Inorganic pyrophosphatase-based detection systems. II. Detection and quantification of cell lysis and cell-lysing activity.
A novel technique, useful for detection of cell lysis and cell-lysing activity, has been developed. The method can be used for detection of cell lysis, both induced and natural, in all types of cells. The technique can be used for detection and quantification of all types of cell-lysing activities, e.g., cell wall hydrolases, toxicants, phospholipases, and antibiotics. The method relies on the detection, by a very sensitive enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA; P. Nyrén and A. Lundin (1985) Anal. Biochem. 151, 504-509) of an enzyme, inorganic pyrophosphatase, which is constitutively expressed in all cells. The fraction of lysed cells in a sample can be assessed by determining the activity in the absence and in the presence of total lysing activity. The technique was used for determination of the effect of storage conditions on the intactness of two different cells, Micrococcus luteticus and Saccharomyces cerevisiae. In a model system, the approach was also used for detection of cell-lysing activity. The activity of a cell wall hydrolase (lysozyme) and a surfactant (Triton X-100) was quantified. M. luteticus cells were incubated with a specific buffer containing the lysing activity and inorganic pyrophosphate. The amount of unhydrolyzed PPi was determined by the ELIDA. The amount of PPi hydrolyzed was proportional to the amount of lysozyme present. The sensitivity of the assay was dependent on several factors, such as amount of cells used, incubation time, and incubation temperature. Lysozyme at concentrations below 5 ng/ml (< 50 pg) could be detected. The possibility of using the approach for detection of other types of cell-lysing activities is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)
Nyrén
P
P
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Edwin
V
V
D001433:Bacteriolysis / Q000502:physiology*; D016923:Cell Death / Q000502:physiology; D043564:Inorganic Pyrophosphatase; D008837:Micrococcus / Q000201:enzymology; D009113:Muramidase / Q000032:analysis; D017830:Octoxynol; D011755:Pyrophosphatases / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012680:Sensitivity and Specificity
D017830:Octoxynol; D009113:Muramidase; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase