Fluctuations in Proline and Other Free Amino Acids during the Mitotic Cycle of the Myxomycete Physarum polycephalum

Abstract

1. During synchronous growth of the acellular slime mould Physarum polycephalum the free amino pool had two maxima, one of 650 units [nmol/plasmodium dry weight (mg)] at metaphase and the other of 780 units in mid G2 with minima of 550 units before and after mitosis. 2.Proline formed 20 -25 % of the total pool with aspartic acid, glutamic acid, threonine, valine, leucine, lysine and arginine making up 55 % of the pool.3. The fluctuation of proline during the mitotic cycle was quite different from that of the other amino acids and was transiently very low during telophase.A knowledge of the composition, size and intracellular distribution of pools of free amino acids is of importance in understanding the control of enzyme repression and synthesis and for the critical evaluation of pulse labeling experiments. One area where such knowledge is particularly needed is in the study of the cell cycle [l]. Distinct changes in free amino acids were found during the cell cycle of the alga Chlorella pyrenoidosa [2] whereas in the fission yeast Schizosaccharomyces pombe [3,4] and in HeLa cells [5] virtually no changes were detected.The macroplasmodium of Physarum polycephalum is a syncytium in which all the nuclei undergo a natural synchronous mitosis [6]. As a plasmodium may contain lo8 nuclei this is an ideal system for studying the biochemistry of the processes of nuclear division and growth. After nuclear division DNA synthesis begins at once, lasts for 3 h and is followed by a G2 period of about 5.5 h before the next mitosis [7]. A study by nuclear magnetic resonance (NMR) of extracts of macroplasmodia revealed a large amount of proline and temporal changes in this and in other amino acids (Dobson, Poulsen and Threlfall, unpublished). This has prompted us to investigate these changes in detail using more sensitive methods of amino acid analysis. MATERIALS AND METHODS Growth of PhysarumStrain M3C was routinely grown as microplasmodia in 500 ml indented conical flasks containing Abbreviation. NMR, nuclear magnetic resonance. Enzyme. Glutamate dehydrogenase (EC 1.4.1.2). 50 ml tryptone citrate medium with haemin in an orbital shaker [8]. Macroplasmodia were established from microplasmodia in the usual way [9]. The second mitosis (M2) after fusion of microplasmodia occured 15-16 h after feeding and the third (M3) about 8 h later. 3 h before M2 the medium was discarded and replaced by fresh. Incubation of all cultures was at 26°C in the dark. The time of metaphase and other mitotic stages was determined by phase-contrast observation of ethanol-fixed smears of small pieces of plasmodium mounted in glycerol/ethanol (111, v/v). The appearance and duration of these stages was the same as described [lo]. Extraction of Amino AcidsA whole plasmodium or 30 s in 1000 ml ice-cold 0.01 buffer pH 4.6. Excess liquid sector was washed for M sodium citrate/HCl was removed and the growing region quickly excised with a spatula to a polypropylene tube cooled in liquid nitrogen until required. The material was homogenised once for 15 s in 30 ...

Tritiation of α-bungarotoxin with N-succinimidyl [2,3-3H]propionate. A useful reagent for labelling proteins

“…0. Dolly, E. A. V. Nockles, M. M. S. Lo and E. A. Barnard metrically (Lowry et al, 1951), was confirmed by the alanine and valine contents determined by amino acid analysis (Threlfall & Thomas, 1979), using pure a-bungarotoxin as standard in both cases. Biological activities of all toxins were assessed from their ability to bind irreversibly to acetylcholine receptor of cat denervated leg muscle and electric organ of Torpedo marmorata (Lo et al, 1981); the receptor was solubilized with Triton X-100 and its binding to the tritiated toxins (15-30nM) was measured at 250C or 370C by the DEAE-cellulose disc assay and appropriate blanks plus controls .…”

Controlling Tumor-Derived and Vascular Endothelial Cell Growth

“…Early reports have demonstrated that significant amino-acid transport occurs in mitosis as well as in interphase. 73,74 Anti-␤E11 probably inhibits amino acid transport during interphase and possibly mitosis. The gross observation that cells inhibited in growth by anti-␤E11 do not arrest at a certain phase of the cell cycle suggests that amino acid transport mediated by the ␤E11/4F2 antigen is not limiting for cell cycle progression and growth.…”

“…A hallmark of many growing tumors is extensive angiogenesis, 70 a process characterized by proliferation of endothelial cells that extend the vascular network so that nutrients may be effectively delivered to rapidly dividing tumor cells. [71][72][73][74] Anti-␤E11 alone or conjugated to a toxin may not only inhibit the growth of tumor cells but may also attenuate angiogenesis. Therefore, in terms of tumor therapeutics, anti-␤E11 may have distinct advantages over other monoclonal antibodies that have been used to inhibit tumor cell growth.…”