Cai Houjian, Hauser Melinda, Naider Fred, Becker Jeffrey M
Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, USA.
Department of Microbiology, University of Tennessee, Knoxville, USA.
Bio Protoc. 2016 Nov 20;6(22). doi: 10.21769/BioProtoc.2025.
We describe an assay for determination of toxicity in involving spotting of a toxic peptide on a lawn of yeast cells. This assay may be generalized to determine toxicity of a variety of compounds by substituting a putative toxic compound in place of the peptide. The general protocol may also be used to determine toxicity of any small compound toward another microorganism by replacing with the target microbe and modifying growth conditions accordingly.
Di-/tripeptides are one of the major sources of nitrogen, carbon, and amino acids for all organisms. Synthetic peptides containing a toxic amino acid residue provide an experimental approach to measure peptide transport and/or utilization in . Hydrolysis of internalized peptides by intracellular peptidases or proteases releases the toxic residue leading to an easily detectable zone (halo) of growth arrest on a lawn of cells plated in a Petri plate. For example, upon intracellular hydrolysis the toxic peptide Ala-Eth releases ethionine (Eth), a methionine antagonist which interferes with the incorporation of amino acids into proteins and with the normal methylation of DNA and other methylation pathways, thereby leading to cell death. When spotted onto a lawn of yeast cells, the transported dipeptide Ala-Eth will inhibit growth, and a clear 'halo' will form in the lawn of cells around the region where the Eth-containing toxic peptide is spotted (Figure 1A). The assay described here for determination of peptide toxicity in may be generalized as follows: (1) it may be modified to determine toxicity of any substrate by simply using a putative toxic compound in place of a peptide containing a toxic amino acid, or (2) it may be modified to determine toxicity of a substrate toward any microorganism by replacing in the assay with the target organism. It is a simple, inexpensive and relatively rapid method for determining substrate toxicities as modified for the specific organism and toxic moiety assayed.
我们描述了一种用于测定毒性的试验，该试验涉及将一种有毒肽点样在酵母细胞平板上。通过用一种假定的有毒化合物替代该肽，此试验可推广用于测定多种化合物的毒性。通过用目标微生物替代并相应地修改生长条件，该通用方案也可用于测定任何小分子化合物对另一种微生物的毒性。
二肽/三肽是所有生物体氮、碳和氨基酸的主要来源之一。含有有毒氨基酸残基的合成肽为测量肽在……中的转运和/或利用提供了一种实验方法。内化的肽被细胞内肽酶或蛋白酶水解会释放有毒残基，从而在培养皿中铺板的细胞平板上形成易于检测到的生长停滞区（晕圈）。例如，在细胞内水解后，有毒肽丙氨酸 - 乙硫氨酸（Ala - Eth）会释放出乙硫氨酸（Eth），乙硫氨酸是一种蛋氨酸拮抗剂，它会干扰氨基酸掺入蛋白质以及DNA的正常甲基化和其他甲基化途径，从而导致细胞死亡。当点样在酵母细胞平板上时，转运的二肽丙氨酸 - 乙硫氨酸会抑制生长，并且在点样含乙硫氨酸的有毒肽区域周围的细胞平板上会形成清晰的“晕圈”（图1A）。此处描述的用于测定……中肽毒性的试验可概括如下：（1）通过简单地用一种假定的有毒化合物替代含有毒氨基酸的肽，可对其进行修改以测定任何底物的毒性，或者（2）通过在试验中用目标生物体替代……，可对其进行修改以测定底物对任何微生物的毒性。这是一种简单、廉价且相对快速的方法，可针对特定生物体和所检测的有毒部分进行修改以测定底物毒性。
