences in a yeast genome ORF, the mutation of which abolishes Ppn1 activity. Null mutants in Ppn1 accumulate long-chain poly P and are defective in growth in minimal media. A double mutant of PPN1 and PPX1 (the gene encoding a potent exopolyphosphatase) loses viability rapidly in stationary phase. Whether this loss is a result of the excess of long-chain poly P or to the lack of shorter chains (i.e., poly P(60) and P(3)) is unknown. Overexpression of the processed form of Ppn1 should provide a unique and powerful reagent to analyze poly P when the chain termini are unavailable to the actions of polyPase and poly P kinase.
来自酵母和动物细胞的内多聚磷酸酶（Ppn1）可将数百个磷酸残基的无机多聚磷酸（多聚P）链水解成更短的长度。极限消化产物主要由60个（P(60)）和3个（P(3)）磷酸残基的链组成。酿酒酵母的Ppn1是一种由35 kDa亚基（约352个氨基酸）组成的同型二聚体，起源于液泡，需要75 kDa（674个氨基酸）前体多肽的蛋白酶激活。Ppn1基因（PPN1）现已被克隆、测序、过表达和缺失。当在GAL启动子下过表达时，Ppn1增加了25倍，以及几个与酵母基因组开放阅读框中的序列完全匹配的肽序列，其突变消除了Ppn1活性，从而证实PPN1编码Ppn1。Ppn1的缺失突变体积累长链多聚P，并且在基本培养基中生长有缺陷。PPN1和PPX1（编码一种强效外多聚磷酸酶的基因）的双突变体在稳定期迅速丧失活力。这种丧失是由于长链多聚P过量还是由于短链（即多聚P(60)和P(3)）缺乏尚不清楚。当多聚磷酸酶和多聚P激酶无法作用于链末端时，Ppn1加工形式的过表达应该为分析多聚P提供一种独特而强大的试剂。
