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粗糙脉孢菌野生型和Rec样突变体中表达的主要细胞内碱性脱氧核糖核酸酶活性。

The major intracellular alkaline deoxyribonuclease activities expressed in wild-type and Rec-like mutants of Neurospora crassa.

作者信息

Chow T Y, Fraser M J

出版信息

Can J Biochem. 1979 Jun;57(6):889-901. doi: 10.1139/o79-109.

Abstract

Over 95% of the deoxyribonuclease (DNase) activity of log-phase mycelia of Neurospora crassa is expressed as single-strand (ss) specific endonucleolytic activity. This activity is associated with three nucleases (D1, D2, and D3) which after partial purification from extracts, express activity with double-strand (ds) DNA as well. All three enzymes also degrade RNA at approximately the same rates that they degrade ss-DNA. D3 has been identified as endoexonuclease, an enzyme previously shown to have endonuclease activity with ss-DNA and RNA and exonuclease activity with ds-DNA, both of which are inhibited by ATP. D3 is inhibited by ATP, is relatively resistant to p-hydroxymercuribenzoate (PHMB), and sediments with an apparent molecular weight of 75 000. D2 has the properties of the previously described mitochondrial nuclease. It is a relatively unstable Mg2+-dependent endonuclease with no appreciable strand specificity for DNA. In addition, it is not inhibited by ATP and is strongly inhibited by PHMB and by the ethylenediamine tetraacetic acid (EDTA). It also sediments with an apparent molecular weight of 75,000. The properties of D1 are quite variable from one preparation to another. Freshly isolated D1 sediments with an apparent molecular weight of 180 000. It often shows some inhibition by ATP, but is relatively resistant to both PHMB and EDTA. However, on 'ageing,' the properties of D1 gradually convert to those of D2 with concomitant decrease in molecular weight, loss of inhibition by ATP, and increase in sensitivities to PHMB and EDTA. The results indicate that D1 is very likely a second form of the mitochondrial enzyme. Evidence was obtained for the presence of protein inhibitor(s) in crude extracts which may account for the masking of the ds-DNase activities of these enzymes in extracts. Two Rec-like mutants of Neurospora (uvs-3, and nuh-4) are deficient mainly inexpressed levels of D3, the endo-exonuclease. However, the levels of inactive endo-exonuclease precursor in these two mutants are higher than in the wild type. There may, therefore, be some defect in the conversion of precursor to active enzyme in these two mutants. Another mutant, which is not sensitive to mutagens relative to the wild (nuh-3), has depressed levels of both endo-exonuclease and the mitochondrial enzyme. Nuh-3 has some defect in the conversion of D1 to D2. Proteinases probably play some role in vivo in these enzyme conversions.

摘要

粗糙脉孢菌对数期菌丝体中超过95%的脱氧核糖核酸酶(DNase)活性表现为单链(ss)特异性内切核酸酶活性。这种活性与三种核酸酶(D1、D2和D3)相关,从提取物中部分纯化后,它们也能对双链(ds)DNA表现出活性。这三种酶降解RNA的速率与降解ss-DNA的速率大致相同。D3已被鉴定为核酸外切内切酶,该酶先前已被证明对ss-DNA和RNA具有内切酶活性,对ds-DNA具有外切酶活性,这两种活性均受ATP抑制。D3受ATP抑制,对对羟基汞苯甲酸(PHMB)相对抗性较强,沉降时表观分子量为75000。D2具有先前描述的线粒体核酸酶的特性。它是一种相对不稳定的依赖Mg2+ 的内切酶,对DNA没有明显的链特异性。此外,它不受ATP抑制,但受PHMB和乙二胺四乙酸(EDTA)强烈抑制。它沉降时表观分子量也为75000。D1的特性在不同制备物之间变化很大。新分离的D1沉降时表观分子量为180000。它常常表现出受ATP一定程度的抑制,但对PHMB和EDTA相对抗性较强。然而,随着“老化”过程进行,D1的特性逐渐转变为D2的特性,同时分子量减小,ATP抑制作用丧失,对PHMB和EDTA的敏感性增加。结果表明D1很可能是线粒体酶的第二种形式。有证据表明粗提取物中存在蛋白质抑制剂,这可能解释了提取物中这些酶的ds-DNase活性被掩盖现象发生的原因。粗糙脉孢菌的两个Rec样突变体(uvs-3和nuh-4)主要在内切外切核酸酶D3的表达水平上存在缺陷。然而,这两个突变体中无活性的内切外切核酸酶前体水平高于野生型。因此,这两个突变体在前体转化为活性酶的过程中可能存在某些缺陷。另一个相对于野生型对诱变剂不敏感的突变体(nuh-3),其内切外切核酸酶和线粒体酶的水平均降低。Nuh-3在D1向D2的转化过程中存在某些缺陷。蛋白酶可能在这些酶的体内转化过程中发挥了一定作用。

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