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  • 1
    Electronic Resource
    Electronic Resource
    Characterization of renatured profilin purified by urea elution from poly-L-proline agarose columns (1989)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 14 (1989), S. 251-262 
    Details
    ISSN: 0886-1544
    Keywords: Acanthamoeba ; affinity chromatography ; Dictyostelium ; NMR spectroscopy ; platelets ; myosin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We present evidence that native profilin can be purified from cellular extracts of Acanthamoeba, Dictyostelium, and human platelets by affinity chromatography on poly-L-proline agarose. After applying cell extracts and washing the column with 3 M urea, homogeneous profilin is eluted by increasing the urea concentration to 6-8 M. Acanthamoeba profilin-I and profilin-II can subsequently be separated by cation exchange chromatography. The yield of Acanthamoeba profilin is twice that obtained by conventional methods. Several lines of evidence show that the profilins fully renature after removal of the urea by dialysis: (1) dialyzed Acanthamoeba and human profilins rebind quantitatively to poly-L-proline and bind to actin in the same way as native, conventionally purified profilin without urea treatment; (2) dialyzed profilins form 3-D crystals under the same conditions as native profilins; (3) dialyzed Acanthamoeba profilin-I has an NMR spectrum identical with that of native profilin-I; and (4) dialyzed human and Acanthamoeba profilins inhibit actin polymerization. We report the discovery of profilin in Dictyostelium cell extracts using the same method. Based on these observations we conclude that urea elution from poly-L-proline agarose followed by renaturation will be generally useful for preparing profilins from a wide variety of cells. Perhaps also of general use is the finding that either myosin-II or alpha-actinin in crude cell extracts, can be bound selectively to the poly-L-proline agarose column depending on the ionic conditions used to equilibrate the column. We have purified myosin-II from both Acanthamoeba and Dictyostelium cell extracts and alpha-actinin from Acanthamoeba cell extracts in the appropriate buffers. These proteins are retained as complexes with actin by the agarose and not by a specific interaction with poly-L-proline. They can be eluted by dissociating the complexes with ATP and separated from actin by gel filtration if necessary.
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970140211
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  • 2
    Electronic Resource
    Electronic Resource
    A Stopped-flow/rapid-freezing machine with millisecond time resolution to prepare intermediates in biochemical reactions for electron microscopy (1990)
    New York, NY : Wiley-Blackwell
    Journal of Electron Microscopy Technique 16 (1990), S. 160-166 
    Details
    ISSN: 0741-0581
    Keywords: Stopped-flow ; Rapid-freezing ; Freeze-fracture ; Electron microscopy ; Rapid reactions ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: We have developed an instrument capable of freezing transient intermediates in rapid biochemical reactions for subsequent freeze-fracturing, replication, and viewing by transmission electron microscopy. The machine combines a rapid mixing unit similar to one widely used in chemical kinetics (Johnson, 1986) with a propane jet freezing unit previously used to prepare static samples for freeze-fracturing (Gilkey and Staehelin, 1986). The key element in the system is a unique thin-walled flow cell of copper that allows for injection and aging of the sample, followed by rapid freezing. During freeze-fracturing, a tangential cut is made along the wall of the flow cell to expose the sample for etching and replication. The dead time required for mixing and injection of the reactants into the flow cell is less than 5 ms. Electronic controls allow one to specify, on a millisecond time scale, any time above 5 ms between initiation of the reaction and quenching by rapid freezing.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jemt.1060160206
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