When normal quiescent (G0) cells are stimulated by mitogens to enter the cell cycle, the metabolic derepression which occurs is similar in a variety of cells. The mechanisms initiating these responses and their relationship to subsequent progression through G1 to DNA synthesis in S phase, however, are generally undefined. The clearest evidence has been obtained in sea urchin eggs, where fertilization by sperm causes a rapid, transient increase in the concentration of free cytoplasmic Ca2+ [(Ca]i), followed by a sustained increase in cytoplasmic pH (pHi). It has been demonstrated clearly that these ionic responses are obligatory for progression to DNA synthesis by the normal pathway after fertilization, although the Ca2+ signal can be bypassed by parthenogenetic agents which elevate directly pHi (for example, NH+4 ions). These observations raise the questions of whether other eukaryotic cells show the same sequence of ionic responses when stimulated by mitogens and whether such signals are an obligatory component of their mitogenic pathways. We show here that a common sequence of [Ca]i and pHi responses occurs in both quiescent mouse thymocytes and Swiss 3T3 fibroblasts stimulated by appropriate mitogens. Furthermore, 'opportunistic' mitogens (those that do not act on the cells in vivo, such as concanavalin A (Con A), the Ca2+ ionophore A23187 and 12-o-tetradecanoyl phorbol 13-acetate CTPA] that are mitogenic for both mouse thymocytes and 3T3 fibroblast, each produce characteristic ionic responses that are the same in both types of cell.
X-ray crystal structures of methylmalonyl-CoA mutase in complexes with substrate methylmalonyl-CoA and inhibitors 2-carboxypropyl-CoA and 3-carboxypropyl-CoA (substrate and product analogues) show that the enzyme-substrate interactions change little during the course of the rearrangement reaction, in contrast to the large conformational change on substrate binding. The substrate complex shows a 5'-deoxyadenine molecule in the active site, bound weakly and not attached to the cobalt atom of coenzyme B12, rotated and shifted from its position in the substrate-free adenosylcobalamin complex. The position of Tyralpha89 close to the substrate explains the stereochemical selectivity of the enzyme for (2R)-methylmalonyl-CoA.
Pure complexes of dipalmitoyllecithin (DPL, 16:0) which Ca2+, Mg2+ dependent ATPase from sarcoplasmic reticulum are unusual in retaining significant ATPase activity down to about 30 degrees C, well below the transition temperature of the pure lipid at 41 degrees C. A minimum of about 35 lipid molecules per ATPase is required to maintain maximal ATPase activity, but the complexes are progressively and irreversibly inactivated at lower lipid to protein ratios. Complexes containing more than the minimum lipid requirement show very similar temperature profiles of activity about 30 degrees C over a wide range of lipid to protein ratios, up to 1500:1. Spin-label studies indicate that, at lipid to protein ratios of less than about 30 lipids per ATPase, no DPL phase transition can be detected, but at all higher ratios, a phase transition occurs at about 41 degrees C. In all of these complexes there are breaks in the Arrhenius plots of ATPase activity at 27--32 degrees C and at 37.5--38.5 degrees C. Experiments with perturbing agents, such as cholesterol and benzyl alcohol which have well-defined effects on the DPL phase transition, indicate that these breaks in the Arrhenius plots of ATPase activity cannot be attributed to a depressed and broadened phase transition in the lipids near the protein molecules. These results are interpreted as evidence for a phospholipid annulus of at least 30 lipid molecules with interact directly with the ATPase and cannot undergo a phase transition at 41 degrees C. This structural interaction of the ATPase with the annular DPL molecules has a predominant effect in determining the form of the temperature-activity profiles. However, the perturbation of the DPL phase transition does not extend significantly beyond the annulus since a phase transition which starts at 41 degrees C can be detected as soon as extraannular lipid is present in the complexes. We suggest that it may be a general feature of membrane structure that penetrant membrane proteins interact with their immediate lipid environment so as to cause only a minimal perturbation of the lipid bilayer.
Symmetrically substituted difluoro derivatives of 1,2-bis(o-aminophenoxy)ethane-NNN',N'-tetraacetic acid (nFBAPTA) show large '9F NMR chemical shifts on chelating divalent cations. The complexes of Ca2+ with 4FBAPTA and 5FBAPTA show fast and slow exchange behavior, respectively, and the chemical shift or the areas of the resonances from the free and complexed forms can be used to determine the free Ca2+ concentration. The measurement of the free Ca2+ concentration by either ligand is unaffected by free Mge+ concentrations <10 mM, bypH 6-8, or by contaminating divalent ions of high affinity (Zn'+, Fe +, Mn2+). The tetraacetozymethyl ester derivative of 5FBAPTA was used to load mouse thymocytes with 5FBAPTA to intracellular concentrations of 1 mM, and the '9F spectrum indicated a free intracellular Ca2+ concentration ([Ca] 7178The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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