Incorporation of [32P]orthophosphate into inorganic polyphosphates by human granulocytes and other human cell types.

Abstract

Vol. 269, p. 9480 Previously, we reported that human granulocytic leukocytes incorporated [32P]orthophosphate into inorganic polyphosphates (i.e. poly(P)), but that the magnitude of incorporation varied greatly in different cell preparations(1Cowling R.T. Birnboim H.C. J. Biol. Chem. 1994; 269: 9480-9485Abstract Full Text PDF PubMed Google Scholar). Further analyses have now revealed that the poly(P) signal likely originated from a low level contamination of Pseudomonas fluorescens and not from the granulocytes. Poly(P) has not been demonstrated previously in this microorganism. Radiolabeled cultures of P. fluorescens contain [32P]poly(P), as verified by its: (i) insolubility in barium acetate, (ii) lability to acid-catalyzed hydrolysis, (iii) hydrolysis to 32Pi by a yeast exopolyphosphatase(2Wurst H. Kornberg A. J. Biol. Chem. 1994; 269: 10996-11001Abstract Full Text PDF PubMed Google Scholar), and (iv) formation of [32P]trimetaphosphate. Stationary phase P. fluorescens contain ~0.5 μg of poly(P)/109 bacteria, with a length of at least 100 phosphate residues. When exposed to high specific activity 32Pi in nutrient-limited solution, 1 bacterium can incorporate about the same amount of radiolabel as 1000 granulocytes, ≥20% of which is [32P]poly(P). Solutions used to isolate and radiolabel the granulocytes were the sources of contamination, but routine microscopic examination failed to detect such low numbers of bacteria. These solutions were filter-sterilized at time of preparation and were stored at 4°C, but they were not handled aseptically thereafter. Human granulocytes handled under strictly sterile conditions contain no detectable [32P]poly(P). Our observation may not be unique. Therefore, we wish to alert other investigators to the potential problem of low level microbial contamination when studying poly(P) in mammalian cells. There are many microorganisms that possess the capacity to synthesize poly(P) and that are common laboratory inhabitants (e.g. Pseudomonas and nonfermentative Gram-negative bacteria). In nutrient-limited solutions that are unfavorable to growth (such as those used to isolate subcellular organelles), these microorganisms can exist undetected and can preferentially synthesize poly(P)(3Harold F.M. Bacteriol. Rev. 1966; 30: 772-794Crossref PubMed Google Scholar). We were unable, however, to detect [32P]poly(P) in Acholeplasma laidlawii, a representative mycoplasma found in tissue cultures. We also attempted to detect [32P]poly(P) in cultured human skin fibroblasts by radiolabeling for 24 h in complete media with 950 μCi of 32Pi (specific activity ≈ 120 mCi/mmol). Poly(P) was isolated using procedures similar to those described previously(1Cowling R.T. Birnboim H.C. J. Biol. Chem. 1994; 269: 9480-9485Abstract Full Text PDF PubMed Google Scholar). By heating Ba2+-precipitable cell extracts at 70°C for 70 h, approximately 1 pmol of phosphate/106 cells was converted to trimetaphosphate. We are unsure of the origin of this small amount of trimetaphosphate, since several unknown phosphorylated species were present in the Ba2+ precipitates. Using the highly specific enzymes poly(P) kinase and exopolyphosphatase, Kumble and Kornberg have identified small, but reproducible quantities of poly(P) in mammalian tissues and cultured cells(4Kumble K.D. Kornberg A. J. Biol. Chem. 1995; 270: 5818-5822Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar), but they have not been able to detect [32P]poly(P) by radiolabeling subcellular fractions (K. D. Kumble and A. Kornberg, personal communication). They are confident that their preparations are free of contaminating microorganisms (K. D. Kumble and A. Kornberg, personal communication). As work progresses in this field, it is imperative that investigators remain wary of the problems that we have encountered. Given the apparently tiny quantities of poly(P) in mammalian cells, contamination by very few microbes may present a significant problem.